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Roles of chaperone-mediated autophagy in the regulation of TDP-43 phosphorylation and aggregation

  • 고승범 (아주대학교 일반대학원)

초록/요약

Abnormal cytoplasmic redistribution, hyperphosphorylation, and aggregation of TAR DNA-binding protein 43 （TDP-43） are hallmark features of amyotrophic lateral sclerosis （ALS） and frontotemporal lobar degeneration （FTLD）. Pathological TDP-43 phosphorylation is mediated by specific kinases, including casein kinase 1 （CK1） isoforms, yet the mechanisms that regulate these kinases and thereby control pathological TDP-43 changes remain poorly understood. Chaperone-mediated autophagy （CMA） is a selective lysosomal degradation pathway that controls the abundance of individual cytosolic proteins through a KFERQ-like motif– and LAMP2A-dependent mechanism. Although reduced CMA activity has been reported in motor neurons of sporadic ALS patients with TDP-43 pathology, whether and how CMA regulates pathological TDP-43 phosphorylation and aggregation has not been defined. In this study, we used H2O2-induced oxidative stress and TDP-43 overexpression to establish cellular models of TDP-43 pathology and investigated the effect of CMA on pathological TDP-43 changes. Pharmacological CMA activation by AR7 treatment and genetic activation by LAMP2A overexpression both reduced TDP-43 phosphorylation, insoluble TDP-43 accumulation, and cytoplasmic mislocalization across multiple experimental conditions. Conversely, CMA inhibition by LAMP2A knockdown increased TDP-43 phosphorylation, insoluble accumulation, and mislocalization, demonstrating that CMA bidirectionally regulates TDP-43 pathology. To define the mechanism underlying CMA-dependent suppression of TDP-43 pathology, we screened candidate TDP-43 phosphorylation-related kinases and identified CK1 family kinases as the most consistently CMA-responsive candidates. Among these, CK1δ fulfilled multiple criteria for a CMA substrate: it interacted with the CMA chaperone Hsc70, its interaction with Hsc70 was reduced by mutation of a KFERQ-like motif, disruption of this motif delayed CK1δ degradation and abolished its responsiveness to CMA activation and inhibition, and endogenous CK1δ accumulated in LAMP1-positive lysosomal vesicles upon lysosomal inhibition. These findings establish CK1δ as a KFERQ-like motif-dependent CMA substrate. Functionally, inhibition of CK1δ/ε activity suppressed the increases in TDP-43 phosphorylation and insoluble TDP-43 accumulation induced by CMA inhibition, indicating that CMA suppresses TDP-43 pathology, at least in part, by promoting CK1δ degradation and thereby limiting CK1δ/ε-dependent TDP-43 phosphorylation. Collectively, these findings define a CMA–CK1δ–TDP-43 regulatory axis and provide a mechanistic basis for understanding how CMA dysfunction may contribute to pathological TDP-43 phosphorylation and aggregation in ALS- and FTLD- associated TDP-43 proteinopathy.

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목차

I. INTRODUCTION 1
1. ALS and FTLD 1
2. TDP-43: biology and pathology 4
3. TDP-43 phosphorylation and aggregation 7
4. Autophagy pathways 8
5. Autophagy dysfunction in neurodegenerative diseases 12
6. Chaperone-mediated autophagy 14
7. Objectives of this study 15
II. MATERIALS AND METHODS 17
1. DNA constructs 17
2. Antibodies 18
3. Chemicals and reagents 18
4. Cell culture and treatments 19
5. Plasmid transfection and RNA interference 19
6. Generation of stable cell lines 20
7. Immunoblotting 20
8. Immunoprecipitation 21
9. Preparation of RIPA-soluble and -insoluble fractions 21
10. Immunofluorescence staining and microscopy 22
11. Quantification and statistical analysis 22
12. Preparation of schematic illustrations 23
III. RESULTS 24
1. Starvation-induced autophagic activation inhibits TDP-43 pathology 24
2. CMA suppresses TDP-43 pathology 38
3. CK1 family kinases are candidate CMA substrates 54
4. CK1δ is selectively degraded via CMA through a KFERQ-like motif 62
5. CMA regulates TDP-43 pathology through CK1δ degradation 73
IV. DISCUSSION 76
V. REFERENCES 82
국문요약 90

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