Clec16a is Critical for Autolysosome Function and Purkinje Cell Survival

We detected no alteration in endoplasmic reticulum morphology in CLEC16A-deficient cells.

“We detected no alteration in endoplasmic reticulum morphology (Fig. 6c), as expected from prior studies of Clec16a-deficient murine islets16.”

• cites [16] Soleimanpour, S. A. et al. The diabetes susceptibility gene clec16a regulates mitophagy. Cell 157, 1577–1590, 10.1016/j.cell.2014.05.016 (2014). sourceDisputed

  The Results section on pancreas-specific Clec16a deficiency reports that mutant murine β-cells contained large vacuoles likely representing dilated ER, contrary to the cited expectation of unchanged ER morphology in murine islets. The paper reports no noticeably abnormal ER morphology in Clec16a-deficient fibroblasts, but that finding does not apply to the islets named in the citing sentence. (LLM, conf=0.89)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Cerebellar Purkinje cells and neurons of the deep cerebellar nuclei express high levels of Clec16a.

“Further in situ hybridization reveals that cerebellar Purkinje cells and neurons of the deep cerebellar nuclei express high levels of Clec16a (Allen Mouse Brain Atlas, http://mouse.brain-map.org/gene/show/50215)242526.”

• cites [24] Lein, E. S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176, 10.1038/nature05453 (2007). sourceNot checked — no full text of the source
• cites [25] Sunkin, S. M. et al. Allen Brain Atlas: an integrated spatio-temporal portal for exploring the central nervous system. Nucleic Acids Res 41, D996–D1008, 10.1093/nar/gks1042 (2013). sourceCould not verify

  The Results (Resource overview, Table 1; page unavailable) describe the Allen Mouse Brain Atlas as a source of brain-wide in situ hybridization images. Searches for Clec16a, Purkinje cells, and deep cerebellar nuclei found no passage in our copy reporting the claimed expression levels; the linked atlas entry itself is not part of the paper copy. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [26] Allen Mouse Brain Atlas (2015), Available at: http://mouse.brain-map.org/gene/show/50215. (Accessed: February 18, 2016)Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Purkinje cell degeneration models such as pcd mice display motor abnormalities that were similar to those observed in B6.Clec16aGT/GT and Clec16aCURT/CURT mice.

“Purkinje cell degeneration models such as pcd mice display motor abnormalities that were similar to those observed in B6.Clec16aGT/GT and Clec16aCURT/CURT mice3031 and staining with the Purkinje cell marker calbindin confirmed loss of these cells in B6.Clec16aGT/GT mice (Fig. 2d) at 8 weeks of age that was dramatically increased by 47 weeks of age (Supplementary Fig. 2e).”

• cites [30] Chakrabarti, L., Eng, J., Ivanov, N., Garden, G. A. & La Spada, A. R. Autophagy activation and enhanced mitophagy characterize the Purkinje cells of pcd mice prior to neuronal death. Mol Brain 2, 24, 10.1186/1756-6606-2-24 (2009). sourceSupported
• cites [31] Wang, Q. J. et al. Induction of autophagy in axonal dystrophy and degeneration. J.Neurosci. 26, 8057–8068 (2006). sourcePartly supported

  The Introduction describes Purkinje cell degeneration in Lurcher mice, and the Methods identify ataxia as a Lurcher phenotype. The paper studies Lurcher mice, not pcd mice, and does not compare their motor abnormalities with those of either Clec16a strain. No page numbers are available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Axons in the deep cerebellar nuclei of mice lacking Atg5 and Atg7 in Purkinje cells displayed increased membranous and vacuole-like structures.

“axons in the deep cerebellar nuclei of mice lacking Atg5 and Atg7 in Purkinje cells displayed increased membranous and vacuole-like structures”

• cites [20] Komatsu, M. et al. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 104, 14489–14494, 10.1073/pnas.0701311104 (2007). sourcePartly supported

  The Results (Fig. 5) report increased aberrant membrane structures in Purkinje-cell axon terminals in the deep cerebellar nuclei after Atg7 deletion. But the Results (Fig. 6) say double-membrane vacuole-like structures were virtually absent in those mice, not increased. The cited study examines Purkinje-cell-specific Atg7 deletion, not Atg5 deletion. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [21] Nishiyama, J., Miura, E., Mizushima, N., Watanabe, M. & Yuzaki, M. Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death. Autophagy 3, 591–596, 4964 (2007). sourcePartly supported

  The ‘Aberrant membranous structures and autophagosome-like organelles’ section reports swollen axons in the deep cerebellar nuclei of mice with Purkinje-cell-specific Atg5 deletion; Figure 4 describes frequent aberrant membranes and autophagosome-like organelles in those axons. The paper does not report this finding for Purkinje-cell-specific Atg7 deletion. Its Introduction mentions Atg7 in other knockout contexts, not in this experiment. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Conditional deletion of the autophagy genes Atg5 or Atg7 in Purkinje cells leads to degeneration of these cells.

