A Conserved Requirement for RME-8/DNAJC13 in Neuronal Autolysosome Reformation

Abstract

Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.

Figure 1.. Loss of RME-8 results in accumulation of elongated lysosomal tubules.

A) Representative drawing of ALM neurons in C. elegans. One of two ALM neurons is shown (other is located laterally opposite). Black dashed box represents the approximate area that is shown in example micrographs. B) Fluorescent micrographs showing pmec-7 promoter-driven mScarletI::RAB-7 labeled (late endosome/ lysosome marker) puncta in soma (left) and proximal axon (right) in ALM neurons in rme-8(+), rme-8(N861S), and rme-8(b1023ts) Day 1 adult backgrounds. White arrowhead indicates grossly elongated tubule. C) Length of RAB-7 puncta/tubule in the proximal axon in rme-8(+), rme-8(N861S) and rme-8(b1023ts) is graphed in Day 1 adult animals. Each data point represents an individual puncta/tubule. A minimum of 15 animals were analyzed per strain. D) Pmec-7 promoter-driven mScarletI::RAB-7 labeled puncta in rme-8(+) and rme-8(N861S) backgrounds in Day 9 adult. E) Length of RAB-7 puncta/ tubule in the proximal axon in rme-8(+), and rme-8(N861S) is graphed in Day 9 adults. Each data point represents an individual puncta/tubule. F) Pmec-7 LMP-1::mNeonGreen (NG) labeled (lysosome marker) puncta. A severely elongated LMP-1 positive tubule can be seen emanating into the axon from puncta in the soma (white arrow), and another dimmer tubule can be seen further out in the axon (white arrowhead). G) Quantification of LMP-1::NG axonal puncta/ tubule length seen in conditions shown in E). Each data point represents an individual puncta/tubule. A minimum of 15 animals were analyzed per strain. H) Graph depicting LMP-1::mNG Integrated intensity (Arbitrary Units; AU) for the same experiment as 1E and 1F. Each data point represents the integrated intensity of all thresholded puncta in one soma in one animal. I) Micrographs of pmec-7 RME-8::oxGFP rescue of LMP-1::mScarleti length in rme-8(b1023ts) background. J) Graph quantifying elongation of LMP-1::mScarletI in rme-8(+), rme-8(b1023ts), and rme-8(b1023ts); pmec-7 RME-8::GFP. Scale bars, 5 um. Abbreviations: rme-8(NS)= rme-8(pw22[N861S]); rme-8(ts)= rme-8(b1023ts). C), E), and J): One-way ANOVA followed with Tukey’s Multiple Comparisons Test. G), H): Student’s unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2.. rme-8 mutants accumulate abnormal autolysosomes.

A) Micrographs are shown for single channels as well as merged images in double-labeled LMP-1::mScarlet-I (lysosome marker); mNG::LGG-1 (autophagosome marker) in rme-8(+) and rme-8(b1023ts) backgrounds. White arrow labels mNG::LGG-1+/ LMP-1::mSc- puncta. White arrowhead labels tubules labeled by LMP-1 and dimly labeled by LGG-1 can be seen in the rme-8(b1023ts) background. B) Graphs quantifying the colocalization of LMP-1:mSc and mNG::LGG-1, measured by %Area Colocalization. Each data point represents average % Area colocalization for one animal. **P < 0.01, ns > 0.05 by two-tailed unpaired t-test. C) Micrograph showing pmec-7::mNG::LGG-1 in neurons of rme-8(+), rme-8(b1023ts), rme8(N861S), and snx-1(tm847) backgrounds. Scale bar: 5um. D) Graph quantifying elongation of LGG-1::mNG in rme-8(+), rme-8(b1023ts), and rme-8(N861S); snx-1(0) backgrounds. Each data point represents an individual puncta/tubule. ****P < 0.0001 by One-way ANOVA followed with Tukey’s Multiple Comparisons Test. E) Graph depicting mNG::LGG-1 Integrated intensity (Arbitrary Units; AU) for the same experiment as 2C and 2D. Each data point represents the integrated intensity of all thresholded puncta in one soma in one animal. ****P < 0.0001, **P<0.01 by One-way ANOVA followed with Tukey’s Multiple Comparisons Test.

