Amyotrophic Lateral Sclerosis (ALS) is a fatal motor neuron (MN) disease with astrocytes implicated as a significant contributor to MN death in familial ALS (fALS)1–5. However, these conclusions, in part, derive from rodent models of fALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene which account for less than 2% of all ALS cases2, 4, 5. Here, we generated astrocytes from post-mortem tissue from both fALS and sporadic ALS (sALS) patients, and show that astrocytes derived from both patient groups are similarly toxic to MNs. In addition, we show that SOD1 is a viable target for sALS, as its knockdown significantly attenuates astrocyte-mediated toxicity towards MNs. Our data highlight astrocytes as a non-cell autonomous component in sALS and provide the first in vitro model system to investigate common disease mechanisms and evaluate potential therapies for sALS and fALS.

Carbohydrates have been shown to mediate or modulate a number of important events in the development of the nervous system; however, there is little evidence that they participate directly in the development of synapses. One carbohydrate structure that is likely to be important in synaptic development of the neuromuscular junction is the CT carbohydrate antigen [GalNAcbeta1,4[NeuAcalpha2,3]Galbeta1(-3GalNAc or -4GlcNAc)]. The synaptic localization of the CT antigen is due to the presence of the terminal beta1,4 GalNAc linkage, and such linkages are localized to the neuromuscular junction in many species. Here we show that an enzyme that can create the synaptic CT structure, the CT GalNAc transferase, is also confined to the neuromuscular junction in mice. Using transgenic mice, we show that overexpression of the CT GalNAc transferase in extrasynaptic regions in skeletal myofibers caused as much as a 60% reduction in the diameter of adult myofibers and an order of magnitude increase in satellite cells. Neuromuscular junctions of transgenic mice had severely reduced numbers of secondary folds, Schwann cell processes were present in the synaptic cleft, and secondary folds were often misaligned with active zones. In addition, multiple presynaptic specializations occurred on individual myofibers. In addition, some normally synaptic proteins, including laminin alpha4, laminin alpha5, utrophin, and NCAM, were expressed along extrasynaptic regions of myofibers. One of the muscle proteins that displayed increased glycosylation with the CT antigen in the transgenic mice was alpha-dystroglycan. These experiments provide the first in vivo evidence that a synaptic carbohydrate antigen has important roles in the development of the neuromuscular synapse and suggest that the CT antigen is involved in controlling the expression of synaptic molecules.

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Comprehensive testing of the CFTR, PRSS1, and SPINK1 genes identified genetic variants in nearly half of all subjects considered by their physicians as candidates for genetic testing. Comprehensive test identified numerous novel variants that would not be identified by standard clinical screening panels.

Cystic fibrosis (CF) is one of the most frequently diagnosed autosomal-recessive diseases in the Caucasian population. For general-population CF carrier screening, the American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists (ACOG) have recommended a core panel of 23 mutations that will identify 49 -98% of carriers, depending on ethnic background. Using a genotyping technology that can rapidly identify disease-causing mutations is important for high-throughput generalpopulation carrier screening, confirming clinical diagnosis, determining treatment options, and prenatal diagnosis. Here, we describe a proof-of-concept study to determine whether the Ion Torrent Personal Genome Machine (PGM) sequencer platform can reliably identify all ACMG/ACOG 23 CF transmembrane conductance regulator (CFTR) mutations. A WT CF specimen along with mutant DNA specimens representing all 23 CFTR mutations were sequenced bidirectionally on the Ion Torrent 314 chip to determine the accuracy of the PGM for CFTR variant detection. We were able to reliably identify all of the targeted mutations except for 2184delA, which lies in a difficult, 7-mer homopolymer tract. Based on our study, we believe PGM sequencing may be a suitable technology for identifying CFTR mutations in the future. However, as a result of the elevated rate of base-calling errors within homopolymer stretches, mutations within such regions currently need to be evaluated carefully using an alternative method.

The deposition of amyloid in senile plaques and along the walls of the cerebral vasculature is a characteristic feature of Alzheimer disease. The peptide comprising the carboxyl-terminal 100 amino acids of the P.amyloid precursor protein (.APP) has been shown to aggregate into amyloid-like fibrils in vito and to be neurotoxic, suggesting that this fragment may play a role in the etiology of Alzhelmer disease.To address this question, we expressed this carboxyl-terminal 100-amino acid peptide of lAPP in transgenic mice under the control of the brain dystrophin promoter. We used an antibody to the principal component of amyloid, p/A4, to demonstrate cell-body and neuropil accumulation of ,/A4 immunoreactivity in the brains of 4-and 6-month-old transgenic mice. Only light cytoplasmic staining with this antibody was visible in control nice. In addition, immunocytochemical analysis of the brains with an antibody to the carboxyl terminus of lAPP revealed abnormal aggregation of this epitope of (IAPP within vesicular structures in the cytoplasm and in abnormalappearing neurites in the CA2/3 region of the hippocampus in transgenic mice, similar to its aggregation in the cells of Alzheimer disease brains. Thiofavin S histochemistry suggested accumulations of amyloid in the cerebrovasculature of transgenic mice with the highest expression of the PAPP-C100 transgene. These observations suggest that expression of abnormal carboxyl-terminal subfragments of JIAPP in vivo may cause amyloidogenesis and specific neuropathology.Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory and declining cognitive function beginning in late life. A prominent feature of AD neuropathology is the deposition of amyloid in senile plaques and along the walls of the cerebrovasculature. The peptide fragment termed 3/A4 (39-43 amino acids; refs. 1 and 2) is the principal constituent of the amyloid deposits, although plaques contain numerous other components. The ,B-amyloid protein precursor (SAPP), from which 8/A4 is derived, is a transmembrane protein in which the /A4 peptide spans the border between the extracellular domain and the transmembrane region (3, 4). Normal cleavage of ,BAPP in the secretory pathway occurs at the 8/A4 Lys16-Leu17 peptide bond (5). Recent data, however, have revealed that multiple 8/A4-containing carboxyl-terminal fragments of 8APP are also produced in the brain, via the endosomallysosomal system (6).Additional evidence has implicated at least one of these fragments, the carboxyl-terminal 100 amino acids ofBAPP, in the development of AD neuropathology. This fragment, which spans the P/A4 and cytoplasmic domains, is amyloidogenic (7-9) and neurotoxic both in vitro (10, 11) and in vivo (12). To test the hypothesis that this neurotoxic fragment may play a role both in amyloidogenesis and in the development of the progressive neuropathology of AD, we introduced into mice a transgene carrying the sequence for the carboxyl-terminal 104 amino acids of (3APP (jAPP-C104) under ...

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