Targeting the glycine-rich domain of TDP-43 with antibodies prevents its aggregation in vitro and reduces neurofilament levels in vivo.

Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1ß, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.

Drug screen in iPSC-Neurons identifies nucleoside analogs as inhibitors of (G4C2)n expression in C9orf72 ALS/FTD.

An intronic (G4C2)n expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G4C2)n foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders.

Gel-like inclusions of C-terminal fragments of TDP-43 sequester stalled proteasomes in neurons.

Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-43 mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD.

Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS.

Expansion of a (G4C2)n repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.

Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD.

The C9orf72 repeat expansion causes amyotrophic lateral sclerosis and frontotemporal dementia, but the poor correlation between C9orf72-specific pathology and TDP-43 pathology linked to neurodegeneration hinders targeted therapeutic development. Here, we addressed the role of the aggregating dipeptide repeat proteins resulting from unconventional translation of the repeat in all reading frames. Poly-GA promoted cytoplasmic mislocalization and aggregation of TDP-43 non-cell-autonomously, and anti-GA antibodies ameliorated TDP-43 mislocalization in both donor and receiver cells. Cell-to-cell transmission of poly-GA inhibited proteasome function in neighboring cells. Importantly, proteasome inhibition led to the accumulation of TDP-43 ubiquitinated within the nuclear localization signal (NLS) at lysine 95. Mutagenesis of this ubiquitination site completely blocked poly-GA-dependent mislocalization of TDP-43. Boosting proteasome function with rolipram reduced both poly-GA and TDP-43 aggregation. Our data from cell lines, primary neurons, transgenic mice, and patient tissue suggest that poly-GA promotes TDP-43 aggregation by inhibiting the proteasome cell-autonomously and non-cell-autonomously, which can be prevented by inhibiting poly-GA transmission with antibodies or boosting proteasome activity with rolipram.

Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model.

The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non-canonical translation of the expanded repeat results in abundant poly-GA inclusion pathology throughout the CNS. (GA)149 -CFP expression in mice triggers motor deficits and neuroinflammation. Since poly-GA is transmitted between cells, we investigated the therapeutic potential of anti-GA antibodies by vaccinating (GA)149 -CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA)10 conjugates and pre-aggregated carrier-free (GA)15 . Only ovalbumin-(GA)10 immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA)10 immunization largely rescued the motor function in (GA)149 -CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA)10 -immunized poly-GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP-43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers.

Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity.

Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)149 and nucleolar (PR)175-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Overexpressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule-like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomes may chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis.

Antibodies inhibit transmission and aggregation of C9orf72 poly-GA dipeptide repeat proteins.

Cell-to-cell transmission of protein aggregates is an emerging theme in neurodegenerative disease. Here, we analyze the dipeptide repeat (DPR) proteins that form neuronal inclusions in patients with hexanucleotide repeat expansion C9orf72, the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Sense and antisense transcripts of the (G4C2)n repeat are translated by repeat-associated non-ATG (RAN) translation in all reading frames into five aggregating DPR proteins. We show that the hydrophobic DPR proteins poly-GA, poly-GP, and poly-PA are transmitted between cells using co-culture assays and cell extracts. Moreover, uptake or expression of poly-GA induces nuclear RNA foci in (G4C2)80-expressing cells and patient fibroblasts, suggesting an unexpected positive feedback loop. Exposure to recombinant poly-GA and cerebellar extracts of C9orf72 patients increases repeat RNA levels and seeds aggregation of all DPR proteins in receiver cells expressing (G4C2)80 Treatment with anti-GA antibodies inhibits intracellular poly-GA aggregation and blocks the seeding activity of C9orf72 brain extracts. Poly-GA-directed immunotherapy may thus reduce DPR aggregation and disease progression in C9orf72 ALS/FTD.