Definition / Time Period

Definition / Time Period

I love too read books and feel the accomplishment of seeing the words "the end". It makes me feel like I completed something, and I think I will struggle with not having an end necessarily.

This is interesting. After reading some poems lately it really puts things innto perspective how the things you read about had to exist in order for them to "belong" to the story.

Human beings often fail to appreciate the good things when they have them, overlooking how amazing a moment can be because it is "normal" or frequently occurring. The metaphor of a "backdrop of abundance" highlights the paradox of excess humans experience: the good, instead of enriching experience, can instead dull perception. Once it is gone is when we truly realize what we had and now do not. This speaks to a broader theme in Feet in Smoke, where memory and love can blur and even lose impact when it has become the "typical" in our everyday lives.

This makes the authors story more human. Him and his brother used music less as a way of speaking and more of a way of bonding. The author also mentions a 7 year age gap his brother and him have and how they started to bond after his brother discovered him playing music later in the essay. This reinforces the way music connects the brothers, it acts less of a form of entertainment and more of a natural force that binds them.

Upon reading the entire essay, its very interesting how music is so deeply important and valued to Worth and something that connects him to his brother, yet his passion for it lead to his "death." It's ironic how a microphone electrocution is what killed a musician.

The many internet cultures and subcultures.

when a TV series has no good progression in the native the series is probably going to be canceled or loss a lot of money

sometimes if a story inconsistency between multiple parts of the story the author\creator will make another story between the OG story clear it up and make it more understandable for audiences

yes These events can be very slow paste to flush out the plot and to introduce interesting ideas and creations

yes this very true anticipated events from soup can be very interesting and entertaining to people

The narrative moves the story forward and causes a problem for that will be solved later in the future of the story

The narrative tells a story through a spoken or written account of connected events; a story.

In TV series, the problem or conflict repeats. ach week returns to the same basic setup (family tensions, workplace drama, etc). New events happen, but the core problem never fully resolves

TV action shows often use small, repeatable segments instead of one big mystery or plot. Each clinch is like its own mini-story, giving the episode structure without needing a strong overall mystery.

TV assumes viewers won’t watch with full focus or remember every detail. People might miss episodes or get distracted. Because of this, TV shows must be easy to follow at any point unlike films, which expect full attention from start to finish.

sitcom = new problem every episode, news = new events each day

TV is usually watched at home, not in full focus. People might be cooking, talking, or doing other things while it’s on. This is kept on mind when making some shows so they give us recaps, and easy to follow segments.

The narrator has follow the rules of the director or author when announcing certain events in a story

Tv series tend to have a more connected and longer story than films even though the episodes are short form due to the fact that the issues created with in the story can be prolonged. This makes the viewer more interested as they want a solution to the problem, but this can lead to a decrease in narrative progression.

In cinema events tend to be more fast paced than the segments that are broadcasted so a story can conclude quickly rather than going on through several episodes of a show.

The narrative in a way drives the story forward and generally requires a problem to be solved to avoid later consequences.

Narratives tell a story and bring segments of a series together through an overarching theme or issue within the series making the segments feel interconnected.

Narration has to follow certain guidelines to be considered proper narration and is very different when compared to the distinctions of fiction and non-fiction.

high cholesterol leads to pathology. in normmal amounts, synthesizes vitamin D (integumentary system, this is the system. hits sunlight in the skin, in cell membranes of epithelial cells which has lots of cholesterol) makes any steroid hormones need cholesterol.

more double bonds, easier to metabolize. single bonds are saturated with hydrogens, dont let enzymes in as easily. linked to pathology in the body like heart disease.

surface tension, due to hydrogen bonds between water. if water gets in lungs, pneumonia, pulmonary edema, have water that will cause hydrogen bonding w other and lungs will collapse and do bad exchange, from amniotic fluid life to air life, they're collapsed and need to expand, release surfactant. breaks down hydrogen bonds, and they open up. analogy is pool party balloons fall in water, cant pry them apart, put detergent, the balloons inflate

have slippery membranes so the heart beating doesnt have friction, pericharditis is life threatening rub.

gallbladder stores bile and releases during fats, pain in upper right quadrant gall stone. appendix in lower right quadrant appendicitis. left upper stomach ache, lower left intestinal issues. a bit of a clue of whats going on to a clinician

e.g. thermoreceptor, produce action potentials, brain receives sensory information through the afferent pathway, control center is hypothalamus brain stem, effector depends on change will affect, e.g. shiver contract muscles rapidly, sweat glands to release sweat and evaporation occurs.

lots of fluctuations in environment

indiferrent gonad. progression to ducts, ovary analagous to testes, germ cell production.

also smooth muscle. bladder has capacity.

involuntary smooth muscle. if ozempic u control argument? stretch receptors satisfaction food in stomach. absorption has different transit times depending on food. protein longer, carbs shorter. microbiome bacteria living in colon, control set point for weight, mood, compelling studies

super spongey.

sometimes fluids accumulate. edema, or swelling. opportunity to cleanse body. deep tissue massage.

different environments. homeostatic mechanisms to not succumb to environment. selectively permeable membrane of the cell. gated channels, so selective, maintains great boundaries

This stood out to me because it connects TV flow to gender roles.Williams imagined one kind of viewer, but Modleski pointed out how women experienced TV differently. It makes me realize how media studies always has to account for whose perspective is being centered.

I like how this shows Williams wasn’t just describing TV scheduling he saw “flow” as shaping ideology. It makes me think about how much the way media is organized and controls how we understand the world.

How do austerity measures reduce public debt?

This might not be the most helpful annotation, but I couldn't help but notice/appreciate Trouillot's word choice. Though obliterate is defined as meaning to utterly wipe out, I think also of the French word “oublier,” which means “to forget.” He goes on to use the terms obliteration/obliterate again later in this chapter. Given the context (Trouillot’s knowledge of French, and the discussion of silences and memory in history) this seems like a deliberate choice meant to evoke a specific meaning. Sans Souci the man was made to have been forgotten, ignored, or to use Trouillot’s terminology, silenced in the historical narrative of the Haitian Revolution.

Watching the news, I sometimes feel informed but also overwhelmed. Its like everything is happening at once but I don’t fully understand any of it. Williams does a great job expelling this feeling. TV doesn’t give me a clear picture of the world, it just gives me the vibe of being “up to date.”

He’s right; TV throws all kinds of stuff at us, but somehow it feels like one whole experience. That’s why it’s so hard to analyze TV the same way we analyze books or films. It’s not about single stories, it’s about the mood or feeling the flow creates.

This is very true. I’ll plan to watch one episode and suddenly it’s three hours late. It makes me feel like TV has always been designed to keep us stuck to the screen.

Williams explains how advertising changes TV itself. Commercials don’t just “interrupt” programs but they become part of the overall flow.

Williams is points out something we almost take for granted, that television isn’t about isolated shows but about a continuous flow of programming. It suggests that TV’s power lies in shaping how we experience time and continuity in everyday life.

In principle I understand but I don't understand how the equation explains the message it's trying to convey @rebelford

This reflects the unanimous decision in Cantwell v. Connecticut, which was decided seven years prior to this case and likely set a precedent. The Fourteenth Amendment, specifically the Equal Protections Clause, prohibits discrimination by local, state, and federal governments (solified by the Cantwell decision) on the basis of religion. With transportation being a public service and religious schools being private institutions, it can be expected that transportation to a private school would be less profit-oriented just as transportation to a public school would be.

I really enjoy how the author is is explaining the meaning of friendship and goodness through the relationships of humans and animals. At this point in the story, it can be assumed that the goodness and love of humanity is the main theme being discussed.

It is interestig to see the mother reply in this manner as earlier, she mentions that animals are not worth receiving education and clearly explains that humans have value over animals, even calling animals inferior. Here, she is expaining that most people who would treat an animal with care would of course already treat their family with kindness as treating the animals well was alreayd a sign of having a good heart. Is she explaining this in agreement, that animals shouldn't be seen as inferior? Or is she simply saying this?

I love this last line, and how it conveys the message shown throughout the story. Seeing how this incredible dog, Apollo, in the beginning of the story is praised for knowledge, you would think he would be the best and most liked dog. However, its the dog that showed more compassion, care, and empathy that was liked the most. I like how this dog comparison serves as a moral lesson for both animals and humans, where sometimes, it is not about what you can do, but who you are on the inside.

When I read this line, I think William is explaining how he thinks it is easier to believe something so out of the ordinary, like a dog talking, rather than believing that a man of higher authority would ever lie. I think this goes hand-in-hand with the time that this story was written, where everyone believed anything that a powerful man (a king, dictator, etc.) was saying. This is a small piece in a large story that shows how these individuals think.

when she was in the Chicago,she was remembered his father and she have good feel because due to his father ,she had good future and happy life ,.she was thankful of his father.

Families sacrifice a lot, especially foreign ones. They want the best for their children and for them to do more with their lives.

She wants to prove that she can have an education and be successful.

Hispanic families often have traditional roles. For women they are expected to stay at home, cook, clean, and care for the children, while their husbands provide.

i.e., scholars are divided by discipline, if they get to claim X is a discipline, so can IS

I like that this article, which discusses religion, emphasizes the life before death rather than life after death. In my personal experience, lots of religious education has been about the life you need to leave before death to enjoy your life after death, which for me was never particularly captivating.

This quote resonates with me, because in my high school AP Psychology class we discussed a phenomenon called confirmation bias, wherein an individual will seek out information to reaffirm their prior beliefs. Now in my everyday life I have become self aware of this human tendency and try my best to avoid it.

I can see how Alice's resist to being an author shows an opposite contrast to her mother, Mrs. Courland. I believe Alice is a realist, and more practical, while her mother is a dreamer. Showing two opposite personalities throughout the story escalates the plot, and I am sure the author did this for that reason.

light molecule because the imfs are easier to break @rebelford

A Norwegian business has developed a method for turning unproductive, sandy, soil, into productive, arable soil, in around 7 hours. It costs around 2 USD per square meter, in 2025, but costa may go down in the future.

a Permanent dipole is a molecule which has an uneven distribution of electrons. When atoms in a molecule have different electronegativities, electrons are pulled towards one atom. Example: H2O aka Water - Oxygen is more electronegative than hydrogen, thus pulls the shared electrons closer to itself, giving it a partial negative charge. This. along with its bent shape creates permanent negative and positive charges at opposite ends.

This shows that these new technologies aren't very versatile, which won't allow for it to thrive. It needs to function in the digital economy and speak numerous languages for it to be fully effective.

IT playing single player games with puzzels and problem solving, while managers played more competitive action roleplay games enhancing problem solving and managerial skills. Engineers played a game where you would build civilizations from scratch. A lot of the games played ties into each profession

Women stayed away from multiplayer confrontations games that is a representation of structural issues with sexual harassment or masked their gender to stay away from scrutiny

recap so far: vietnam land divided with a mountain range running through the country north to south...in history vietnamese land expansion was seen in two phases: resisting china to the north, and expansion to the south...south vietnam seen as a place throuhout history as a place to banish and exile people

This question portrays an ongoing theme throughout the text, which is power and authority. Asking who is really the slave and who is really the mistress kind of messes with the normal, more traditional roles in a slave-owning house. Adding this question made me stop and think to myself, do slave-owners have as much control as they think they do? I thought that was cool to ponder on.

I am unsure the definition of this term.

A document of a students academic records.

To work at the school you are enrolled in.

When a student is given something in reward for an achievement.

Funds given to a student for accomplishments that do not have to be payed back.

I am unsure of the definiton.

An application to receive financial aid?

Money loaned from the government to help pay for school.

A career choice a student chooses to pursue for the rest of their working years.

i am unsure the definiton.

The classes you must take and pass in order to advance to your major of choice.

A form of completion for a course.

A class that the student chooses that sparks their interests.

The cost it takes to attend a certain class or program.

The required amount of time you must spend enrolled in a class.

I am unsure the definition of this phrase.

Student put on a trial to monitor how their doing.

A number that is used to identify you and verify you attend a certain school.

To be accepted to a school.

To successfully attend a school.

Signing up for classes of your choice.

The two semesters that make up an entire school year.

Is their a limit to how many notes you can make a text and what the difference between notes and tags and highlighting which one is better

Viewers have control of what becomes popular and help connect television to more traditional art through the reinforcement the broadcast system brings. When people start talking about their favorite shows or films they can describe it as an art and find it similar to more traditional forms of art.

By crossing the lines of fantasy and reality viewers are forced to look at media differently which creates a deeper story through something other than the conventional narrative American tv offers.

To bring more customers and to interest them boxes were typically designed to match the content of a series in hopes it would intrigue the viewer and convince them to purchase it. Boxes also were memorable for those who have already seen these shows.

The 1950s was a time for many technological changes and changed how television is seen today still leaving an impact today.

This is interesting to note, since legality isn't something that can be figured out by itself. This has to be done by government figures who are paying attention to these issues.

