DOI: 10.1111/jnc.16205

Resource: MMRRC_050600-UNC

Curator: @dhovakimyan1

SciCrunch record: RRID:MMRRC_050600-UNC

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What is this?

DOI: 10.1111/ejn.16510

Resource: (RGD Cat# 13508588,RRID:RGD_13508588)

Curator: @dhovakimyan1

SciCrunch record: RRID:RGD_13508588

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What is this?

DOI: 10.1101/2024.08.28.610136

Resource: (IMSR Cat# JAX_032864,RRID:IMSR_JAX:032864)

Curator: @olekpark

SciCrunch record: RRID:IMSR_JAX:032864

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What is this?

DOI: 10.1101/2024.08.28.610136

Resource: (IMSR Cat# JAX_029591,RRID:IMSR_JAX:029591)

Curator: @olekpark

SciCrunch record: RRID:IMSR_JAX:029591

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What is this?

DOI: 10.1101/2024.08.28.610136

Resource: (IMSR Cat# JAX_030541,RRID:IMSR_JAX:030541)

Curator: @olekpark

SciCrunch record: RRID:IMSR_JAX:030541

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What is this?

DOI: 10.1101/2024.08.28.610136

Resource: RRID:IMSR_JAX:000664

Curator: @olekpark

SciCrunch record: RRID:IMSR_JAX:000664

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What is this?

DOI: 10.1093/nar/gkae673

Resource: RRID:Addgene_42230

Curator: @olekpark

SciCrunch record: RRID:Addgene_42230

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What is this?

DOI: 10.1007/s00401-024-02804-z

Resource: RRID:Addgene_132972

Curator: @olekpark

SciCrunch record: RRID:Addgene_132972

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What is this?

DOI: 10.1007/s00401-024-02804-z

Resource: RRID:Addgene_23971

Curator: @olekpark

SciCrunch record: RRID:Addgene_23971

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What is this?

One of the three pillars of Dual Language Education from the book, The Guiding Principles of Dual Language Education, is bilingualism and biliteracy.

I am always seeking articles that provide instructional implications that improve or help to build a pedagogical strategy.

Data collection method where student explanations of strategies utilized is a strength because it engaged the participants in metacognition.

The sixty-three participants is a good amount of students to collect data from and the data collection from students in different grades was also interesting.

Again this goes back to the idea of fiction in philosophy. (Same as previous comment).

Doesn't this contradict the idea of fiction in philosophy? The idea of the family dying won't actually happen and we are just testing out the fields of waters so the idea of fiction is actually very important in philosophy right? Fiction gives us the pathway to imagination which will help us to determine what are the pro and cons of the situation.

I think that thinking critically definitely helps us see things that were not quite visible and as obvious. But being able to take this new found insight and consider how we might bear able to change or find an alternative is what makes philosophy great. I still have questions whether the applications are as talked about as the theories and hypotheticals because often times things are much more difficult to do then said.

I think this is such a generic way of framing something so big that it gives so many interpretations. This gives so much interpretations since it seems to say that philosophy is the study of the world from OUR perspectives. Does this mean that philosophy is different to each person?

I think in order to reconstruct the foundation of knowledge, we have to break everything down in bite sized pieces and become familiar with these building blocks before we can proceed with our philosophical construction. I wonder if we need to reconstruct our views of the world around us or if other courses that are required for students provide a sufficient framework for which we can stand upon.

Clearly goes against the author's first paragraph, just doesn't make sense to me why the author would give such a specific example for the first paragraph when the author's students don't even fit into this category besides a few of them.

Realistically though the chances of this happening are very little and especially for a person like me, a 17 year old man, this is pretty unrealistic. Yes it does set a good start to the article, but personally I feel that it should be more generic and a better hook or something to get me captured.

Coming from two different perspectives allows for more discussion and critical thinking

Philosophy has many definitions, one being that there are multiple ways to dive deeper into what is being asked or told

skepticism (scepticism?) - questioning your own knowledge as well as other assumed truths. Descartes' method relies on creating imaginary scenarios to believe as truths in order to use them as a basis for questioning.

Jayden Perkins: I think this is interesting because some parents who teach the "Hard way" in life will tell you about this reality from a young age, that you take what youre dealt and make the best out of it. If they studied philosophy would this mindset change, is that the goal?

Jayden Perkins: In a world where someone can think this world is perfect, would philosophy still be something they can learn from? Doesnt really apply to me because I think the world is the furthest thing from perfect, but in the case someone did, could they still benefit from the subject?

key idea

plato's cave

govt should be made for the people, by the people, but that means ALL people

something feels a little messed up here but I can't put my finger on it. it would be nice to make billionaires have to pay rent for once though, so maybe we should do this in real life

a little narcissistic?

but how can they determine that their "knowledge" is superior/the "right" knowledge?

should we question reality as people born into certain religions tend to question their beliefs at an older age/as they become disillusioned?

ability to study philosophy as a measure of class divide?

teaching critical thinking and to question the world around you

asking how we know what we know?

E. coli; MDR E. coli; extended spectrum beta-lactamase (ESBL) E. coli strain; non-MDR E. coli; carbapenem-resistant strain; non-carbapenem-resistant strain; E. coli causes uti; E. coli causes IBD;

Note: an update was removed, see Archive.org 30 Aug 2023 SUN - UL 1973, Batteries for Use in Stationary, Vehicle Auxiliary Power and Light Electric Rail (LER) Applications [ANSI/CAN/UL 1973:2022 Ed 3] Check Archive.org

Trabajaron juntos muchos años, cumpliendo y aceptando la dirección, del cuerpo gobernante, llevando las cartas, y decisiones de éstos hermanos para que esas congregaciones obedecieran y se mantengan unidas y organizadas bajó la dirección y guía de Jehová y Jesús mediante el espíritu Santo.

Timoteo era sincero y desarrollo amor muy fuerte por lo que aprendia de Jehová y su Organización. Al punto que éso lo impulsaba a aceptar su responsabilidad de predicar y enseñar, lo mismo que otras responsabilidades de servicio, cómo acompañar a Pablo, dar discursos, corregir y nombrar ancianos. Y desarrollar esa clase de amor está al alcance de los jóvenes hoy, porque lo vemos en muchos jóvenes de la congregación, que el entrenamiento y enseñanza de sus padres u otros hermanos les llegó al corazón, y hoy son ejemplo y columnas de la congregación, por su preparación, enseñanza y disposición a aceptar responsabilidades

Unfortunately, many different animals can carry various kinds of diseases. Which can consist of bacterial infections (Salmonella, Leptospirosis, Cat-Scratch Disease (Bartonella)), viral infections (Rabies, Lymphocytic Choriomeningitis Virus), parasitic infections (Toxoplasmosis, Roundworm and Hookworm, Fleas and Ticks), and fungal infections (Ringworm). The level of concern is understandable since many of these diseases can be transmitted to humans.

To understand what these questions might have been talking about, I thought it could be useful to look up what the different definitions are for each of them/what they consist of. Intrusion items: These assess unwanted and distressing thoughts, memories, or flashbacks of a traumatic event that intrude into a person's mind.

Avoidance items: These measure efforts to avoid thoughts, feelings, people, places, or situations that remind someone of the trauma.

Hyperarousal items: These evaluate heightened states of alertness, irritability, difficulty sleeping, and being easily startled following a traumatic experience.

This condition is characterized by the formation of a blood clot (thrombus) in a deep vein, usually in the legs or thighs. This can lead to death and multiple other health problems if it goes untreated for a long period of time.

In this era, people took their oath to their faith seriously, and he was outpouring his thoughts about how they do not attend as often as they should/like, and he contributes that to their misfortunes.

This gives a glimpse of hope to what is to come for Maria.

It is good to see some holding onto to their heritage, as it is portrayed through their display of clothing.

This sounds to be part of a farmer's almanac!

You can definitely tell by the names, that this was from a different time and era than we are in now.

To gather a picture, Peribonka is a municipality situated in the Maria-Chapdelaine Regional County Municipality of the Canadian province of Quebec. It is located on the north bank of Lac Saint-Jean, at the mouth of the Peribonka River, where it forms a bay.

desired outcome

agree

interesting data to compare

easy follow up study opportunity

good consideration

important for everything

need larger studies to compare these

if only

self regulation and self management seem like the foundation, yet they get less attention. easier to see when social skills are lacking?

I have seen this as well in my district

all important outcomes

importance of adult SEL

unfortunately

suggestion for future research

good to note, potential explanation

consistent with existing research

major finding with implications for practice

there are mixed results in this paragraph. some expected parallels, and others not so much

convergent parallel

useful visual

interesting, why?

not comfortable enough with quant to understand this

would be interesting to see by state within USA

summary of quant approach

maybe this is more motivating

does not need to be this way

tough without support

focused only on tier 2-3

this does not need to be zero sum

what if they lead the work? why does it need to start from the school?

non-academic but important

blending of SEL and academic instruction

disconnect between what they have and what they want

can we teach skills we are lacking? adult SEL focus is vital

lines up with the data from earlier

aren't all moments teachable for SEL?

practical experience tied into the research, even though they were unaware of research findings

this makes sense

direct quotes from participants used throughout

helpful to note

we used this

4 categories for coding qual.

explains type of analysis

good to note

will this be valid?

explains why an adaptation was used

semi structured

helpful for the reader

no ethical concerns

non-probability

no ethical concerns

explains the 13 instead of 14

gives some background info on participants

population for qual.

purposeful sampling

benefits of existing measure

integration phase

why interviews

type of mixed methods study

RQ3

assumptions/hypotheses

rq2

RQ 1a, 1b, 1c

shows a big need in teacher prep, clearly flows down to schools

gap

always!

explore these articles for my own research

what would count as SEL for this?

so where does the training happen?

interesting finding, makes sense

noteworthy

example of how mixed methods could benefit this research space

important

how current? country/state/city level differences?

interesting point to explore via study referenced

SEL is not only for elementary aged students

social awareness definition

self management definition

self awareness definiton and subcomponents

implications for teaching these skills?