“For example, the conditional deletion of the autophagy genes Atg5 or Atg7 in Purkinje cells leads to degeneration of these cells2021.”

• cites [20] Komatsu, M. et al. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 104, 14489–14494, 10.1073/pnas.0701311104 (2007). sourcePartly supported

  The Results report Purkinje cell-specific deletion of Atg7, and the paper states that the resulting axonal degeneration was followed by Purkinje cell death. The Discussion mentions Atg5 deletion in mutant mouse brains, but does not establish that Purkinje cell-specific Atg5 deletion caused degeneration of those cells. No page number is available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [21] Nishiyama, J., Miura, E., Mizushima, N., Watanabe, M. & Yuzaki, M. Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death. Autophagy 3, 591–596, 4964 (2007). sourcePartly supported

  The section “Deletion of Atg5 in Purkinje cells caused progressive and cell-autonomous neurodegeneration” reports that Purkinje-cell-specific Atg5 deletion led to cell loss. A discussion passage also reports neurodegeneration and Purkinje-cell axonal swelling in Atg7-deficient mice, but those mice had nestin-Cre deletion across neural cells, not Purkinje-cell-specific deletion. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

In several in vivo models, neurons, particularly Purkinje cells, require functional autophagy to survive.

“In several in vivo models, neurons, and in particular Purkinje cells, demonstrate a striking requirement for functional autophagy in order to survive.”

• cites [20] Komatsu, M. et al. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 104, 14489–14494, 10.1073/pnas.0701311104 (2007). sourceSupported
• cites [21] Nishiyama, J., Miura, E., Mizushima, N., Watanabe, M. & Yuzaki, M. Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death. Autophagy 3, 591–596, 4964 (2007). sourceSupported

The mutations in the gene analyzed here are likely more disruptive of Clec16a function than alleles associated with human disease risk.

“Importantly, the mutations in the gene analyzed here are likely more disruptive of Clec16a function than alleles associated with human disease risk12163342.”

• cites [12] Tooze, S. A., Abada, A. & Elazar, Z. Endocytosis and Autophagy: Exploitation or Cooperation? Csh Perspect Biol 6, 10.1101/cshperspect.a018358 (2014). sourceCould not verify

  The passages on autophagy machinery and the References section (pages not provided) do not address Clec16a mutations or human disease-risk alleles. Searches for “Clec16a” and “Clec16a mutations human disease risk” found no passage comparing their effects on Clec16a function. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [16] Soleimanpour, S. A. et al. The diabetes susceptibility gene clec16a regulates mitophagy. Cell 157, 1577–1590, 10.1016/j.cell.2014.05.016 (2014). sourcePartly supported

  The Results report that pancreas-specific Clec16a deletion reduced islet transcript by 70%, while the human disease-risk SNP was associated with a dose-dependent decrease in CLEC16A expression. This supports the suggested distinction between a strong experimental disruption and a human risk allele, but the paper does not assess the specific mutations analyzed in the citing document or directly compare their effects with human alleles. No page numbers are available in our copy. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [33] van Luijn, M. M. et al. Multiple sclerosis-associated CLEC16A controls HLA class II expression via late endosome biogenesis. Brain 138, 1531–1547, 10.1093/brain/awv080 (2015). sourcePartly supported

  The Introduction identifies CLEC16A-region SNPs associated with human disease risk, and the knockdown results show that substantially reducing CLEC16A expression disrupts late-endosome biogenesis. The paper does not assess the mutations analyzed in the citing document or compare their effects with those of human risk alleles, so it does not support the claimed relative severity. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The phenotype of flies mutant in ema can be complemented by expression of human or mouse CLEC16A.

“the phenotype of flies mutant in ema can be complemented by expression of human or mouse CLEC16A”

• cites [47] Kim, S., Wairkar, Y. P., Daniels, R. W. & DiAntonio, A. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation. J Cell Biol 188, 717–734, 10.1083/jcb.200911126 (2010). sourcePartly supported

  The Results section, “Ema orthologues are functionally conserved” (page unavailable), reports that expression of human CLEC16A rescues lethality and other phenotypes in ema-mutant flies. The paper does not report a rescue experiment using mouse CLEC16A, so the mouse portion of the claim is unsupported. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

The function of CLEC16A is highly conserved.

“indicating that the function of this protein is highly conserved47.”