This is really interesting to think about since AI only knows what it is given. This ends up making people of color less represented by AI, since they don't have information surrounding these groups of people.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Response to reviewers

We thank the reviewers for their constructive feedback, which has greatly improved the clarity and rigor of our manuscript. We have carefully addressed each comment below, indicating changes made to the text, figures, or supplementary material where appropriate. References to line numbers correspond to the revised version of the manuscript.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

* In this paper, the authors focus on the role of Reticulon-1C in concert with Spastin in response to axonal injury. In data mining, they find axonal mRNAs encoding for ER-associated proteins including Rtn-1. They establish a knockdown targeting both Rtn-1 isoforms Rtn-1A and Rtn-1C. They observe decreased beta-3-Tubulin levels in the soma while axonal protein levels are unchanged. In microfluidic devices, they characterise the effect of a compartment-specific Rtn-1 KD on axonal outgrowth in the axonal compartment. The authors quantify axonal outgrowth, seeing increased outgrowth in an axonal compartment-specific Rtn-1 KD, while the effect seems to be reversed when applying the KD construct in the somatic compartment. When focussing on the axonal growth cone, they find the Rtn-1 KD shows differences in several morphological features of the growth cone. They find an increase in Tubulin levels in an axonal compartment-specific, but a decrease in a somatic compartment-specific Rtn-1 KD. Colocalisation of Rtn-1C and Spastin is shown to be monolaterally increased following axotomy. Combining axotomy with the Rtn-1 KD shows increases in dynamic microtubule growth rates and track lengths. In another model system, neuron balls, they show Rtn1-C, but not Rtn1-A to be present in the axon. In a puro-PLA assay they also show it can be synthesised in the axonal compartment. To investigate the mechanism enabling the cooperation between Spastin and Rtn-1C, they move to a cell line model in which they see a correlating distribution between Spastin and Rtn-1C but not Rtn-1A. Finally, they use in silico modelling to speculate on binding between Spastin domains and Rtn-1 isoforms.*

Major comment:

The rationale behind the work is convincing, however some interpretations are presented as more robust than some data allow. Most notably, while the interaction between Rtn-1 and Spastin has been shown prior to this study, it is only presented here through in silico analysis. In figure 5, an increase in the growth rate of dynamic microtubules is observed in either a Rtn-1C KD or by using a Spastin-inhibitor. Due to a described increase in colocalisation between Rtn-1C and Spastin (5A), the increase in growth rate is displayed as caused by Rtn-1 promoting Spastin's severing ability. This result might however be correlative. Further in the injured samples, Spastin-levels seemingly increase (in the representative images) and it is thus not surprising that the level of Rtn-1C colocalising with Spastin increases as well. This might not be indicative of a cooperation and further experimental evidence are required.

R: We thank the reviewer for this thoughtful comment. We agree that our interpretation should be more cautious, and we have revised the Title, Results and Discussion sections accordingly. In particular:

1. Following yours and other reviewer comments, we have analyzed a new set of experiments regarding the STED images of non-injured and injured axons. To eliminate the risk of artifactual descriptions, we have avoided deconvolution and worked directly with raw STED images (Figure 5A). Under these conditions, the distribution of Spastin and its intensity in distal axons are not modified by injury, nor those of Rtn-1C and Spastin (Supplementary figure 4). We emphasize in the revised text that the in silico modeling we present is supportive, but not definitive, of a direct interaction. To address this concern, we clarify that our study builds on prior evidence of biochemical interaction between Rtn-1C and Spastin (Mannan et al., 2006), and that our own data demonstrate: i) compatible subcellular distribution in axons by super-resolution (STED microscopy, Figure 5A);ii) a potential functional interplay in axons (rescue of β3-tubulin levels by Spastin inhibition, Figure 5B), and iii) isoform-specific co-distribution with Spastin in heterologous cells that is associated with changes on microtubule integrity (see improved Figure 7). Together, these results go beyond correlative localization, but we acknowledge that they do not directly demonstrate a molecular complex in axons. Thus, we now indicate that "Although we did not directly test their molecular association, these results are consistent with Rtn-1C and Spastin sharing a similar subcellular localization, potentially enabling their functional interaction in distal axons" (lines 285-287)

We would like to clarify a possible misunderstanding: in our experiments, the increase in microtubule growth rate was observed after axonal Rtn-1 KD. Spastazoline (SPTZ) only prevented the reduction in β3-tubulin levels induced by Rtn-1 KD, while leaving the KD-driven increase in growth rate and track length unaffected (Figures 5B-E). Thus, our interpretation is that axonal Rtn-1 KD correlates with increased Spastin function. (lines 307-309)

Other comments:

• Generally, graphs would benefit from individual values plotted as well as the summary. Font sizes and types (but rarely) are sometimes inconsistent. Proteins should be consistently written (capitalised or not).

__R: __ We agree with the reviewer and thank for taking the time for noticing these inconsistencies as it significantly affects the quality of the work. We have improved several figures and added graphs plotting individual values (Figures: 2 C, 2E; 4 (A-E); 5E; 6D). We have reviewed the Font size and types more carefully and capitalized the proteins accordingly.

• *Table 1 and figure 1 present data collected from a vast amount of resources. It should be highlighted that datasets from which data was obtained includes many different models, different DIVs and neuronal cell types. Figure 1B may benefit from a different colour scheme. "Ex-vivo" should be "Ex vivo". For "ER mRNAs are a relevant category" it is not described what "relevant" would mean in this context. The title might remove this small part or describe it in the text. It should be described how it is decided that mRNAs are "common". *

• *

__R: __We have now highlighted in the result section the diverse origins of the analyzed samples; We removed the indicated part from the text and explained that common mRNAs were chosen based on the Benjamini-Hochberg (Ben) analysis. (Page 33, lines 1299-1304).

* - Figure 2: add description to y-axis to describe what fold change is displayed, applies to multiple figures. Will improve readability of the figures. In 2C, the ROI showing neuronal somata should be increased to show part of the axon and not cut off the soma.*

• *

__R: __We thank the reviewer for taking the time to highlight this. We have included this modification in figure 2 and throughout the article. We have also enlarged the indicated ROIs in figure 2C as requested. (Page 34)

• *Figure 3: Three out of four axonal compartments seem to be comprised of dying or damaged axons. Especially the axonal KD scrambled image. It should be ensured that neuronal cultures are healthy. *

• *

__R: __We completely agree with the reviewer that the selected images were not describing the general good health of axons which has been accredited by the lack of fragmentation and functional responsiveness shown in (Figure 4 and 5 B, C, E). Thus, we have now replaced the previous axonal fields by more representative ones (Figure 3). (page 36)

• *

Typo in "intersections". The schematic of 3B is a great addition to explain the graphs above. Perhaps it could be a bit refined as it is currently hard to see whether this is a neuron or a growth cone without context. Maybe show where the axon connects to the depicted growth cones and change the third icon which looks like it was crossed out. Small formatting issues: remove additional space bar before "Figure 3." And add after "Bar"

__R: __Many thanks for these great suggestions. We have now improved the figures as suggested and changed the indicated formatting issues. (page 36)

- Figure 4: If not misunderstanding what is depicted, in 4A and B, different lookup tables are used to depict the same signal. Only one of each images is necessary. Do the axons have more tiny branches in the Rtn-1 KD condition in 4A? Unclear why Rtn-1 levels are increased in the Rtn-1 KD (4C), please clarify.

• *

__R: __We thank the reviewer for these observations. The reviewer is correct that different lookup tables were initially applied to the same image. Our intention was to highlight the fine distribution of axonal Rtn-1, but since this aspect is already clearly shown in previous figures, we now retain only a single lookup table. The appearance of tiny branches in the Rtn-1 KD condition represents an isolated observation and does not reflect a consistent or robust phenotype associated with Rtn-1 KD.

As the reviewer points out, the increase of Rtn-1 in the cell bodies of injured neurons following axonal KD was initially surprising to us. However, this was a consistent phenomenon, as shown in the improved Figure 4. Of note, previous studies have reported that total Rtn-1C (but not Rtn-1A) levels increase in response to injury in cortical neurons(Fan et al., 2018). In our case, we interpret this as a compensatory somatic response triggered by the local reduction of Rtn-1 in injured axons. This interpretation is also consistent with the apparent lack of effect of siRNA on distal axonal Rtn-1 levels when applied locally after injury (while somatic application of the same siRNA does reduce axonal Rtn-1). Thus, after 24 hours of KD, the somatic upregulation of Rtn-1 may partially compensate for its expected local synthesis decrease. We have clarified this assumption in the revised text. (lines 247-251)

- Figure 5: It may be easier to understand what "axotomy" samples are if just referred to as "injured" as later in the same figure. The procedure could also very briefly be explained in the results. 5C should depict AUC in µm2 not µm. 5D Spastin is barely visible, brightness and contrast should be adjusted to enhance visibility.

• *

__R: __We thank the reviewer for these helpful suggestions and have implemented the requested changes in Figure 5. Specifically:

We now consistently refer to "axotomy" samples as "injured" throughout the figure and article. In addition, a brief explanation of the axotomy procedure has been added before Figure 2 and before figure 5, also the description has been clarified in Materials and methods. (lines 191-192) and (lines 289-290) and (lines 779-787)

To improve the reproducibility of our outgrowth measurements, we revised this analysis approach. Based on previous work from a co-autor (McCurdy et al., 2019), instead of reporting the "relative number of intersections," we now present the total counts obtained from Sholl analysis of binarized axons (see Methods). To this end, we took advantage of the NeuroAnatomy plugin of FIJI, which more precisely tracks axon trajectories and makes the measurement more independent of axon width. Also, this new approach avoids the conflict we had with what we considered the "first line" after the groove ends, which was a bit of arbitrary. Accordingly, the correct term is now "summation of intersections (sum.)" at different distance bins, as reflected in Figure 5D. (page 40)

For the former Figure 5D (now Figure 5B), we have improved the acquisition of representative images and applied a different set of lookup tables to enhance visibility. (page 40)

- Figure 6: It should be made clear why it is necessary to switch to another model system just for 6A, please indicate this in the text. PCR bands seem very pixelated, check the quality. It is unclear why soma genes/proteins were only tested with either PCR or WB others with both. Rtn-1C and Rtn1-A should be presented in the same order in the PCR and WB panel. Correct "Rtn1-1A" typo. In 6D, 1.5 dots per soma seems like a low number. When normalized to the area the soma vs the axon occupies, the compartmentalization does not work? Maybe it makes sense to refine analysis or apply puromycin in the somatic compartment and analyze the axonal compartment as comparison?

__R: __Many thanks for these observations. We have now included the following clarification in the text: "We sought to characterize the isoform expression of Rtn-1 mRNA and protein in both axons and cell bodies. Because microfluidic chambers yield only limited cellular material, we adopted an alternative culture approach using 'neuronballs.' This method enables the segregation of an axon-enriched fraction by mechanically separating axons from somato-dendritic structures" (lines 375-376).

The resolution of PCR bands has been improved in the revised figure. Note that because the amount of cellular material is relatively scarce, we did not obtain too strong bands.

The difference in the genes/proteins used for characterizing RNA and protein samples reflects our intention to treat both approaches as complementary. The PCR markers were primarily included to confirm sample purity, which also applies to the WB samples since they derive from the same preparation. In both assays, we used MAP2 as a dendritic marker to demonstrate axonal purity. While we acknowledge that the same genes could have been tested by both methods, we believe the results as presented adequately demonstrate the effective isolation of axons.

We have switched the order of Rtn-1C/1A for consistency across PCR and WB panels and corrected the indicated typo in Figure 6A.

We agree with the reviewer that an average of 1.5 puncta per soma initially appeared low. We have identified at least three reasons for this:

First, the signal derives from only a 15-minute puromycin pulse, which is a very short labeling window. Second, our puro-PLA assay is particularly stringent, as ligation relies directly on puromycin- and Rtn-1C-labeled primary antibodies, without the additional spacing normally introduced by secondary antibodies. In standard PLA, the critical distance for amplification is ~30-40 nm, whereas in our assay this distance is even more restrictive. Third, in our initial analysis we applied an overly cautious threshold to define "true" amplification. We have now refined this threshold using a baseline defined by the absence of puromycin stimulation. With this improved criterion, we now quantify an average of ~5 puncta per soma and ~10 puncta per 1000 µm² of axonal area (Figure 6D and Supplementary Figure 3D). Assuming a neuronal soma diameter of 15 µm (area ≈ 176.71 µm²), this yields ~0.028 puncta per µm² in soma. In comparison, axons display ~0.01 puncta per µm², approximately one-third of the soma value, which is compatible with the idea thar cell bodies dominate neuronal protein synthesis.

Following the reviewer's valuable suggestion, we performed additional quantifications in which puromycin was applied exclusively to the somatic compartment. Under these conditions, we still observed amplification in axons (~5 puncta per 1000 µm²), although this value was significantly lower than when puromycin was applied directly to axons. This analysis provided a novel appreciation of the puro-PLA technique in neurons: at least half of the signal originates in the axonal compartment, while a portion may reflect proteins synthesized in soma and transported anterogradely to the axon through yet-unknown mechanisms (potentially involving rapid anterograde transport) (Figure 6D). (page 42)

• Figure 7: 7A shows two images depicting the same information that may not be needed. Can probably be removed. In 7B there is no negative (or any) correlation between Spastin levels and Tubulin, however later it is mentioned that Rtn-1C transports Spastin thus causing a decrease in Tubulin at certain locations? It is nclear if Spastin levels vary intensely between different samples. Mean intensity of the somatic area may be beneficial to rule this out. 7B Tubulin on the right top panel seems to have a decrease in Tubulin levels which is not visible due to the Y axis of Tubulin being set to a different range than the middle and lower panel. The average of line scans from multiple cells may be helpful to determine whether there is indeed no colocalization between Rtn-1A and Spastin. The provided representative images seem to show similar degrees of colocalization between Spastin and Rtn-1A/C.

