GCP II inhibition rescues neurons from gp120IIIB-induced neurotoxicity.

Excessive glutamate neurotransmission has been implicated in neuronal injury in many disorders of the central nervous system (CNS), including human immunodeficiency virus (HIV)-associated dementia. Gp120IIIB is a strain of a HIV glycoprotein with specificity for the CXCR4 receptor that induces neuronal apoptosis in in vitro models of acquired immunodeficiency syndrome (AIDS)-induced neurodegeneration. Since the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) by glutamate carboxypeptidase (GCP) II increases cellular glutamate, an event associated with excitotoxicity, we hypothesized that inhibition of GCP II may prevent gp120IIIB-induced cell death. Furthermore, through GCP II inhibition, increased NAAG may be neuroprotective via its agonist effects at the mGlu(3) receptor. To ascertain the therapeutic potential of GCP II inhibitors, embryonic day 17 hippocampal cultures were exposed to gp120IIIB in the presence of a potent and highly selective GCP II inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). 2-PMPA was found to abrogate gp120IIIB-induced toxicity in a dose-dependent manner. Additionally, 2-PMPA was neuroprotective when applied up to 2 h after the application of gp120IIIB. The abrogation of apoptosis by 2-PMPA was reversed with administration of mGlu(3) receptor antagonists and with antibodies to transforming growth factor (TGF)-beta. Further, consistent with the localization of GCP II, 2-PMPA failed to provide neuroprotection in the absence of glia. GCP II activity and its inhibition by 2-PMPA were confirmed in the hippocampal cultures using radiolabeled NAAG and high-performance liquid chromatography (HPLC) analysis. Taken together, these data suggest that GCP II is involved in mediating gp120-induced apoptosis in hippocampal neurons and GCP II inhibitors may have potential in the treatment of neuronal injury related to AIDS.

Activation of c-Jun N-terminal kinase mediates gp120IIIB- and nucleoside analogue-induced sensory neuron toxicity.

Peripheral neuropathy is the most common neurological symptom in patients with acquired immunodeficiency syndrome (AIDS). Here, we examine possible mechanisms of gp120 and nucleoside reverse transcriptase inhibitors (NRTIs) in the pathogenesis of AIDS peripheral neuropathy. Neonatal dorsal root ganglion (DRG) neurons were found to undergo apoptosis in response to chronic treatment with gp120IIIB, an effect enhanced by the co-application of hCD4, as well as upon exposure to the nucleoside reverse transcriptase inhibitor (NRTI), 2',3'-dideoxyinosine (ddI). DRG neurons were rescued from the neurotoxic effects of these agents by CEP-1347, an inhibitor of the mixed lineage kinases (MLKs), upstream activators of the c-Jun N-terminal kinase (JNK) signaling pathway. In addition, gp120- or ddI-mediated toxicity were also inhibited by neuronal expression of dominant negative versions of the MLKs. Our results suggest that both gp120 and the NRTIs cause sensory neuron apoptosis through the activation of the JNK pathway, and that CEP-1347-like compounds may serve as a therapeutic option in patients with AIDS-associated peripheral neuropathy.

CD4 dependence of gp120IIIB-CXCR4 interaction is cell-type specific.

The HIV-1 envelope protein gp120IIIB is selective for the CXCR4 chemokine receptor and has been shown to induce apoptosis in neurons both in vivo and in vitro. We examined the ability of gp120IIIB to signal through the rat CXCR4 (rCXCR4) receptor and its dependence on the presence of the human CD4 (hCD4) protein in a number of cell systems. SDF-1alpha potently inhibited N-type Ca channels in cultured HEK293 cells expressing both the Ca channel subunits and rCXCR4 receptors. However, gp120IIIB was ineffective in producing either Ca channel inhibition or in blocking the effects of SDF-1alpha. However, when hCD4 was coexpressed with rCXCR4 and Ca channel subunits, gp120IIIB also produced Ca channel inhibition. Similarly, in PC12 cells transfected with the rCXCR4, SDF-1alpha produced mobilization of intracellular Ca, while gp120IIIB was only effective when hCD4 was coexpressed. SDF-1alpha induced endocytosis of Yellow Fluorescent Protein (YFP)-tagged rCXCR4 expressed in PC12 cells, as did gp120IIIB, an effect which was enhanced by hCD4 coexpression. When tagged rCXCR4 was expressed in F-11 cells or in rat DRG neurons, SDF-1alpha produced extensive receptor endocytosis. However, the ability of gp120IIIB to produce endocytosis was dependent on the coexpression of hCD4. Our results demonstrate that the degree of hCD4 dependence of the agonist effects of gp120IIIB at the rCXCR4 receptor is cell-type specific.

Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis models.

Approximately 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and approximately 20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc 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.

Mixed lineage kinase 3 mediates gp120IIIB-induced neurotoxicity.

Overexpression of gp120, the major coat protein of the HIV-1 virus, in central glial cells, or treatment of neurons with gp120 in culture, produces apoptotic neuronal death. Here we demonstrate that CEP-1347 (KT7515), an inhibitor of mixed lineage kinase 3 (MLK3), an upstream activator of JNK, inhibits gp120IIIB-induced apoptosis of hippocampal neurons. Furthermore, expression of wild type MLK3 in hippocampal pyramidal neurons enhanced gp120IIIB-induced neurotoxicity, whereas expression of a dominant negative MLK3 protected neurons from the toxic effects of the glycoprotein. These results indicate a role for MLK3 signaling in gp120IIIB-induced neuronal death, and suggest potential clinical utility of CEP-1347 in inhibiting the progression of AIDS dementia.