• cites [47] Kim, S., Wairkar, Y. P., Daniels, R. W. & DiAntonio, A. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation. J Cell Biol 188, 717–734, 10.1083/jcb.200911126 (2010). sourceSupported

Lack of reported neurodegeneration in parkin-deficient and Nrdp1 mutant mice makes it unlikely that the Nrdp1 interaction explains the finding of neurodegeneration in Clec16a mutant mice.

“The lack of reported neurodegeneration in parkin-deficient and Nrdp1 mutant mice171819 makes it unlikely that this interaction explains our finding of neurodegeneration in Clec16a mutant mice.”

• cites [17] Perez, F. A. & Palmiter, R. D. Parkin-deficient mice are not a robust model of parkinsonism. Proc Natl Acad Sci USA 102, 2174–2179, 10.1073/pnas.0409598102 (2005). sourceSupported
• cites [18] Goldberg, M. S. et al. Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons. J Biol Chem 278, 43628–43635, 10.1074/jbc.M308947200 (2003). sourceNot checked — no full text of the source
• cites [19] Wang, C. et al. The E3 ubiquitin ligase Nrdp1 ‘preferentially’ promotes TLR-mediated production of type I interferon. Nat Immunol 10, 744–752, 10.1038/ni.1742 (2009). sourceNot checked — no full text of the source

The Drosophila CLEC16A homolog ema is required for efficient recruitment of the Golgi complex protein Lava lamp (Lva) to autophagic structures in fat body cells.

“The Drosophila CLEC16A homolog ema is required for efficient recruitment of the Golgi complex protein Lava lamp (Lva) to autophagic structures in fat body cells”

• cites [9] Kim, S., Naylor, S. A. & DiAntonio, A. Drosophila Golgi membrane protein Ema promotes autophagosomal growth and function. Proc Natl Acad Sci USA 109, E1072–1081, 10.1073/pnas.1120320109 (2012). sourceSupported
• cites [47] Kim, S., Wairkar, Y. P., Daniels, R. W. & DiAntonio, A. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation. J Cell Biol 188, 717–734, 10.1083/jcb.200911126 (2010). sourcePartly supported

  The Results section, “Ema orthologues are functionally conserved” (page unavailable), identifies human CLEC16A as an orthologue of Drosophila ema. The paper also lists a Lava lamp antibody in Materials and methods, but the retrieved Results passages concern endosomal maturation in garland cells, not Lva recruitment to autophagic structures in fat body cells. The claim’s central recruitment finding is therefore unsupported by our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Gross cerebellar architecture was normal in B6.Clec16aGT/GT mice despite decreased brain size.

“However, while gross cerebellar architecture was normal in B6.Clec16aGT/GT mice despite decreased brain size (Fig. 2a,b), loss of Purkinje cells was readily detectable (Fig. 2c,d) at eight weeks of age before development of neurological deficits in most mice (Fig. 1b,c).”

Bielschowsky staining revealed characteristic 'empty baskets' in areas of Purkinje cell loss.

“Bielschowsky staining revealed characteristic ‘empty baskets’ (basket cell processes) in areas of Purkinje cell loss, indicating that the Purkinje cells had developed normally but were then lost (Supplementary Fig. 2f)32.”

Western blotting revealed increased p62 protein levels in HeLa-CLEC16A∆ cells.

“Immunofluorescence analysis of both independent clones of HeLa-CLEC16A∆ cells demonstrated increased p62+ and LC3+ puncta/cell compared to control cells (Fig. 4c–f) and western blotting revealed increased p62 protein levels in these cells (Fig. 4g,h).”

Staining with the Purkinje cell marker calbindin confirmed loss of Purkinje cells in B6.Clec16aGT/GT mice at 8 weeks of age that was dramatically increased by 47 weeks of age.

“Purkinje cell degeneration models such as pcd mice display motor abnormalities that were similar to those observed in B6.Clec16aGT/GT and Clec16aCURT/CURT mice3031 and staining with the Purkinje cell marker calbindin confirmed loss of these cells in B6.Clec16aGT/GT mice (Fig. 2d) at 8 weeks of age that was dramatically increased by 47 weeks of age (Supplementary Fig. 2e).”

Clec16a transcripts across exons 2-3 were comparable in wild-type and B6.Clec16aGT/GT MEFs.

“While amplification of Clec16a transcripts across exons 2–3 were comparable in wild-type and B6.Clec16aGT/GT murine embryo fibroblast cells (MEFs), there was a greater than 90% reduction in Clec16a transcript using primers targeting exons 23–24 (Supplementary Fig. 1e), indicating that the mRNA for this gene was interrupted by the GT cassette.”

The transcription of neighboring genes, Ciita, Dexi and Socs-1, in B6.Clec16aGT/GT MEFs was unaffected by gene trap insertion in Clec16a.