CASEL 5

Test

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Reply to the reviewers

Response to Reviewer Comments:

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

*Glaucoma-associated optineurin mutations increase transmitophagy in vertebrate optic nerve.

Summary In Jeong et al., the authors perform live imaging of the X. laevis optic nerve to track neuronal mitochondrial movement and expulsion in an intact nervous system. The authors observe similar mitochondrial dynamics in vivo as previously described in other systems. They find that stationary mitochondria are more likely to be associated with OPTN, suggestive of mitochondria undergoing mitophagy. Forced expression of OPTN mutations results in a larger pool of stationary mitochondria that colocalize withLC3B, and OPTN. Finally, the authors argue that extra-axonal mitochondria are observed more frequently in OPTN mutants, suggesting that mutations in OPTN that are associated with disease can lead to an increase in the expulsion of mitochondria through exopher-like structures.

Major Findings and impact: • The authors establish that mitochondria dynamics can be tracked in the X. laevis optic nerve. • OPTN mutations increase the stationary pool of mitochondria and likely result in increased rates of mitophagy. • Exopher-like structures containing mitochondria and LC3 can be expelled from the optic nerve and increase in the presence of OPTN mutations. These structures were observed in a living system and have interesting implications in the context of disease.

Concerns: • The authors state in their results that the secreted blebs are exophers. While these initial observations are consistent with exophers, additional data are needed to strengthen this claim. For example: what are the sizes of secreted vesicles? Do all express LC3? How frequently do these occur? From where are they expelling? Alternatively, the discussion of exophers could be moved to the discussion.*

We agree that calling the axon shedding intermediates “exophers” was an overreach on our part. While we believe that in all probability time will demonstrate this to be the case, reviewers are correct in stating that putting our work in the context of exophers is best left to the discussion. We have removed all mention of exophers from the results and graphical abstract and now use the term only once in the discussion. We do provide detail as to the frequency of the structures, what fraction contain mitochondria, and morphological parameters of the contained mitochondria. And while all of these new data support them being exophers, the point remains that the use of the nomenclature “exopher” in the results section was inappropriate.

• Quantifications in sparse labeling experiments seem quite surprising and concerns related to these findings should be addressed. As the authors used LC3b expression to represent axonal volume, the authors should demonstrate that this is the case using an axonal fill or membrane marker in both the wt and E50K conditions. This is important as it is unclear whether LC3b expression is consistent between the wild type and the E50K conditions. Lower expression of LC3b in E50K could account for the large changes in axonal width that seem to be observed and could confound the measured amount of expelled mitochondria.*
• *

We agree that using EGFP-LC3b as a “cell fill” was problematic in a situation where the interventions likely perturb autophagy/mitophagy and therefore might have also perturbed LC3b. We do provide some axon width and LC3b-EGFP intensity data for a partial dataset that had been imaged side-by-side, showing that expression of LC3b is not different in the two conditions. We also provide independent measures of extra-axonal mitochondria based on a membrane-GFP reporter. While in principle there would be value to repeat the studies of Wt vs. E50K in the context of the membrane-GFP reporter, these experiments would involve new constructs and new breedings, and would likely take months to years to complete.

• • Could large amounts of exogenous mitochondria in explant experiments be from cells that died during the plantation?* The concern that some of the exogenous mitochondria signal might derive from degenerating axons is one that we worry much about, and not only in the transplantation experiments. In our sparse labeling experiments we do occasionally see axons undergoing Wallerian degeneration, but it is rare and does not appear to be more common in the expression of the mutated OPTN, at least not at the stage after transgene expression that the analyses were performed. We do provide new data that expression of E50K OPTN does not compromise vision at the time that experiments were carried out, ruling out that extra-axonal mitochondria are the result of large-scale degeneration. However, from other data we know that axon loss would likely need to be very extensive to manifest itself in functional vision loss in our behavioral assay, so milder axon loss contributing some noise to the measures cannot be excluded. But, the point raised is heard, and now we include a sentence in the discussion acknowledging that some of the signal outside of axons could have been due to degenerating axons, but still contend that our documentation of shedding intermediates support the view that many of the axonal mitochondria outside of axons were shed from otherwise intact axons.

Suggested experiments/quantifications: • In OPTN/MITO/LC3b trafficking experiments, does flux/number of events change? Representative kymograph in Figure 2D seems to show far more OPTN-positive mitochondria which is opposite of what is shown in Figure 2C.

Multiple reviewers rightfully point out that we did not carry out the flux experiments which would be necessary to make definitive statements regarding the amount of mitophagy. New experiments show that inhibiting lysosomal activity through chloroquine does increase the amount of astrocytic autophagosomes not yet acidified as expected, and that they contain axonal mitochondria signal, supporting the idea that astrocytes are involved in the degradation of axonal mitochondria. However, they did not show changes in the amount of stopped mitochondria, supporting the view that the co-localization of OPTN and mitochondria in axons is not conventional autophagy. This is a very important point that affects the interpretation of our results, and we thank reviewers for suggesting this experiment.

• Demonstrate that axonal width measured with LC3B is representative of axonal fill/membrane marker in wt and E50K. Axonal area appears to change, is this accurate? This appears to be the case for both figure 3 and figure 4.* Addressed above.

• Raw images in addition to the reconstruction would be beneficial.* Now include raw images beside the reconstruction at the first use of reconstructions.

• Further characterization of exopher-like structures.**

* Addressed above.

***Referees cross-commenting**

I agree with the concerns of the other reviewers, and perhaps was over-optimistic about a timeline for revision. However, I do think the work is worth the effort, and I hope to see a revised manuscript published somewhere, as these observations are novel

Reviewer #1 (Significance (Required)):

This work reports potentially novel biology, and thus will be of interest to the field. The strength of the study is that it is an initial description of this biology, rather than a complete analysis. The work raises many more questions than it answers, and much further work on this topic is required to support these initial findings, but the manuscript will likely be of interest to many. Revisions are required to improve the rigor and clarity of the work, but following these revisions we recommend publication to facilitate follow-up work.*

Fully agree that our study raises far more questions than it answers. Believe that the revisions made to address reviewer comments go a long way to improve rigor and clarity of the work. We hope that the reviewers agree and deem the changes sufficient.

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

Summary: This article studied transmitophagy in xenopus optic nerves in the context of overexpressing glaucoma-associated optineurin mutations. Using a series of labeling, imaging and transplantation techniques, the authors found that overexpressing mutated optineurins stops mitochondria movements and potentially induces transmitophagy, and that astrocytes are responsible for taking up the extra-axonal mitochondria. Below are my comments on this article.

Major comments: 1. Identifying extra-axonal mitochondria is key to this research. In Figure 3, the authors used EGFP-LC3B as a marker for RGC boundaries. However, it is unconvincing how perfect LC3B is as a cell membrane marker. Particularly in the case of OPTN E50K OE, it seems that the optic nerve is thinner than the WT condition, which makes the quantification of extra-axonal OPTN less convincing. The authors should detect extra-axonal mitochondria with an RGC membrane marker or cytosolic marker. In addition, in Figure 3, the extra-axonal mitochondria seem to localize mostly on the dorsal surface. Why is there such a polarity?*

As stated above, we acknowledge that the use of LC3b as both an autophagosome marker and a cell fill was somewhat problematic and now provide additional experiments ruling out that the LC3b expression or axon thickness in our sparse axon labeling experiments, or that E50K might affect the thickness of the optic nerve. In addition, we also provide additional new data using a bona fide membrane marker together a transgenic labeling or RGC mitochondria that also shows under the “baseline state” extensive mitochondria signal outside the axons on the surface of the optic nerve (New Fig. 6A and new Suppl Fig. 3D). All the new data are consistent with the previous data and support the view that using LC3b potentially could have been problematic, for the reasons reviewers state, but in practice it was not.

The reviewer observes that the E50K optic nerve appears thinner--this observation is not a consistent difference in optic nerves across the experimental groups. The images we show are always near the mean values for the quantitative results presented, and we rather not include prettier nerves that are not representative of the whole datasets.

As for why the extra-axonal mitochondria localize mostly to the dorsal surface, it remains undetermined. There are dorsoventral differences in the optic nerve established during development, as developmental Sonic hedgehog signaling emanating from the midline appears to affect dorsoventral aspects of the optic nerve differentially. Early axon loss in humans and some models of glaucoma do show a dorsal bias, and there may be optic nerve lymphatic structure reported in mice that also may be preferentially dorsal. However, it is not known whether any of these observations are connected, so we did not want to speculate beyond what the data say. We do now explicitly mention the dorsoventral difference in the discussion, and state why we think it may be worth further study.

• The experiment in Figure 5 is very important as it gives direct evidence of transmitophagy. However, one caveat is that the mitotracker injection is done after the transplantation. If in rare cases the dye is leaky after injection and is taken up by astrocytes directly, then the conclusion that mitochondria from RGCs are phagocytosed by astrocytes will be flawed. The authors should either use a transgene in the donor to label mitochondria or inject mitotracker into the donor before the transplantation and repeat the experiments. In addition, in Figure 5E, what is the large membranous structure inside the highlighted astrocyte? Is it associated with phagocytosis?*

We fully agree that MitoTracker is an imperfect tool, both for the reason stated here that the dye may get into the astrocytes directly (or may label astrocyte mitochondria after it is released from degrading RGC mitochondria), and, also as stated by reviewer 3, that it requires healthy mitochondria for labeling. For this reason, we have added new datasets that rely on RGC mitochondria labeling not by Mitotracker but through a genetic reporter. As to identity of the conspicuous structure shown inside the astrocytes, it remains an open question, and we are avidly pursuing what astrocytic organelles are involved through additional transgenic reporters and correlated-light-EM studies, but those are complicated experiments that are beyond the scope of the current manuscript.

