The contribution of proteasomal impairment to autophagy activation by C9orf72 poly-GA aggregates.

BACKGROUND: Poly-GA, a dipeptide repeat protein unconventionally translated from GGGGCC (G4C2) repeat expansions in C9orf72, is abundant in C9orf72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9orf72-ALS/FTD). Although the poly-GA aggregates have been identified in C9orf72-ALS/FTD neurons, the effects on UPS (ubiquitin-proteasome system) and autophagy and their exact molecular mechanisms have not been fully elucidated. RESULTS: Herein, our in vivo experiments indicate that the mice expressing ploy-GA with 150 repeats instead of 30 repeats exhibit significant aggregates in cells. Mice expressing 150 repeats ploy-GA shows behavioral deficits and activates autophagy in the brain. In vitro findings suggest that the poly-GA aggregates influence proteasomal by directly binding proteasome subunit PSMD2. Subsequently, the poly-GA aggregates activate phosphorylation and ubiquitination of p62 to recruit autophagosomes. Ultimately, the poly-GA aggregates lead to compensatory activation of autophagy. In vivo studies further reveal that rapamycin (autophagy activator) treatment significantly improves the degenerative symptoms and alleviates neuronal injury in mice expressing 150 repeats poly-GA. Meanwhile, rapamycin administration to mice expressing 150 repeats poly-GA reduces neuroinflammation and aggregates in the brain. CONCLUSION: In summary, we elucidate the relationship between poly-GA in the proteasome and autophagy: when poly-GA forms complexes with the proteasome, it recruits autophagosomes and affects proteasome function. Our study provides support for further promoting the comprehension of the pathogenesis of C9orf72, which may bring a hint for the exploration of rapamycin for the treatment of ALS/FTD.

FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions.

C9ORF72 repeat expansion is the most common genetic mutation in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Abnormal dipeptide repeat proteins (DPRs) generated from repeat-associated non-AUG (RAN) translation of repeat-containing RNAs are thought to be pathogenic; however, the mechanisms are unknown. Here we report that (GR)80 and (PR)80 are toxic in neuronal and non-neuronal cells in Drosophila. In contrast to reported shorter poly(GR) forms, (GR)80 is mostly localized throughout the cytosol without detectable accumulation in the nucleolus, accompanied by suppression of Notch signaling and cell loss in the wing. Some Notch target genes are also downregulated in brains and iPSC-derived cortical neurons of C9ORF72 patients. Increased Notch expression largely suppressed (GR)80-induced cell loss in the wing. When co-expressed in Drosophila, HeLa cells, or human neurons, (GA)80 recruited (GR)80 into cytoplasmic inclusions, partially decreasing the toxicity of (GR)80 and restoring Notch signaling in Drosophila. Thus, different DPRs have opposing roles in cell loss and we identify the Notch pathway as one of the receptor signaling pathways that might be compromised in C9ORF72 FTD/ALS.

Overexpression of Toxic Poly(Glycine-Alanine) Aggregates in Primary Neuronal Cultures Induces Time-Dependent Autophagic and Synaptic Alterations but Subtle Activity Impairments.

The pathogenic expansion of the intronic GGGGCC hexanucleotide located in the non-coding region of the C9orf72 gene represents the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation leads to the accumulation of toxic RNA foci and dipeptide repeats (DPRs), as well as reduced levels of the C9orf72 protein. Thus, both gain and loss of function are coexisting pathogenic aspects linked to C9orf72-ALS/FTD. Synaptic alterations have been largely described in C9orf72 models, but it is still not clear which aspect of the pathology mostly contributes to these impairments. To address this question, we investigated the dynamic changes occurring over time at the synapse upon accumulation of poly(GA), the most abundant DPR. Overexpression of this toxic form induced a drastic loss of synaptic proteins in primary neuron cultures, anticipating autophagic defects. Surprisingly, the dramatic impairment characterizing the synaptic proteome was not fully matched by changes in network properties. In fact, high-density multi-electrode array analysis highlighted only minor reductions in the spike number and firing rate of poly(GA) neurons. Our data show that the toxic gain of function linked to C9orf72 affects the synaptic proteome but exerts only minor effects on the network activity.

Poly-GA immunohistochemistry is a reliable tool for detecting C9orf72 hexanucleotide repeat expansions.

Poly-GA immunohistochemistry (A) on formalin fixed paraffin embedded cerebellum sections shows a similar distribution to p62 antibody (B) and reliably identifies neuronal cytoplasmic inclusions and neurites in cases with known C9orf72 repeat expansion. This is useful in the research setting where genetic testing has not been performed in life or suitable tissue is not avilable post-mortem.