• *

__R: __We thank the reviewer for these valuable observations and acknowledge that Figure 7 may have caused confusion. We have eliminated the fluorescence line-scan traces, as they can be biased depending on the region of the cell analyzed. Although this may not have been sufficiently emphasized in the text, we had already performed a quantitative colocalization analysis across multiple cells and independent experiments, using Mander's coefficients (Figure 7B). These analyses showed higher colocalization between Rtn-1C and Spastin compared to Rtn-1A. Regarding the concerns about variability in Spastin levels or possible bias from Y-axis scaling, we have eliminated those traces by the risk of bias. Also, we had already quantified the total tubulin fluorescence intensity across all the z-stacks and from multiple cells from independent experiments as shown in Figure 7C. To further rule out artifacts caused by variable transfection efficiency, we quantified total fluorescence intensity in both RFP and GFP channels across conditions. As shown in Supplementary Figure 6, no significant differences were observed, suggesting that the changes in tubulin reflect specific effects of Spastin/Rtn-1C co-expression rather than variability in expression levels.

Results: - It would be helpful to reiterate the hypothesis at the start to ease the reading flow.

__ R: __Many thanks, we have introduced a line reiterating the hypothesis as suggested (lines 117-118)

- There seems to be minor redundancy in lines 132-138.

• *

__R: __Indeed, we have now removed the indicated phrase.

• There are several spellings, proof-reading is recommended. For example, in line 136 should be "promotes". 160 "localla", 192 should be "the actin cytoskeleton".,194 should be "we first examined", 195 should be "Different", 223 "using", 259 "axons".

__R: __We apologize for the spellings; we have now performed a careful proof-reading and introduced these corrections.

- 154-155: Unclear, why the lower MW Rtn-1C was seen as more important.

__R: __We apologize for not being clear enough. It is not necessarily more important, but we just took the Rtn-1C molecular weight as reference for the analysis considering that this isoform is the predominant in axons. In any case we have found a significant effect for both isoforms at least on siRNA 2 (data not shown), which is now expressed in the text (line 165-169) : "We also examined the 180 kDa band and found that siRNA 1 reduced expression to a mean of 0.41 relative to Scr, showing a strong trend that did not reach statistical significance (p = 0.05; N = 3; Wilcoxon test compared to 1, data not shown). In contrast, siRNA 2 further reduced expression to a mean of 0.29, which was statistically significant (p = 0.04; N = 3; Wilcoxon test compared to 1, data not shown)."

- 167 results of 2E not stated before interpreting them.

• *

__R: __We have corrected this mistake.

- 181 would suggest "outline" instead of "perimeter".

• *

__R: __We have considered this suggestion and included "outline", nevertheless the morphometric parameter is defined as perimeter, so we retained the term, but with the suggested clarification.

• *

- 183-184 "longest shortest path" is a confusing term.

__R: __We agree that it is a confusing term, thus have now introduced multiple clarifications for the term in the leyend of figure 3 (page 36), and with more detail in a new section of Materials and methods (lines 697-699).

• figure 4B should be referenced earlier in the sentence.

__R: __We have corrected the sentence in the text.

- 243-244 may be correlation. Rtn-1 and Spastin do not necessarily interact so that this result is achieved.

• *

__R: __Thanks for the clarification, we are aware that so far in the manuscript the conclusion is not correct, thus now we have stated at the end of the paragraph: "Together, these observations suggest that axonal Rtn-1 KD correlates with higher Spastin microtubule severing" (lines 307-309)

- 246: In figure 1 the KD seemed to influence both Rtn-1 isoforms, why not here anymore? 259 "axons". 284 "counteract" instead of "suppress"?

• *

__R: __We acknowledge the confusion at this point of the article because of measuring a specific isoform. We now indicate that we will focus on Rtn-1C because of previous evidence of the literature pointing to an interaction of Rtn-1C with Spastin (line 264-267). Later we show that Rtn-1C is the predominant isoform in axons (Figure 6). We have corrected all the suggestions in the manuscripts.

- 485: rephrase as the interaction between Rtn-1C with Spastin has not been shown directly in these experiments.

__R: __Many thanks for the relevant clarification. Now, we have corrected:" Here, we have described an emerging mechanism relating Rtn-1C with the activity of Spastin, which is the most frequently mutated isoform in HSP (Hazan et al., 1999; Mannan et al., 2006)." (line 632-634). * Methods: 535 "in PBS". 543 citation error. 689-699 is it necessary to add a gaussian blur?*

• *

__R: __We have corrected the words and removed the wrong reference. Regarding the use of Gaussian blur, it is a very important point. We used this approach because, in our experimental conditions, it was critical to highlight moving particles that otherwise would go unnoticed by the noise. This was particularly manifest for the seemingly more "unorganized" movements of axonal microtubules after injury.

References: Mannan, A U et al. appears twice in the citation list (36 and 44).

* *R: Many thanks for the observation. Now we have corrected it.

Reviewer #1 (Significance (Required)):

Overall, this manuscript describes novel fundings which will be interesting to the neuronal cell biology community and scientists working on the field of neuronal injury and regeneration. It is well structured, and the data are mostly well presented but sometimes conclusions are over-interpreted. However, several points need to be addressed in a more convincing way.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Axonal mRNA localization and localized translation support many neuronal functions and is an important determinant of the regenerative potential of axons after injury. How this works mechanistically remains unclear. The authors present a well performed and technically challenging study in which they identify RTN-1 as a regulator of axonal outgrowth after injury. They provide evidence using experiments in microfluidic chambers that RTN1 is locally synthesized in axons. Interestingly, they identify a (local) interplay between RTN1 and Spastin which affects microtubules and thereby regulates the outgrowth of cortical axons after injury. This study provides an interesting new link between a locally synthesized protein (RTN1) and a microtubule-regulating protein Spastin that is changed upon axon injury. This provides an advance in our understanding in axon regeneration after injury and provides the basis for new studies that can further investigate this interplay. Although interesting, I have several concerns that should be clarified and are needed to substantiate the findings and model presented in this study.

Major concerns:

2. In figure 1, the authors provide an analysis of overlapping axonal mRNAs. There are more axonal transcriptome studies and a recent study by von Kugelgen and Chekulaeva (2020; doi: 10.1002/wrna.1590) already performed such an analysis, which included more studies. It would be good to mention this. It can be perceived that studies were now chosen to get the outcome that Rtn-1 is present in all studies. For example, von Kugelgen finds mRNA coding for RTN3, another ER structural protein, as present in 16 out of 20 studies analyzed. That said, the authors present more reasons to look at Rtn-1, so the selection to continue with this protein remains valid but can be written up differently so not to present it as the 'sole' ER-shaping protein consistently present in axonal transcriptomes. __R: __We appreciate this important observation to enrich the article; we are aware that the transcriptome data can be even further expanded to more recent studies. Thus, we have now included this reference in the main text and highlighted the relevant finding of RTN3. However, Kugelgen and Chekulaeva used data from dendrites/axons (neurites). Thus, we indicate that "...On a similar approach, but combining data from dendrites and axons, it was found that Reticulon-3 *mRNA is present in 16 out of 20 studies, further suggesting a wider presence of other mRNAs coding for ER structural proteins in axons " (line 128-131)

3. The description of methods is currently insufficient and incomplete and does not allow for reproducibility of this study. For example, different Rtn-1 antibodies seem to be used in this study. Is the same antibody used for staining and WB? There is no listing of any of the antibodies used in the study and which one is used for which technique/experiment. This should be clarified and should be easy to do so in the methods section (antibody name, origin/company, dilution used) to enhance reproducibility of this study. This is not limited to primary antibodies and any information on secondary antibodies, including what was used for STED is completely missing.*

4. *

__R: __Thanks for these critical comments. First, we apologize for the former method version which was mistakenly not as accurate as it should. We have now revisited it and improved several points throughout this section. Regarding the use of primary and secondary antibodies, plasmids, siRNAs, and general reagents, they are all indicated in the Supplementary material, including company and dilution ("Reagent tables").

• The timeline of KD experiments in Figures 2 and 3 are unclear. For the Western blot KD is performed at DIV7 and collected 48 hours later. However, this is not specified for the stainings done in Figure 2C-E. Is this also at DIV7 and then for 48 hours? In figure 3 the siRNA is added at DIV8 (together with axotomy) and outgrowth is measured 24 hours later. Is 24 hours sufficient to achieve knockdown? Is this also what was done for stainings? Later on in Figure 5B, 48 hours of KD is again used. It is unclear what the rationale of these differing timepoints is. Why was this chosen? Is the timeline also the reason for the difference in segment lengths chosen? In Figure 3, there is a significant effect on outgrowth in the KD in the 'mid-range' which is not present in Figure 5.*

__R: __We regret the confusion, now all this information is explicitly clarified in the main text (lines 297-299) and the corresponding figure legends. We have strong reasons to have used these different time points. Figure 2 A-B is aimed at validating the siRNA against Rtn-1 thus we treated 7 DIV cultures for 48 hours to be sure of revealing a global effect by WB. In figure 2 C-D, we used the same 7 DIV cultures, but only for 24 hours. The reason for this is that, once the RNAi was validated, we explored its control on local synthesis in a shorter period based in previous literature supporting that axonal KD for 24 hours is sufficient for regulating axonal transcripts (Batista et al., 2017; Gracias et al., 2014; Lucci et al., 2020). We are also confident of using this time point based in the new supplementary figure 3D that shows a significant decrease on puro-PLA signal (indicative of Rtn-1C synthesis) 24 hours after axonal KD.

In figure 3, we performed axotomy thus we had to wait a longer period for axons to grow (8 DIV) before fully cut them out, in this case we performed axonal KD from 8 to 9 DIVs. This is the same period used for the staining and quantifications shown in figure 4. All this is properly clarified in the main text and figures.

In Figure 5 we performed a more challenging experiment that required to transfect cells with an EB3-GFP plasmid, then perform axotomy along with axonal KD as well as pharmacological treatment selectively in axonal compartment. First, we tried to measure microtubule dynamics under the same temporal frame of figure 3. Nevertheless, expression levels of EB3-GFP were not adequate for axonal measurements by live-cell imaging. Therefore, compared to figure 3, we increased the time frame after axotomy 24 hours (from 9 to 10 DIV) by this technical reason, but also to explore whether the changes on tubulin intensity might be revealed more clearly (which was the case, figure 5B). These considerations are now included in the main text

Regarding the significant effect on outgrowth in the KD in the 'mid-range' which is not present in Figure 5. Given that in figure 5D axons are left growing for two days instead of one, the number of intersections and the differences between conditions is modified compared to figure 3, while retaining the overall trends. Note that to improve the reproducibility of our outgrowth measurements, we revised this analysis approach. Based on previous work of a co-autor (McCurdy et al., 2019), instead of reporting the "relative number of intersections," we now present the total counts obtained from the Sholl analysis of binarized axons (see Materials and methods). To this end, we took advantage of the NeuroAnatomy plugin of FIJI, which precisely tracks axon trajectories and makes the measurements more independent of axon width segmentation. Also, this new approach avoids the conflict we had with what we considered the "first line" after the groove ends, which was a bit of arbitrary. Accordingly, the correct term is now "summation of intersections (sum.)" at different distance bins, as reflected in the new Figure 5D.

Could the authors provide a rescue condition for their siRNA (using a siRNA-resistant construct) to show that their siRNA is specific for RTN1. They nicely show the efficiency of the siRNA but not its specificity. This is crucial because if not specific, this will affect a large part of their study. They already have RTN1A and RTN1C constructs available. Such a rescue experiment should ideally also be performed for one or more of their phenotypic experiments, such as the one presented in Figure 3A or 5 to show that the phenotype is really RTN1 dependent. If done by re-expressing either RTN1A or RTN1C, this could provide insightful information on the relevant isoforms.