• This research is entirely based on overexpression of OPTN. Since overexpressing WT OPTN does seem to affect mito trafficking (Figure S2G, and the description in the manuscript is often inconsistent with this result), it is unclear what the increased stalled mitochondria really mean when overexpressing mutated OPTN. Similarly, the authors examined extra-axonal mitochondria in Figures 3 and 4 all in overexpressing conditions, and made the connection that increased stalled mitochondria lead to transmitophagy. However, this conclusion will be better supported by using mutant animals rather than overexpression. The authors should consider using OPTN mutant xenopus if available or using CRISPR to introduce the specific mutations and repeat mitochondria trafficking and transmitophagy.*

• *

We thank this reviewer by pointing out an important detail that we failed to highlight, namely that transgenic overexpression of Wt OPTN (and/or Wt LC3B) does have a small but significant effect on mitochondria trafficking. Interestingly, it is affecting just the speed of retrogradely transported mitochondria, which based on the elegant work of Holzbaur and colleagues, include mitochondria destined for degradation. So, we now acknowledge more explicitly that, since our studies involve expression of OPTN and LC3b transgenes (fluorophore tagged human genes, no less), that some caution should be exercised in not overinterpreting the results. Nonetheless, since we show that expression of Wt OPTN behaves similarly to expression of a mitochondria reporter (Tom20-mCherry) in not affecting either stopped mitochondria or extra-axonal mitochondria, we believe that our results still stand. Nonetheless, we now make mention of the effect Wt OPTN on retrograde mitochondria movement. We have embarked on OPTN loss-of-function studies and have some founder animals carrying CRISPR-generated mutations; however, these experiments will take additional time, and based on the results in mammals may or may not show any measurable effects in our assays, not only because of possible redundancy by the other damaged mitochondria adaptors that we mention in the introduction, but also because the mutations that affect the shedding process (as well as cause glaucoma) are thought to be gain-of-function mutations. However, we decided not to dwell on these complexities in the discussion, as the discussion was previously quite extensive and now is even moreso with the added discussion on how our studies relate to those of exophers.

• On Page 12, the authors claim that even overexpressing WT OPTN causes extra-axonal mitochondria in the optic nerve. However, there is no control condition without OE to support this conclusion. It is thus unclear to what extent extra-axonal mitochondria occur at baseline and how many extra-axonal mitochondria can be induced by overexpression. The authors should include, in Figure 3 and 4, controls without overexpression.*

We acknowledge that our language was confusing and somewhat misleading on this point. With the caveat mentioned above that WT OPTN expression does perturb the system somewhat (by increasing the speed of mitochondria retrograde transport, perhaps by increasing the proportion of retrograde moving mitochondria tagged for degradation), we still contend that the state observed after WT OPTN expression is close to the “baseline” state. In support of that, in the new data included in response to the LC3b concern, we observe plentiful shedding events in the absence of any OPTN or LC3b transgenes. Indeed, what may be the most surprising finding of our studies is that in the absence of any significant perturbation of OPTN, there is already a large fraction of axonal mitochondria that are outside of axons and inside of astrocytes, which is consistent with what we previously observed in the optic nerve head of mice; however, the current studies provide much more rigorous quantification of the process and live imaging of intermediates, but also provide for an intervention that increases the process. While there are many more questions to answer, we do believe our studies contribute mechanistic insights.

• A technical question regarding kymographs: Based on Figure 2C, it looks that OPTN and LC3B labeling are pretty diffuse in axons and this makes sense since they may only be associated with damaged mitos. But this raises a question about how accurate the kymograph assay is. It may significantly underestimate the fraction of OPTN/LC3B that is stationary since they appeared diffusedon the kymograph. This may explain why the percentage of stationary OPTN/LC3B is so small when the authors OE WT OPTN in Figure 2E and 2E', compared to the percentage of moving mitochondria shown in Figure 1E.*

We fully agree that the kymograph studies likely underestimate the amounts of stationary mitochondria for the reasons stated. However, we interpret the discrepancy between Figure 1E and 2E and 2E’ differently. We believe that the value of stopped mitochondria in the sparse labeling experiments are actually more accurate, as the value of stopped mitochondria in the whole nerve experiments likely include mitochondria stopped within the axons, but also mitochondria recently shed either by those or nearby axons which are perceived to be in axons due to limitations of imaging resolution. In the discussion we now make very explicit that all the measures we provide need should be interpreted as estimates, as every experiment relies on assumptions and is subject to technical limitations.

Minor: 1. Figure 2E and 2E' do not agree with the text on page 7 and page 8. Not only F178A, but also H486R and D474N have no effect on OPTN trafficking. The authors should make their conclusions more accurate.

F178 was the only mutation that had no effect on either OPTN or LC3b in either F0 or F1 experiments. However, we agree that our language should have been clearer, and now we have made our description of the results (and conclusions) more accurate.

• Figure S2E-F: why does OE of mutated OPTN in F1s but not in F0s reduce trafficking speed compared to WT?*

We do not know the reason for this discrepancy. Though it does not wholly agree with the rest of the story, we felt it important to include all relevant data, not only that which perfectly fit our interpretation. One possible reason may be that the F1 data derives from a single integration event, which is the reason why we trust more the F0 data that derive from multiple integrations, in what are essentially outbred animals, which is the reason we present the F0 data as the primary results where possible.

* In movie 5, fusion of exopher with other structures is not clear and also the GFP signal does not disappear, which is in contrast to the statement in the text that the GFP signal is quenched in acidified environment. To confirm that LC3B leaves RGC axons in exophers, the authors should consider switching the fluorophores and examine LC3B localization during exopher formation.*

This too is a valid point, and we have amended our description of these results. While swapping fluorophores between OPTN and LC3b is a highly worthy experiment, for technical reasons it likely would take many months to carry out just because of how involved it is to make the relevant constructs (recombineering details provided in the methods section).

• In figure 6, to better show exopher formation and the pinching-off step, the authors should consider labeling the membrane and mitochondria instead of using the LC3B and OPTN marker.*

This arguably was the biggest weakness of our initial submission, and now provide new experiments using a bona fide membrane marker. We have not yet captured a pinching-off event with these better reporters, but that is not surprising given how rare they are, which we now quantify. Indeed, a membrane reporter and a mitochondria transgene in sparsely labeled axons are the ideal tool for figuring out the frequency of these structures and what fraction contain mitochondria, data which we now provide.

***Referees cross-commenting**

Generally agree with the criticisms voiced by the other reviewers; in aggregate the reviews indicate the manuscript needs more than just a quick fix.

Reviewer #2 (Significance (Required)):

Previous literature has already described the transmitophagy process in the optic nerve. The significance of this paper lies in the observation that overexpressing glaucoma-associated OPTN mutants can induce increased transmitophagy through astrocytes, which points to a potential role of OPTN in glaucoma. A highlight of this paper is the use of correlated light SBEM to directly show transmitophagy in astrocytes. However, the significance of this paper may be limited for the following reasons: 1. everything is based on overexpression of mutated OPTN, which makes it hard to translate the results to real disease conditions; 2. The consequence of increased transmitophagy on RGC survival or visual functions is unclear.

*

While we agree that much of the paper is based on OPTN overexpression, we did have experiments and now provide more that that were not based on OPTN overexpression. Some of these still involve expression of a different transgene (Tom20-mCherry) that might in principle perturb the system, though we show that expression of Tom20-mCherry does not affect mitochondria movement parameters as measured by Mitotracker. As to “the consequence of increased transmitophagy”, we do now provide data showing that there is no vision loss suggestive of axon loss or severe dysfunction at the time that the imaging studies were carried out. Whether longer term expression of these OPTN transgenes lead to axon degeneration and visual dysfunction are studies that are ongoing, but those studies involve extensive characterizations and controls that are beyond what could be included in this study.

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

Summary In this work, Jeong et al describe the effect of Optineurin (OPTN) mutations in the transcellular degradation of retinal ganglion cell (RGC) mitochondria by astrocytes at the Optic Nerve (ON), a process previously described this group and referred as "transmitophagy" (Davis et al 2014). Here, authors use Xenopus laevis animal model to image the optic nerve of animals carrying different OPTN mutations associated to disease or with compromised function and explore its effect in mitochondria dynamics at the RGC axons. They find that OPTN mutants lead to increased stationary mitochondria in the nerve and affect their co-localization with mitophagy-related markers, suggesting alterations in this pathway. Finally, they found that mitochondria co-localizing with OPTN can be found in the periphery of the ON under different conditions and this is particularly increased in glaucoma-associated E50K mutation. This extracellular mitochondria are transferred in vesicles to astrocytes, as they previously described in mice (Davis 2014), where they are presumably degraded. Major comments - OPTN levels at a given time point cannot be used as readout for mitophagy level/flux. Both OPTN and LC3b are degraded upon fusion with acidic compartment (i.e. lysosomes, PMID: 33783320, 33634751) and that is the reason why the field of autophagy /mitophagy blocks lysosomal activity to measure autophagy/mitophagy flux (PMID: 33634751). In this document, authors claim that there is low levels of mitophagy in RGC axons at baseline and increased levels of mitophagy in glaucoma associated perturbations just based on increased presence of OPTN+ mitochondria in this condition. This could be also interpreted as an accumulation of non-degraded defective mitochondria due to a mitophagy block in neurons carrying the glaucoma associated mutation, which is the opposite of what they propose. If authors want to evaluate mitophagy levels in this system, mitophagy/autophagy flux experiments should be performed.*

In response to reviewers, we do now include “lysosome inhibition” experiment, using chloroquine at doses modestly above those used in aquaculture as an anti-parasitic. After testing various chemical means to inhibit lysosome activity, it was the only one that did not adversely affect the animals. We know the chloroquine intervention works because we see the expected increase in autophagosomes using the standard LC3b-tandem reporter, and in those unacidified astrocytic autophagosomes we do indeed find axonal mitochondria signal. However, since the amount of mitochondria signal there is small relative to the total amount of axonal mitochondria in the astrocytes, we do not feel it would be appropriate to make mechanistic claims, for example claiming this to be related to LC3b associated phagocytosis; much more work would be needed to make that claim. However, we were surprised to find no alteration in either stopped mitochondria in axons or axonal mitochondria material within the astrocytes. There are technical reasons why this result might be difficult to interpret, but now having done it (as we should have before), we are even more careful in describing the process as transcellular degradation rather than transmitophagy. We elaborate further on this point in the next response.