“The transcription of neighboring genes, Ciita, Dexi and Socs-1, in B6.Clec16aGT/GT MEFs was unaffected by gene trap insertion in Clec16a (Supplementary Fig. 1e).”

Neurons in the deep cerebellar nuclei showed accumulation of the autophagy substrate p62 in both B6.Clec16aGT/GT and Clec16aCURT/CURT mice.

“Neurons in the deep cerebellar nuclei showed accumulation of the autophagy substrate p62 in both B6.Clec16aGT/GT and Clec16aCURT/CURT mice (Fig. 3b, Supplementary Fig. 3a).”

The morphology of synapses between neuronal processes in both the Purkinje cell layer and the deep cerebellar nuclei were similar between B6.Clec16aGT/GT mice and littermate controls.

“The morphology of synapses between neuronal processes in both the Purkinje cell layer and the deep cerebellar nuclei were similar between B6.Clec16aGT/GT mice and littermate controls (data not shown).”

In myelinated axons passing through the deep cerebellar nuclei, we observed increased autophagosome- and autolysosome-like structures in B6.Clec16aGT/GT mice.

“However in myelinated axons passing through the deep cerebellar nuclei, a region dense in calbindin-positive Purkinje cell axons (Fig. 3b), we observed increased autophagosome- and autolysosome-like structures (Fig. 3e, Supplementary Fig. 3b).”

Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells.

“Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells.”

Loss of Purkinje cells was readily detectable in B6.Clec16aGT/GT mice at eight weeks of age before development of neurological deficits in most mice.

“However, while gross cerebellar architecture was normal in B6.Clec16aGT/GT mice despite decreased brain size (Fig. 2a,b), loss of Purkinje cells was readily detectable (Fig. 2c,d) at eight weeks of age before development of neurological deficits in most mice (Fig. 1b,c).”

Starting at seven to eight weeks of age, male and female B6.Clec16aGT/GT mice displayed abnormal hind limb clasping.

“Starting at seven to eight weeks of age, male and female B6.Clec16aGT/GT mice displayed abnormal hind limb clasping (Fig. 1a,b)23 and were unable to maintain their balance or grip the bars of an inverted metal cage for a normal period of time (Fig. 1c)28.”

There was a greater than 90% reduction in Clec16a transcript using primers targeting exons 23-24 in B6.Clec16aGT/GT MEFs.

“While amplification of Clec16a transcripts across exons 2–3 were comparable in wild-type and B6.Clec16aGT/GT murine embryo fibroblast cells (MEFs), there was a greater than 90% reduction in Clec16a transcript using primers targeting exons 23–24 (Supplementary Fig. 1e), indicating that the mRNA for this gene was interrupted by the GT cassette.”

Clec16aGT/GT mice displayed motor impairment.

“Clec16aGT/GT mice on a mixed 129/SvEv-C57BL/6 genetic background averaged 42% of the weight of control mice (Supplementary Fig. 1b,c) and displayed motor impairment.”

Immunofluorescence analysis of both independent clones of HeLa-CLEC16A∆ cells demonstrated increased p62+ and LC3+ puncta/cell compared to control cells.

“Immunofluorescence analysis of both independent clones of HeLa-CLEC16A∆ cells demonstrated increased p62+ and LC3+ puncta/cell compared to control cells (Fig. 4c–f) and western blotting revealed increased p62 protein levels in these cells (Fig. 4g,h).”

Western blotting demonstrated an increase in p62 and the lipidated form of LC3, termed LC3-II, in B6.Clec16aGT/GT MEFs.

“Western blotting demonstrated an increase in p62 and the lipidated form of LC3, termed LC3-II, in B6.Clec16aGT/GT MEFs (Supplementary Fig. 4c).”

Many Clec16a mutant mice, but not control mice, exhibited hind limb paralysis.

“While many of the Clec16a mutant mice, but not control mice, exhibited hind limb paralysis, we did not separately quantify this observation over time.”

Clec16aGT/GT mice on a mixed 129/SvEv-C57BL/6 genetic background averaged 42% of the weight of control mice.

“Clec16aGT/GT mice on a mixed 129/SvEv-C57BL/6 genetic background averaged 42% of the weight of control mice (Supplementary Fig. 1b,c) and displayed motor impairment.”

Immunofluorescence analysis of B6.Clec16aGT/GT MEFs revealed a 5.7 fold increase in LC3+ puncta/cell at baseline compared to control cells.

“Immunofluorescence analysis of B6.Clec16aGT/GT MEFs revealed a 5.7 fold increase in LC3+ puncta/cell at baseline compared to control cells and this did not result in a statistically significant change when blocking autolysosome degradation through treatment with chloroquine (CHQ) (Fig. 4a,b).”

