Note originally posted by Nellie Song on 2020-02-18</p>

A detailed version of a method used in this article can be found here, doi.org/10.21769/BioProtoc.3041

Note originally posted by Hans Moldenhauer on 2019-08-21

Corresponding author Andrea Meredith, PhD, and the subject KCNMA1-Linked channelopathy were featured in in The New York Times/Netflix documentary series Diagnosis in Episode four “Looking For A Village.” In the episode, Dr. Meredith explains the function of the KCNMA1 ion channel. Filming in her lab shows the mice she created with transgenic modifications to the KCNMA1 gene and her research on the patient mutations in KCNMA1. Watch the episode here: https://www.netflix.com/title/80201543.

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Note originally posted by Aliaksei Chareshneu on 2019-06-29</p> (potential) mistake in residue numbering

There is no <span style="color: rgb(0, 0, 0);">R219 in epsilon subunit of mouse muscle nAChR</span>

Annotation originally posted by Eduardo Rios on 2018-04-02

Thanking collaborators for images shared in this article left a feeling of omission, as other coworkers were equally responsible for the development of ideas that inform this "Milestone". As partial amendment, I take advantage of the Remarq tool to acknowledge the contributions of Enrico Stefani, Bob Fitts, Adom González, Laszlo Csernoch, Jianjie Ma, Gonzalo Ferreira, Wolfgang Kirsch, Peace Cheng, Daniela Rossi, Alessandra Nori, Pompeo Volpe, Vincenzo Sorrentino, Mike Fill, Lothar Blatter, Isaac Pessah and more recently Paul Allen, Susan Hamilton, Feliciano Protasi and Monika Sztretye, with whom I had the privilege of working in these matters.

Annotation originally posted by Nellie Song on 2020-02-18</p>

A detailed version of a method used in this article can be found here, <span style="color: black;">doi.org/10.21769/BioProtoc.3178</span>

Annotation originally posted by Shona Lang on 2018-11-28

Annotation originally posted by John Vincent on 2019-12-06

In reference to the recent paper in this journal by Xu and colleagues (Xu et al., 2018), whilst we cannot fault the authors’ functional characterizations of effects of the p.Y1087C variant (Chr1:10397567A>G; NM_015074.3:c.3260A>G) in the KIF1B protein at the cellular level, we would like to address the authors’ assertion that this variant is a disease-causing or disease-associated mutation. According to the genome aggregation database (gnomAD: https://gnomad.broadinstitute.org/), this specific missense variant, with the single nucleotide polymorphism (SNP) identification rs2297881, has an overall allele frequency of 3.3% (9217 alleles out of 138,616 population control individuals, making its prevalence in the population far too frequent to be considered a disease-causing mutation; and in particular for a rare autosomal dominant disorder such as Charcot-Marie-Tooth type 2A (CMT2A; MIM 118210). In whole exome sequencing data of an East Asian control population of 7487 individuals from the non-neurological sub-population (“gnomAD v2.1.1 non-neuro”), Y1087C alleles were present at an allele frequency 7.8%, including 1070 heterozygotes and 47 homozygotes. If the Y1087C allele were indeed causative for CMT2A in heterozygous form, then one would expect that homozygous Y1087C would manifest a much more severe phenotype, and thus would not be present, let alone so frequently, among non-neurological controls. Since knockout of Kif1b in mice is reported as postnatally lethal (Zhao et al., 2001), and ablates both isoforms alpha and beta, and since Y1087C is only present in the beta isoform, we would suggest that a better model for the Xu et al study would be an isoform restricted knockout, or even better, a Y1087C knock-in model.

We also note the clinical discrepancy between the two brothers in the family reported by the authors (Xu et al., 2018), in which Patient 1 presents with clinical symptoms of progressive neuropathy, whereas Patient 2 presents with intellectual disability, bipolar disorder and hypertension, but in whom the diagnosis of CMT2 appears to be based only on laboratory testing of nerve conduction velocity and electromyography. It should also be noted that one of the most common CMT2 genes, mitofusin 2 (MFN2: MIM 608507), is within 2 Mb from KIF1B, and is within the original disease linkage peak reported by Zhao et al (2001).

CMT is a heterogeneous group of neurological disorders, with an estimated total prevalence, including all subtypes, of 1 in 2,500 individuals (0.04%; Krajewski et al., 2000; https://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Charcot-Marie-Tooth-Disease-Fact-Sheet#265923092). Thus, we believe that the assertions made in the paper including the title and abstract, associating this specific variant to the CMT2A neuropathy subtype, are contradicted and refuted by the widely available population data. The characterization of a polymorphism—which rs2297881 quite clearly is—as a disease-associated ‘mutation’, and presenting molecular evidence in support of this characterization, is likely to lead to clinical mis-interpretation, which could potentially have serious consequences in the diagnostic and genetic counselling system.