- I find inappropriate the use of the term "transmitophagy". Although this term transmits very well the message that the authors try to strength, the term "mitophagy" refers to the specific elimination of mitochondria through autophagy (PMID: 21179058). There are many reasons why I think that "transmitophagy" is not adequate to describe this phenomena but I will just refer to these three: First, authors do not provide data showing that this mechanism is specific for mitochondria as they have never checked for the presence of other type of cargo in the vesicles produced by RGCs. If these are related to exophers as they suggest in the document, is very probable that they contain other type of cargo; Second, if the final destiny for those particles is the acidic compartment of astrocytes, this process may have nothing to do with autophagy/mitophagy and just share some molecular mediators with those pathways; Third, they should explore if other canonical mitophagy molecular mediators (i.e. Parkin/Pink) are regulating the production or the mitochondria recruitment to this extracellular particles.

We too struggle with our own “transmitophagy” term, for the very reasons stated. To address this concern, we now refer to the process as “transcellular degradation of mitochondria”, which is how we described it initially in mice as well. We do present new data that show that while the majority of axonal outpocketings contain mitochondria, not all do. This suggests that the others may contain other cargo, which supports the view that what we are dealing with in axons are indeed exophers. And yet, since what we measure is mitochondria, we think most appropriate to describe the process narrowly and not extrapolate to other types of exophers. We agree that what we originally discovered in mice and now live image and perturb in frog, may not be “autophagy” according to the strict definition of the term, but rather a process that uses some of the same molecular machinery, which given the evolutionary link between autophagy and phagocytosis that should be no surprise. Terminology can be tricky, and we thank the reviewer for calling us out on this point. We now use the term “transmitophagy” only once in the discussion section making the link between our work and the emerging field of exopher biology, and use that occasion to elaborate the point that the more descriptive term “transcellular degradation of mitochondria” is more appropriate in our case.

*- In several experiments, authors use Mitotracker instead of genetic tools to quantify the amount of mitochondria co-localizing with OPTN (Fig2, Fig3) or being transferred to astrocytes (Fig4). A problem here is that Mitotracker needs the mitochondria to be active at the time of injection in order to label them (PMID: 21807856) and it has a clear effect in mitochondria dynamics in their setting, as pointed by the authors. Since most mitochondria transferred to astrocytes would be presumably damaged and not able to import Mitotracker, I am concern about how this is affecting their quantifications and the conclusions.

*

We agree. The use of Mitotracker to label the RGC mitochondria can be problematic for the reasons stated by reviewers 1 and 3. Indeed, our opinion is that many of the studies out there that claim to demonstrate transfer of mitochondria between cells likely are just showing the transfer of the dye rather than the mitochondria. While the previous submission included a number of controls to address this concern, we now provide multiple new experiments that measure the transfer of mitochondria through a transgene rather than Mitotracker. The provided experiments use a new Tom20-mCherry transgene which is highly specific to mitochondria due to the use of an SOD2 UTR. We have similar data using RGC-expressed Mito-mCherry and Mito-EGFP-mCherry (using the commonly used Cox8 mitochondria matrix targeting sequence); we do not include such data because we find the provided data sufficiently compelling, and the story is already sufficiently long and complicated.

- Some conclusions are based on single images with no quantifications or statistics. This is the case for: 1) Page 6) "Most of the mCherry and Mitotracker objects colocalized with each other both in the merged images (Fig. S1C) and kymographs (Fig. S1D), indicating that the mitochondria-targeted transgene and Mitotracker similarly label the RGC axonal mitochondria".

That is a fair comment. After reanalyzing the original dataset used, it would be very difficult to quantify that statement, largely because the Tom20-mCherry expression was relatively weak in those particular animals. We are confident that we could generate a new dataset to provide support for this statement, but instead chose to just provide side-by-side movies of mitochondria labeled by Mitotracker or the Tom20-mCherry transgenes, which we believe is far more compelling than any quantification we could provide.

2) Page 8) "In the nerves labeled by Mitotracker, visual inspection of the raw images (Fig. 2C) and the derived kymographs (Fig. 2D) showed that OPTN and the Mitotracker labeled mitochondria often co-localized, particularly in the stopped populations, and more so in the animals expressing E50K OPTN, further suggesting that at least a fraction of the stopped LC3b, OPTN and mitochondria might represent mitophagy occurring in the axons".

While we have made a minor change to this sentence, we feel that it is appropriate given that it serves just as a justification to carry out the quantitative studies that follow. We would not have quantified the process had it not been obvious to the eye. However, we do not interpret the results as supporting that mitophagy occurs in axons, for the reasons explained above.

3) Page 14) "We also observed similar axonal dystrophies and exopher-like structures in E50K OPTN under similar imaging settings, but with 2-min intervals and additional Mitotracker labeling (Mov. 6), demonstrating that these structures not only contain OPTN but also mitochondria or mitochondria remnants". Image in video is not clear and there is not quantification for OPTN or OPTN+ mitochondria.*

*

We have removed Mov. 6.

*Minor comments

• In Figures showing the reconstruction of OPTN+ mitochondria outside nerve (Fig.3 and Fig.4), those seem to be present only in one lateral of the nerve. Is this process polarized in any way (i.e. faced to astrocytes) or is the result of a technical issue (i.e. difference in laser penetration for blue vs Yellow lasers)? I think it will be important to include this in the discussion.*

This was also pointed out by reviewer 1, and we agree that it is worth including in the discussion, which we now do. While we do not believe it to be a light penetration issue (based on fluorescence intensities and apparent spatial resolution), we also do not yet have an explanation. Having studied dorsoventral differences in the visual pathway both during my graduate and post-doctoral years, I am very interested in this asymmetry, and we have some theories that might explain it, mentioned above. The asymmetry is obvious and thus we think it would have been inappropriate not to show, but it also be inappropriate to be overly speculative.

- In Pag.13 authors claim "OPTN and mitochondria leave RGC axons in the form of exophers". After "exophers" were coined by the Driscoll lab in 2017, too few people has adopted this terminology and the molecular machinery involved in this process is still under research. It is clear that the particles described here share some similarities with exophers like size (in the range of microns) and cargo (mitochondria), but you have not demonstrated if they share the same origin or are part of the same phenomena. For that reason, I recommend to be more cautious with this statement and point these limitations in the discussion. Additionally, since Exophers are not a consensus or well defined particles, authors should include an introductory paragraph at the beginning of this section for readers to understand what they are talking about.

We wholly agree with all points. We now have moved all mention of exophers to just the discussion.

- Exophers described by Monica Driscoll and Andres Hidalgo laboratories are presented as "garbage bags" that help cells to stay fit through elimination of unwanted material. If the extracellular vesicles presented here are part of the same mechanism and potentially beneficial for the RGCs, why are they increased in OPTN mutants? Is it part of RGCs response to a proteomic stress generated by malfunctioning OPTN? I think that is critical to understand this to figure out the relevance of your findings.

• *

Our personal opinion is that the OPTN mutants most likely lead to stress focally in the axons, thus triggering exopher generation. We are carrying additional experiments to determine whether too much exopher generation or their insufficient degradation by astrocytes might be deleterious (by causing inflammation). However, those are big stories that would not stand on their own were we not able to first rigorously demonstrate that certain OPTN mutants increase exopher generation, which I believe our study demonstrates, albeit now without calling them exophers.

- Related to Fig.5G, authors say "The soma of the astrocytes were located at the optic nerve periphery but had processes that extended deep into the parenchyma". This is very interesting and opens the possibility that many mitochondria are directly transferred to astrocytes through that processes instead of the lateral of the nerve, meaning that your quantifications of "transmitophagy" may be underestimated.

* *We also agree that this. Our limited optical resolution, and limitations intrinsic to carrying out quantifications with Imaris software, are likely the main reasons for the discrepancy between the whole nerve and sparse-labelled-axon estimates of how much axonal material is outside of axons. Our view is that most of the transcellular degradation occurs within fine astrocyte processes, and that only in the case of failure to degrade material in these fine processes that significant amounts accumulate in the cell body (optic nerve periphery), and that in the cell body additional or different degradative pathways are utilized. Experiments using various transgenes and correlated EM as well as perturbation experiments are ongoing attempting to firmly establish what organelles are used in processes versus soma. However, we believe that such studies are well beyond the scope of this manuscript..

- Reference to Fig. S2G is missing. Now mentioned twice. Thank you.

- I cannot find in Fig.5 E-I legends what are the cells/structures labelled in Green and Red. Thank you.

***Referees cross-commenting**

In agreement with my colleagues, I think that a revision is needed to support some important points of the paper. The the work is interesting and I think it deserves a chance for revision. Having that said, I am not familiar with the breeding and experimental times when working with Xenopus but, considering the amount of work requested, it may require more than 3 months to have the work done.