Western blots confirmed increased expression of the lipidated form of the autophagy protein LC3 in the cerebellum of B6.Clec16aGT/GT mice.

“Western blots confirmed increased expression of the lipidated form of the autophagy protein LC3 in the cerebellum of B6.Clec16aGT/GT mice (Fig. 3a).”

Neurons from Clec16a-mutant mice exhibited abnormal Golgi morphology.

“We found that cell bodies of cerebellar and deep nuclei neurons from B6.Clec16aGT/GT mice contained enlarged, vacuolated membranous structures and lacked defined Golgi stacks (Fig. 3c).”

These abnormal dystrophic axons were not observed in control mice.

“These abnormal dystrophic axons were not observed in control mice.”

An experienced neuropathologist found no evidence that mutation in Clec16a resulted in neurodegeneration in the pons/medulla, hippocampus, frontal lobe and basal ganglia at this time point.

“In order to characterize the specificity of the neurodegeneration seen in B6.Clec16aGT/GT mice an experienced neuropathologist analyzed multiple areas of the brain, including the pons/medulla, hippocampus, frontal lobe and basal ganglia, and found no evidence that mutation in Clec16a resulted in neurodegeneration in these regions of the brain at this time point (Supplementary Fig. 2a–d).”

Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents.

“Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents.”

Two independent mouse strains carrying homozygous mutations in Clec16a developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells.

“Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells.”

• Also stated in: § Neurologic disease and Purkinje cell loss in Clec16a mutant mice · hedged

  “Therefore, mutation of Clec16a was consistent with the development of neurologic disease in two independent mouse strains.”

Autophagosome formation was unimpaired in Clec16a-deficient cells.

“However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation.”

Chloroquine treatment did not result in a statistically significant change in LC3+ puncta/cell in B6.Clec16aGT/GT MEFs.

“Immunofluorescence analysis of B6.Clec16aGT/GT MEFs revealed a 5.7 fold increase in LC3+ puncta/cell at baseline compared to control cells and this did not result in a statistically significant change when blocking autolysosome degradation through treatment with chloroquine (CHQ) (Fig. 4a,b).”

The accumulation of p62 also occurred in calbindin-positive axons, derived from Purkinje cells, in the same region.

“The accumulation of p62 also occurred in calbindin-positive axons, derived from Purkinje cells, in the same region (Fig. 3b, Supplementary Fig. 3a).”

Transcript levels of Clec16a were significantly reduced in Clec16aCURT MEFs using primers targeting the mRNA both 5′ and 3′ of the mutation in exon 21.

“Transcript levels of Clec16a were significantly reduced in Clec16aCURT MEFs using primers targeting the mRNA both 5′ and 3′ of the mutation in exon 21, indicating that this mutation significantly destabilizes Clec16a mRNA(s) (Supplementary Fig. 1e).”

Clec16a-deficient cells displayed abnormal bulk autophagy.

“However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation.”

Lack of Atg5 did not lead to altered Golgi or ERGIC morphology.

“These abnormalities were not due to defective autophagy in Clec16a-deficient cells since lack of the essential autophagy gene Atg5 did not lead to altered Golgi or ERGIC morphology (Supplementary Fig. 6d).”

Chloroquine elevated lysosomal pH in B6.Clec16aGT/GT MEFs and control cells.

“B6.Clec16aGT/GT MEFs and control cells had a comparable lysosomal pH of 4.8 and as expected this was elevated by treatment with chloroquine (Fig. 8c).”

Knock-down of CLEC16A in HeLa cells did not alter the rate of endocytic internalization and endolysosomal degradation of the EGF receptor following ligand binding.

“Knock-down of CLEC16A in HeLa cells reduced the amount of epidermal growth factor (EGF) receptor at steady-state but did not alter the rate of endocytic internalization and endolysosomal degradation of the receptor following ligand binding (Fig. 5b,c).”

B6.Clec16aGT/GT MEFs and control cells had a comparable lysosomal pH of 4.8.

“B6.Clec16aGT/GT MEFs and control cells had a comparable lysosomal pH of 4.8 and as expected this was elevated by treatment with chloroquine (Fig. 8c).”

Peptide-specific CD4+ T cells co-cultured with peptide-pulsed macrophages from B6.Clec16aGT/GT mice were equivalently activated across a range of peptide doses.

“Peptide-specific CD4+ T cells co-cultured with peptide-pulsed macrophages from B6.Clec16aGT/GT mice were equivalently activated across a range of peptide doses indicating normal cell surface expression of functional MHC class II (Fig. 5d).”

