Approximately 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and Ϸ20% of these cases of familial ALS (FALS) are caused by mutations of copper͞zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptide N-acetylaspartylglutamate to glutamate, could protect MNs in an in vitro and animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.A myotrophic lateral sclerosis (ALS) is a progressive and fatal degeneration of motor neurons (MNs) in the spinal cord and cerebral cortex. About 10% of ALS cases are inherited in an autosomal dominant fashion, and Ϸ20% of these cases of familial ALS (FALS) are caused by a mutation in copper͞zinc superoxide dismutase type 1 (SOD1) (1). Mice and rats that carry mutant (MT) SOD1 as a transgene manifest a progressive MN degeneration similar to that in patients with ALS (2-4). Several lines of evidence suggest that glutamate excitotoxicity is a pathogenic mechanism in both sporadic .N-acetylaspartylglutamate (NAAG) is one of the most abundant peptides in the mammalian central and peripheral nervous system (14), is present in neuronal vesicles, is released from neurons in a calcium-dependent manner, and functions as a high-affinity agonist at the group II metabotropic glutamate receptor subtype 3 (mGluR3). Activation of mGluR3 by NAAG has been shown to inhibit glutamate release (15), increase release of transforming growth factor ␤ (TGF␤) from glial cells, and provide neuroprotection (16). NAAG is hydrolyzed to N-acetylaspartate and glutamate by glutamate carboxypeptidase II (GCPII) (EC 3.4.17.21; also termed N-acetylated-␣-linked acidic dipeptidase or NAALADase; ref. 17), an enzyme localized on the plasma membrane of glial cells with its catalytic region facing the synapse (18). Therefore, inhibition of GCPII would be expected to provide neuroprotection by means of both decreasing glutamate and increasing NAAG (19).We hypothesized that GCPII inhibition would protect MNs expressing MT SOD1 from cell death, as well as ameliorate the MN degeneration seen in FALS transgenic mouse. For in vitro studies, we used 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a potent and selective GCPII inhibitor (K i ϭ 0.2 nM; ref. 20) that has been shown to selectively reduce ischemic glutamate and provide neuroprotection in cell culture and animal models of ischemia (19), diabetic neuropathy (21), and drug abuse (22, 23). For the animal studies, we used a recently discovered thiol-based GCP II inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA) (24). Unlike 2-PMPA, 2-MPPA is o...

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Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease and this system serves as a relevant infectious model for human multiple sclerosis. It was previously shown that beta2M-deficient C57BL/6 mice lacking functional CD8+ T cells display increased viral persistence and enhanced susceptibility to TMEV-induced demyelination, and yet the majority of mice are free of clinical signs. To understand the mechanisms involved in this general resistance of C57BL/6 mice in the absence of CTL responses, mice (muMT) deficient in the B-cell compartment lacking membrane IgM molecules were treated with anti-CD8 antibody and then infected with TMEV. Although little difference in the proliferative responses of peripheral T cells to UV-inactivated TMEV and the resistance to demyelinating disease was observed between virus-infected muMT and control B6 mice, the levels of CD4(+) T cells were higher in the CNS of muMT mice. However, after treatment with anti-CD8 antibody, 100% of the mice displayed clinical gray matter disease and prolonged viral persistence in muMT mice, while only 10% of B6 mice showed clinical symptoms and very low viral persistence. Transfusion of sera from TMEV-infected B6 mice into anti-CD8 antibody-treated muMT mice partially restored resistance to virus-induced encephalitis. These results indicate that the early anti-viral antibody response is also important in the protection from TMEV-induced encephalitis particularly in the absence of CD8+ T cells.

The butyrate-induced responses of the U138B cell line provide a convenient model system for studying the molecular events accompanying the differentiation of glioblastoma cells. Multiparameter flow cytometry is a useful technique for characterizing such differentiation.