*

*Reviewer #3 (Significance (Required)):

Until not very long ago, it was thought that mitochondria could not cross cell barriers. In recent years however, there has been an explosion in the number of works showing mitochondria transfer between different cell types in vivo. This may happen either as an organelle donation to improve energy production or as a quality control mechanism to get rid of damaged mitochondria, as it is the case in this work. The laboratory of Nicholas Marsh-Armstrong was pioneer in this field with a foundational work in 2014 where they show how RGC-derived mitochondria are captured and eliminated by astrocytes in mice (PMID: 24979790). This work was particularly relevant because it proposed for the first time that mitochondrial degradation can occur in RGC axons far from the cell soma, and surrogated in a different cell type, something that changed completely the view of how quality control is maintained in neurons and other cell types. In the present study, Jeong and collaborators explore how Glaucoma-associated Optineurin mutations affect this process, which is of potential interest for the broad cell biologist community due to its possible implications in other tissues and cell types (OPTN is broadly expressed), but especially for those researchers interested in neurobiology, quality control mechanisms and mitochondria biology. Since some OPTN mutations studied here cause disease, they are also relevant for the clinic. This work provides a thorough characterization of how relevant Optineurin mutations affect mitochondria dynamics in RGCs and their transference to astrocytes, as fairly claimed in the title. However, the mechanism by which they result in pathology is not either explored or carefully discussed, making this a descriptive work with no much conceptual insight. In addition, conclusions are often not unambiguously stated and the results part contains a lot of large sentences and unnecessary technical data that hinders reading and difficult the transmission of the key messages. Even if it stands as a descriptive work, the physiological and clinical relevance of these findings is not clear. There are some claims related with mitophagy activity that may require more sophisticated experiments (mitophagy flux with lysosomal inhibitors). Please see comments above. A critical point to understand the relevance of this work would be to demonstrate if alterations in transmitophagy are either causing or involved in the disease generated by these OPTN mutations in any way, or just a correlative phenomenon. To help authors contextualize my point of view, my field of expertise includes cell biology, imaging, quality control pathways, mitochondria biology and phagocytosis, among others. I am not familiar with Xenopus Laevis genetics or the limitations to work with this animal model.*

• *

We appreciate both the complements and the critiques. To a fault, we rather undersell than oversell. We are actively pursuing the possibility that dysregulation of this process is disease causing, and not just for glaucoma. However, those studies will not stand without a strong foundation, which we believe this study provides.

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Referee #3

Evidence, reproducibility and clarity

Summary

In this work, Jeong et al describe the effect of Optineurin (OPTN) mutations in the transcellular degradation of retinal ganglion cell (RGC) mitochondria by astrocytes at the Optic Nerve (ON), a process previously described this group and referred as "transmitophagy" (Davis et al 2014). Here, authors use Xenopus laevis animal model to image the optic nerve of animals carrying different OPTN mutations associated to disease or with compromised function and explore its effect in mitochondria dynamics at the RGC axons. They find that OPTN mutants lead to increased stationary mitochondria in the nerve and affect their co-localization with mitophagy-related markers, suggesting alterations in this pathway. Finally, they found that mitochondria co-localizing with OPTN can be found in the periphery of the ON under different conditions and this is particularly increased in glaucoma-associated E50K mutation. This extracellular mitochondria are transferred in vesicles to astrocytes, as they previously described in mice (Davis 2014), where they are presumably degraded.

Major comments

• OPTN levels at a given time point cannot be used as readout for mitophagy level/flux. Both OPTN and LC3b are degraded upon fusion with acidic compartment (i.e. lysosomes, PMID: 33783320, 33634751) and that is the reason why the field of autophagy /mitophagy blocks lysosomal activity to measure autophagy/mitophagy flux (PMID: 33634751). In this document, authors claim that there is low levels of mitophagy in RGC axons at baseline and increased levels of mitophagy in glaucoma associated perturbations just based on increased presence of OPTN+ mitochondria in this condition. This could be also interpreted as an accumulation of non-degraded defective mitochondria due to a mitophagy block in neurons carrying the glaucoma associated mutation, which is the opposite of what they propose. If authors want to evaluate mitophagy levels in this system, mitophagy/autophagy flux experiments should be performed.
• I find inappropriate the use of the term "transmitophagy". Although this term transmits very well the message that the authors try to strength, the term "mitophagy" refers to the specific elimination of mitochondria through autophagy (PMID: 21179058). There are many reasons why I think that "transmitophagy" is not adequate to describe this phenomena but I will just refer to these three: First, authors do not provide data showing that this mechanism is specific for mitochondria as they have never checked for the presence of other type of cargo in the vesicles produced by RGCs. If these are related to exophers as they suggest in the document, is very probable that they contain other type of cargo; Second, if the final destiny for those particles is the acidic compartment of astrocytes, this process may have nothing to do with autophagy/mitophagy and just share some molecular mediators with those pathways; Third, they should explore if other canonical mitophagy molecular mediators (i.e. Parkin/Pink) are regulating the production or the mitochondria recruitment to this extracellular particles.
• In several experiments, authors use Mitotracker instead of genetic tools to quantify the amount of mitochondria co-localizing with OPTN (Fig2, Fig3) or being transferred to astrocytes (Fig4). A problem here is that Mitotracker needs the mitochondria to be active at the time of injection in order to label them (PMID: 21807856) and it has a clear effect in mitochondria dynamics in their setting, as pointed by the authors. Since most mitochondria transferred to astrocytes would be presumably damaged and not able to import Mitotracker, I am concern about how this is affecting their quantifications and the conclusions.
• Some conclusions are based on single images with no quantifications or statistics. This is the case for:
  1. Page 6) "Most of the mCherry and Mitotracker objects colocalized with each other both in the merged images (Fig. S1C) and kymographs (Fig. S1D), indicating that the mitochondria-targeted transgene and Mitotracker similarly label the RGC axonal mitochondria".
  2. Page 8) "In the nerves labeled by Mitotracker, visual inspection of the raw images (Fig. 2C) and the derived kymographs (Fig. 2D) showed that OPTN and the Mitotracker labeled mitochondria often co-localized, particularly in the stopped populations, and more so in the animals expressing E50K OPTN, further suggesting that at least a fraction of the stopped LC3b, OPTN and mitochondria might represent mitophagy occurring in the axons".
  3. Page 14) "We also observed similar axonal dystrophies and exopher-like structures in E50K OPTN under similar imaging settings, but with 2-min intervals and additional Mitotracker labeling (Mov. 6), demonstrating that these structures not only contain OPTN but also mitochondria or mitochondria remnants". Image in video is not clear and there is not quantification for OPTN or OPTN+ mitochondria.

Minor comments

• In Figures showing the reconstruction of OPTN+ mitochondria outside nerve (Fig.3 and Fig.4), those seem to be present only in one lateral of the nerve. Is this process polarized in any way (i.e. faced to astrocytes) or is the result of a technical issue (i.e. difference in laser penetration for blue vs Yellow lasers)? I think it will be important to include this in the discussion.
• In Pag.13 authors claim "OPTN and mitochondria leave RGC axons in the form of exophers". After "exophers" were coined by the Driscoll lab in 2017, too few people has adopted this terminology and the molecular machinery involved in this process is still under research. It is clear that the particles described here share some similarities with exophers like size (in the range of microns) and cargo (mitochondria), but you have not demonstrated if they share the same origin or are part of the same phenomena. For that reason, I recommend to be more cautious with this statement and point these limitations in the discussion. Additionally, since Exophers are not a consensus or well defined particles, authors should include an introductory paragraph at the beginning of this section for readers to understand what they are talking about.
• Exophers described by Monica Driscoll and Andres Hidalgo laboratories are presented as "garbage bags" that help cells to stay fit through elimination of unwanted material. If the extracellular vesicles presented here are part of the same mechanism and potentially beneficial for the RGCs, why are they increased in OPTN mutants? Is it part of RGCs response to a proteomic stress generated by malfunctioning OPTN? I think that is critical to understand this to figure out the relevance of your findings.
• Related to Fig.5G, authors say "The soma of the astrocytes were located at the optic nerve periphery but had processes that extended deep into the parenchyma". This is very interesting and opens the possibility that many mitochondria are directly transferred to astrocytes through that processes instead of the lateral of the nerve, meaning that your quantifications of "transmitophagy" may be underestimated.
• Reference to Fig. S2G is missing.
• I cannot find in Fig.5 E-I legends what are the cells/structures labelled in Green and Red.

Referees cross-commenting

In agreement with my colleagues, I think that a revision is needed to support some important points of the paper. The the work is interesting and I think it deserves a chance for revision. Having that said, I am not familiar with the breeding and experimental times when working with Xenopus but, considering the amount of work requested, it may require more than 3 months to have the work done.

Significance

Until not very long ago, it was thought that mitochondria could not cross cell barriers. In recent years however, there has been an explosion in the number of works showing mitochondria transfer between different cell types in vivo. This may happen either as an organelle donation to improve energy production or as a quality control mechanism to get rid of damaged mitochondria, as it is the case in this work. The laboratory of Nicholas Marsh-Armstrong was pioneer in this field with a foundational work in 2014 where they show how RGC-derived mitochondria are captured and eliminated by astrocytes in mice (PMID: 24979790). This work was particularly relevant because it proposed for the first time that mitochondrial degradation can occur in RGC axons far from the cell soma, and surrogated in a different cell type, something that changed completely the view of how quality control is maintained in neurons and other cell types.

In the present study, Jeong and collaborators explore how Glaucoma-associated Optineurin mutations affect this process, which is of potential interest for the broad cell biologist community due to its possible implications in other tissues and cell types (OPTN is broadly expressed), but especially for those researchers interested in neurobiology, quality control mechanisms and mitochondria biology. Since some OPTN mutations studied here cause disease, they are also relevant for the clinic.

This work provides a thorough characterization of how relevant Optineurin mutations affect mitochondria dynamics in RGCs and their transference to astrocytes, as fairly claimed in the title. However, the mechanism by which they result in pathology is not either explored or carefully discussed, making this a descriptive work with no much conceptual insight. In addition, conclusions are often not unambiguously stated and the results part contains a lot of large sentences and unnecessary technical data that hinders reading and difficult the transmission of the key messages.

Even if it stands as a descriptive work, the physiological and clinical relevance of these findings is not clear. There are some claims related with mitophagy activity that may require more sophisticated experiments (mitophagy flux with lysosomal inhibitors). Please see comments above. A critical point to understand the relevance of this work would be to demonstrate if alterations in transmitophagy are either causing or involved in the disease generated by these OPTN mutations in any way, or just a correlative phenomenon. To help authors contextualize my point of view, my field of expertise includes cell biology, imaging, quality control pathways, mitochondria biology and phagocytosis, among others. I am not familiar with Xenopus Laevis genetics or the limitations to work with this animal model.

