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Aberrant hexanucleotide repeat expansions in C9orf72are the most common genetic change underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). RNA transcripts containing these expansions undergo repeat-associated non-ATG translation (RAN-T) to form five dipeptide repeat proteins (DPRs). DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients, and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Using different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and exosome-independent pathways, which may be relevant to disease.

Little is known about global gene expression patterns in the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). To address this, we used high-density oligonucleotide microarray technology to compare expression levels of ∼6,800 genes in postmortem spinal cord gray matter obtained from individuals with ALS as well as normal individuals. Using Fisher discriminant analysis (FDA) and leave-one-out cross-validation (LOOCV), we discerned an ALS-specific signature. Moreover, it was possible to distinguish familial ALS (FALS) from sporadic ALS (SALS) gene expression profiles. Characterization of the specific genes significantly altered in ALS uncovered a pro-inflammatory terminal state. Moreover, we found alterations in genes involved in mitochondrial function, oxidative stress, excitotoxicity, apoptosis, cytoskeletal architecture, RNA transcription and translation, proteasomal function, and growth and signaling. It is apparent from this study that DNA microarray analysis and appropriate bioinformatics can reveal distinct phenotypic changes that underlie the terminal stages of neurodegeneration in ALS.

We have investigated the functional impact of a naturally occurring mutation of the human glutamate transporter GLT1 (EAAT2), which had been detected in a patient with sporadic amyotrophic lateral sclerosis. The mutation involves a substitution of the putative N-linked glycosylation site asparagine 206 by a serine residue (N206S) and results in reduced glycosylation of the transporter and decreased uptake activity. Electrophysiological analysis of N206S revealed a pronounced reduction in transport rate compared with wild-type, but there was no alteration in the apparent affinities for glutamate and sodium. In addition, no change in the sensitivity for the specific transport inhibitor dihydrokainate was observed. However, the decreased rate of transport was associated with a reduction of the N206S transporter in the plasma membrane. Under ionic conditions, which favor the reverse operation mode of the transporter, N206S exhibited an increased reverse transport capacity. Furthermore, if coexpressed in the same cell, N206S manifested a dominant negative effect on the wild-type GLT1 activity, whereas it did not affect wild-type EAAC1. These findings provide evidence for a role of the N-linked glycosylation in both cellular trafficking and transport function. The resulting alteration in glutamate clearance capacity likely contributes to excitotoxicity that participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O⨪2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O⨪2levels appears to activate or modulate cell death, as a recombinant SV expressing the O⨪2scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O⨪2rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O⨪2by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.

The phosphorylation state of two identified neural-specific protein kinase C substrates (the presynaptic protein B-50 and the postsynaptic protein neurogranin) was monitored after the induction of long term potentiation in the CA1 field of rat hippocampus slices by quantitative immunoprecipitation following 32Pilabeling in the recording chamber. B-50 phosphorylation was increased from 10 to 60 min, but no longer at 90 min after long term potentiation had been induced, neurogranin phosphorylation only at 60 min. Increased phosphorylation was not found when long term potentiation was blocked with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate, when only low frequency stimulation was applied or tetanic stimulation failed to induce long term potentiation. Our data show that both B-50 and neurogranin phosphorylation are increased following the induction of long term potentiation, thus providing strong evidence for pre- and postsynaptic protein kinase C activation during narrow, partially overlapping, time windows after the induction of long term potentiation.