__R: __We agree with the reviewer that this is a critical point. A major challenge in demonstrating the functional role of axonally synthesized proteins using a KD approach is that the rescue may also need to occur locally. Since axonal Rtn-1 appears to play a distinct role compared to its somato-dendritic counterpart (Figure 3), a siRNA-resistant construct would ideally require an axon-targeting sequence to restore local synthesis. As this is technically demanding, we have not yet been able to perform such an experiment, but we are actively working on identifying the optimal sequence to direct Rtn-1C to axons. Importantly, studies performing axonal KD typically rely on at least two independent siRNA sequences, thereby minimizing the likelihood that a phenotype arises from off-target effects. Thus, we have now validated a third siRNA (siRNA 3), which selectively downregulates Rtn-1C. Then, following the same experimental frame of figure 3, we performed axonal Rtn-1 KD after injury and observed that siRNA 3 also significantly increases the outgrowth of injured axons (Supplementary figure 2). This suggests that, at least this phenotype, is not product of an off-target effect. Complementarily, pharmacological rescue with the Spastin inhibitor SPTZ mitigated both the reduction in distal axonal β3-tubulin and the increase on axon outgrowth, supporting that the observed phenotypes are unlikely to arise from off-target effects. If these effects were due to random interference with unrelated mRNA targets, inhibition of an ostensibly independent target such as Spastin would not be expected to yield such a consistent rescue. Accordingly, SPTZ treatment alone did not increase β3-tubulin, indicating that its action is specifically contingent upon Rtn-1 KD. Taken together, the pharmacological rescue in axons (Figure 5B) and the Rtn-1C/Spastin co-distribution in heterologous cells, which correlates with preserved microtubules (improved Figure 7), provide converging evidence to suggest that Rtn-1C-Spastin interplay may underly the observed phenotypes in axons.

• I find the data presented in Figure 4A/B confusing. Axonal RTN-1 KD does not reduce axonal RTN1 levels, but somatic KD does. I understand that this implies most protein comes from the soma, and the authors indeed present an explanation that increased somatic RTN1 occurs after axonal KD as a compensation mechanism. However, this can also be interpreted that there is no axonal synthesis of RTN1 after injury and axonal KD has indirect or even aspecific effects. Their model depends on this difference. Their data in Figure 6 could provide supporting evidence if it shows RTN1 puro-PLA after injury. Along these same lines, in Figure 6, they nicely include a compartment control for puro-PLA. It therefore seems doable to include a somatic puromycin control for their axonal puro-PLA, to exclude and diffusion/transport of the newly synthesized peptides. This is especially considering two recent papers reporting on this possible phenomenon, although these studies were not performed in neurons.*

__R: __We consider the possibility that after injury there is no axonal Rtn-1 synthesis as a plausible and relevant appreciation. Unfortunately, we could not perform a puro-PLA experiment after injury, which would have provided a more definite answer. However, now we are more confident of regulating Rtn-1 synthesis before injury as supported by a new supplementary figure 3D that shows a significant decrease on puro-PLA signal (indicative of Rtn-1C synthesis) 24 hours after axonal KD. Thus, based on the similar phenotypes observed before and after injury, we consider our results are still compatible with Rtn-1 axonal synthesis being downregulated, but not absent after injury. First, axonal Rtn-1 KD decreased β3-tubulin levels before and after injury according to figure 5B and the improved statistical analysis performed on figure 2E. Similarly, axonal Rtn-1KD significantly increases microtubule growth rate before and after injury according to the current statistical comparisons (Figure 5E). Second, if β3-tubulin decrease was a merely unspecific siRNA targeting, it is unlikely that SPTZ treatment should increase and restore β3-tubulin levels only in the context of axonal Rtn-1 KD (Figure 5B). We have now included these considerations in the discussion (lines 537-543). Although on a different track, the mechanistic relationship between Rtn-1C and Spastin suggested in Figure 7 could make more plausible that a similar phenomenon regarding the control of tubulin levels may occur locally in axons.

Following the reviewer's valuable suggestion, we performed additional quantifications in which puromycin was applied exclusively to the somatic compartment. Under these conditions, we still observed amplification in axons (~4 puncta per 1000 µm²), although this value was significantly lower than when puromycin was applied directly to axons (~10 puncta per 1000 µm²). This analysis provided a novel appreciation of the puro-PLA technique in neurons: at least half of the signal originates in the axonal compartment, while a portion may reflect proteins synthesized in soma and transported anterogradely to the axon through yet-unknown mechanisms (potentially involving rapid anterograde transport). Note that we revised the criteria for detecting true amplification spots based in staining without puromycin, which increased true amplification numbers. Still, these seemingly low values are compatible with reflecting a limited amount of time (only 15´ of puromycin pulse) and the stringent conditions of this experiment in which secondary antibodies were avoided by directly labeling primary ones. This approach makes the classical 30-40nm distance for PLA even narrower, thus reducing signal. In any case, assuming a neuronal soma diameter of 15 µm (area ≈ 176.71 µm²), this yields ~0.028 puncta per µm² in somata. In comparison, axons display ~0.01 puncta per µm², approximately one-third of the soma value, which makes sense for the expected difference in ribosome density.

• In Figure 5A the authors find an increased co-localization (RTN1/Spastin) after axotomy. From their images, it seems that the amount of Spastin is hugely increased, which would by default increase the chance of (random) colocalization of RTN1 on Spastin. Could the authors comment on this?*

__R: __Thanks for this relevant and constructive critique. We formerly based our colocalization analysis on deconvolved images. However, after performing several quantifications through different deconvolution parameters, we were not convinced about the robustness of this finding and the performed staining. Thus, we performed a new set of experiments and found that non-deconvolved images from the STED microscope were more informative about the expected tubular morphology of the axonal ER. Thus, we improved figure 5A, and now the main conclusion is just that both proteins are closely distributed in distal axons before and after injury.

• In figure 5E and 5F, the condition of scr + SPTZ is omitted. What is the reason for this? The explanation of results in these figures is confusing. The authors report a 'clear trend' in increase in comet track length and lifetime upon addition of SPTZ to axonal RTN-1 KD. This is however not significant. The comparisons that are made afterwards are confusing (e.g. increase in comet lifetime of SPTZ in non-injured axons with RTN1 KD compared to Scr+DMSO and KD + DMSO in injured axons). Their conclusion is axonal RTN-1 synthesis in injured axons (see my concern in the points above on this) governs microtubules growth rate beyond Spastin activity yet blocking Spastin activity still completely blocks the effect of KD on outgrowth.*

* *__R: __We thank this observation and fully agree that the general description provided in figure 5 E wasn't satisfactory. We have re-organized the descriptions of these results and performed more relevant statistical comparisons (lines 338-359). Based on the reviewer observation, we now conclude: "Together, these results suggest that axonal Rtn-1 synthesis controls microtubule dynamics in both non-injured and injured axons, mostly independently of Spastin-mediated microtubule severing." (lines 357-359).

Other/minor concerns:

- The gene ontology analysis in Figure 1A contains the category 'Endoplasmic reticulum'. In this category are mainly ribosomal proteins. Although in a gene ontology analysis these proteins will be included in this category, it is misleading in this respect since they are just as likely to be coming from cytoplasmic ribosomes. Although it cannot be excluded that these are ER-bound ribosomes, not in the last place because a recent study (Koppers et al., 2024, doi: 10.1016/j.devcel.2024.05.005) found ribosomes attached to the ER in axons, I believe the category should be adapted or at the least clarified in the text.

• *

__R: __Many thanks for the suggestion, which is now included in the text. "Note that several of the identified transcripts in the category 'endoplasmic reticulum' code for cytoplasmic ribosomal components, which indeed can be attached to the axonal ER (Koppers et al., 2024) and be locally synthesized in axons (Shigeoka et al., 2019)." (lines 125-128)

- Is RTN-1C isoform still an ER-shaping protein or rather an ER protein with alternative functions? The final sentence in the abstract makes a statement that a locally synthesized ER-shaping protein lessens microtubule dynamics. Could the authors provide a clearer description and discussion of the evidence in literature for this? RTN1C has been suggested to perform alternative functions in which case the statement that the local synthesis of an ER-shaping protein is important for axonal outgrowth should be adapted.

R: We agree with the reviewer and are aware that some non-canonical roles of Rtn-1C may partially explain the observed phenotypes. Thus, we have rephrased the last statement of the abstract: "These findings uncover a mechanism by which axonal protein synthesis provides fine control over the microtubule cytoskeleton in response to injury.". Also, we have modified the discussion section introducing new references accordingly..." Some studies have pointed to a non-canonical role for Rtn-1C in the nucleus, including DNA binding and histone deacetylase inhibition (Nepravishta et al., 2010, 2012). It is tempting to speculate that these still emerging roles may also contribute to the observed phenotypes. Of note, different axonally synthesized proteins exert transcriptional control in response to injury or local cues (Twiss et al., 2016)." (lines 576-580).

• Is there a difference in RTN1 distribution or levels pre- and post-axotomy?

R: Thanks for the suggestion, with the new analysis we have only found slight reorganization of Rtn-1C and Spastin in distal axons (Figure 5A). We have also included now quantification of their levels and found no significant differences for both proteins (Supplementary figure 4)

- Line 100/101 states 'the interactome of the axonal ER provides...'. To my knowledge there has been no study looking at the interactome of the axonal ER specifically. Surely axonal ER proteins are known but there is a difference.

• *

__R: __We agree with the reviewer that the phrase was misleading, so we rephrased it in the introduction "...Different lines of evidence support that the protein components of the axonal ER may interact with proteins that regulate microtubule dynamics"

* - Typo line 160 'localla'*

• *

__R: __Thanks for taking the time, we have now corrected it.

- In Figure S1 B, please add the DIVs to make it clearer what each graph corresponds to. The legend of S1B states different distances from the cell body but the graph shows distances from the tip.

• *

__R: __We have now corrected the legend accordingly.

- Figure 2C, why does B3 tubulin decrease in soma, aspecific effect of siRNA?

• *

__R: __This was indeed an unexpected finding. However, we do not observe unspecific or global changes in β3-tubulin levels (see Figure 2A and Supplementary Figure 2). Considering our other results linking Rtn-1 to the regulation of the microtubule cytoskeleton, we interpret this decrease as an indirect effect of Rtn-1 depletion rather than an off-target action of the siRNA. Moreover, if the effect were unspecific, both proteins would likely be reduced in the cell body, given that the siRNA was specifically designed to target Rtn-1 as its primary sequence-specific target.

- What is the rationale on the opposite effect found in outgrowth in Figure 3?

• *

__R: __The apparent opposite outcomes observed in Figure 3 - where axonal versus somatic Rtn-1 knockdown leads to divergent effects on axonal outgrowth - can be explained by compartment-specific environments and isoform distribution. The siRNA targets the conserved RHD region, reducing both Rtn-1A and Rtn-1C. Axons are enriched in Rtn-1C. Thus, axonal KD preferentially reduces Rtn-1C. In contrast, somatic KD reduces both isoforms. Rtn-1A, predominant in cell bodies, may probably engage other signaling pathways (Kaya et al., 2013). Interestingly, it was reported by Nozumi et al. (2009b) that global Rtn-1 depletion reduces axonal outgrowth in developing cortical neurons. This aligns with the notion that somatic KD mimics a more global loss of function, whereas axonal KD reveals a compartmentalized, pro-regenerative effect due to local Rtn-1C regulation. (All the references indicated here are in the main manuscript). These considerations are now included in the discussion ( lines 581-593).

* - Missing word 'we' on line 194*

• *

__R: __ We have corrected it.

- Typo line 629 'witmn h', please proofread the entire manuscript carefully.

• *

__R: __ We apologize for the spellings, now we have carefully revised the manuscript.

- Could the authors comment on why, in Figure 7B/C, GFP only is colocalizing with Spastin-RFP? In general, GFP should be diffusive and not display punctate colocalization with Spastin.

• *

We appreciate the reviewer's comment. Under normal conditions, GFP displays a diffuse cytoplasmic distribution. However, in our experimental setup, we observed punctate GFP signals only in the context of co-expression with Spastin-RFP. This is consistent with prior reports showing that soluble GFP can occasionally be sequestered into late endosomal structures (Sahu et al., 2011), which are also known to harbor the M87 Spastin isoform (Allison et al., 2013; Allison et al., 2019). To rigorously exclude the possibility of unspecific fluorescence crosstalk, we independently acquired each fluorophore channel and confirmed that GFP puncta were genuine and not due to bleed-through (Supplementary Figure 5). Further, cells expressing only GFP or only Spastin-RFP did not show overlapping puncta, and co-expression of GFP with Rtn-1A-RFP did not produce any apparent overlap, indicating that the punctate GFP pattern is specifically associated with Spastin co-expression. Thus, the observed GFP colocalization with Spastin reflects a biological phenomenon potentially linked to the endosomal localization of M87 Spastin, and not an artifact of imaging or fluorophore bleed-through.