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Referee #2

Evidence, reproducibility and clarity

Summary: This article studied transmitophagy in xenopus optic nerves in the context of overexpressing glaucoma-associated optineurin mutations. Using a series of labeling, imaging and transplantation techniques, the authors found that overexpressing mutated optineurins stops mitochondria movements and potentially induces transmitophagy, and that astrocytes are responsible for taking up the extra-axonal mitochondria. Below are my comments on this article.

Major comments:

1. Identifying extra-axonal mitochondria is key to this research. In Figure 3, the authors used EGFP-LC3B as a marker for RGC boundaries. However, it is unconvincing how perfect LC3B is as a cell membrane marker. Particularly in the case of OPTN E50K OE, it seems that the optic nerve is thinner than the WT condition, which makes the quantification of extra-axonal OPTN less convincing. The authors should detect extra-axonal mitochondria with an RGC membrane marker or cytosolic marker. In addition, in Figure 3, the extra-axonal mitochondria seem to localize mostly on the dorsal surface. Why is there such a polarity?
2. The experiment in Figure 5 is very important as it gives direct evidence of transmitophagy. However, one caveat is that the mitotracker injection is done after the transplantation. If in rare cases the dye is leaky after injection and is taken up by astrocytes directly, then the conclusion that mitochondria from RGCs are phagocytosed by astrocytes will be flawed. The authors should either use a transgene in the donor to label mitochondria or inject mitotracker into the donor before the transplantation and repeat the experiments. In addition, in Figure 5E, what is the large membranous structure inside the highlighted astrocyte? Is it associated with phagocytosis?
3. This research is entirely based on overexpression of OPTN. Since overexpressing WT OPTN does seem to affect mito trafficking (Figure S2G, and the description in the manuscript is often inconsistent with this result), it is unclear what the increased stalled mitochondria really mean when overexpressing mutated OPTN. Similarly, the authors examined extra-axonal mitochondria in Figures 3 and 4 all in overexpressing conditions, and made the connection that increased stalled mitochondria lead to transmitophagy. However, this conclusion will be better supported by using mutant animals rather than overexpression. The authors should consider using OPTN mutant xenopus if available or using CRISPR to introduce the specific mutations and repeat mitochondria trafficking and transmitophagy.
4. On Page 12, the authors claim that even overexpressing WT OPTN causes extra-axonal mitochondria in the optic nerve. However, there is no control condition without OE to support this conclusion. It is thus unclear to what extent extra-axonal mitochondria occur at baseline and how many extra-axonal mitochondria can be induced by overexpression. The authors should include, in Figure 3 and 4, controls without overexpression.
5. A technical question regarding kymographs: Based on Figure 2C, it looks that OPTN and LC3B labeling are pretty diffuse in axons and this makes sense since they may only be associated with damaged mitos. But this raises a question about how accurate the kymograph assay is. It may significantly underestimate the fraction of OPTN/LC3B that is stationary since they appeared diffusedon the kymograph. This may explain why the percentage of stationary OPTN/LC3B is so small when the authors OE WT OPTN in Figure 2E and 2E', compared to the percentage of moving mitochondria shown in Figure 1E.

Minor:

1. Figure 2E and 2E' do not agree with the text on page 7 and page 8. Not only F178A, but also H486R and D474N have no effect on OPTN trafficking. The authors should make their conclusions more accurate.
2. Figure S2E-F: why does OE of mutated OPTN in F1s but not in F0s reduce trafficking speed compared to WT?
3. In movie 5, fusion of exopher with other structures is not clear and also the GFP signal does not disappear, which is in contrast to the statement in the text that the GFP signal is quenched in acidified environment. To confirm that LC3B leaves RGC axons in exophers, the authors should consider switching the fluorophores and examine LC3B localization during exopher formation.
4. In figure 6, to better show exopher formation and the pinching-off step, the authors should consider labeling the membrane and mitochondria instead of using the LC3B and OPTN marker.

Referees cross-commenting

Generally agree with the criticisms voiced by the other reviewers; in aggregate the reviews indicate the manuscript needs more than just a quick fix.

Significance

Previous literature has already described the transmitophagy process in the optic nerve. The significance of this paper lies in the observation that overexpressing glaucoma-associated OPTN mutants can induce increased transmitophagy through astrocytes, which points to a potential role of OPTN in glaucoma. A highlight of this paper is the use of correlated light SBEM to directly show transmitophagy in astrocytes. However, the significance of this paper may be limited for the following reasons: 1. everything is based on overexpression of mutated OPTN, which makes it hard to translate the results to real disease conditions; 2. The consequence of increased transmitophagy on RGC survival or visual functions is unclear.

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Referee #1

Evidence, reproducibility and clarity

Glaucoma-associated optineurin mutations increase transmitophagy in vertebrate optic nerve.

Summary

In Jeong et al., the authors perform live imaging of the X. laevis optic nerve to track neuronal mitochondrial movement and expulsion in an intact nervous system. The authors observe similar mitochondrial dynamics in vivo as previously described in other systems. They find that stationary mitochondria are more likely to be associated with OPTN, suggestive of mitochondria undergoing mitophagy. Forced expression of OPTN mutations results in a larger pool of stationary mitochondria that colocalize withLC3B, and OPTN. Finally, the authors argue that extra-axonal mitochondria are observed more frequently in OPTN mutants, suggesting that mutations in OPTN that are associated with disease can lead to an increase in the expulsion of mitochondria through exopher-like structures.

Major Findings and impact:

• The authors establish that mitochondria dynamics can be tracked in the X. laevis optic nerve.
• OPTN mutations increase the stationary pool of mitochondria and likely result in increased rates of mitophagy.
• Exopher-like structures containing mitochondria and LC3 can be expelled from the optic nerve and increase in the presence of OPTN mutations. These structures were observed in a living system and have interesting implications in the context of disease.

Concerns:

• The authors state in their results that the secreted blebs are exophers. While these initial observations are consistent with exophers, additional data are needed to strengthen this claim. For example: what are the sizes of secreted vesicles? Do all express LC3? How frequently do these occur? From where are they expelling? Alternatively, the discussion of exophers could be moved to the discussion.
• Quantifications in sparse labeling experiments seem quite surprising and concerns related to these findings should be addressed. As the authors used LC3b expression to represent axonal volume, the authors should demonstrate that this is the case using an axonal fill or membrane marker in both the wt and E50K conditions. This is important as it is unclear whether LC3b expression is consistent between the wild type and the E50K conditions. Lower expression of LC3b in E50K could account for the large changes in axonal width that seem to be observed and could confound the measured amount of expelled mitochondria.
• Could large amounts of exogenous mitochondria in explant experiments be from cells that died during the plantation?

Suggested experiments/quantifications:

• In OPTN/MITO/LC3b trafficking experiments, does flux/number of events change? Representative kymograph in Figure 2D seems to show far more OPTN-positive mitochondria which is opposite of what is shown in Figure 2C.
• Demonstrate that axonal width measured with LC3B is representative of axonal fill/membrane marker in wt and E50K. Axonal area appears to change, is this accurate? This appears to be the case for both figure 3 and figure 4.
• Raw images in addition to the reconstruction would be beneficial.
• Further characterization of exopher-like structures.

Referees cross-commenting

I agree with the concerns of the other reviewers, and perhaps was over-optimistic about a timeline for revision. However, I do think the work is worth the effort, and I hope to see a revised manuscript published somewhere, as these observations are novel

Significance

This work reports potentially novel biology, and thus will be of interest to the field. The strength of the study is that it is an initial description of this biology, rather than a complete analysis. The work raises many more questions than it answers, and much further work on this topic is required to support these initial findings, but the manuscript will likely be of interest to many. Revisions are required to improve the rigor and clarity of the work, but following these revisions we recommend publication to facilitate follow-up work.

nota 2

La institución más grade de atención a la salud en México

The reason for this can be along the lines of what is happening to coral right now, and also the benefits of coral. As of the last 30 years, 50% of corals have been lost, and with projections to come by the year 2050 70-90% could be lost. Along with that, just looking at what corals do for ocean inhabitants, coral reefs provide essential habitat, breeding grounds, and food sources for a diverse array of marine species, supporting biodiversity and ecosystem health in ocean environments. This is disregarding what else it does for economic benefits, social benefits, and climate regulation.

I looked into this a little bit more as to why they last such a short period of time, and the main reasons that I found where: rapid changes in environment, resource limitations, biological factors, research focus, and funding cycles. But even with that information, I have to wonder what would be found if they were able to keep the research going for an extended period of time (longer than 2 years).

I have to wonder what exactly made so many of them become dislodged from where they were placed. I'd have to assume that because they are transplants they are less stable than natural forming coral. I would have just assumed that with time they'd be better able to remain where they were planted.

compare to state average in virginia- bodes well

this needs an edit

This section is an example of more tools out there that I have never heard of that now I want to investigate to inspire creative options.

I think this was highlighted really well in Maha's Organizational Development Class. This like pursuit of perfect operationalization tends to stifle innovation and lead to an organizations that's incapable of pivoting which can often lead to the collapse of the principles that once made it great.

I do kind of appreciate this being called out

Ok but like.... me, you, the girlypops and an off the grid farm full of goats and shared produce does in fact sound like a vibe I'd be down to pursue.

The weird sad irony of joining a radicalized group to escape capitalism feels like it needs more lines

Has anyone watched the Secret Lives of Mormon Wives? It's definitely trash TV but I actually think it surprisingly does a really good job of showing how this kind of thinking separates people within their own in-group and becomes a way of breeding distrust internally to keep people from experiencing alternative perspectives. I also really just want to talk about this show because I need to know if Taylor stays with Dakota for the baby lol.

Proud member of 'normal' society, apparently. Without being overly judgmental, I welcome the bigotry of the very religious. My secular viewpoint is as valid as theirs. As the author explains it, at least in the US there is a remarkable hypocrisy to assert that secular people cause inequalities and rampant injustice. That is culturally absurd in the US.

