One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function. In contrast to this postulate, we have established previously that a heterogeneous population of mRNAs and biologically active polyribosomes exist in the giant axon and presynaptic nerve terminals of the photoreceptor neurons in squid. We report that these mRNA populations contain mRNAs for nuclear-encoded mitochondrial proteins to include: cytochrome oxidase subunit 17, propionyl-CoA carboxylase (EC 6.4.1.3), dihydrolipoamide dehydrogenase (EC 1.8.1.4), and coenzyme Q subunit 7. The mRNA for heat shock protein 70, a chaperone protein known to be involved in the import of proteins into mitochondria, has also been identified. Electrophoretic gel analysis of newly synthesized proteins in the synaptosomal fraction isolated from the squid optic lobe revealed that the large presynaptic terminals of the photoreceptor neuron contain a cytoplasmic protein synthetic system. Importantly, a significant amount of the cycloheximide resistant proteins locally synthesized in the terminal becomes associated with mitochondria. PCR analysis of RNA from synaptosomal polysomes establishes that COX17 and CoQ7 mRNAs are being actively translated. Taken together, these findings indicate that proteins required for the maintenance of mitochondrial function are synthesized locally in the presynaptic nerve terminal, and call attention to the intimacy of the relationship between the terminal and its energy generating system. J. Neurosci. Res. 64:447-453, 2001. Published 2001 Wiley-Liss, Inc.

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na؉ -dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS. Amyotrophic lateral sclerosis (ALS)4 is a fatal neurodegenerative disease, resulting from a progressive death of cortical and spinal motor neurons. About 90% of ALS cases are sporadic, whereas the remaining 10% are inherited in a dominant manner (familial ALS). Transgenic expression of high levels of human mutant SOD1 (mutSOD1) proteins in mice and rats leads to a progressive motor neuron disease that shares most of the clinical features of ALS (1, 2). Astrocytes are essential partners of motorneurons, providing them with trophic support (3) and mediating rapid clearance of synaptic glutamate mainly through the action of glial glutamate transporters. The EAAT2 (excitatory amino acid transporter 2) glial isoform of glutamate transporters accounts for ϳ95% of total glutamate uptake activity in the brain (4). Studies in mutSOD1 mice and in in vitro models of ALS affirmed a role for astroglial involvement in motorneuron death and suggested that motorneuron loss is also the result of toxic contributions of nonneuronal cells of the spinal cord (5-8). Reactive astrocytes surrounding motorneurons contain protein inclusions; express inflammatory markers, such as the inducible forms of nitric oxide synthase and cycloxygenase-2; and display increased neural growth factor synthesis (9), nitrotyrosine immunoreactivity, and down-regulation of EAAT2. Excitotoxicity caused by consistent reduction in expres...

Mutations in PARK7 DJ-1 have been associated with autosomal-recessive early-onset Parkinson's disease (PD). This gene encodes for an atypical peroxiredoxin-like peroxidase that may act as a regulator of transcription and a redox-dependent chaperone. Although large gene deletions have been associated with a loss-of-function phenotype, the pathogenic mechanism of several missense mutations is less clear. By performing a yeast two-hybrid screening from a human fetal brain library, we identified TRAF and TNF receptor-associated protein (TTRAP), an ubiquitin-binding domain-containing protein, as a novel DJ-1 interactor, which was able to bind the PD-associated mutations M26I and L166P more strongly than wild type. TTRAP protected neuroblastoma cells from apoptosis induced by proteasome impairment. In these conditions, endogenous TTRAP relocalized to a detergent-insoluble fraction and formed cytoplasmic aggresome-like structures. Interestingly, both DJ-1 mutants blocked the TTRAP protective activity unmasking a c-jun N-terminal kinase (JNK)-and p38-MAPK (mitogen-activated protein kinase)-mediated apoptosis. These results suggest an active role of DJ-1 missense mutants in the control of cell death and position TTRAP as a new player in the arena of neurodegeneration. Cell Death and Differentiation (2009) Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder, affecting 1-2% of all individuals above the age of 65 years. Its neuropathological hallmark is the selective degeneration of subsets of mesencephalic dopaminergic cells and the formation of proteinaceous cytoplasmic aggregates called Lewy bodies. 1 Several studies implicate the ubiquitin-proteasome system in PD pathogenesis. 2 Synthetic proteasome inhibitors preferentially affect catecholaminergic neurons, leading to cell death. Furthermore, key ubiquitin-proteasome system elements are altered in PD post-mortem brains. [3][4][5] The identification of genes (PARK1-14) associated with familial PD has provided crucial insights into the pathogenic mechanisms. 1 The ubiquitin ligase parkin (PARK2) and the ubiquitin C-terminal hydrolase-L1 (UCH-L1) (PARK5) have been implicated in the ubiquitin-proteasome system function. Interestingly, upon treatment with proteasome inhibitors, they formed insoluble aggregates and were recruited to a juxtanuclear aggresome-like inclusion that resembled the Lewy body. 6,7 Autosomal-recessive early-onset PD has been associated with mutations in PARK7/DJ-1. 8 Functional DJ-1 is a dimer that acts as an atypical peroxiredoxin-like peroxidase as well as a regulator of transcription and a chaperone. 9-12 Interestingly, ectopic DJ-1 expression protects cells from death induced by a variety of insults. 13 DJ-1 loss in humans causes PD. 14 DJ-1 knock-out (KO) mice and flies showed increased vulnerabilities to neurotoxic agents but no signs of dopaminergic cell death. [15][16][17] PD families may also present missense mutations of DJ-1 in homozygous (L166P, M26I and E64D) and heterozygous forms (A104T and D149A)....

It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.