We observed normal expression of the lysosomal protein LAMP-1 by western blot analysis in Clec16a-mutant cells.

“As described above, we observed normal expression of the lysosomal protein LAMP-1 by western blot analysis in Clec16a-mutant cells (Fig. 6f, Supplementary Fig. 6e).”

Purkinje cell axons in the deep cerebellar nuclei of B6.Clec16aGT/GT mice accumulated autolysosome-like structures.

“Purkinje cell axons in the deep cerebellar nuclei of B6.Clec16aGT/GT mice accumulated autolysosome-like structures2021.”

The distribution and number of lysosomes as marked by the lysosomal membrane protein LAMP-1 was not altered by loss of CLEC16A expression in human cells.

“Further, the distribution and number of lysosomes as marked by the lysosomal membrane protein LAMP-1 was not altered by loss of CLEC16A expression in human cells (Fig. 8a,b).”

Western blotting of EndoH- and PNGase- digested lysates of B6.Clec16aGT/GT MEFs and HeLa-CLEC16A∆ cells demonstrated that the highly glycosylated lysosomal membrane protein LAMP-1 was present at equivalent levels in wild-type and CLEC16A-deficient cells.

“Western blotting of EndoH- and PNGase- digested lysates of B6.Clec16aGT/GT MEFs and HeLa-CLEC16A∆ cells demonstrated that the highly glycosylated lysosomal membrane protein LAMP-1 was present at equivalent levels in wild-type and CLEC16A-deficient cells.”

Knock-down of CLEC16A did not affect secretion.

“However knock-down of CLEC16A did not affect secretion (Fig. 6d,e).”

B6.Clec16aGT/GT MEFs and CLEC16A-mutant HeLa cells contained large numbers of single membrane bound structures containing cellular material not seen in control cells.

“B6.Clec16aGT/GT MEFs and CLEC16A-mutant HeLa cells contained large numbers of single membrane bound structures containing cellular material not seen in control cells (Fig. 7a, Supplementary Fig. 7a,b).”

The rate of endolysosomal degradation of the receptor following ligand binding was unimpaired in CLEC16A-deficient HeLa cells.

“The rate of endolysosomal degradation of the receptor following ligand binding was also unimpaired in CLEC16A-deficient HeLa cells (Supplementary Fig. 5b,c), indicating that a failure to observe changes in siRNA-treated cells was not due to residual low levels of protein.”

LAMP-1 acquired appropriate N-linked glycans in CLEC16A-deficient cells.

“Further, LAMP-1 acquired appropriate N-linked glycans3839 (Fig. 6f, Supplementary Fig. 6e).”

• cites [38] Carlsson, S. R., Roth, J., Piller, F. & Fukuda, M. Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem 263, 18911–18919 (1988). sourceNot checked — no full text of the source
• cites [39] Fukuda, M., Viitala, J., Matteson, J. & Carlsson, S. R. Cloning of cDNAs encoding human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Comparison of their deduced amino acid sequences. J Biol Chem 263, 18920–18928 (1988). sourceNot checked — no full text of the source

Staining with other markers such as the trans-Golgi network protein, TGN-46, and the Golgi tethering protein Giantin confirmed the dispersion of the Golgi in CLEC16A-mutant cells.

“Staining with other markers such as the trans-Golgi network protein, TGN-46, and the Golgi tethering protein Giantin confirmed the dispersion of the Golgi in CLEC16A-mutant cells (Supplementary Fig. 6c).”

Knock-down of CLEC16A in HeLa cells reduced the amount of epidermal growth factor (EGF) receptor at steady-state.

“Knock-down of CLEC16A in HeLa cells reduced the amount of epidermal growth factor (EGF) receptor at steady-state but did not alter the rate of endocytic internalization and endolysosomal degradation of the receptor following ligand binding (Fig. 5b,c).”

Bafilomycin A1 treatment did not increase the colocalization of LC3 and LAMP-1 in CLEC16A-mutant cells.

“Importantly, while treatment of control cells with bafilomycin A1 increases the colocalization of LC3 and LAMP-1+ in puncta consistent with its known role in inhibiting lysosome and autophagolysosome function, bafilomycin A1 (BAF) treatment did not increase the colocalization of these markers in CLEC16A-mutant cells (Fig. 7c,d).”

Treatment of control cells with bafilomycin A1 increases the colocalization of LC3 and LAMP-1+ in puncta.

“Importantly, while treatment of control cells with bafilomycin A1 increases the colocalization of LC3 and LAMP-1+ in puncta consistent with its known role in inhibiting lysosome and autophagolysosome function, bafilomycin A1 (BAF) treatment did not increase the colocalization of these markers in CLEC16A-mutant cells (Fig. 7c,d).”