References

Krajewski, K. M. 2000. Neurological dysfunction and axonal degeneration in Charcot-Marie-Tooth disease type 1A. Brain. 123:1516–1527. https//doi.org/10.1093/brain/123.7.1516

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Xu, F., Takahashi, H., Tanaka, Y., Ichinose, S., Niwam S., Wicklundm M.P., Hirokawa, N. 2018. KIF1Bβ mutations detected in hereditary neuropathy impair IGF1R transport and axon growth. J. Cell Biol. 217:3480-3496. https//doi.org/10.1016/s0092-8674(01)00363-4

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Zhao, C., Takita, J., Tanaka, Y., Setou, M., Nakagawa, T., Takeda, S., Yang, H.W., Terada, S., Nakata, T., Takei, Y., Saito, M., Tsuji, S., Hayashi, Y., Hirokawa, N. 2001. Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta. Cell. 105:587–597. https//doi.org/10.1016/s0092-8674(01)00363-4

Annotation originally posted by Susan Mango on 2018-12-01

Annotation originally posted by Samuel Landry on 2018-09-26

Annotation originally posted by Journal of Cell Biology on 2018-11-02

Annotation originally posted by John Hewitt on 2018-07-07

Photoreceptor anaerobic glycolysis "Although it was initially suggested that in analogy to the ANLS model for the central nervous system, the radial glial cells of the retina (Müller cells) might supply lactate to photoreceptors (Poitry-Yamate et al., 1995; Hurley et al., 2015), the opposite appears to be true: photoreceptors perform aerobic glycolysis and export lactate, which is then oxidized by Müller cells."

Especially in rods it seems, as we now know; https://phys.org/news/2018-06-metabolism-functionthe-extreme-photoreceptors.html

Posted on behalf of Xiaofan Wei and Hongquan Zhang by JCB:

Thanks for the comments. We were also confused about the conflicting results which may be caused by the different tagged constructs or different concentration of plasmids used for the transfection. However, we cannot repeat the observation that Smurf1 has obvious effects on Talin-head degradation even though we performed the experiments at many different conditions and using several cell lines. Although Dr. Huang et al. found that Smurf1 induced slightly reduced levels of Talin-H as shown in Nat Cell Biol. 2009 May;11(5):624-30, Fig. S6a, we tend to think that Talin-head is not a direct target of Smurf1. If Talin-head is a genuine target of Smurf1, it should be degraded to the same extent as Kindlin-2 is degraded by Smurf1. Thus, it needs further investigation to elucidate the mechanism of Talin-head degradation.

Annotation originally posted by Mark Ginsberg on 2018-05-24</p>

We noted with surprise that Wei et al. (J Cell Biol. 2017 May 1;216(5):1455-1471) reported in Fig. 4 that they could not repeat our observation that expression of SMURF1 induced reduced expression of the talin head domain (THD) (Huang et. al. Nat Cell Biol. 2009 May;11(5):624-30, Fig. S6a). Blinded investigators in both of our labs have repeated this experiment as described in Nat Cell Biol. 2009 May;11(5):624-30, Fig. S6a and have confirmed our reported results. We cannot account for the differing results; however we did note that expression of GFP-labelled talin head domain (THD) (used by Wei et. al.) was relatively resistant to reduction by co-expression of SMURF1, compared with the HA-tagged THD used in our paper. We therefore repeated the experiment with untagged THD and again confirmed that co-expression of SMURF1 reduced expression.

Cai Huang and Mark H. Ginsberg

Annotation originally posted by Stephanie Gupton on 2018-05-08</p>

Full resolution z-stack tiled images allow discernment of axon branches versus axon crossings. The published images in Figure 8B are maximum projections, and the video files (Video 6) were necessarily compressed to meet size restrictions. However, this makes it difficult for the reader to judge axon branching versus crossings. We include a link to full resolution files here (https://www.dropbox.com/sh/rcey7a6g8210hdq/AADF_aR1LWfH4E-B9iJjMma-a?dl=0) and on our lab website (http://guptonlab.web.unc.edu/movies). These raw data are multi-GB 3D stacks (~25-40 planes) of 3x3 stitched images (3072x3072 pixels each) encompassing the corpus callosum. Scoring of branches was performed blind to genotype, by marking potential branches/crossings as regions of interest (ROI) in the max projection. Inspection of each ROI in individual planes at full resolution resolved between a branch (GFP+ axon terminating in a GFP+ axon, full resolution example in Fig.8B’ of publication1) or a crossing in 3D.