Reviewer #2 (Significance (Required)):

* Axonal mRNA localization and localized translation support many neuronal functions and is an important determinant of the regenerative potential of axons after injury. How this works mechanistically remains unclear. The authors present a well performed and technically challenging study in which they identify RTN-1 as a regulator of axonal outgrowth after injury. They provide evidence using experiments in microfluidic chambers that RTN1 is locally synthesized in axons. Interestingly, they identify a (local) interplay between RTN1 and Spastin which affects microtubules and thereby regulates the outgrowth of cortical axons after injury. This study provides an interesting new link between a locally synthesized protein (RTN1) and a microtubule-regulating protein Spastin that is changed upon axon injury. This provides an advance in our understanding in axon regeneration after injury and provides the basis for new studies that can further investigate this interplay. Although interesting, I have several concerns that should be clarified and are needed to substantiate the findings and model presented in this study.*

*

The audience for this study will be mainly basic research in the fields of both axonal protein synthesis and axon regeneration. My expertise is in the field of mRNA localization and local protein synthesis.*

Batista, A. F. R., Martínez, J. C., & Hengst, U. (2017). Intra-axonal synthesis of SNAP25 is required for the formation of presynaptic terminals. Cell Reports, 20(13), 3085. https://doi.org/10.1016/J.CELREP.2017.08.097

Fan, X. xuan, Hao, Y. ying, Guo, S. wen, Zhao, X. ping, Xiang, Y., Feng, F. xue, Liang, G. ting, & Dong, Y. wei. (2018). Knockdown of RTN1-C attenuates traumatic neuronal injury through regulating intracellular Ca2+ homeostasis. Neurochemistry International, 121, 19-25. https://doi.org/10.1016/J.NEUINT.2018.10.018

Gracias, N. G., Shirkey-Son, N. J., & Hengst, U. (2014). Local translation of TC10 is required for membrane expansion during axon outgrowth. Nature Communications 2014 5:1, 5(1), 1-13. https://doi.org/10.1038/ncomms4506

Lucci, C., Mesquita-Ribeiro, R., Rathbone, A., & Dajas-Bailador, F. (2020). Spatiotemporal regulation of GSK3β levels by miRNA-26a controls axon development in cortical neurons. Development (Cambridge), 147(3). https://doi.org/10.1242/DEV.180232,

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This manuscript investigates the relationship between the endoplasmic reticulum morphogen reticulon-1 (Rtn-1) and the microtubule severing protein spastin in axons after injury. The main message and conclusion of the paper is that local axonal synthesis of Rtn-1 plays a role in regulating the microtubule severing activity of spastin by interacting with spastin and inhibiting its activity. This mechanism would be important after injury by regulating axonal growth.

* The conclusions of the paper are based on the following claims:*

* 1) Rtn-1 is synthesized locally in axons.*

* 2) Specific downregulation in Rtn-1 in axons using microfluidic chambers affects microtubules abundance (measured by beta-3 tubulin) and promotes axon growth after injury.*

* 3) Inhibition of spastin MT-severing activity with a specific drug rescues the growth effect induced by axonal downregulation of Rtn-1.*

* 4) Rtn-1c interacts with spastin-M87 to limit its MT-severing activity in a cellular system upon overexpression.*

*

*

Major comments:

1) Evidence that Rtn-1 is synthesized in axons comes from two experiments. Initially, the authors show that Rtn-1 siRNA transfection in the axonal compartment of microfluidic chambers reduces Rtn-1 levels in axons, suggesting that there is some local synthesis. Although this method is very attractive, I am concerned about the statistical analysis. The graphs show bars rather than individual data points from the average of many neurons (about 300). The plots also show the SEM instead of the SD, thus covering all the variability that is inherent in this type of experiment. The statistics are probably not performed on the 3 biological replicates, but consider the individual neurons as N. This is obviously not correct, since neurons in an experiment may all be affected by the same technical problem and are not independent replicates. For this reason, I am a bit skeptical about this quantification. Another problem is that the quantification of the fluorescence intensity of the sample does not take the nuclei into account. Are the nuclei removed for analysis? Are the images single planes? Addressing the quantification issues is crucial also for data in Figure 4, where the authors show a different effect of Rtn-1 axonal KD after injury.

* The second experiment is the Puro-PLA in Figure 6D. This experiment shows an average of 1.5 dots of signal per soma, which is a very low level of translation for this compartment where most of the synthesis should be taking place. In the axons, it is not clear how they calculate the axonal area. Again, the number of dots detected is very low and the physiological significance is questionable. A control with a known mRNA translated in axons would be important.*

* Finally, as an important control, the authors should show the presence of Rtn-1 mRNA by FISH in their experimental system.*

__R: __We appreciate the critical points addressed here as they moved us to improve the quality of the findings. We analyzed cells/axons as statistical units to increase statistical power given the subtle nature of these local changes. We agree with the reviewer that this approach may increase the risk of finding false positives. To address this point, i) we plotted the individual data points and colored them according with the different experimental dates (all the dates showed a similar trend) ii) We indicated SD instead of SEM iii) We analyzed our data using linear mixed-effects models, with experimental date included as a random effect. This approach allows to preserve the granularity and statistical power, while avoiding pseudoreplication. To exclude artifactual changes, we now analyzed the intensity fold change of total fluorescence normalized to Scr. Our former quantifications were based on the corrected fluorescence intensity used to construct the plot profiles, which could be adding some distortion to the measurements. These changes were applied throughout figures 2 and 4 (pages 34 and 38, respectively). After these new analyses the formerly presented results remain valid.

We thank the reviewer for raising concerns about the quantification of fluorescence intensity in cell bodies. We now specify in Materials and methods that fluorescence intensity analysis of distal axons (always isolated by the microfluidic chambers) and of cell bodies was performed using the wide-field configuration of the microscope. In all the cases, a single (epifluorescent) plane was analyzed to reflect the total fluorescence of a cell or axon. We did not exclude the nuclear region from the quantifications, as this would also remove cytoplasmic signal located above or below the nucleus.

We also understand the concerns about puro-PLA experiments. We agree with the reviewer that an average of 1.5 puncta per soma initially appeared low. We have identified at least three reasons for this. First, the signal derives from only a 15-minute puromycin pulse, which is a short labeling window. Second, our puro-PLA assay is particularly stringent, as ligation relied directly on puromycin- and Rtn-1C-labeled primary antibodies, without the additional spacing normally introduced by secondary antibodies. In standard PLA, the critical distance for amplification is ~30-40 nm, whereas in our assay this distance is even more restrictive. Third, in our initial analysis we applied an overly cautious threshold to define "true" amplification. We have now refined this threshold using a baseline defined by the absence of puromycin stimulation. With this improved criterion, we now quantify an average of ~5 puncta per soma and ~10 puncta per 1000 µm² of axonal area (Supplementary Figure 3D). As it is now included in methods, we calculated the axonal area by binarizing β3-tubulin staining and only counted the true amplification spots inside this region. Assuming a neuronal soma diameter of 15 µm (area ≈ 176.71 µm²), this yields ~0.028 puncta per µm² in somata. In comparison, axons display ~0.01 puncta per µm², approximately one-third of the soma value which seems more reasonable. This is also compatible with most of Rtn-1C synthesis comes from the cell body.

Unfortunately, we could not be able to perform puro-PLA of other axonally synthesized proteins. Nevertheless, to further validate our puro-PLA signal, we tested the specificity of the Rtn-1C antibody we used for this assay by WB, IF, and Rtn-1 KD (Supplementary figure 3 A-C). In addition, we performed axonal Rtn-1 KD in microfluidic chambers for twenty-four hours, which elicited a significant decrease in puro PLA signal compared to Scr (Supplementary figure 3D). Together, these results strongly indicate that the quantified signal reflects Rtn-1C synthesis. To prove that Rtn-1 mRNA is present in these conditions, we now included a RT-PCR performed on RNA isolated from the somato-dendritic and pure axonal fractions of 8 DIV microfluidic chambers (Supplementary figure 3D). Note that the presence of this mRNA in axons has been supported by several studies, one of them using cortical neurons of similar DIV and cultured in microfluidic chambers (Table I and figure 1).

2) The effects on tubulin following Rtn-1 downregulation in axons is potentially very interesting, but the authors should be careful because it could also mean that the axons are suffering. Can they also stain for other cytoskeletal markers?

R: Regarding this concern, we are aware that in the former Figure 3 we mistakenly selected axonal fields that did not display healthy axons, which was not the dominant trend. This is accredited by the lack of fragmentation and by the functional responsiveness (microtubule dynamics) shown in Figures 4 and 5B, C, E. We have now replaced the previous axonal fields in Figure 3 with more representative axons (healthy), devoid of varicosities and fragmentation (page 37)

3) The results using SPTZ are very interesting and implicate spastin microtubule severing activity in the observed phenotype. In my opinion these experiments however do not prove that "axonal Rtn-1 is indeed promoting the severing of microtubules by spastin", but simply that the blocking spastin activity prevents the appearance of the microtubular phenotype (which appears still with a mysterious mechanism). What happens if they try to stabilize the cytoskeleton by another mean (with taxol for example?). The authors should rephrase this conclusion.

__R: __We completely agree with the reviewer's appreciation. We now explicitly indicate in the main text that this is (so far in the manuscript) a still correlative phenomenon that suggests an interplay with Spastin activity "..Together, these results suggest that locally synthesized Rtn-1 normally acts to suppress the outgrowth of injured axons, a process that could involve the microtubule-severing activity of Spastin." (lines 321-323). Later in the article, with the improved Figure 7, we further propose that these findings may reflect a causal relationship, although this mechanism has not yet been directly demonstrated in axons.

4) The last experiment (Figure 7) that aims to connect Rtn-1 and spastin function is very artificial, since it is based on overexpression. Why should spastin M87 interact with an ER morphogen? Endogenously it is conceivable that spastin M1 which localizes to the ER would interact with Rtn-1. Moreover, this experiment needs further controls and quantifications. First, it is quite obvious from panel 7C that there is crossover of signal in the two fluorescence channels (see GFP and spastin). Controls need to be shown, where only one of the two fluorescent proteins is expressed, and the specificity of the laser is tested. This experiment is based on only 1 cell shown where co-localisation is detected based on a line that is placed in a specific area of the cell. The effects on the microtubular network needs quantification.

__R: __We have now improved Figure 7 and added the requested controls to rule out crosstalk as indicated in Supplementary Figure 5 and in the main text. We agree that under normal conditions GFP should display a diffuse cytoplasmic distribution. However, in our experimental setup, we observed punctate GFP signals only in the context of co-expression with Spastin-RFP. This is consistent with prior reports showing that soluble GFP can occasionally be sequestered into late endosomal structures (Sahu et al., 2011), which are also known to harbor the M87 Spastin isoform (Allison et al., 2013; Allison et al., 2019). To exclude the possibility of unspecific fluorescence crosstalk, we independently acquired each fluorophore channel and confirmed that GFP puncta were genuine and not due to bleed-through (Supplementary Figure 5). Further, cells expressing only GFP or only Spastin-RFP did not show overlapping puncta (arrowheads), and the co-expression of GFP with Rtn-1A-RFP did not produce any apparent overlap, indicating that the punctate pattern of GFP is specifically associated with Spastin co-expression. Thus, we consider that the observed GFP colocalization with Spastin potentially reflects a true phenomenon and not an artifact of imaging or fluorophore bleed-through.

We thank for these observations and apologize for the confusion in the outline of the former figure 7 and the lack of a better description. As the reviewer indicates, one interesting aspect of the M87 isoform is that lacks the ER morphogen domain (so is soluble or cytoplasmic in principle). However, it also harbors endosome and microtubule binding domains which according to previous literature (now included in the main text) may render it a punctate rather than a homogeneous pattern. Also, M87 is the most abundant isoform in the nervous system, particularly at early development. This is the reason why we selected this isoform to test our model. To clarify this point, we based our colocalization analysis in different cells and experimental dates and analyzed all the z-stacks for each cell (see new figure 7B and methods), the intensity plots (now removed) were only for graphical purposes. Similarly, we had already quantified the total tubulin intensity in COS cells based on many cells from different dates and included the sum projections of all the z-stacks from these cells (see new figure 7C). Thus, we removed the intensity profiles as they were clearly misleading (see new figure 7).

We agree that over-expressing constructs may force interactions or co-distribution of proteins. However, in this case, if the observed results were mainly due to over-expression, we should see a similar trend with isoform A as both constructs are under the control of the same strong promoter (CMV) and harbor the same ER morphogen domain (RHD). Nevertheless, the distribution of M87 tightly mirrors Rtn-1C, which is not the case for Rtn-1A. Only as a theoretical prediction, our molecular modeling suggests that Rtn-1C may be associated with Spastin through its microtubule binding domain (Figure 7E). This would suppose that Spastin "decorates" ER-tubules rather than being in the same ER membranous structure. This discrete pattern of Spastin is more coherent with the distribution of both proteins that is now more clearly observed in distal axons by STED super-resolution (new figure 5A). So, despite a bit unexpected, these results suggest a novel interaction mechanism between these two proteins that deserves further validation.

5) What is exactly the model proposed? The title implies that axonal synthesis of Rtn-1 is important during injury, but the data in the paper rather suggest that upon injury the majority of Rtn-1 is not locally synthesized. If the levels of Rtn-1 do not change, why the effect on the microtubules should be specific? Why would a siRNA against Rtn-1 in axons not affect the levels of Rtn-1, but those of tubulin? The authors should be careful, and test other control siRNAs, and Rtn-1 siRNAs, since it is well known even in more simple cellular systems that the toxicity of individual siRNAs can vary greatly.