We did not observe alterations in the endosomal network as detected by staining of endosomes for Rab5, Rab7 or EEA1 in Clec16a-deficient cells.

“Knock-down or loss of Clec16a in human and murine cells has been described to alter the morphology of late endosomes1633 but we did not observe alterations in the endosomal network as detected by staining of endosomes for Rab5, Rab7 or EEA1 in Clec16a-deficient cells (Fig. 5a, Supplementary Fig. 5a).”

This was confirmed by labeling these structures with both LC3 and LAMP-1 by immuno-electron microscopy.

“This was confirmed by labeling these structures with both LC3 and LAMP-1 by immuno-electron microscopy (Fig. 7b, Supplementary Fig. 7c).”

CLEC16A-deficient human and mouse cells demonstrated a striking dispersion of the Golgi apparatus and ER-Golgi intermediate compartment (ERGIC) as detected by staining for cis-Golgi matrix protein GM130 and ERGIC protein 53 (ERGIC-53).

“CLEC16A-deficient human and mouse cells demonstrated a striking dispersion of the Golgi apparatus and ER-Golgi intermediate compartment (ERGIC) as detected by staining for cis-Golgi matrix protein GM130 and ERGIC protein 53 (ERGIC-53) (Fig. 6a,b, Supplementary Fig. 6a,b).”

CLEC16A-deficient cells have increased colocalization of LC3 and LAMP-1 in puncta compared to control cells.

“CLEC16A-deficient cells have increased colocalization of LC3 and LAMP-1 in puncta compared to control cells, consistent with the increased number of autolysosomes observed by confocal microscopy (Fig. 7c,d).”

Clec16a is required for Purkinje cell survival.

“Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival.”

• Also stated in: § Neurologic disease and Purkinje cell loss in Clec16a mutant mice · hedged

  “These data indicate that Clec16a is important for Purkinje cell survival.”

Clec16a is important for Purkinje cell survival.

“These data indicate that Clec16a is important for Purkinje cell survival.”

The role of Clec16a in the central nervous system has not been defined.

“Thus the role of Clec16a in the central nervous system (CNS) has not been defined.”

Mutation of Clec16a was consistent with the development of neurologic disease in two independent mouse strains.

“Therefore, mutation of Clec16a was consistent with the development of neurologic disease in two independent mouse strains.”

The mRNA for Clec16a was interrupted by the GT cassette.

“While amplification of Clec16a transcripts across exons 2–3 were comparable in wild-type and B6.Clec16aGT/GT murine embryo fibroblast cells (MEFs), there was a greater than 90% reduction in Clec16a transcript using primers targeting exons 23–24 (Supplementary Fig. 1e), indicating that the mRNA for this gene was interrupted by the GT cassette.”

Clec16a-deficiency was not associated with major alterations in mitophagy in cells cultured under our conditions.

“We did not detect a change in total mtDNA, or ATP levels in Clec16a-deficient cells, indicating that Clec16a-deficiency was not associated with major alterations in mitophagy in cells cultured under our conditions, and that abnormalities in autophagy were not due to inadequate intracellular energy stores (Supplementary Fig. 4d,e).”

The structures within Purkinje cell axons of B6.Clec16aGT/GT mice were likely of autophagic origin as they labeled for LC3 expression by immuno-electron microscopy.

“The structures within Purkinje cell axons of B6.Clec16aGT/GT mice were likely of autophagic origin as they labeled for LC3 expression by immuno-electron microscopy (Fig. 3f).”

The Clec16aCURT mutation significantly destabilizes Clec16a mRNA.

“Transcript levels of Clec16a were significantly reduced in Clec16aCURT MEFs using primers targeting the mRNA both 5′ and 3′ of the mutation in exon 21, indicating that this mutation significantly destabilizes Clec16a mRNA(s) (Supplementary Fig. 1e).”

Clec16a has a unique role in the function and clearance of autolysosomes.

“Our findings regarding the loss of Purkinje cells and impaired autophagy point to a potential in vivo side-effect of pharmacological inhibition of CLEC16A protein function or autophagy, and support the conclusion that Clec16a has a unique role in the function and clearance of autolysosomes.”

One dominant effect of Clec16a deficiency in the whole mouse is related to a role in Purkinje cells.

“These results show that one dominant effect of Clec16a deficiency in the whole mouse is related to a role in Purkinje cells.”

Golgi abnormalities in Clec16a-deficient cells were not due to defective autophagy.

“These abnormalities were not due to defective autophagy in Clec16a-deficient cells since lack of the essential autophagy gene Atg5 did not lead to altered Golgi or ERGIC morphology (Supplementary Fig. 6d).”

