Dataset Information

Distinct sub-cellular autophagy impairments occur independently of protein aggregation in aged induced neurons from patients with Huntington’s disease

ABSTRACT: Huntington’s disease (HD) is an inherited neurodegenerative disorder that is caused by CAG expansions in the huntingtin (HTT) gene. Modelling HD in the lab has proven challenging as rodent models poorly reproduce the disease process and cellular models fail to include age-dependent processes crucial to the expression of the disease clinically. Here we generated induced neurons (iNs) through direct reprogramming of skin fibroblasts from HD-patients. This resulted in the generation of patient-derived HD neurons that retained age-dependent epigentic characteristics of the donors. Using these cells, we then undertook a proteomic analysis and demonstrated there was an alteration in the CAMKK-AMPK pathway and autophagy in these HD-iNs, compared to healthy control iN cells. In line with this, HD-iNs displayed a distinct subcellular alteration in autophagy, specifically in the neurites, characterized by a reduction in the transport of late autophagic structures between neurites and the soma and subsequent cargo degradation. In addition to which the HD-iNs displayed shorter, smaller and fewer neurites. CRISPRi-mediated silencing of HTT (both wt and mutant) did not rescue the morphology nor could this rescue the autophagic defect in HD-iNs and indeed resulted in additional autophagy alterations when done using ctrl-iNs, highlighting the importance of wild type HTT in neuronal autophagy. In summary, our results have identified a distinct subcellular autophagy impairment in aged patient derived HD-neurons. In addition, we have shown a role for normal wt Htt in autophagy. Together this work provides a new rational for future development of autophagy activation therapies while also highlighting problems that may arise using non allele specific htt silencing approaches, some of which are now in clinical trials.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

DISEASE(S): Huntington Disease

SUBMITTER: Jeovanis Gil

LAB HEAD: Johan Jakobsson

PROVIDER: PXD024286 | Pride | 2022-10-14

REPOSITORIES: Pride

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Source:

			Action	DRS
	20190618_FBC-_1_.msf	Msf
	20190618_FBC_C11-P9_R1.raw	Raw
	20190618_FBC_C11-P9_R2.raw	Raw
	20190618_FBC_C11-P9_R3.raw	Raw
	20190618_FBC_C12-P7_R1.raw	Raw

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Distinct subcellular autophagy impairments in induced neurons from patients with Huntington's disease.

Pircs Karolina K Drouin-Ouellet Janelle J Horváth Vivien V Gil Jeovanis J Rezeli Melinda M Garza Raquel R Grassi Daniela A DA Sharma Yogita Y St-Amour Isabelle I Harris Kate K Jönsson Marie E ME Johansson Pia A PA Vuono Romina R Fazal Shaline V SV Stoker Thomas T Hersbach Bob A BA Sharma Kritika K Lagerwall Jessica J Lagerström Stina S Storm Petter P Hébert Sébastien S SS Marko-Varga György G Parmar Malin M Barker Roger A RA Jakobsson Johan J

Brain : a journal of neurology 20220901 9

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterize ...[more]

PMID: 34936701

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Project description:Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer lifespans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyglutamine length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions. Gene expression profiles were analyzed to examine the effects of p62 depletion in mouse with or without mutant huntingtin exon 1 To examine the effect of p62 depletion on the transcriptome of Huntington's disease model mice, we crossed p62 knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with p62 knockout (HD_p62KO), HD mice with normal p62 (HD_p62WT), non-HD-transgenic mice with p62 knockout (NT_p62KO), and non-HD-transgenic mice with normal p62 (NT_p62WT).