Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activityinduced neurodegeneration and injury-induced axonal degeneration 1,2 . Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heatshock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions. Injury-induced axonal degeneration is dramatically delayed in wallerian degeneration slow (Wld S ) mice, a mutant strain that over-expresses a chimaeric protein containing the NAD synthase NMNAT 3,4 . The Wld S chimaeric protein offers neuroprotection against axonal degeneration 2,5-7 as well as a variety of neurodegenerative conditions [8][9][10][11] . Wld S protein contains the amino (N)-terminal 70-amino-acid fragment of ubiquitination factor E4B ©2008 Nature Publishing GroupCorrespondence and requests for materials should be addressed to R.G. 4,14,15 . Drosophila contains only one NMNAT gene, whose overexpression delays axonal degeneration 2 . This study and our finding that NMNAT functions as a maintenance factor to protect against activity-induced neurodegeneration 1 suggest that NMNAT alone can protect against multiple neurodegenerative insults. Our recent finding that enzymatically inactive NMNAT retains neuroprotective capabilities also exposed a hitherto unknown molecular function 1 .To test if NMNAT is a general factor required for neuronal maintenance and protection, we first examined the effects of NMNAT overexpression in a Drosophila model for SCA1. Overexpression of wild-type NMNAT or enzyme-inactive NMNAT (NMNAT-WR) 1 suppresses the degenerative phenotypes induced by overexpression of Drosophila ataxin-1 (dAtx-1). It also offers moderate protection against the severe phenotypes caused by overexpression of human ataxin-1 with an expanded (82) poly-glutamine tract (hAtx-1[82Q]) 16 (Fig. 1). These findings, and the observations that NMNAT protects from axonal injury 2 , from photoreceptor injury caused by intense light, and that its loss c...

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2a, 4E and 3g, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia. Keywords: brain ischemia, chaperone, heat-shock cognate protein 70, protein aggregation, protein synthesis, ribosomal proteins, stress granule, ubiquitin. Although substantial progress has been made, cellular and molecular mechanisms underlying focal ischemic neuronal damage are still incompletely understood. Focal brain ischemia leads to acute cell death in the core area and a slow type of neuronal death in the penumbra that can begin hours after ischemia and continue to mature for days (Siesjo et al. 1995;Lee et al. 2002;Wang et al. 2004). During this maturation period, protein synthesis is persistently inhibited in these penumbral neurons destined to die (Mies et al. 1991;Hossmann 1993;Kokubo et al. 2003). Activation of PKRlike ER eIF-2a kinase (PERK) may be responsible, at least in part, for the increase in eukaryotic initiation factor 2a (eIF2a) phosphorylation and for the transient suppression of protein synthesis early in reperfusion after transient brain ischemia. However, the nature of the transient change in phosphorylation of PERK and eIF2a may suggest that eIF2a phosphorylation cannot account for irreversible inhibition of protein synthesis after brain ischemia (Hu and Wieloch 1993;Burda et al. 1994;Althausen et al. 2001;DeGracia 2004;Owen et al. 2005).Cellular proteins in non-native states, i.e. those newly synthesized, misfo...

Transient cerebral ischemia leads to irreversible translational inhibition which has been considered as a hallmark of delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether irreversible translational inhibition is due to abnormal aggregation of translational complex, i.e. the ribosomes and their associated nascent polypeptides, initiation factors, translational chaperones and degradation enzymes after ischemia. Translational complex aggregation was studied by electron microscopy, as well as by biochemical analyses. A duration of 15 or 20 min of cerebral ischemia induced severe translational complex aggregation starting from 30 min of reperfusion and lasting until the onset of delayed neuronal death at 48 h of reperfusion. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons. After ischemia, most ribosomes were clumped into large abnormal aggregates in neurons destined to die. Translational complex components consisting of small ribosomal subunit protein 6, large subunit protein 28, eukaryotic initiation factor-3eta, co-translational chaperone heat shock cognate protein 70 and co-chaperone HSP40-Hdj1, as well as co-translational ubiquitin ligase c-terminus of hsp70-interacting protein were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational components were also highly ubiquitinated. To our knowledge, irreversible aggregation of translational components has not been reported after brain ischemia. This study clearly indicates that ischemia damages co-translational chaperone and degradation machinery, resulting in irreversible destruction of protein synthesis machinery by protein aggregation after ischemia.

Charcot-Marie-Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre-clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock-in mouse model expressing Mfn2(R94W), which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2(R94W) heterozygous mice show histopathology and age-dependent open-field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations.

Background and Purpose Over-assembly of synaptic glutamate receptors leads to excitotoxicity. The goal of this study is to investigate phosphorylation and assembly of AMPA and NMDA receptors after brain ischemia with reperfusion (I/R). Methods Rats were subjected to 15 min of global ischemia followed by 0.5, 4, and 24 h of reperfusion. Phosphotyrosine (Ptyr) peptides of glutamate receptors in synaptosomal fraction after I/R were identified and quantified by state-of-the-art immuno-affinity purification of Ptyr peptides followed by LC-MS/MS analysis (IAP-LC/MS/MS). Glutamate receptor phosphorylation and synaptic assembly after I/R were studied by biochemical methods. Results Numerous Ptyr sites of AMPA and NMDA were upregulated by about 2- to 37-fold after I/R. A core glutamate receptor kinase, Src kinase, was significantly activated. GluR2/3 and NR2A/B were rapidly clustered from extrasynaptic to synaptic membrane fractions after I/R. GluR2/3 was then translocated into the intracellular pool, whereas NR2A/B remained in the synaptic fraction for as long as 24 h. Consistently, trafficking-related phosphorylation of GluR2/3-S880 was significantly but transiently upregulated, whereas NR2A/B-Y1246 and -Y1472 were significantly and persistently upregulated after I/R. Conclusions Phosphorylation of glutamate receptors at synapses may lead to over-assembly of glutamate receptors, probably via activation of Src family kinases, after I/R. This study provides “global” proteomic information about glutamate receptor tyrosine phosphorylation after brain ischemia.