Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies.

N-Methyl-D-aspartate receptors (NMDARs) have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca(2+) influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca(2+) homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases.

Astrocytic dynamin-like protein 1 regulates neuronal protection against excitotoxicity in Parkinson disease.

Mitochondrial dynamics has recently become an area of piqued interest in neurodegenerative disorders, including Parkinson disease (PD); however, the contribution of astrocytes to these disorders remains unclear. Here, we show that the level of dynamin-like protein 1 (Dlp1; official name DNM1L), which promotes mitochondrial fission, is lower in astrocytes from the brains of PD patients, and that decreased astrocytic Dlp1 likely represents a relatively early event in PD pathogenesis. In support of this conclusion, we show that Dlp1 knockdown dramatically affects mitochondrial morphological characteristics and localization in astrocytes, impairs the ability of astrocytes to adequately protect neurons from the excitotoxic effects of glutamate, and increases intracellular Ca(2+) in response to extracellular glutamate, resulting from compromised intracellular Ca(2+) buffering. Taken together, our results suggest that astrocytic mitochondrial Dlp1 is a key protein in mitochondrial dynamics and decreased Dlp1 may interfere with neuron survival in PD by disrupting Ca(2+)-coupled glutamate uptake.

Evidence of calcium-permeable AMPA receptors in dendritic spines of CA1 pyramidal neurons.

GluA2-lacking, calcium-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) have unique properties, but their presence at excitatory synapses in pyramidal cells is controversial. We have tested certain predictions of the model that such receptors are present in CA1 cells and show here that the polyamine spermine, but not philanthotoxin, causes use-dependent inhibition of synaptically evoked excitatory responses in stratum radiatum, but not s. oriens, in cultured and acute hippocampal slices. Stimulation of single dendritic spines by photolytic release of caged glutamate induced an N-methyl-d-aspartate receptor-independent, use- and spermine-sensitive calcium influx only at apical spines in cultured slices. Bath application of glutamate also triggered a spermine-sensitive influx of cobalt into CA1 cell dendrites in s. radiatum. Responses of single apical, but not basal, spines to photostimulation displayed prominent paired-pulse facilitation (PPF) consistent with use-dependent relief of cytoplasmic polyamine block. Responses at apical dendrites were diminished, and PPF was increased, by spermine. Intracellular application of pep2m, which inhibits recycling of GluA2-containing AMPARs, reduced apical spine responses and increased PPF. We conclude that some calcium-permeable, polyamine-sensitive AMPARs, perhaps lacking GluA2 subunits, are present at synapses on apical dendrites of CA1 pyramidal cells, which may allow distinct forms of synaptic plasticity and computation at different sets of excitatory inputs.

The role of glutamate in the morphological and physiological development of dendritic spines.

Dendritic spines form the postsynaptic half of the synapse but how they form during CNS development remains uncertain, as are the factors that promote their morphological and physiological maturation. One hypothesis posits that filopodia, long motile dendritic processes that are present prior to spine formation, are the precursors to spines. Another hypothesis posits that they form directly from the dendritic shaft. We used microphotolysis of caged glutamate to stimulate individual dendritic processes in young hippocampal slice cultures while recording their morphological and physiological responses. We observed that brief trains of stimuli delivered to immature processes triggered morphological changes within minutes that resulted, in about half of experiments, in a more mature, spine-like appearance such as decreased spine neck length and increased spine head width. We also observed that glutamate-induced inward currents elicited from immature processes were mostly or entirely mediated by NMDARs, whereas responses in those processes with a more mature morphology, regardless of actual developmental age, were mediated by both AMPARs and NMDARs. Consistent with this observation, glutamate-induced morphological changes were largely, but not entirely, prevented by blocking NMDARs. Our observations thus favor a model in which filopodia in the developing nervous system sense and respond to release of glutamate from developing axons, resulting in physiological and morphological maturation.

Longitudinal assessment of tau and amyloid beta in cerebrospinal fluid of Parkinson disease.