Just a small pleasant reminder that ethnic nationalism is distinctively different from shared ethnic cultural identities. A shared culture heritage, although it can and is perhaps often associated with historic ties to geographic locations has existed for most of humanity but the concept of a nation-state as well as the concept of national borders is something that is rather unique to modern history!!! And although there have definitely been ethnic clashes, empires, and war prior to the "start," of modern history, the systematic and unified understanding of blood as it pertains to a country is uniquely tied to the past few hundred years.

Ah yeah I spoke before I read the rest of the paragraph, but it seems the author is on a similar page about potentially subverting that expectation

This to me, feels like a perhaps skewed perception of community. I think there are fundamental shared values that create community -- like, perhaps the queer community assumes that those that are part of it will not discriminate against other queer people but it fails to understand the nuance of people's perceptions of what that means. After all, there are plenty of members of the queer community that identify as queer but have troubling opinions on who is queer or what counts as queer. There are transphobes and people that don't recognize bisexuality in queerness yet still choose to identify with the community. I don't think the phrase "As we all know," is intended to inspire groupthink or be a rallying cry for unanimity but I do think it is a means of creating an outgroup if not exclusively due to the Niavety of believing in a shared assumption without the understanding that assumptions are beholden to the nuance of the beholder's perspective.

It is useful, but not useful to be used as the only form of writing in highschool.

This type of writing demands your attention.

The 5 paragraph essay is chosen as an assignment as a way to protect a student from failure. If you do the same thing for years, how is a student then going to mess it up

These sorts of essays are so point-driven they kill the creativity in students.

These sorts of essays have no personality, are boring, and create bad habits.

The author see's the 5 paragraph essay structure as a bad habit, he is arguing that teaching this is negative.

Porque el ejemplo de Pedro nos hizo meditar en que si bajamos la guardia, puede sorprendernos una tentación que ya creíamos controlada o vencida.

Tener un buen programa espiritual es de gran ayuda

Orar Estudiar y meditar Reuniones Predicación

• Primero vigilar lo que pensamos para no ser tentados por nuestro propio deseo

• debemos tener cuidado con quienes nos asociamos, que lugares frequentamos ya sea lugares físicos o virtuales en internet

Jehová nos ayudará, porque nos ama, puede y quiere hacerlo.

Jehová está ahí para ayudarnos (lea 2 Corintios 4:7). Eso sí, fijémonos en que él nos da el poder que va más allá de lo normal. Pero a nosotros nos corresponde poner el poder normal, es decir, los esfuerzos diarios para no caer en la tentación.

Una conciencia limpia, la alegría de poder hacer feliz a Jehová, por nuestra obediencia y la posibilidad de disfrutar de vida eterna.

Sería tonto poner en riesgo todo eso por no vencer una tentación que nos lleve a un pecado grave.

.h2

Seguir el consejo de Jesús de mantenernos en guardia para resistir la tentación rápidamente cuando quiera sorprendernos

Evitar situaciónes de riesgo que puedan llevarnos a caer en tentación

Y continuar aplicando los métodos que personalmente nos dan resultado con la bendición de Jehová

Puede pasarnos lo mismo que a Pablo, puede que vuelva a atraparnos alguna debilidad que creíamos que teníamos dominada.

Con el miedo al hombre.

Recordemos que negó tres veces conocer a Jesús por temor.

Luego enfrentó al sanedrín y habló con valor

Pero después volvió a demostrar temor al hombre cuando dejó de comer con cristianos no judíos.

Parece que está debilidad lo persiguió por mucho y a veces era vencido por ella.

.h2

Como vimos nuestra imperfección puede llevarnos a dar pasos que terminen en pecado.

Pero estar ocupados dando pasos que agraden a Jehová, son la mejor protección

Participar en las reuniones y predicamos, se fortalece nuestra amistad con Dios, nos prepara cómo mejores maestro y estimulamos a nuestros hermanos (Mat. 28:19, 20; Heb. 10:24, 25).

Si estamos solos Leer y estudiar la Palabra de Dios y meditar en ella nos ayudará a alejarnos de lo malo y al mismo tiempo fortalecerá nuestro amor por la ley de Jehová (Jos. 1:8; Sal. 1:2, 3; 119:97, 101).

Y recordar que Jesús nos aconsejó orar constantemente para resistir las tentaciones (Mat. 26:41). Y nosotros sabemos que Jehová bendice ésas oraciones y al mismo tiempo lo hacen Feliz

---

Seguir un buen programa de actividades espirituales nos ayuda a rechazar las tentaciones

(Amós 5:15). Nos da la clave, no podemos pensar en dos cosas al mismo tiempo, por éso debemos desarrollar amor por lo que es bueno, cambiando el pensar en algo inapropiado, por hacer lo correcto en el momento que nos damos cuenta.

Tener la firme convicción de que cumplir las normas de Jehová, siempre al 100%.

Porque Jehová es santo y perfecto, entonces lo que nos pide siempre es para nuestro beneficio, muy diferente de nosotros que somos imperfectos y por ese motivo podemos desarrollar pensamientos y deseos diferente a los de él.

Porque podemos atraer lo que pensamos, en el sentido que. Con el tiempo se fortalece el deseo de cumplir esos pensamientos.

Por éso debemos resistir y evitar esos pensamientos cuando surjan. Para evitar que crezcan hasta convertirse en deseos intensos, y difíciles de resistir y que pueden llevarnos a cometer un pecado grave (Filip. 4:8; Col. 3:2; Sant. 1:13-15).

they have no fear of the uncle?

potential divorce/husband is abusive?

symbolism for kids?

1 - Method to measure current state of ECC quality

There wasn't a set order where the Udl was going be used to support educators originally to understand the guide better.

Why do Guidelines help with tech? Guidelines help by providing security while using different apps or websites.

There is some of the educators that are already teaching about the how they should use guidelines and having them practice them in the classroom. There is a few students that are struggling with guidelines and it creating barriers and since some educators are teaching that makes some barriers as well.

Although the Uld is something new in a way it is now up to date and the new version is now more accurate to the flow of the three large networks.

During the pandemic I was very much into MBSR and meditation and felt a lot of clarity mentally and emotionally from my practice.

People in teams typically have a common shared goal together, however have different drives or means of achieving them

They seem to have an amazing community, I have had similar experiences so I can relate to this

Can understand the story and what's going on simply through the pictures, they tell a story of their own.

Having quotes with large impact like this is something I want to implement in my photo essay

Describing the scene is usually what happens for the hook in most of the stories we've read. I don't think it's that captivating, so I'm going to try something different for my essay.

This sentence lets you imagine the president leading everybody to the people that caused the event.

vivid imagery

I would be interested to read about fent in this section of the textbook.

It's extremely disappointing to me how addiction is stigmatized in this country. Addiction is a disease that requires treatment.

This reminds me of a video I watched about a way that scientists can recreate visual images of what someone imagines in their mind's eye.

Freud seems like a freak

Beta > Alpha > Theta > Delta

I know that REM sleep is affectted cannabis use as well as alcohol use.

Cono Elliot's work on notational design and his influential papers - Cono got his PhD at Carnegie Mellon University in the '90s under Frank Fenny working on higher order unification. - Cono has devoted his life to thinking and refining graphic computation and tools behind it, and has published influential papers on various topics related to functional programming and notational design.

Living in a forest setting with deep connection to nature. - Conor lives on 20 acres next to his family's 60 acres and has a deep emotional connection to the place because of his parents' presence. - He sees a connection between nature and technology, highlighting the non-sequential nature of computation and neurology.

We are in a pre-scientific age of thinking about computation. - Humans have created thinking organisms that think systematically, leading to computation. - We are in an awkward phase of thinking about computation in a clumsy and pre-scientific way.

Humans are driven by curiosity to understand the universe. - We have a limited ability to perceive the universe due to our evolutionary constraints. - Through the advancement of science and technology, we have developed tools like telescopes, microscopes, high-speed cameras, and time-lapse to enhance our perception.

Elegance and wonder in computer science - Elegance is the deepest value in computer science, inspiring a sense of play and wonder. - Computer science is in a deeply inelegant phase, but there is potential for Elegance and Beauty in the field.

Elegance as a guiding value in theoretical physics - Elegance guided Einstein in developing the special and general theory of relativity. - Modern civilization is built on general relativity and quantum physics; GPS system corrects for relativity.

Elegance and simplicity in formalizing concepts in computer science - Elegance and simplicity in formalizing concepts are related - People often mistake familiarity for simplicity in programming

Academia today lacks time for critical thinking - Focused on churning out papers and credentials - Issue of education accessibility affecting teaching quality

Semantics is crucial in programming - Meanings are called semantics - The relationship between a program and its meaning is important

Dana Scott answered the crucial question of the mathematical meaning of Lambda calculus in 1970. - Lambda calculus was originally intended for encoding high order logic and quantifiers, not for programming. - Peter Landon realized the potential of Lambda calculus for programming and introduced the concept of executing Lambda calculus on a machine.

Languages convey meanings, computation looks at meanings. - Languages and programming languages serve the same purpose: to convey meanings. - Computation and technological tools help us observe and understand meanings in various forms, from stars and quasars to microorganisms and atoms.

Euclid revolutionized geometry with his conceptual approach - Introduced a new way of thinking about geometry with axioms and postulates - Plato's influence on the idea of mathematical space and its relation to the physical world

Mathematics describes real truth and possibly taps into platonic truth. - Platonist perspective considers mathematics as a way to describe truth beyond us. - Success of mathematical fantasy or story inspires acting as if tapping into platonic truth.