We consider the possibility that after injury there is no axonal Rtn-1 synthesis as a plausible and relevant appreciation. Unfortunately, we could not perform a puro-PLA experiment after injury, which would have provided a more definite answer. However, now we are more confident of regulating Rtn-1 synthesis before injury as supported by a Supplementary figure 3D that shows a significant decrease on puro-PLA signal (indicative of Rtn-1C synthesis) 24 hours after axonal KD. Thus, based on some similar phenotypes before and after injury, we consider our results are still compatible with Rtn-1 axonal synthesis being downregulated, but not fully absent (the mRNA is still detected, as described by Taylor 2009). As such, axonal Rtn-1 KD decreased β3-tubulin levels before and after injury according to figure 5B and the improved statistical analysis performed on figure 2E. Similarly, axonal Rtn-1KD significantly increases microtubule growth rate before and after injury according to the current statistical comparisons (Figure 5E). in complement, if β3-tubulin decrease was merely due to unspecific siRNA targeting, it is unlikely that SPTZ treatment should restore β3-tubulin only in the context of axonal Rtn-1 KD (Figure 5B). Although on a different track, the mechanistic relationship between Rtn-1C and Spastin suggested in Figure 7 could make more plausible that a similar phenomenon regarding the control of tubulin levels could be occurring locally in axons. We have now included these considerations in the discussion (lines 535-543).

To discard off-targets effects, we have now validated a third siRNA sequence (siRNA 3) specifically designed against Rtn-1 and showed that it selectively downregulates Rtn-1C but not β3-tubulin in cultured cortical neurons. Then, following the same experimental frame of figure 3, we performed axonal Rtn-1 KD after injury and observed that siRNA 3 also significantly increases the outgrowth of injured axons (Supplementary figure 2). This suggests that, at least this phenotype, is not product of an off-target effect. Thus, the pharmacological rescue of β3-tubulin levels by SPTZ (Figure 5B) and the Rtn-1C/Spastin co-distribution in heterologous cells, which correlates with preserved microtubules (improved Figure 7), provide converging evidence to suggest that Rtn-1C-Spastin interplay may underly the observed phenotypes in axons.

Minor comments:

In Figure 5A, it would be helpful to indicate the border of the axon. The figure is not really convincing.

Following yours and other reviewer comments, we have analyzed a new set of experiments regarding the STED images of non-injured and injured axons. To eliminate the risk of artifactual descriptions, we have avoided deconvolution and worked directly with raw STED images (Figure 5A). Under these conditions, distribution of Spastin and its intensity in distal axons are not modified by injury, nor those of Rtn-1C and Spastin (Supplementary figure 4). Despite these results, data still supports that both proteins are restricted to similar domains subcellular domains before and after injury.

Reviewer #3 (Significance (Required)):

The manuscript uses complex methods to address an interesting cell biological question of relevance to understand axonal growth regulation upon injury. A limitation of the study is the statistical analysis, which triggers some doubts about the reproducibility of the data. Further experiments and the addition of controls would be important to support the claims of the authors.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

This manuscript investigates the relationship between the endoplasmic reticulum morphogen reticulon-1 (Rtn-1) and the microtubule severing protein spastin in axons after injury. The main message and conclusion of the paper is that local axonal synthesis of Rtn-1 plays a role in regulating the microtubule severing activity of spastin by interacting with spastin and inhibiting its activity. This mechanism would be important after injury by regulating axonal growth.

The conclusions of the paper are based on the following claims:

1. Rtn-1 is synthesized locally in axons.
2. Specific downregulation in Rtn-1 in axons using microfluidic chambers affects microtubules abundance (measured by beta-3 tubulin) and promotes axon growth after injury.
3. Inhibition of spastin MT-severing activity with a specific drug rescues the growth effect induced by axonal downregulation of Rtn-1.
4. Rtn-1c interacts with spastin-M87 to limit its MT-severing activity in a cellular system upon overexpression.

Major comments:

1. Evidence that Rtn-1 is synthesized in axons comes from two experiments. Initially, the authors show that Rtn-1 siRNA transfection in the axonal compartment of microfluidic chambers reduces Rtn-1 levels in axons, suggesting that there is some local synthesis. Although this method is very attractive, I am concerned about the statistical analysis. The graphs show bars rather than individual data points from the average of a large number of neurons (about 300). The plots also show the SEM instead of the SD, thus covering all the variability that is inherent in this type of experiment. The statistics are probably not performed on the 3 biological replicates, but consider the individual neurons as N. This is obviously not correct, since neurons in an experiment may all be affected by the same technical problem and are not independent replicates. For this reason, I am a bit skeptical about this quantification. Another problem is that the quantification of the fluorescence intensity of the sample does not take the nuclei into account. Are the nuclei removed for analysis? Are the images single planes? Addressing the quantification issues is crucial also for data in Figure 4, where the authors show a different effect of Rtn-1 axonal KD after injury. The second experiment is the Puro-PLA in Figure 6D. This experiment shows an average of 1.5 dots of signal per soma, which is a very low level of translation for this compartment where most of the synthesis should be taking place. In the axons, it is not clear how they calculate the axonal area. Again, the number of dots detected is very low and the physiological significance is questionable. A control with a known mRNA translated in axons would be important. Finally, as an important control, the authors should show the presence of Rtn-1 mRNA by FISH in their experimental system.
2. The effects on tubulin following Rtn-1 downregulation in axons is potentially very interesting, but the authors should be careful because it could also mean that the axons are suffering. Can they also stain for other cytoskeletal markers?
3. The results using SPTZ are very interesting and implicate spastin microtubule severing activity in the observed phenotype. In my opinion these experiments however do not prove that "axonal Rtn-1 is indeed promoting the severing of microtubules by spastin", but simply that the blocking spastin activity prevents the appearance of the microtubular phenotype (which appears still with a mysterious mechanism). What happens if they try to stabilize the cytoskeleton by another mean (with taxol for example?). The authors should rephrase this conclusion.
4. The last experiment (Figure 7) that aims to connect Rtn-1 and spastin function is very artificial, since it is based on overexpression. Why should spastin M87 interact with an ER morphogen? Endogenously it is conceivable that spastin M1 which localizes to the ER would interact with Rtn-1. Moreover, this experiment needs further controls and quantifications. First, it is quite obvious from panel 7C that there is crossover of signal in the two fluorescence channels (see GFP and spastin). Controls need to be shown, where only one of the two fluorescent proteins is expressed and the specificity of the laser is tested. This experiment is based on only 1 cell shown where co-localisation is detected based on a line that is placed in a specific area of the cell. The effects on the microtubular network needs quantification.
5. What is exactly the model proposed? The title implies that axonal synthesis of Rtn-1 is important during injury, but the data in the paper rather suggest that upon injury the majority of Rtn-1 is not locally synthesized. If the levels of Rtn-1 do not change, why the effect on the microtubules should be specific? Why would a siRNA against Rtn-1 in axons not affect the levels of Rtn-1, but those of tubulin? The authors should be careful, and test other control siRNAs, and Rtn-1 siRNAs, since it is well known even in more simple cellular systems that the toxicity of individual siRNAs can vary greatly.

Minor comments:

In Figure 5A, it would be helpful to indicate the border of the axon. The figure is not really convincing.

Significance

The manuscript uses complex methods to address an interesting cell biological question of relevance to understand axonal growth regulation upon injury. A limitation of the study is the statistical analysis, which triggers some doubts about the reproducibility of the data. Further experiments and the addition of controls would be important to support the claims of the authors.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Axonal mRNA localization and localized translation supports many neuronal functions and is an important determinant of the regenerative potential of axons after injury. How this works mechanistically remains unclear. The authors present a well performed and technically challenging study in which they identify RTN-1 as a regulator of axonal outgrowth after injury. They provide evidence using experiments in microfluidic chambers that RTN1 is locally synthesized in axons. Interestingly, they identify a (local) interplay between RTN1 and Spastin which affects microtubules and thereby regulates the outgrowth of cortical axons after injury. This study provides an interesting new link between a locally synthesized protein (RTN1) and a microtubule-regulating protein Spastin that is changed upon axon injury. This provides an advance in our understanding in axon regeneration after injury and provides the basis for new studies that can further investigate this interplay. Although interesting, I have several concerns that should be clarified and are needed to substantiate the findings and model presented in this study.

Major concerns:

1. In figure 1, the authors provide an analysis of overlapping axonal mRNAs. There are more axonal transcriptome studies and a recent study by von Kugelgen and Chekulaeva (2020; doi: 10.1002/wrna.1590) already performed such an analysis, which included more studies. It would be good to mention this. It can be perceived that studies were now chosen to get the outcome that Rtn-1 is present in all studies. For example, von Kugelgen finds mRNA coding for RTN3, another ER structural protein, as present in 16 out of 20 studies analyzed. That said, the authors present more reasons to look at Rtn-1, so the selection to continue with this protein remains valid but can be written up differently so not to present it as the 'sole' ER-shaping protein consistently present in axonal transcriptomes.
2. The description of methods is currently insufficient and incomplete and does not allow for reproducibility of this study. For example, different Rtn-1 antibodies seem to be used in this study. Is the same antibody used for staining and WB? There is no listing of any of the antibodies used in the study and which one is used for which technique/experiment. This should be clarified and should be easy to do so in the methods section (antibody name, origin/company, dilution used) to enhance reproducibility of this study. This is not limited to primary antibodies and any information on secondary antibodies, including what was used for STED is completely missing.
3. The timeline of KD experiments in Figure 2 and 3 are unclear. For the Western blot KD is performed at DIV7 and collected 48 hours later. However, this is not specified for the stainings done in Figure 2C-E. Is this also at DIV7 and then for 48 hours? In figure 3 the siRNA is added at DIV8 (together with axotomy) and outgrowth is measured 24 hours later. Is 24 hours sufficient to achieve knockdown? Is this also what was done for stainings? Later on in Figure 5B, 48 hours of KD is again used. It is unclear what the rationale of these differing timepoints is. Why was this chosen? Is the timeline also the reason for the difference in segment lengths chosen? In Figure 3, there is a significant effect on outgrowth in the KD in the 'mid-range' which is not present in Figure 5.
4. Could the authors provide a rescue condition for their siRNA (using a siRNA-resistant construct) to show that their siRNA is specific for RTN1. They nicely show the efficiency of the siRNA but not its specificity. This is crucial because if not specific, this will affect a large part of their study. They already have RTN1A and RTN1C constructs available. Such a rescue experiment should ideally also be performed for one or more of their phenotypic experiments, such as the one presented in Figure 3A or 5 to show that the phenotype is really RTN1 dependent. If done by re-expressing either RTN1A or RTN1C, this could provide insightful information on the relevant isoforms.
5. I find the data presented in Figure 4A/B confusing. Axonal RTN-1 KD does not reduce axonal RTN1 levels but somatic KD does. I understand that this implies most protein comes from the soma and the authors indeed present an explanation that increased somatic RTN1 occurs after axonal KD as a compensation mechanism. However, this can also be interpreted that there is no axonal synthesis of RTN1 after injury and axonal KD has indirect or even aspecific effects. Their model depends on this difference. Their data in Figure 6 could provide supporting evidence if it shows RTN1 puro-PLA after injury. Along these same lines, in Figure 6, they nicely include a compartment control for puro-PLA. It therefore seems doable to include a somatic puromycin control for their axonal puro-PLA, to exclude and diffusion/transport of the newly synthesized peptides. This is especially in light of two recent papers reporting on this possible phenomenon, although these studies were not performed in neurons.
6. In Figure 5A the authors find an increased co-localization (RTN1/Spastin) after axotomy. From their images, it seems that the amount of Spastin is hugely increased, which would by default increase the chance of (random) colocalization of RTN1 on Spastin. Could the authors comment on this?
7. In figure 5E and 5F, the condition of scr + SPTZ is omitted. What is the reason for this? The explanation of results in these figures is confusing. The authors report a 'clear trend' in increase in comet track length and lifetime upon addition of SPTZ to axonal RTN-1 KD. This is however not significant. The comparisons that are made afterwards are confusing (e.g. increase in comet lifetime of SPTZ in non-injured axons with RTN1 KD compared to Scr+DMSO and KD + DMSO in injured axons). Their conclusion is axonal RTN-1 synthesis in injured axons (see my concern in the points above on this) governs microtubules growth rate beyond Spastin activity yet blocking Spastin activity still completely blocks the effect of KD on outgrowth.

Other/minor concerns:

• The gene ontology analysis in Figure 1A contains the category 'Endoplasmic reticulum'. In this category are mainly ribosomal proteins. Although in a gene ontology analysis these proteins will be included in this category, it is misleading in this respect since they are just as likely to be coming from cytoplasmic ribosomes. Although it cannot be excluded that these are ER-bound ribosomes, not in the last place because a recent study (Koppers et al., 2024, doi: 10.1016/j.devcel.2024.05.005) found ribosomes attached to the ER in axons, I believe the category should be adapted or at the least clarified in the text.
• Is RTN-1C isoform still an ER-shaping protein or rather an ER protein with alternative functions? The final sentence in the abstract makes a statement that a locally synthesized ER-shaping protein lessens microtubule dynamics. Could the authors provide a clearer description and discussion of the evidence in literature for this? RTN1C has been suggested to perform alternative functions in which case the statement that the local synthesis of an ER-shaping protein is important for axonal outgrowth should be adapted.
• Is there a difference in RTN1 distribution or levels pre- and post-axotomy?
• Line 100/101 states 'the interactome of the axonal ER provides...'. To my knowledge there has been no study looking at the interactome of the axonal ER specifically. Surely axonal ER proteins are known but there is a difference.
• Typo line 160 'localla'
• In Figure S1 B, please add the DIVs to make it more clear what each graph corresponds to. The legend of S1B states different distances from the cell body but the graph shows distances from the tip.
• Figure 2C, why does B3 tubulin decrease in soma, aspecific effect of siRNA?
• What is the rationale on the opposite effect found in outgrowth in Figure 3?
• Missing word 'we' on line 194
• Typo line 629 'witmn h', please proofread the entire manuscript carefully.
• Could the authors comment on why, in Figure 7B/C, GFP only is colocalizing with Spastin-RFP? In general, GFP should be diffusive and not display punctate colocalization with Spastin.