Tau gene has been consistently associated with the risk of Parkinson disease in recent genome wide association studies. In addition, alterations of the levels of total tau, phosphorylated tau [181P], and amyloid beta 1-42 in cerebrospinal fluid have been reported in patients with sporadic Parkinson disease and asymptomatic carriers of leucine-rich repeat kinase 2 mutations, in patterns that clearly differ from those typically described for patients with Alzheimer disease. To further determine the potential roles of these molecules in Parkinson disease pathogenesis and/or in tracking the disease progression, especially at early stages, the current study assessed all three proteins in 403 Parkinson disease patients enrolled in the DATATOP (Deprenyl and tocopherol antioxidative therapy of parkinsonism) placebo-controlled clinical trial, the largest cohort to date with cerebrospinal fluid samples collected longitudinally. These initially drug-naive patients at early disease stages were clinically evaluated, and cerebrospinal fluid was collected at baseline and then at endpoint, defined as the time at which symptomatic anti-Parkinson disease medications were determined to be required. General linear models were used to test for associations between baseline cerebrospinal fluid biomarker levels or their rates of change and changes in the Unified Parkinson Disease Rating Scale (total or part III motor score) over time. Robust associations among candidate markers are readily noted. Baseline levels of amyloid beta were weakly but negatively correlated with baseline Unified Parkinson Disease Rating Scale total scores. Baseline phosphorylated tau/total tau and phosphorylated tau/amyloid beta were significantly and negatively correlated with the rates of the Unified Parkinson Disease Rating Scale change. While medications (deprenyl and/or tocopherol) did not appear to alter biomarkers appreciably, a weak but significant positive correlation between the rate of change in total tau or total tau/amyloid beta levels and the change of the Unified Parkinson Disease Rating Scale was observed. Notably, these correlations did not appear to be influenced by APOE genotype. These results are one of the very first pieces of evidence suggesting that tau and amyloid beta are critically involved in early Parkinson disease progression, potentially by a different mechanism than that in Alzheimer disease, although their applications as Parkinson disease progression markers will likely require the addition of other proteins.

Suppressed pro-inflammatory response of microglia in CX3CR1 knockout mice.

Neuronal fractalkine acts via its receptor, CX3CR1, on microglia to regulate neuroinflammation. Conflicting results have been reported in studies employing CX3CR1 deficient (Cx3cr1(-/-)) mice. Here, compared to wild-type, endotoxin-treated neuron-glial Cx3cr1(-/-)cultures produced less TNF-α, nitric oxide and superoxide; however, fractalkine treatment inhibited the release of pro-inflammatory factors in wild-type and BV-2 cell cultures. Furthermore, endotoxin-treated BV-2 cells expressing siRNA against CX3CR1 increased nitric oxide and TNF-α production. We hypothesize that CX3CL1-CX3CR1 signaling is neuroprotective and propose that the reduced production of pro-inflammatory signals in Cx3cr1(-/-)microglia may result from compensatory mechanisms and not be the direct result of CX3CR1 deficiency.

Palmitoylation at two cysteine clusters on the C-terminus of GluN2A and GluN2B differentially control synaptic targeting of NMDA receptors.

Palmitoylation of NMDARs occurs at two distinct cysteine clusters in the carboxyl-terminus of GluN2A and GluN2B subunits that differentially regulates retention in the Golgi apparatus and surface expression of NMDARs. Mutations of palmitoylatable cysteine residues in the membrane-proximal cluster to non-palmitoylatable serines leads to a reduction in the surface expression of recombinant NMDARs via enhanced internalization of the receptors. Mutations in a cluster of cysteines in the middle of the carboxyl-terminus of GluN2A and GluN2B, leads to an increase in the surface expression of NMDARs via an increase in post-Golgi trafficking. Using a quantitative electrophysiological assay, we investigated whether palmitoylation of GluN2 subunits and the differential regulation of surface expression affect functional synaptic incorporation of NMDARs. We show that a reduction in surface expression due to mutations in the membrane-proximal cluster translates to a reduction in synaptic expression of NMDARs. However, increased surface expression induced by mutations in the cluster of cysteines that regulates post-Golgi trafficking of NMDARs does not increase the synaptic pool of NMDA receptors, indicating that the number of synaptic receptors is tightly regulated.

Applying bioinformatics to proteomics: is machine learning the answer to biomarker discovery for PD and MSA?

Bioinformatics tools are increasingly being applied to proteomic data to facilitate the identification of biomarkers and classification of patients. In the June, 2012 issue, Ishigami et al. used principal component analysis (PCA) to extract features and support vector machine (SVM) to differentiate and classify cerebrospinal fluid (CSF) samples from two small cohorts of patients diagnosed with either Parkinson's disease (PD) or multiple system atrophy (MSA) based on differences in the patterns of peaks generated with matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PCA accurately segregated patients with PD and MSA from controls when the cohorts were combined, but did not perform well when segregating PD from MSA. On the other hand, SVM, a machine learning classification model, correctly classified the samples from patients with early PD or MSA, and the peak at m/z 6250 was identified as a strong contributor to the ability of SVM to distinguish the proteomic profiles of either cohort when trained on one cohort. This study, while preliminary, provides promising results for the application of bioinformatics tools to proteomic data, an approach that may eventually facilitate the ability of clinicians to differentiate and diagnose closely related parkinsonian disorders.

Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: Role of astrocytes and extracellular matrix proteins.

In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1mM for 24h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol.