The Atlanta VA Health Care System: Morehouse School of Medicine Core Recruiting Site-A Strategy to Increase Diversity in the VA Scientific Workforce.

The Department of Veterans Affairs (VA) initiative to enhance recruitment of diverse biomedical scientists from Historically Black Colleges and Universities (HBCUs) through the VA Career Development Program has provided a unique opportunity for HBCUs to partner with VA to strengthen diversity recruitment efforts. The Atlanta VA Health Care System and the Morehouse School of Medicine (MSM) enjoy a productive and growing interinstitutional collaboration. The partnership between the Atlanta VA and MSM provides the unique opportunity for MSM to increase research opportunities for faculty and students while providing a pipeline of diverse candidates for the Atlanta VA to enhance recruitment of diverse HCBU biomedical scientists. This relationship led to the creation of an inaugural HBCU Core Recruitment Site (CRS) at MSM and the Atlanta VA. The CRS provides a pathway to identify and recruit young diverse investigators who are eligible to compete for VA Career Development Award funding. This Atlanta VA/MSM CRS initiative established a pipeline program to further enhance diversity in the VA scientific workforce. In this review, the Atlanta VA/MSM CRS is presented as a potential model for maximizing the VA initiative to enhance the recruitment of diverse candidates from HBCUs.

Reduced vesicular monoamine transport disrupts serotonin signaling but does not cause serotonergic degeneration.

We previously demonstrated that mice with reduced expression of the vesicular monoamine transporter 2 (VMAT2 LO) undergo age-related degeneration of the catecholamine-producing neurons of the substantia nigra pars compacta and locus ceruleus and exhibit motor disturbances and depressive-like behavior. In this work, we investigated the effects of reduced vesicular transport on the function and viability of serotonin neurons in these mice. Adult (4-6 months of age), VMAT2 LO mice exhibit dramatically reduced (90%) serotonin release capacity, as measured by fast scan cyclic voltammetry. We observed changes in serotonin receptor responsivity in in vivo pharmacological assays. Aged (months) VMAT2 LO mice exhibited abolished 5-HT1A autoreceptor sensitivity, as determined by 8-OH-DPAT (0.1 mg/kg) induction of hypothermia. When challenged with the 5HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (1 mg/kg), VMAT2 LO mice exhibited a marked increase (50%) in head twitch responses. We observed sparing of serotonergic terminals in aged mice (18-24 months) throughout the forebrain by SERT immunohistochemistry and [(3)H]-paroxetine binding in striatal homogenates of aged VMAT2 LO mice. In contrast to their loss of catecholamine neurons of the substantia nigra and locus ceruleus, aged VMAT2 LO mice do not exhibit a change in the number of serotonergic (TPH2+) neurons within the dorsal raphe, as measured by unbiased stereology at 26-30 months. Collectively, these data indicate that reduced vesicular monoamine transport significantly disrupts serotonergic signaling, but does not drive degeneration of serotonin neurons.

Age-related changes in regulator of G-protein signaling (RGS)-10 expression in peripheral and central immune cells may influence the risk for age-related degeneration.

Inflammation in the aging brain increases risk for neurodegenerative disease. In humans, the regulator of G-protein signaling-10 (RGS10) locus has been associated with age-related maculopathy. Chronic peripheral administration of lipopolysaccharide in the RGS10-null mice induces nigral dopaminergic (DA) degeneration, suggesting that RGS10 modulates neuroimmune interactions and may influence susceptibility to neurodegeneration. Because age is the strongest risk factor for neurodegenerative disease, we assessed whether RGS10 expression changes with age and whether aged RGS10-null mice have altered immune cell profiles. Loss of RGS10 in aged mice does not alter the regulation of nigral DA neurons but does alter B-cell, monocyte, microglial, and CD4+ T-cell populations and inflammatory cytokine levels in the cerebrospinal fluid. These results suggest that loss of RGS10 is associated with an age-dependent dysregulation of peripheral and central immune cells rather than dysregulation of DA neuron function.

Direct regulation of complex I by mitochondrial MEF2D is disrupted in a mouse model of Parkinson disease and in human patients.

