The major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a C9orf72 GC repeat expansion. Proposed mechanisms by which the expansion causes c9FTD/ALS include toxicity from repeat-containing RNA and from dipeptide repeat proteins translated from these transcripts. To investigate the contribution of poly(GR) dipeptide repeat proteins to c9FTD/ALS pathogenesis in a mammalian in vivo model, we generated mice that expressed GFP-(GR) in the brain. GFP-(GR) mice developed age-dependent neurodegeneration, brain atrophy, and motor and memory deficits through the accumulation of diffuse, cytoplasmic poly(GR). Poly(GR) co-localized with ribosomal subunits and the translation initiation factor eIF3η in GFP-(GR) mice and, of importance, in c9FTD/ALS patients. Combined with the differential expression of ribosome-associated genes in GFP-(GR) mice, these findings demonstrate poly(GR)-mediated ribosomal distress. Indeed, poly(GR) inhibited canonical and non-canonical protein translation in HEK293T cells, and also induced the formation of stress granules and delayed their disassembly. These data suggest that poly(GR) contributes to c9FTD/ALS by impairing protein translation and stress granule dynamics, consequently causing chronic cellular stress and preventing cells from mounting an effective stress response. Decreasing poly(GR) and/or interrupting interactions between poly(GR) and ribosomal and stress granule-associated proteins may thus represent potential therapeutic strategies to restore homeostasis.

No treatment for frontotemporal dementia (FTD), the second most common early-onset dementia, is available but therapeutics are being investigated to target the two main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hamstrung by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human iPSC-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease sub-types marked by TDP-43 inclusions. Lastly, we validated that truncated STMN2 RNA is elevated in the frontal cortex of a cohort of FTLD-TDP cases but not in controls or cases with progressive supranuclear palsy (PSP), a type of FTLD-tau. Further, in FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

INTRODUCTION: Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that share clinical and neuropathological features. Furthermore, the most common genetic cause of both FTD and ALS is a GGGGCC (G4C2) repeat expansion in the C9orf72 gene. This repeat expansion leads to several abnormalities, including C9orf72 haploinsufficiency, the accumulation of repeat RNA, and the production of five aggregation-prone proteins composed of repeating dipeptides. However, the contribution of these abnormalities to disease pathogenesis remains unresolved. RATIONALE: Among the five dipeptide repeat proteins nonconventionally translated from expanded G4C2 repeats, proline-arginine (PR) repeat proteins [poly(PR) proteins] have proven especially toxic in various model systems. Their involvement in C9orf72-associated FTD and ALS (c9FTD/ALS) has nevertheless been questioned because poly(PR) pathology is relatively infrequent in c9FTD/ALS patient brains. Postmortem tissues, however, represent end-stage disease and do not necessarily reflect early events in the disease process. Therefore, we generated mice that express poly(PR) in the brain to evaluate the temporal consequences of its expression in a mammalian in vivo model. More specifically, we engineered mice to express green fluorescent protein (GFP)–conjugated (PR)50 (a 50-repeat PR protein) or GFP via intracerebroventricular administration of adeno-associated viral vectors and then performed behavioral, pathological, and transcriptomic characterizations of poly(PR) mice in comparison with control GFP mice. RESULTS: We found that ~60% of poly(PR)- expressing mice died by 4 weeks of age and had significantly decreased brain and body weights at death compared with age-matched GFP control mice. Poly(PR) mice that escaped premature death developedmotor andmemory impairments, likely as a consequence of their progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis. In investigating the mechanisms by which poly(PR) caused neurodegeneration and functional deficits, we found that poly(PR) localized to heterochromatin (highly condensed regions of transcriptionally silent chromatin) and caused abnormal histone H3 methylation, features that we also detected in brain tissues from patients with c9FTD/ALS. Additionally, we observed aberrations in nuclear lamins and heterochromatin protein 1α (HP1α), key proteins thatmaintain heterochromatin structure and regulate gene silencing. Nuclear lamina invaginations and decreased HP1a protein expression were seen in poly(PR)-positive cells in poly(PR) mice, and in vitro studies demonstrated that poly(PR) disrupted HP1α liquid phases. Because poly(PR)-induced histone H3 posttranslational modifications, lamin invaginations, and decreased HP1α levels could profoundly affect gene expression, we compared transcriptome profiles between control and poly(PR) mice. As well as analyzing differentially expressed genes, we examined repetitive element expression given that repetitive...

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G4C2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G4C2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G4C2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G4C2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G4C2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G4C2 RNA and downstream G4C2 RNA–mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G4C2 RNA–based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention.

Significant transcriptome alterations are detected in the brain of patients with amyotrophic lateral sclerosis (ALS), including carriers of the C9orf72 repeat expansion and C9orf72-negative sporadic cases. Recently, the expression of repetitive element transcripts has been associated with toxicity and, while increased repetitive element expression has been observed in several neurodegenerative diseases, little is known about their contribution to ALS. To assess whether aberrant expression of repetitive element sequences are observed in ALS, we analysed RNA sequencing data from C9orf72-positive and sporadic ALS cases, as well as healthy controls. Transcripts from multiple classes and subclasses of repetitive elements (LINEs, endogenous retroviruses, DNA transposons, simple repeats, etc.) were significantly increased in the frontal cortex of C9orf72 ALS patients. A large collection of patient samples, representing both C9orf72 positive and negative ALS, ALS/FTLD, and FTLD cases, was used to validate the levels of several repetitive element transcripts. These analyses confirmed that repetitive element expression was significantly increased in C9orf72-positive compared to C9orf72-negative or control cases. While previous studies suggest an important link between TDP-43 and repetitive element biology, our data indicate that TDP-43 pathology alone is insufficient to account for the observed changes in repetitive elements in ALS/FTLD. Instead, we found that repetitive element expression positively correlated with RNA polymerase II activity in postmortem brain, and pharmacologic modulation of RNA polymerase II activity altered repetitive element expression in vitro. We conclude that increased RNA polymerase II activity in ALS/FTLD may lead to increased repetitive element transcript expression, a novel pathological feature of ALS/FTLD.