Figure 3.. Loss of RME-8 phenocopies autolysosome reformation mutants.

A) Micrographs are shown of pmec-7 LMP-1::mNG in ALM neurons in rme-8(+), rme-8(ts), vps-15(ts), bec-1(0), and dyn-1(ts) backgrounds. B) Quantification of LMP-1 tubule length (um) in ALM proximal axons in the backgrounds shown in 3A. Each data point represents an individual puncta/tubule. ***P < 0.001, **P < 0.01, *P < 0.05, ns > 0.05 by One-way ANOVA followed with Tukey’s Multiple Comparisons Test. C) Micrographs are shown of pmec-7 LMP-1::mNG in ALM neurons in rme-8(+), rme-8(ts), atg-18(0), epg-1(0), epg-6(0), epg-8(0), backgrounds. Example images and graphs are separate from 3A and 3B because data was obtained during separate experiments. D) Quantification of LMP-1 tubule length (um) in ALM proximal axons in the backgrounds shown in 3C. Each data point represents an individual puncta/tubule. ***P < 0.001, **P < 0.01, *P < 0.05, ns > 0.05 by One-way ANOVA followed with Tukey’s Multiple Comparisons Test.

Figure 4.. RME-8 knockdown in mouse cortical neurons causes elongated autolysosome tubules and enlarged autolysosomes.

A) Micrographs of double-labeled GFP-LAMP1, mRFP-LC3 in primary mouse cortical neurons transfected with control (top panel) or RME8/DNAJC13 (bottom two panels) shRNA. Images are shown for single channel labeling and merged. Arrows (top panel, control shRNA) indicate lysosomes (GFP-LAMP1 positive; mRFP-LC3 negative), arrowheads indicate autolysosomes (GFP-LAMP1 positive; mRFP-LC3 positive). Scale bar, 10 um. B) The average LAMP1 tubules length of each soma is quantified in control and RME-8/DNAJC13 shRNA conditions. Each puncta represents the average length in one neuron. C) Frequency distribution of average tubule length per soma in control and RME-8/DNAJC13 shRNA. D) Frequency distribution of LAMP-1 positive (lysosomes and autolysosomes) vesicle area per soma in control and RME-8/DNAJC13 shRNA. B), F), E): Student’s unpaired t-test. ****P<0.0001.

Figure 5.. RME-8 localizes primarily to endosomes.

A) Fluorescent single channel and merged micrographs of double labeled strains LMP-1::mScarlet; RME-8::GFP and mScarlet::SNX-1; RME-8::GFP in wild-type background. Scale bar, 5um. B) Quantification of colocalization between strains shown in A) using % Area Colocalization.

Figure 6.. Loss of RME-8 causes decreased autophagosome levels like known ALR mutants.

A), D) Micrographs of pmec-7 LGG-1::mNG in C. elegans ALM neurons. Area shown includes soma (left) and proximal axon (right). Example images and graphs are separate because data was obtained during separate experiments. Scale bars, 5 um. B), E) Quantification of integrated intensity of LGG-1::mNG puncta in backgrounds shown in A) and D) (AU; arbitrary units). C), F) Average Intensity of LGG-1 positive puncta in axons and somas of backgrounds shown in A) and D). ***P < 0.001, ****P < 0.0001 by One-way ANOVA followed with Tukey’s Multiple Comparisons Test.

Figure 7.. RME-8/DNAJC13 knock down in mouse cortical neurons reduces autophagic flux.

A) Micrographs are shown of in vitro mouse cortical neurons transfected with GFP-LC3. A 2×2 factorial design was used, conditions were treatment with either control or RME-8/DNAJC13 shRNA, and +/− pepstatin/ E64D (lysosome inhibitors; LIs). Scale bar, 10 um. B) Autophagic vacuole (AV) density is quantified in neurons for the 2×2 factorial design shown in A). ****P < 0.0001 by Student’s t-test.

Figure 8.. RME-8 is required for efficient clathrin recruitment to lysosomes.

A) Single-channel and merge images of double-labeled LMP-1::mSc and CLIC-1::mNG in C. elegans’ ALM neurons. B) Quantification of the percent of LMP-1::mSc that has CLIC-1::mNG present on it. Scale bar, 5 um. One-way ANOVA followed with Tukey’s Multiple Comparisons Test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.