The transcription factors in the myocyte enhancer factor 2 (MEF2) family play important roles in cell survival by regulating nuclear gene expression. Here, we report that MEF2D is present in rodent neuronal mitochondria, where it can regulate the expression of a gene encoded within mitochondrial DNA (mtDNA). Immunocytochemical, immunoelectron microscopic, and biochemical analyses of rodent neuronal cells showed that a portion of MEF2D was targeted to mitochondria via an N-terminal motif and the chaperone protein mitochondrial heat shock protein 70 (mtHsp70). MEF2D bound to a MEF2 consensus site in the region of the mtDNA that contained the gene NADH dehydrogenase 6 (ND6), which encodes an essential component of the complex I enzyme of the oxidative phosphorylation system; MEF2D binding induced ND6 transcription. Blocking MEF2D function specifically in mitochondria decreased complex I activity, increased cellular H(2)O(2) level, reduced ATP production, and sensitized neurons to stress-induced death. Toxins known to affect complex I preferentially disrupted MEF2D function in a mouse model of Parkinson disease (PD). In addition, mitochondrial MEF2D and ND6 levels were decreased in postmortem brain samples of patients with PD compared with age-matched controls. Thus, direct regulation of complex I by mitochondrial MEF2D underlies its neuroprotective effects, and dysregulation of this pathway may contribute to PD.

A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone.

Brain-derived neurotrophic factor (BDNF), a cognate ligand for the tyrosine kinase receptor B (TrkB) receptor, mediates neuronal survival, differentiation, synaptic plasticity, and neurogenesis. However, BDNF has a poor pharmacokinetic profile that limits its therapeutic potential. Here we report the identification of 7,8-dihydroxyflavone as a bioactive high-affinity TrkB agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signaling. 7,8-Dihydroxyflavone protected wild-type, but not TrkB-deficient, neurons from apoptosis. Administration of 7,8-dihydroxyflavone to mice activated TrkB in the brain, inhibited kainic acid-induced toxicity, decreased infarct volumes in stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson disease. Thus, 7,8-dihydroxyflavone imitates BDNF and acts as a robust TrkB agonist, providing a powerful therapeutic tool for the treatment of various neurological diseases.

Highly pathogenic H5N1 influenza virus can enter the central nervous system and induce neuroinflammation and neurodegeneration.

One of the greatest influenza pandemic threats at this time is posed by the highly pathogenic H5N1 avian influenza viruses. To date, 61% of the 433 known human cases of H5N1 infection have proved fatal. Animals infected by H5N1 viruses have demonstrated acute neurological signs ranging from mild encephalitis to motor disturbances to coma. However, no studies have examined the longer-term neurologic consequences of H5N1 infection among surviving hosts. Using the C57BL/6J mouse, a mouse strain that can be infected by the A/Vietnam/1203/04 H5N1 virus without adaptation, we show that this virus travels from the peripheral nervous system into the CNS to higher levels of the neuroaxis. In regions infected by H5N1 virus, we observe activation of microglia and alpha-synuclein phosphorylation and aggregation that persists long after resolution of the infection. We also observe a significant loss of dopaminergic neurons in the substantia nigra pars compacta 60 days after infection. Our results suggest that a pandemic H5N1 pathogen, or other neurotropic influenza virus, could initiate CNS disorders of protein aggregation including Parkinson's and Alzheimer's diseases.

Nonmotor symptoms of Parkinson's disease revealed in an animal model with reduced monoamine storage capacity.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, culminating in severe motor symptoms, including resting tremor, rigidity, bradykinesia, and postural instability. In addition to motor deficits, there are a variety of nonmotor symptoms associated with PD. These symptoms generally precede the onset of motor symptoms, sometimes by years, and include anosmia, problems with gastrointestinal motility, sleep disturbances, sympathetic denervation, anxiety, and depression. Previously, we have shown that mice with a 95% genetic reduction in vesicular monoamine transporter expression (VMAT2-deficient, VMAT2 LO) display progressive loss of striatal dopamine, L-DOPA-responsive motor deficits, alpha-synuclein accumulation, and nigral dopaminergic cell loss. We hypothesized that since these animals exhibit deficits in other monoamine systems (norepinephrine and serotonin), which are known to regulate some of these behaviors, the VMAT2-deficient mice may display some of the nonmotor symptoms associated with PD. Here we report that the VMAT2-deficient mice demonstrate progressive deficits in olfactory discrimination, delayed gastric emptying, altered sleep latency, anxiety-like behavior, and age-dependent depressive behavior. These results suggest that the VMAT2-deficient mice may be a useful model of the nonmotor symptoms of PD. Furthermore, monoamine dysfunction may contribute to many of the nonmotor symptoms of PD, and interventions aimed at restoring monoamine function may be beneficial in treating the disease.

Reduced vesicular storage of dopamine exacerbates methamphetamine-induced neurodegeneration and astrogliosis.

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5-10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.

Response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) differs in mouse strains and reveals a divergence in JNK signaling and COX-2 induction prior to loss of neurons in the substantia nigra pars compacta.

Parkinson's disease (PD) is a neurodegenerative disease whose hallmark pathological features include a selective loss of dopaminergic neurons in the midbrain. Recent studies have described the activation of a stress-induced signal cascade, c-Jun N-terminal kinase (JNK)-mediated activation of c-Jun, and an increase in the expression of a downstream effector, cyclooxygenase 2 (COX-2), in postmortem PD brains. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces selective neuronal loss in the midbrain similar to that seen in PD, also induces JNK-mediated activation of c-Jun and generates a COX-2 response in C57BL/6J mice. However, mice exhibit a strain-dependent susceptibility to MPTP. Identifying the point(s) of molecular divergence in the MPTP-induced response may provide insight into the cause of PD or a means to identify susceptibility to PD in humans. Here we examined JNK signaling and COX-2 induction in two strains of mice, the MPTP-sensitive C57BL/6J and the MPTP-resistant Swiss Webster (SW). We show that C57BL/6J and SW strains differ in JNK and c-Jun activation in response to MPTP. In addition, the MPTP-induced COX-2 response occurs exclusively in C57BL/6J mice. Furthermore, strain-specific responses to MPTP are not due to differences in MPP(+) levels and are not secondary to cell death. These results provide evidence toward a mechanism of strain-dependent sensitivity to MPTP.

GSTpi expression mediates dopaminergic neuron sensitivity in experimental parkinsonism.

The cause of 95% of Parkinson's disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GSTpi, in dopaminergic neuron death. GSTpi is the only GST family member expressed in substantia nigra neurons. GSTpi reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GSTpi contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD.

Glia cell number modulates sensitivity to MPTP in mice.

Free radical damage has been shown to play a significant role in the pathogenesis of a number of neurodegenerative diseases including Parkinson's disease. One model of experimental parkinsonism is the loss of substantia nigra cells following administration of MPTP. Previously, it has been shown that a number of inbred strains of mice have differential responses to this toxin, and this difference is dependent on glial cells. In this study, the number of glial cells in the substantia nigra pars compacta of C57Bl/6J (MPTP-sensitive) and Swiss Webster (MPTP-resistant) strains of mice was examined. The C57Bl/6J mice have an approximately 50% lower number of GFAP+ and S-100beta glial cells than the Swiss Webster mice. C57Bl/6J mice have a 25% increased number of resident nonactivated microglial cells. To determine whether this difference in cell number has functional significance, we used an in vitro SN culture system that allowed us to manipulate the number of glial cells. When C57Bl/6 neurons were grown on a glial mat plated with twice the number of cells, we were able to rescue the MPTP-sensitive neurons from toxin-induced cell death. This suggests that the number of glial cells in the SNpc may be an important factor in the survival of dopaminergic neurons following exposure to xenobiotics.

Environmental enrichment in adulthood eliminates neuronal death in experimental Parkinsonism.

Idiopathic Parkinson's disease (PD) affects 2% of adults over 50 years of age. PD patients demonstrate a progressive loss of dopamine neurons in the substantia nigra pars compacta (SNpc). One model that recapitulates the pathology of PD is the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Here we show that exposure to an enriched environment (EE) (a combination of exercise, social interactions and learning) or exercise alone during adulthood, totally protects against MPTP-induced Parkinsonism. Furthermore, changes in mRNA expression would suggest that increases in glia-derived neurotrophic factors, coupled with a decrease of dopamine-related transporters (e.g. dopamine transporter, DAT; vesicular monoamine transporter, VMAT2), contribute to the observed neuroprotection of dopamine neurons in the nigrostriatal system following MPTP exposure. This non-pharmacological approach presents significant implications for the prevention and/or treatment of PD.

Inhibitory effects of lysophosphatidylcholine on the dopaminergic system.

Lysophosphatidylcholine (lyso-PTC) is formed by phospholipase A2 (PLA2) from phosphatidylcholine (PTC), that is produced through phosphatidylethanolamine (PTE) methylation. 1-Methyl-4-phenyl-pyridinium (MPP+), a Parkinson's disease (PD) inducing agent, and S-adenosylmethionine (SAM), a biological methyl donor, increase lyso-PTC formation and both induce PD-like changes in animal models. In the current study, we investigated the effect of lyso-PTC on the dopaminergic system to determine the modulating role of lyso-PTC in dopaminergic neurotransmission. The results of these experiments show that lyso-PTC has a remarkable inhibitory effect on dopamine D1 and D2 receptor binding activities in the striatal membrane prepared from Sprague-Dawley rats. Lyso-PTC decreased the Bmax values of both D1 and D2 receptor binding activities. The Kd values for D1 and D2 receptors were not changed, but lyso-PTC also inhibited dopamine transporter and decreased striatal dopamine turnover rate. MPP+ showed similar, but less potent effects. The current studies suggest that lyso-PTC significantly impair the dopaminergic system and might play a role in MPP+ and SAM induced PD-like changes through its inhibitory effects on dopaminergic neurotransmission.

