The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.

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BackgroundOxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches.ResultsIn this report, using in vitro neuronal cultures, ex vivo organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between in vivo vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons.ConclusionLow energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons.

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-b (Ab) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Ab toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Ab peptides, inhibits Abinduced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Ab-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Ab-induced activation of the cytosolic cdk5-p25 complex, but not the membraneassociated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Ab toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Ab-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Ab toxicity and neurofibrillary pathology in AD and other tauopathies.

Deposition of ␤-amyloid peptide (A␤) and hyperphosphorylation of the protein are associated with neuronal dysfunction and cell death in Alzheimer's disease. Although the relationship between these two processes is not yet understood, studies have shown that both in vitro and in vivo exposure of neurons to A␤ leads to hyperphosphorylation and neuronal dystrophy. We previously reported that the microtubule-stabilizing drug paclitaxel (Taxol) protects primary neurons against toxicity induced by the A␤ [25][26][27][28][29][30][31][32][33][34][35] peptide. The studies in this report were undertaken to characterize the actions of paclitaxel more fully, to assess the effectiveness of structurally diverse microtubulestabilizing agents in protecting neurons, and to determine the time course of the protective effects of the drugs. Primary neurons were exposed to A␤ in the presence or absence of several agents shown to interact with microtubules, and neuronal survival was monitored. Paclitaxel protected neurons against A␤ 1-42 toxicity, and paclitaxel-treated cultures exposed to A␤ showed enhanced survival over A␤-only cultures for several days. Neuronal apoptosis induced by A␤ was blocked by paclitaxel. Other taxanes and three structurally diverse microtubule-stabilizing compounds also significantly increased survival of A␤-treated cultures. At concentrations below 100 nM, the drugs that protected the neurons did not produce detectable toxicity when added to the cultures alone. Although multiple mechanisms are likely to contribute to the neuronal cell death induced by oligomeric or fibrillar forms of A␤, low concentrations of drugs that preserve the integrity of the cytoskeletal network may help neurons survive the toxic cascades initiated by these peptides.

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