Ancient beliefs about movement of stars and planets - Stars and planets thought to move in circular paths due to perfection/God concept - Some stars behaved differently, known as 'The Wanderers' or planets

Kepler discovered planetary laws - Planets move in an ellipse, not a circle - Kepler's explanation lacked why planets move in an ellipse

Scientific theories evolve with enhanced observations - Newton's theory successful until discrepancies discovered in the 20th century - Einstein's theory validated through observations of planet Mercury during solar eclipse

Scientific exploration is an unending journey - Science aims to understand what we don't know - In academia, the system often fails to reward wonder and not knowing

Denotational semantics helps distinguish beauty and elegance from complexity - Beauty or elegance in theory is described precisely in terms of mathematics - Fortran, led by John Backus, introduced expressions, advancing from Von Neumann style sequential programming

Functional programming emphasizes expressions over statements - Fortran blends statements and expressions but still leans towards statements - Functional programming eliminates everything except expressions

Hardware limitations led to sequential model prototyping - John Von Neumann's experiment from 1947 is still relevant in 2022 - John Backus discussed fundamental problems in computing during the war

Von Neumann bottleneck affects computer performance. - Physical bottleneck slows computers due to high heat generation. - Mental bottleneck limits brain capacity and mental efficiency.

Breaking out of the Von Neumann bottleneck - The Von Neumann style of programming forces us to think small and is fundamentally sequential and mechanistic. - The lecture emphasizes the importance of thinking in larger, powerful notions and focusing on functions rather than words.

Functions as building blocks for knowledge - Functions built from other functions allow for scalability and creation of complex systems - Importance of denotational semantics in designing new languages rather than just explaining existing ones

Backus emphasized fixing defects and learning from mistakes. - Using denotational semantics reveals detailed defects in existing languages. - Advancement in computer science involves replacing outdated concepts like go-to with structured and functional programming.

The cost of focusing on education and progress is losing the ability to make significant advances in science. - The speaker expresses disappointment with the impact of Academia on progress and science. - The speaker remains dedicated to truth and beauty, advocating for the importance of denotational semantics in making aesthetic distinctions.

Ideas are expressions of beauty or ugliness which give deep insights across fields. - Denotational semantics serves as a reliable guide to beauty and elegance in ideas. - Beauty and elegance are valuable guides for understanding the universe and computation.

Passion for mathematics and computer graphics - Attended undergrad in math at UC Santar with a small group of math students in a nurturing environment - Transitioned to grad school at Carnegie Mellon for computer science and pursued computer graphics due to love for geometry and math

Had to change plans at Carnegie Mellon - Arrived at CMU to study computer graphics, but found out the people I wanted to study with had left - Discovered a group focusing on reasoning about programs, which became the focus of my PhD work

Transition to computer graphics and involvement in group projects - Worked with notable advisors like Dana Scott, John Reynolds, and Frank - Focused on exploring the next advancements in programming interfaces and data structures at Sun Microsystems

Introduction to denotational semantics in understanding language meanings - Studied denotational semantics under Stevenh Brooks and Dana Scott in grad school, leading to a revelation on language meanings - Believes language meanings should be independent of specific machines and analyzed compositionally for better understanding

Graphics programs are sequential commands organizing video memory for visual output. - Graphics programs are different from traditional software due to their focus on organizing instructions for video memory. - Alternative design paradigms focus on conveying meanings and inventing tools to help users view desired content through a computer.

Designing a language library for geometry and colors - Creating a composable vocabulary of geometry and colors, similar to modern linguistic frameworks - Developing a rich system of types for three-dimensional geometry and adding a time component to the design

Rendering graphics offscreen to build up incrementally for a correct answer. - Rendering offscreen allows showing previous true things before replacing them incrementally. - Temporal discreetness in computer graphics breaks compositionality and introduces fundamental bugs.

Compositional models with approximations lose accuracy when composed - Compositional models incorporating approximations result in gross inaccuracies upon composition - Functional reactive programming involves composing before approximating for accurate results

Outline fonts are resolution independent - Outline fonts are continuous and do not have pixels when zoomed in - Switching from bitmap graphics to outline fonts improves efficiency and clarity

Transition from discrete to continuous programming in space and time. - Examples of continuous programming in space like fonts, 2D and 3D geometry, vector graphics. - Applying continuous programming principles to time requires a fundamental shift in implementing and describing things that vary with time within the Von Neumann model.

John Reynolds introduced the idea of using functions from the reals instead of sequences for solving time interpolation problems - This approach helped in resolving issues with interpolations and time manipulation - Continuous time modeling was found to be more effective than discrete modeling for things that vary with time

Functional programming requires a shift from loops to lazy lists - Functional programming involves describing the mathematical model behind the data manipulation - The common reasoning that input and output data should have the same nature is wrong in functional programming

Functional reactive programming is about understanding concepts in the simplest, most elegant compositional terms. - It emphasizes denotational semantics, where types have a mathematical model. - It focuses on fully explaining operations in terms of the model, independent of implementation.

Programming expresses ideas with clear understanding before implementation - Category Theory is appreciated for its precise and elegant tools in mathematics - Functional Reactive Programming lacks denotational and compositional principles, leading to fundamental misunderstandings in programming

Algebraic patterns like monoids and distributivity are powerful for organizing reasoning - There are different types of monoids like addition and multiplication each with their own properties - Multiplication distributes over addition and zero plays a special role in this interaction

Algebra and category theory provide reusability and reasoning in mathematics and programming. - Algebra allows for reasoning that is parameterized and applicable to different mathematical scenarios. - Category theory generalizes various algebraic concepts and is important for correctness in programming.

The complexity of Python programs and limited cognitive abilities can lead to a lack of understanding. - Options include quitting the profession or divorcing what you've seen from what you do. - Another option is switching to a language with simple semantics, such as purely functional or denotative languages.

Denotative programming allows for proving program correctness. - Denotative programming enables answering questions about the multiple meanings of programs. - Functional programs can have meanings within a cartesian closed category.

Tropical semi-rings relate to timing analysis of parallel computations. - Understanding operations of plus and max in relation to semi-rings. - Realization of dot products and matrix multiplication pattern in timing analysis.

Timing analysis can be described compositionally using the language of categories - I realized the parallel sequential composition is the fundamental building blocks of functions computation - The type Lambda calculus has more than one model, and the mathematical values it describes can have different interpretations

Realizing the connection between HCLL and lambda calculus led to successful compilation to hardware. - HCLL translates to a small core lambda calculus - Interpreting lambda calculus in cartesian closed categories enabled successful compilation to hardware

Exploring unconventional categories for computation - Discovering powerful ideas by compiling categories since 1980 - Seeking beauty in solutions to drive innovation and never settling for unsatisfactory answers

Geometry and the introduction to proof changed my life - The systematic way of exploring what is true and growing knowledge in geometry was a life-changing concept for me. - Discovering computers at Lawrence Hall of Science through the Star Trek Club in high school eventually led me to computation.

Introduction to programming through games on teletypes - Experiencing games and printing out results on rolls of paper as souvenirs - Discovering source code hidden in the printed paper, initiating an interest in programming

Started college with no computer science department, emphasized logic and enjoyed math contests - Computer science classes offered in math department or College of Engineering - Discovered talent and passion for math despite discouragement from elementary school teacher

The origin of computer science in universities and its impact on its development - Initial classes were labeled as Computer Science or logic, sparking a debate on department placement. - Placement in engineering rather than mathematics influenced the practical nature of computer science education.

Transition from imperative to functional programming - Discovered Haskell as a better alternative to imperative programming languages - Applied Haskell in programming for 25+ years and mentorship in hardware design for machine learning

Realizing the power of category theory in simplifying automatic differentiation - Changed vocabulary to be more symmetric with respect to composition - Describing automatic differentiation in the language of categories simplifies and generalizes it

Denotational design is key for software implementation - HLL was not effective for teaching denotational design - Inner guidance essential for understanding and using HLL effectively

Struggling with teaching denotations and homomorphisms in programming - Encountered issues with students not understanding correct implementations - Wanted compiler to indicate errors instead of personally correcting

Understanding the question is more important than answering it correctly - Operational thinking is about biases in answering problems and questions - The most important thing is to understand the question in the most beautiful way

Realization about teaching and learning process - Programmers differ in their attitude towards being told they're wrong - Importance of being open to feedback for growth in programming

Automation has benefits but limited scalability - SMT automation has advantages in problem-solving but faces scaling limitations - Despite advancements, SMT technology cannot achieve unlimited scalability

Agda is the most tasteful tool for working with dependent types. - Agda offers beauty, consistency, simplicity, and tremendous power. - Agda contributes to an incredibly beautiful story about the equivalence of computation, logic, and the foundations of mathematics.

Exploring if all of mathematics can be built on logic - David Hilbert's attempt to formalize logic in the early stages - Can natural numbers be understood via logic as a foundation?

Natural numbers are a profound and important concept - Natural numbers are a product of human construction on top of other systems - Piano numbers are a significant concept in mathematics

Constructive logic allows expression of proofs as either A or B - In constructive logic, every proof of A or B can be expressed as a proof of A or a proof of B - Brower's logic allows for this expression without the law of excluded middle, leading to simple answers for negation, implication, truth, and falsehood in terms of types.

De bruyne pioneered logic computable through computers - Exploration of dependent typing and realization of logic and types - Mechanization of information and manipulation, leading to modern programming languages

The power of math and knowledge in programming - Manipulating from the bones is a powerful and beautiful concept - Embracing sequential stateful notion of computation limits insights and learning

Written language enabled deep reflection and improvement of ideas. - Written language allowed ideas to be examined and improved over time. - Written language initiated a feedback loop for continuous enhancement of concepts.

Continuous improvement through iterative optimization - Iteratively refining program logic and expressions for efficiency and clarity. - Enhanced abstraction and reusability through denotational design and parameterization.

The debate on using formal proofs in industry - Industry perspective often argues against formal proofs due to perceived time constraints and impracticality. - Decision to use formal proofs depends on the objectives and the value placed on accuracy and thoroughness.

Achieving 100% correctness is the only way to reach 95%. - Errors compound, leading to significant deviations in calculations. - Approximations and probable correctness can lead to overall incorrectness in complex projects.

Inspired by deep conversation - The conversation has been engaging and has touched on major topics of interest. - The speaker hopes to discuss denotational design and its application in software design.

Create space for contemplation in the age of instant information - Encourage meditation and reflection on content - Announcement about a dedicated email for audience feedback and inquiries