Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis.

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.

The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo.

Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-4310-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43's extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins.

Neuronal inclusions of poly(GA), a protein unconventionally translated from G4C2 repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology.

Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits.

The major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis is a G4C2 repeat expansion in C9ORF72. Efforts to combat neurodegeneration associated with "c9FTD/ALS" are hindered by a lack of animal models recapitulating disease features. We developed a mouse model to mimic both neuropathological and clinical c9FTD/ALS phenotypes. We expressed (G4C2)66 throughout the murine central nervous system by means of somatic brain transgenesis mediated by adeno-associated virus. Brains of 6-month-old mice contained nuclear RNA foci, inclusions of poly(Gly-Pro), poly(Gly-Ala), and poly(Gly-Arg) dipeptide repeat proteins, as well as TDP-43 pathology. These mouse brains also exhibited cortical neuron and cerebellar Purkinje cell loss, astrogliosis, and decreased weight. (G4C2)66 mice also developed behavioral abnormalities similar to clinical symptoms of c9FTD/ALS patients, including hyperactivity, anxiety, antisocial behavior, and motor deficits.