Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin ß1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

Misfolded SOD1 is not a primary component of sporadic ALS.

A common feature of inherited and sporadic ALS is accumulation of abnormal proteinaceous inclusions in motor neurons and glia. SOD1 is the major protein component accumulating in patients with SOD1 mutations, as well as in mutant SOD1 mouse models. ALS-linked mutations of SOD1 have been shown to increase its propensity to misfold and/or aggregate. Antibodies specific for monomeric or misfolded SOD1 have detected misfolded SOD1 accumulating predominantly in spinal cord motor neurons of ALS patients with SOD1 mutations. We now use seven different conformationally sensitive antibodies to misfolded human SOD1 (including novel high affinity antibodies currently in pre-clinical development) coupled with immunohistochemistry, immunofluorescence and immunoprecipitation to test for the presence of misfolded SOD1 in high quality human autopsy samples. Whereas misfolded SOD1 is readily detectable in samples from patients with SOD1 mutations, it is below detection limits for all of our measures in spinal cord and cortex tissues from patients with sporadic or non-SOD1 inherited ALS. The absence of evidence for accumulated misfolded SOD1 supports a conclusion that SOD1 misfolding is not a primary component of sporadic ALS.

Beta Frequency Oscillations in the Subthalamic Nucleus Are Not Sufficient for the Development of Symptoms of Parkinsonian Bradykinesia/Akinesia in Rats.

Substantial correlative evidence links the synchronized, oscillatory patterns of neural activity that emerge in Parkinson's disease (PD) in the beta (ß) frequency range (13-30 Hz) with bradykinesia in PD. However, conflicting evidence exists, and whether these changes in neural activity are causal of motor symptoms in PD remains unclear. We tested the hypothesis that the synchronized ß oscillations that emerge in PD are causal of symptoms of bradykinesia/akinesia. We designed patterns of stimulation that mimicked the temporal characteristics of single unit ß bursting activity seen in PD animals and humans. We applied these ß-patterned stimulation patterns along with continuous low-frequency and high-frequency controls to the subthalamic nucleus (STN) of intact and 6-OHDA-lesioned female Long-Evans and Sprague-Dawley rats. ß-Patterned paradigms were superior to low-frequency controls at induction of ß power in downstream substantia nigra reticulata (SNr) neurons and in ipsilateral motor cortex. However, we did not detect deleterious effects on motor performance across a wide battery of validated behavioral tasks. Our results suggest that ß frequency oscillations (BFOs) may not be sufficient for the generation of bradykinesia/akinesia in PD.

Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs.

Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.

Neuropathology of Amyotrophic Lateral Sclerosis and Its Variants.

The neuropathologic molecular signature common to almost all sporadic amyotrophic lateral sclerosis (ALS) and most familial ALS is TDP-43 immunoreactive neuronal cytoplasmic inclusions. The neuropathologic and molecular neuropathologic features of ALS variants, primarily lateral sclerosis and progressive muscular atrophy, are less certain but also seem to share the primary features of ALS. Genetic causes, including mutations in SOD1, TDP-43, FUS, and C9orf72, all have distinctive molecular neuropathologic signatures. Neuropathology will continue to play an increasingly key role in solving the puzzle of ALS pathogenesis.