The inhibitory role of methylation on the binding characteristics of dopamine receptors and transporter.

Excess methylation has been suggested to play a role in the pathogenesis of Parkinson's disease (PD), since the administration of S-adenosylmethionine (SAM), a biological methyl donor, induces PD-like changes in rodents. It was proposed that SAM-induced PD-like changes might be associated with its ability to react with the dopaminergic system. In the present study the effects of SAM on dopamine receptors and transporters were investigated using rats and cloned dopamine receptor proteins. Autoradiographic examination of SAM indicated its tendency to be localized and accumulated in rat striatal region after the intracerebroventricular injection into rat brain. Moreover, results showed that SAM significantly decreased dopamine D1 and D2 receptor binding activities by decreasing the Bmax and increasing the Kd values. At concentrations of 0.1, 0.25 and 0.5 mM, SAM was able to reduce the Bmax from the control value of 848.1 for dopamine D1-specific ligand [3H] SCH 23390 to 760.1, 702.6 and 443.0 fmol/mg protein, respectively. At the same concentrations, SAM was able to increase the Kd values from 0.91 for the control to 1.06, 3.84 and 7.01 nM of [3H] SCH 23390, respectively. The effects of SAM on dopamine D2 binding were similar to those of dopamine D1 binding. SAM also decreased dopamine transporter activity. The interaction of SAM with dopamine receptor proteins produced methanol from methyl-ester formation and hydrolysis. We propose that the SAM effect might be related to its ability to react with dopamine receptor proteins through methyl-ester formation and methanol production following the hydrolysis of the carboxyl-methylated receptor proteins.

S-adenosyl-methionine-induced apoptosis in PC12 cells.

Our previous studies showed that S-adenosyl-methionine (SAM) induced Parkinson's disease-like changes in rat. It caused death to dopamine neurons in the substantia nigra, which appeared shrunken and fragmented, indicative of apoptosis-like changes (Charlton and Crowell [1995] Mol. Chem. Neuropathol. 26:269-284; Charlton [1997] Life Sci. 61:495-502). In this study, we investigated whether SAM causes apoptosis in both undifferentiated PC12 (PC12) cells and nerve growth factor (NGF)-differentiated PC12 (D-PC12) cells. S-adenosyl-homocysteine (SAH), the nonmethyl analog of SAM, was also tested. SAM and SAH (1.0 nM to 10.0 microM) caused lactate dehydrogenase (LDH) release from the PC12 cells and D-PC12 cells; cells with morphological changes and fluorescent DNA fragmentation staining were detected among both PC12 cell and D-PC12 cell. Compared with the PC12 cell, the D-PC12 cell, a postmitotic cell, was more sensitive to the toxic effects of SAM or SAH and presented much greater LDH release, suggesting a lethal effect; surprisingly, the amounts of apoptotic cells did not differ significantly between the two kinds of cells. In medium deprived of exogenous methionine, a decline in LDH release was observed in PC12 and D-PC12 cells. Also, lower levels of intracellular SAM and SAH were observed in the methionine-deleted media, which were reversed by the addition of either SAM or SAH. An antivitamin B(12) monoclonal antibody was added to methionine-depleted medium, resulting in deficiency of both endogenous and exogenous methionine, which caused further decreases in LDH release and reduction in the levels of intracellular SAM and SAH. The preliminary data showed different sensitivities to SAM or SAH between PC12 cell and D-PC12 cells, which suggests that PC12 cell may be more stable as a metabolic model. Apoptosis of PC12 cells was also assessed by PARP cleavage detection, Western blot analysis of Bax and Bcl-2 proteins, and DNA laddering on agarose gel electrophoresis. The proapoptoic protein Bax was dominantly expressed, whereas Bcl-2 was slightly down-regulated by SAM. SAH weakly induced the expression of Bax and slightly decreased Bcl-2 levels. The effects of SAM and its analog, SAH, were demonstrated conclusively to induce apoptosis in PC12 cells.