Propranolol reduces the accumulation of cytotoxic aggregates in C9orf72-ALS/FTD in vitro models.

Mutations in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathogenetic mechanisms linked to this gene are a direct consequence of an aberrant intronic expansion of a GGGGCC hexanucleotide located between the 1a and 1b non-coding exons, which can be transcribed to form cytotoxic RNA foci or even translated into aggregation-prone dipeptide repeat proteins. Importantly, the abnormal length of these repeats affects also the expression levels of C9orf72 itself, which suggests haploinsufficiency as additional pathomechanism. Thus, it appears that both toxic gain of function and loss of function are distinct but still coexistent features contributing to the insurgence of the disease in case of C9orf72 mutations. In this study, we aimed at identifying a strategy to address both aspects of the C9orf72-related pathobiochemistry and provide proof-of-principle information for a better understanding of the mechanisms leading to neuronal loss. By using primary neurons overexpressing toxic poly(GA), the most abundant protein product of the GGGGCC repeats, we found that the antiarrhythmic drug propranolol could efficiently reduce the accumulation of aberrant aggregates and increase the survival of C9orf72-related cultures. Interestingly, the improved catabolism appeared to not depend on major degradative pathways such as autophagy and the proteasome. By analyzing the proteome of poly(GA)-expressing neurons after exposure to propranolol, we found that the drug increased lysosomal degradation through a mechanism directly involving C9orf72 protein, whose levels were increased after treatment. Further confirmation of the beneficial effect of the beta blocker on aggregates' accumulation and survival of hiPSC-derived C9orf72-mutant motoneurons strengthened the finding that addressing both facets of C9orf72 pathology might represent a valid strategy for the treatment of these ALS/FTD cases.

Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation.

The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.

Repeated repeat problems: Combinatorial effect of C9orf72-derived dipeptide repeat proteins.

A microsatellite expansion mutation in C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). The expansion mutation leads to C9orf72 loss of function, RNA foci formation, and generation of five species of non-AUG RAN translated dipeptide repeat proteins (DPRs), such as poly(GA), poly(GP), poly(GR), poly(PA), and poly(PR). Although one cell can contain more than type of DPRs, information about interplay between different DPR species is limited. Here we show that the combined expression of distinct C9orf72-derived dipeptide repeat species produces cellular outcomes and structural differences that are unique compared to the expression of a single DPR species, suggesting the complex biological interactions that occur when multiple DPR variants are simultaneously expressed. Our data highlights the need for further analysis of how combined expression of different DPRs affects the disease state.

HR23B pathology preferentially co-localizes with p62, pTDP-43 and poly-GA in C9ORF72-linked frontotemporal dementia and amyotrophic lateral sclerosis.

Human homologue of yeast UV excision repair protein Rad23b (HR23B) inclusions are found in a number of neurodegenerative diseases, including frontotemporal dementia (FTD), Huntington's disease (HD), spinocerebellar ataxia type 3 and 7 (SCA3/7), fragile X associated tremor/ataxia syndrome (FXTAS) and Parkinson's disease (PD). Here, we describe HR23B pathology in C9ORF72 linked FTD and amyotrophic lateral sclerosis (ALS) cases. HR23B presented in neuropils, intranuclear inclusions and cytoplasmic and perinuclear inclusions and was predominantly found in cortices (frontal, temporal and motor), spinal cord and hippocampal dentate gyrus. HR23B co-localized with poly-GA-, pTDP-43- and p62-positive inclusions in frontal cortex and in hippocampal dentate gyrus, the latter showing higher co-localization percentages. HR23B binding partners XPC, 20S and ataxin-3, which are involved in nucleotide excision repair (NER) and the ubiquitin-proteasome system (UPS), did not show an aberrant distribution. However, C9ORF72 fibroblasts were more sensitive for UV-C damage than healthy control fibroblasts, even though all factors involved in NER localized normally to DNA damage and the efficiency of DNA repair was not reduced. HR23Bs other binding partner NGly1/PNGase, involved in ER-associated degradation (ERAD) of misfolded proteins, was not expressed in the majority of neurons in C9FTD/ALS brain sections compared to non-demented controls. Our results suggest a difference in HR23B aggregation and co-localization pattern with DPRs, pTDP-43 and p62 between different brain areas from C9FTD/ALS cases. We hypothesize that HR23B may play a role in C9ORF72 pathogenesis, possibly by aberrant ERAD functioning.

Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration.

BACKGROUND: The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. METHOD: We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence. RESULTS: Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP. CONCLUSION: These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.