The specificity of this function of Clec16a for autophagy was further supported by the lack of detectable changes in another lysosomal degradation pathway, the recycling of membrane proteins through the endolysosomal system.

“The specificity of this function of Clec16a for autophagy was further supported by the lack of detectable changes in another lysosomal degradation pathway, the recycling of membrane proteins through the endolysosomal system.”

CLEC16A has an important role in the survival of cerebellar Purkinje neurons in two mutant mouse models.

“We report here that CLEC16A, which has been linked by GWAS studies to multiple sclerosis and other autoimmune diseases, has an important role in the survival of cerebellar Purkinje neurons in two mutant mouse models.”

The Golgi functions normally in CLEC16A-deficient cells as measured by these assays.

“Together these data indicated that the Golgi functions normally in CLEC16A-deficient cells as measured by these assays.”

Abnormal autolysosome accumulation, rather than disrupted Golgi morphology, is the proximate cause of Clec16a-dependent autophagic abnormalities.

“Together these data argue for abnormal autolysosome accumulation, rather than disrupted Golgi morphology, as the proximate cause of Clec16a-dependent autophagic abnormalities reported here.”

CLEC16A deficiency results in a failure of autolysosome function or clearance at a step in autophagy downstream of autophagosome and lysosome fusion.

“These data indicate that CLEC16A deficiency results in a failure of autolysosome function or clearance at a step in autophagy downstream of autophagosome and lysosome fusion.”

Abnormalities in autophagy in Clec16a-deficient cells were not due to inadequate intracellular energy stores.

“We did not detect a change in total mtDNA, or ATP levels in Clec16a-deficient cells, indicating that Clec16a-deficiency was not associated with major alterations in mitophagy in cells cultured under our conditions, and that abnormalities in autophagy were not due to inadequate intracellular energy stores (Supplementary Fig. 4d,e).”

These data demonstrated impaired bulk autophagy in Clec16a-deficient human and murine cells.

“These data demonstrated impaired bulk autophagy in Clec16a-deficient human and murine cells.”

Pharmacological inhibition of CLEC16A protein function or autophagy may have a potential in vivo side-effect.

“Our findings regarding the loss of Purkinje cells and impaired autophagy point to a potential in vivo side-effect of pharmacological inhibition of CLEC16A protein function or autophagy, and support the conclusion that Clec16a has a unique role in the function and clearance of autolysosomes.”

These data provide, in a small animal model, insight into the effects of full disruption of the gene in the CNS.

“Thus these data provide, in a small animal model, insight into the effects of full disruption of the gene in the CNS.”

The high level of LC3-positive structures at baseline, combined with a failure of chloroquine to increase the number of these structures, was most consistent with Clec16a playing a role in autophagy after the formation of autophagosomes.

“The high level of LC3-positive structures at baseline, combined with a failure of chloroquine to increase the number of these structures, was most consistent with Clec16a playing a role in autophagy after the formation of autophagosomes.”

These assays did not detect abnormalities in the endosomal or endolysosomal system in Clec16a-mutant cells.

“These assays did not detect abnormalities in the endosomal or endolysosomal system in Clec16a-mutant cells.”

Cell surface expression of functional MHC class II was normal in B6.Clec16aGT/GT macrophages.

“Peptide-specific CD4+ T cells co-cultured with peptide-pulsed macrophages from B6.Clec16aGT/GT mice were equivalently activated across a range of peptide doses indicating normal cell surface expression of functional MHC class II (Fig. 5d).”

The phenotype of Clec16a mutation observed in this study is remarkably similar to the phenotype caused by conditional deletion of Atg5 or Atg7 in Purkinje cells.

“This phenotype is remarkably similar to what we observed here for mutation of Clec16a.”

These structures were most consistent with being autolysosomes rather than autophagosomes as they had a single membrane.

“These structures were most consistent with being autolysosomes rather than autophagosomes as they had a single membrane.”

Failure to clear autolysosomes and to properly digest their contents plays a role in neuronal survival which may contribute to central nervous system disease.

“Studies reported here suggest that a failure to clear autolysosomes and to properly digest their contents plays a role in neuronal survival which may contribute to central nervous system disease.”

Abnormal autophagy, likely as a result of abnormal autophagic flux, leads to neurodegeneration when Clec16a is mutated.

“supports the concept that it is abnormal autophagy, likely as a result of abnormal autophagic flux, that leads to neurodegeneration when Clec16a is mutated.”

Clec16a plays a fundamentally important role in autophagy by acting at a stage in the process for which specific required genes have not previously been identified.

“Clec16a plays a fundamentally important role in autophagy by acting at a stage in the process for which specific required genes have not previously been identified.”