Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1 G93A rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1 G93A rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1 G93A astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1 G93A astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1 G93A rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1 G93A astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1 G93A astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (ϳ10 nM) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1 G93A astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75 NTR ) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75 NTR was inhibited with blocking antibodies.In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75 NTR +/+ mouse embryos but had no effect in p75 NTR -/-knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75 NTR blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75 NTR -dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75 NTR neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75 NTR -dependent mechanism.

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Velo-cardio-facial syndrome/DiGeorge syndrome/22q11.2 deletion syndrome (22q11.2DS) is caused by meiotic non-allelic homologous recombination events between flanking low copy repeats termed LCR22A and LCR22D, resulting in a 3 million base pair (Mb) deletion. Due to their complex structure, large size and high sequence identity, genetic variation within LCR22s among different individuals has not been well characterized. In this study, we sequenced 13 BAC clones derived from LCR22A/D and aligned them with 15 previously available BAC sequences to create a new genetic variation map. The thousands of variants identified by this analysis were not uniformly distributed in the two LCR22s. Moreover, shared single nucleotide variants between LCR22A and LCR22D were enriched in the Breakpoint Cluster Region pseudogene (BCRP) block, suggesting the existence of a possible recombination hotspot there. Interestingly, breakpoints for atypical 22q11.2 rearrangements have previously been located to BCRPs To further explore this finding, we carried out in-depth analyses of whole genome sequence (WGS) data from two unrelated probands harbouring a de novo 3Mb 22q11.2 deletion and their normal parents. By focusing primarily on WGS reads uniquely mapped to LCR22A, using the variation map from our BAC analysis to help resolve allele ambiguity, and by performing PCR analysis, we infer that the deletion breakpoints were most likely located near or within the BCRP module. In summary, we found a high degree of sequence variation in LCR22A and LCR22D and a potential recombination breakpoint near or within the BCRP block, providing a starting point for future breakpoint mapping using additional trios.