Significance

Axonal mRNA localization and localized translation supports many neuronal functions and is an important determinant of the regenerative potential of axons after injury. How this works mechanistically remains unclear. The authors present a well performed and technically challenging study in which they identify RTN-1 as a regulator of axonal outgrowth after injury. They provide evidence using experiments in microfluidic chambers that RTN1 is locally synthesized in axons. Interestingly, they identify a (local) interplay between RTN1 and Spastin which affects microtubules and thereby regulates the outgrowth of cortical axons after injury. This study provides an interesting new link between a locally synthesized protein (RTN1) and a microtubule-regulating protein Spastin that is changed upon axon injury. This provides an advance in our understanding in axon regeneration after injury and provides the basis for new studies that can further investigate this interplay. Although interesting, I have several concerns that should be clarified and are needed to substantiate the findings and model presented in this study.

The audience for this study will be mainly basic research in the fields of both axonal protein synthesis and axon regeneration. My expertise is in the field of mRNA localization and local protein synthesis.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

In this paper, the authors focus on the role of Reticulon-1C in concert with Spastin in response to axonal injury. In data mining, they find axonal mRNAs encoding for ER-associated proteins including Rtn-1. They establish a knockdown targeting both Rtn-1 isoforms Rtn-1A and Rtn-1C. They observe decreased beta-3-Tubulin levels in the soma while axonal protein levels are unchanged. In microfluidic devices, they characterise the effect of a compartment-specific Rtn-1 KD on axonal outgrowth in the axonal compartment. The authors quantify axonal outgrowth, seeing increased outgrowth in an axonal compartment-specific Rtn-1 KD, while the effect seems to be reversed when applying the KD construct in the somatic compartment. When focussing on the axonal growth cone, they find the Rtn-1 KD shows differences in several morphological features of the growth cone. They find an increase in Tubulin levels in an axonal compartment-specific, but a decrease in a somatic compartment-specific Rtn-1 KD. Colocalisation of Rtn-1C and Spastin is shown to be monolaterally increased following axotomy. Combining axotomy with the Rtn-1 KD shows increases in dynamic microtubule growth rates and track lengths. In another model system, neuron balls, they show Rtn1-C, but not Rtn1-A to be present in the axon. In a puro-PL assay they also show it can be synthesised in the axonal compartment. To investigate the mechanism enabling the cooperation between Spastin and Rtn-1C, they move to a cell line model in which they see a correlating distribution between Spastin and Rtn-1C but not Rtn-1A. Finally, they use in silico modelling to speculate on binding between Spastin domains and Rtn-1 isoforms.

Major comment:

The rationale behind the work is convincing, however some interpretations are presented as more robust than some data allow. Most notably, while the interaction between Rtn-1 and Spastin has been shown prior to this study, it is only presented here through in silico analysis. In figure 5, an increase in the growth rate of dynamic microtubules is observed in either a Rtn-1C KD or by using a Spastin-inhibitor. Due to a described increase in colocalisation between Rtn-1C and Spastin (5A), the increase in growth rate is displayed as caused by Rtn-1 promoting Spastin's severing ability. This result might however be correlative. Further in the injured samples, Spastin-levels seemingly increase (in the representative images) and it is thus not surprising that the level of Rtn-1C colocalising with Spastin increases as well. This might not be indicative of a cooperation and further experimental evidence are required.

Other comments:

• Generally, graphs would benefit from individual values plotted as well as the summary. Font sizes and types (but rarely) are sometimes inconsistent. Proteins should be consistently written (capitalised or not).
• Table 1 and figure 1 present data collected from a vast amount of resources. It should be highlighted that datasets from which data was obtained includes many different models, different DIVs and neuronal cell types. Figure 1B may benefit from a different colour scheme. "Ex-vivo" should be "Ex vivo". For "ER mRNAs are a relevant category" it is not described what "relevant" would mean in this context. The title might remove this small part or describe it in the text. It should be described how it is decided that mRNAs are "common".
• Figure 2: add description to y-axis to describe what fold change is displayed, applies to multiple figures. Will improve readability of the figures. In 2C, the ROI showing neuronal somata should be increased to show part of the axon and not cut off the soma.
• Figure 3: Three out of four axonal compartments seem to be comprised of dying or damaged axons. Especially the axonal KD scrambled image. It should be ensured that neuronal cultures are healthy. Typo in "intersections". The schematic of 3B is a great addition to explain the graphs above. Perhaps it could be a bit refined as it is currently hard to see whether this is a neuron or a growth cone without context. Maybe show where the axon connects to the depicted growth cones and change the third icon which looks like it was crossed out. Small formatting issues: remove additional space bar before "Figure 3." And add after "Bar"
• Figure 4: If not misunderstanding what is depicted, in 4A and B, different lookup tables are used to depict the same signal. Only one of each images is necessary. Do the axons have more tiny branches in the Rtn-1 KD condition in 4A? Unclear why Rtn-1 levels are increased in the Rtn-1 KD (4C), please clarify.
• Figure 5: It may be easier to understand what "axotomy" samples are if just referred to as "injured" as later in the same figure. The procedure could also very briefly be explained in the results. 5C should depict AUC in µm2 not µm. 5D Spastin is barely visible, brightness and contrast should be adjusted to enhance visibility.
• Figure 6: It should be made clear why it is necessary to switch to another model system just for 6A, please indicate this in the text. PCR bands seem very pixelated, check the quality. It is unclear why soma genes/proteins were only tested with either PCR or WB others with both. Rtn-1C and Rtn1-A should be presented in the same order in the PCR and WB panel. Correct "Rtn1-1A" typo. In 6D, 1.5 dots per soma seems like a low number. When normalised to the area the soma vs the axon occupies, the compartmentalisation does not work? May be it make sense to refine analysis or apply puromycin in the somatic compartment and analyse the axonal compartment as comparison?
• Figure 7: 7A shows two images depicting the same information that may not be needed. Can probably be removed. In 7B there is no negative (or any) correlation between Spastin levels and Tubulin, however later it is mentioned that Rtn-1C transports Spastin thus causing a decrease in Tubulin at certain locations? It is nclear if Spastin levels vary intensely between different samples. Mean intensity of the somatic area may be beneficial to rule this out. 7B Tubulin on the right top panel seems to have a decrease in Tubulin levels which is not visible due to the Y axis of Tubulin being set to a different range than the middle and lower panel. The average of line scans from multiple cells may be helpful to determine whether there is indeed no colocalization between Rtn-1A and Spastin. The provided representative images seem to show similar degrees of colocalization between Spastin and Rtn-1A/C.

Results:

• It would be helpful to reiterate the hypothesis at the start to ease the reading flow.
• There seems to be minor redundancy in lines 132-138.
• There are several spellings, proof-reading is recommended. For example, in line 136 should be "promotes". 160 "localla", 192 should be "the actin cytoskeleton".,194 should be "we first examined", 195 should be "Different", 223 "using", 259 "axons". ...
• 154-155: Unclear, why the lower MW Rtn-1C was seen as more important.
• 167 results of 2E not stated before interpreting them.
• 181 would suggest "outline" instead of "perimeter".
• 183-184 "longest shortest path" is a confusing term.
• figure 4B should be referenced earlier in the sentence.
• 243-244 may be correlation. Rtn-1 and Spastin do not necessarily interact so that this result is achieved.
• 246: In figure 1 the KD seemed to have an effect on both Rtn-1 isoforms, why not here anymore? 259 "axons". 284 "counteract" instead of "suppress"?
• 485: rephrase as the interaction between Rtn-1C with Spastin has not been shown directly in these experiments.

Methods: 535 "in PBS". 543 citation error. 689-699 is it necessary to add a gaussian blur?

References: Mannan, A U et al. appears twice in the citation list (36 and 44).

Significance

Overall, this manuscript describes novel fundings which will be interesting to the neuronal cell biology community and scientists working on the field of neuronal injury and regeneration. It is well structured, and the data are mostly well presented but sometimes conclusions are over-interpreted. However, several points need to be addressed in a more convincing way.

I feel like this is like punctuation at the end of a sentence or like a comma.

Its like leaving someone of a cliff hanger at the end of a chapter or show.

It is more prevalent now with newer digital systems and commerce have been created and exchanged for value. The phrase nothing is required has become more literal since the tulip mania which lead to transaction with tulips that have not bloomed or sprouted with promises and IOU's not guaranteed.

Civil liberties have been consistently fought for and has been a victim for many subjects, topics, areas, and of course people. Civil liberties should be something that should not be easily squashed or ignored but, it is a subject that comes up more times in courts in relation to big energy, business among other corporations and one we will always here about in technical systems.

Autonomy of politics requires the system and need to be separate, it is practical and one that needs to be used but, not abused. Autonomy of any system has lead to impartial results and ones not controlled but voted with majority. It is not easy but it is necessary, just like technological systems.

When a system is done properly there is positive growth and it requires highly skilled managers who can handle the systems that create and form many facets of life. As a system gets more complex the more skilled people are required to operate and maintain them.

Social patterns have actually been used in both ways stated it is unavoidable social response and imposed by a governing body for its own purpose. Both get so mixed that they can't be distinguished in some cases. Its one thing when it happens naturally it another thing when it is rigged. To identify them is the first step to making changes if necessary.

Modern technology has always have both intended and unintended effects on systems. Unfortunately politically they can have very desirable effects for the people in politics who can benefit than the average person, where it continues is up for debate.

Authoritarian life has been enhanced and developed more with time especially with technical systems that are introduced especially to the basis of the system. Being at the top of the of social hierarchy has unfortunately forever been sought out with these systems as it benefits the ones more in control than the rest.

The use of machines has always had what is the original desired effect but unfortunately it has been used against rather than to help in some cases. Other cases they can help the existing people and structures if they are used correctly and for a more positive effect. What the intended outcome is what eventually happens.

The social inequality of development when it comes to infrastructure and transit has always shown who values the individual or the people, the ones who can afford and the ones who cannot. We don't see it because it becomes the landscape and most people don't see it any different because to them it is normal compared to other cities and states.

Social and economic systems define how technical artifacts are used and created, they are not inherent in nature but, only applied to them after and before their creation. When it is embedded it is then used and defined to what people see it as and where it will continue to be in the system.

Its true about technical systems have changed exercise of power and experience of citizenship they have been involved as they have evolved turning them more interwoven with time such as mentioned politics, production, communication, and warfare. Technical systems will always be created for various areas with what is asked and demanded.

Power and authority has in essence have always been there when it comes to machines, structure, and systems of modern material culture. IT has been used and displayed as such in its own way just as someone shows off a sports car. It can have contributions both positive and negative but these things are always there.

Your lines are really important for your poems, weather you want them to be or not

Do poets do this for "dramatic" effect or what is the purpose for the verses?

I think this sentence is useful to contextualize just how important and useful the French language can be.

I think this is a strong way to end the article since it emphasizes just how wide spread the French language is.

I find it interesting how Quebec is now listed here but Canada is not on the list at all. This shows how densely spoken French is in Quebec.

I find it interesting that Sub saharan Africa has nearly the same percentage of French speakers as Europe.

I find it interesting how French is not in the top 5 most spoken languages but is more wide spread than almost all of them.

It's interesting how the graph shows how French has grown so much the past couple of years.

I think its interesting that french is all around the globe and its made an impression in cultures everywhere

What the author is trying to say by saying "Galaxie francophone" is that french is all around the world and isnt going to slow down any time

I think this text is an informative text because its throwing out a lot of data points and numbers so thats why I believe it is going to the informative.

O*Net explains the duties that are given when you become a medical assistant

I think a social because I love being around others as well as helping other people

A job is temporary, need no experience, and anyone can work there

A career is something you want to do, you have to go to school for, not everyone can study or work in a dream career

So for an already know issue to be considered a social problem there must be attention from policymakers based on public protest? Is that how it would work?

Social problems only get attention when enough public outrage or protest is brought to light about the injustice.

Based on the definition could this be anything that has a large number of consequences of any topics?

An example of this as stated in the Mills article assignment just below this is mass shooting and gun violence control. Society acknowledges the problems with mass shooting but little speak of the preventative measures that we need

So true, I am a Chinese student and my major is ISE. During high school, there were almost only Chinese and Indian people in my POE, DE, math, and physical classes. Most of my friends chose the engineering major when we applied for college. I just found that solving math problems is much easier than reading a whole English article full of words I don't understand, and my interest in reading is not enough to overcoming my dyslexia, though I have liked reading before in Chinses.

This stood out to me because I definitely code switch depending on the different groups I am in.

Technology and education has been intertwined with many issues and to be honest will continue to do so as well. It currently is advancing social struggles in unique ways such as social media oddly distancing people socially in some areas such as in person, but also liberate from domination as history has shown in various ways as well.

Education through technology is something that is truly been pushed for decades now as they continue to implement it in more and new creative ways. Critical analysis however should be done against desire to see if the work is sound and helps not hinders or does nothing.

You can believe with your heart a positive outlook can have results in its own right as it wills us and people forward but, you need to think with your head because believing fire won't burn you doesn't mean its not going to. Both sides work in tandem to create spectacular things using belief and logic to find and ask great things.

Rockstar-ism is a good term and one to be listened to not the person but the topic. People everywhere look up to people to have all the answers in every subject but its true, sometimes we don't have the answers and sometime I don't know or it depends have a lot of weight behind them because it tells us to look harder.

Established norms are common thing but, various areas must be challenged because they can evolve an area and develop it more for the better if challenged correctly. Being content is to be stagnant and while it can be good it also needs to be challenged.

When it comes to pushing boundaries you must be willing to accept and receive criticism, it comes with challenging views that may conflict with established ideas. It is an area that is getting more of these types as long as they can receive criticism.

This was so interesting to see. I thought back on my teachers and, whether intentional or not, I felt sometimes that their point of view and others similar to their own were the only ones being discussed. So hearing this part of the code of ethics for this was interesting to hear.

This is, I feel, the primary goal of most teachers so it's interesting to see it highlighted in such an official code of ethics.

I think that this is an important thing to keep in mind for us future teachers. For me personally, teaching about a wide variety of perspectives and life experiences is incredibly important. So, I need to make sure I'm asking myself this on a daily basis once I start teaching.

I partially agree with this and partially don't. I think the first part is true, that most teachers that go into teaching have a deep respect and understanding of the importance of teacher's job. But I don't overly think that most teachers ethics come from the desire for respect from their students/coworkers/parents/etc.

I thought this was an interesting point to bring up and an idea that I hadn't really thought about before. Of course it makes sense that teachers would have ethics, but it's not really the word I would really use to describe what teachers would do.

Claims of value is a type of claim that evaluates the value of something, or make a comparison to other subjects. Due to the nature of claims of value, claims of value are easier to spot than others.

Claims of fact is a type of claim that does not encourage the readers to take action, instead sharing their idea of reality on a subject, which means that the author shares these ideas as fact. This may make claims of fact harder to spot than other claims.

Claims of policy encourages the audience to take action. Also, claims of policy can take the form of a direct statement. However, these actions do not have to be grand gestures, as the author can suggest their audience to discuss, research, and write on the topic.

This is. such a true statement. I have been unaware of the impact I was having on people, until I received some honest feedback from coworkers. It was really valuable- I never would have known if they hadn't told me and would have been unable to change my behavior if I didn't realize it.

Tools have always been crafted so that the operator can do what they can easily and in their control, this process has lead them to continuously to come up with better tools which has caused civilizations to become more advanced over time.

A fact that every worker has worried is the eventual loss of their job to machines that can do it better and faster, a fact corporations love for it save them money at the cost of the operator. IT has become that even if not done all by itself they can always replace the operator.

People have always wanted control over their tools so that a certain perfection can be achieved consistently without people in some cases getting in the way. Mass production evolved out of this with consistency of the products to the point anybody can do it.

Technology evolving and its control by management is ever constant and technology is always never perfect as it upgrades eventually to be better so management can be better for the company.

Industry and technological advancement has determined what companies survive, thrive, and die. The industry back in the day had many companies and workers and only the ones who could afford the new technology have survived longer than the rest, a sight many still see today.

This is an excellent list. It's important to be mentored by teachers who are proficient at classroom management, model deeper learning, and all of the other traits mentioned in the list. Having a competent mentor teacher is imperative. Then you can gain the insight and support you need.

May depend on different universities, I feel like depending on the place people are more or less willing to use AI because they don’t fear the repercussions of it

Gutenbergs improvements made it easier and cheaper to print books in Europe. That helped more people learn to read and be able to share more ideas which leads to big changes like the Renaissance and the spread of science and new knowledge.

This is interesting to see how one leader, Ivan III, helped turn Russia from a group of small states ruled by outsiders into a powerful empire. By standing up to Mongols and claiming that Moscow was the new "Rome", he gave Russia a big new role in the world.

This is really important, it shows how Shah Abbas made Isfahan a safe place for refugees By welcoming Armenians, he helped the city grow and become stronger. It also shows he followed old traditions and helped people in need.

This was a good thing because it helped keep the empire peaceful and untied together. When people were allowed to keep their own culture, language, and religion, they are waya more likely to be happy and stay loyal. This meant fewer arguments and upbringings and more cooperation. It also most likely made the empire stronger because different groups could share ideas, skills, and trade with each other.

My second additional source which relates to the provided article is the YouTube video titled "Perestroika & Glasnost (The End of the Soviet Union)" published by the channel Simple History (February, 2021).

https://www.youtube.com/watch?v=femvIHkVQG8

While the video does a similar coverage of the article, discussing the way in which Gorbachev contributed to the dismantling of the Soviet Union, it gave a greater explanation of smaller details in the history of this period. It explained how there was three deaths of political leaders during this time which lead to even greater instability in the government. It also discussed how Gorbachev proposed a new market model that gave more freedom to businesses, which he hoped in turn would lead to a restoration in the balance of the economy. These small details give a closer look into the changes he implemented during his serving for Russia's government, and makes it feel more insightful to imagine the differences he was able to make for the regular citizens of the country.

My first additional source which relates to the provided article is titled "Mikhail Gorbachev Championed ‘Glasnost’ and ‘Perestroika.’ Here’s How They Changed the World" , written by Olivia B. Waxman of Time Magazine (October, 2022).

https://time.com/5512665/mikhail-gorbachev-glasnost-perestroika/

Both Waxman and Battle discuss the general strategy of Gorbachev to utilize glasnot (openness) and perestroika (restructuring) to reinvent the political and economic systems of Russia. The plan allowed for more freedom amongst its citizens, and led to the rattling of Russia's control over its people. The main addition that the Time article gave to this conversation was the long-term results of Gorbachev's efforts. While Battle's article highlighted the risky nature of his plans, Waxman confirms that his methods were successful and led to the reformation of the Soviet Union.

Though AI does not have any kind of point of view or discriminatory intent, people do and the data AI is trained on does have a point of view and may have discriminatory intent. The information online is not non-biased and the output of an gen AI tool is also not non-biased. The article brings a good point on the importance of transparency in the type of data AI is trained on.

This is quite optimistic, because although many people are not ready to be violent themselves, most of the white population would allow or even advocate for violence from others (ex. police, lynch mobs, those in power). They are bystanders, they see violence and they do nothing to stop it.

I believe it's also important to teach safety and the proper use of AI, but it’s also essential to cover the basics, like how to use Excel and understand file formats such as PDF, JPEG, and PNG. I've witnessed firsthand how these skills impact students.

Does it matter if we are in a simulation or not? I think the concept of being in a simulation is something that is talked about a lot in philosophy and other subjects. But if we are in a simulation, how much does it really affect our day to day lives. If we discovered that we are in a simulation, who's to say that there is anything outside of it?

Chalmers' main claim in this book is that virtual realities are real, and that they are just as real as life right now, and there is no way of knowing that the life we are living right now is not a simulation. When I ask myself “Can you lead a good life in a virtual world?” I automatically think no. To me, in a virtual world, there is no real good you can do. If you help other people in the virtual world, you are only helping what I would think to be pixels. Anything you do in the virtual reality exists only in the simulation, but if you were to step out of it, anything you did is lost. It reminds me of having a high score in a game, but if you deleted the game, the high score goes away and it's like you never achieved the high score.

This quote makes me think about violent video games. In today's world, these video games are normalized and are not considered actually harmful, because you are not actually killing anyone. However, Chalmers continues to talk about how he thinks things that happen in virtual reality are real. Is Chalmers saying that if someone was to kill someone else in a virtual reality simulation, they actually committed murder? However, this goes back to how murder is defined. Would murder be defined the same as in reality, as in a virtual reality, or would it have a different definition?

I think this connects to what we were talking about in class about gamification. Gamification is a concept that intersects with philosophy. Is it ethical to make certain concepts into a game? This text is trying to do the same thing, and interconnect philosophy and technology, by asking questions about emerging technology (ethics, privacy concerns etc), through a philosophical lens.

kantian idealism

emerson experience coded

Interesting that it took Rhetoric studies took almost 30 years to even attempt defining cultural rhetoric. What does this delay say about who gets to define terms in the field?

I personally have never liked these rubrics unless each score explains what is being asked to achieve the score. If you make a rubric like this say for an assignment, each score should discuss something relating to the assignment not just general "some, basic, good, complete understanding"

Three Main Points: 1. Our goal and Gods goal is so that we may become like him. This is why we have spiritual gifts and talents, all things that will allow us to gain and learn spiritual qualities that help us become more like Him. 2. The more we become like God, the more able we are to serve God and others. 3. By understanding our divinity and eternal potential, we will have more motivation and direction in how to achieve ours and Gods goal.

I always like to ask people self reflective questions when they do something that bothers me. A good example is "why did you decide to...."

How can teachers help students recognize natural consequences so they can avoid and prevent them and grow maturely?

In my Pre I internship there was a student that would often shout things in class and no other student in the class responded and it was very brief so the teacher ignored it, but I personally found it odd. I can't forget it to this day.

This is such a spot on statement. Building capacity in others is the necessary first step-learning how to increase someone's capacity as a professional problem solver and decision maker is an art and truly important.

Before starting this class, I was not someone who knew much about tech. After week one I am already seeing so much I knew nothing about before and I am very excited to learn more this semester. As of right now the websites are a little hard to follow but I am sure with practice this is going to be easier and I am excited to unpack it all.

This is true, we are blessed with the ability to control our thoughts after our automatic assumptions.

Super common in people who have experienced trauma, I have learned in therapy that, that can be a coping mechanism.

I LOVE that mindset, there is always room for improvement. No matter what, whether it's your health or mindset or socializing skills, if everyone worked to better themselves society would be a lot better as a whole.

In the video, "Are there universal expressions of emotion?" it elaborates on how we use our prior knowledge to make sense of the world too. It says that people in foreign tribes even gave off facial expression we would understand or predict due to our understanding of the social world.

That answer will depend on the person 100%. Everyone is different so whether your morals feel more accessible or the obtainment of money.

It would be next to impossible for everyone to process and remember everything at once. Hence why it is recommended that the studying you take breaks and divide the study material into easier and more manageable information.

This is true, it is uncommon for humans to just sit on information. We almost always share it or act on it by changing our behavior or others behavior.

Speaking if things go how we expect them to, in the video "Advertising-Priming Demo." The man was almost completely able to predict what the advertising 'experts' would come up with. It is crazy to see how easy humans are able to predict other humans sometimes. Whether that be ideas, phrases, actions, habits, etc. it is all a crazy concept.

That is a crazy thought. Imagine a world though where no one had gaps of knowledge, there would no longer be a need for research or science, and I wonder with AI if we will ever get to that point like mentioned earlier in the chapter.

Speaking of automatically knowing something, in the video "Derren Brown Astrology" it is discussed that we think we know ourselves well, but when someone points something out that we do, like a habit or personality trait, it makes us take a step back and reflect like wow. Like sitting here thinking about how it is true that no matter what fast restaurant I go into I know exactly what to do.

That is an interesting thought but I personally do think it would be super difficult to end the human race considering there are over 8 billion people.

lack of staking claim in the embeddedness of infrastructure — tfw your job is the most politicized and yet you claim apoliticalness?

To go back to my first annotation: If I write about New Mexican food as a lineage, am I reducing it to textual analysis? Cultural rhetorics seems to push me to honor it as lived, embodied, relational practice (cooking, memory, story) and not just history-as-text. That being said, how can scholars write about it without flattening it?

Rhetoric isn’t just speeches and writing; it’s cultural practice itself. So rhetoric is study and practice. But what are the boundaries of calling practice “rhetorical”?

What I gather from scene 1 and scene 2 is that storytelling is not strictly academic and not strictly everyday but instead a method that recognizes both as valid and interconnected.

Is this because story functions as method, research, teaching? In other words, storytelling = how knowledge circulates? This reminds me of my grandma teaching me how to make New Mexican food. She’s not just giving a recipe. Through her stories and methods, I learn family history (research), skills (method), and culture (teaching). Is this the kind of everyday practice they mean, or are they pointing more toward academic contexts?

The Low > is unclear

Maybe bolding the relevant parts of the bullets could work here.

NTU GPTI 的編輯應該注意了。

既然po文一開始就是中英雙語，"English to follow" 是錯誤用法，這意思是：現在沒有英文，稍後提供。

Keep in mind when upcoming writing task occur