The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Parkinson's disease is a complex neurodegenerative disorder characterized by the death of brain dopamine neurons. In mammals, dopamine neuronal degeneration can be triggered through exposure to neurotoxins accumulated by the presynaptic dopamine transporter (DAT), including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium. We have established a system for the pharmacological and genetic evaluation of neurotoxin-induced dopamine neuronal death in Caenorhabditis elegans. Brief (1 h) exposure of green fluorescent protein-tagged, living worms to 6-OHDA causes selective degeneration of dopamine neurons. We demonstrate that agents that interfere with DAT function protect against 6-OHDA toxicity. 6-OHDA-triggered neural degeneration does not require the CED-3/CED-4 cell death pathway, but is abolished by the genetic disruption of the C. elegans DAT.
Brain regional oxidative damage is thought to be a central mechanism in the pathogenesis of Alzheimer's disease (AD). Recent studies of cerebrospinal fluid (CSF) have suggested that increased lipid peroxidation of CSF and CSF lipoproteins also may occur in AD patients. In the present study, we determined the susceptibility of human CSF to ex vivo lipid peroxidation and tested the hypothesis that oxidized CSF lipoproteins may be neurotoxic. Whole CSF or a CSF lipoprotein fraction (d < 1.210 g/mL) was oxidized with 2,2'-azobis(2-amidino-propane)dihydrochloride (AAPH), a hydrophilic free-radical generator. Kinetics of CSF lipid peroxidation were followed by a standard fluorescence product accumulation assay. Oxidation of AD CSF yielded significantly shorter fluorescent lag times than controls, indicating reduced antioxidant capacity. Electrophoretic mobilities of CSF apolipoproteins were specifically reduced upon oxidation of CSF with AAPH, suggesting that lipoproteins are primary targets of CSF lipid peroxidation. Cultured neuronal cells were exposed to physiological concentrations of isolated CSF lipoproteins oxidized with increasing concentrations of AAPH; the resulting neurotoxicity showed a significant linear AAPH concentration-response relationship. These results suggest that oxidized CSF lipoproteins may contribute to the pathogenesis of neurodegeneration in AD.
Previous studies have shown selective binding of the neurotoxicant 2,5-hexanedione (2,5-HD) to carboxyl-terminal domains of rat neurofilament (NF) M and H proteins in vitro. The present study was designed to further localize this binding in native rat NF preparations exposed to [14C]2,5-HD. Purified M and H proteins from 2,5-HD-treated NFs were subjected to cyanogen bromide (CNBr) cleavage, and the resultant peptides were separated by Tris-tricine SDS-PAGE and electroblotted to PVDF membranes. Peptides were identified by direct sequencing of stained bands and the relative radiolabeling of each peptide was determined by comparing band intensities in fluorographed blots. For NF-M, the highest label was found in CNBr 10, a peptide corresponding to residues 678-846 at the extreme carboxyl terminus. This region of the protein includes three highly conserved lysine-containing sequences believed to be critical to its function. For NF-H, the greatest binding was localized in CNBr 7 + 8, representing an incomplete cleavage product of residues 390-810. This peptide contains essentially all of the phosphorylation sites in the carboxyl terminus of NF-H, a domain believed to control NF interactions in the axon. Only minor radiolabeling was observed in other M or H peptides. Extensive dephosphorylation of NFs prior to 2,5-HD exposure had no effect on relative adduct levels in each protein. These results provide additional support for limited and specific binding of 2,5-HD to neurofilaments and indicate that the phosphorylation state of the protein may not substantially influence this binding.
Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainate neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainate-induced neurons degeneration. We found DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainate (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainate-induced death. While the morphological and biochemical features of neuronal death induced by kainate resembled low-K(+)-induced apoptosis in cerebellar granule cells; the time interval from the institution of the death-promoting condition to neuronal death was briefer with kainate and did not require new protein or RNA synthesis. These results demonstrate that kainate receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainate receptor activation with apoptosis.
Coactivation of metabotropic glutamate receptors (mGluRs) and beta-adrenergic receptors causes a synergistic increase in cAMP formation in the rat hippocampus. Increases in cAMP are known to have many actions in the hippocampus via activation of cAMP-dependent protein kinase. We now report that coactivation of mGluRs and beta-adrenergic receptors induces an acute depression of EPSCs at the Schaffer collateral-CA1 synapse. Interestingly, this depression of EPSCs is dependent upon increases in cAMP levels but independent of protein kinase activity. A series of studies suggests that cAMP-mediated depression of EPSCs is dependent on metabolism of cAMP and release of adenosine or 5'-AMP into the extracellular space with resultant activation of presynaptic adenosine receptors. These studies suggest that cAMP can have local hormone-like effects in the hippocampal formation which are independent of cAMP-dependent protein kinase.
Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to Gs. It has been suggested that mGluR-mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR-mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1S,3R-ACPD-stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1S,3R-ACPD induced a cAMP response that was greater than additive. Finally, (RS)-3,5-dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR-mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.
After treatment with the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), there was a severe loss of dopamine (DA) at all examined sites in the striatum, both in those monkeys which remained asymptomatic (77-99%) and in one monkey which developed severe parkinsonian disability (94-99%). However, the asymptomatic animals had normal DA concentration in the substantia nigra (SN); yet in the symptomatic animal DA was largely depleted in the central (86%) and medial (94%), but not lateral (8%) regions of the SN. The HVA/DA ratio was raised in the striatum of all MPTP-treated animals. In the SN though, this ratio was elevated only in the symptomatic animal, in the central and medial, but not lateral regions. The contralateral half of these brains were examined for DA histofluorescence. The SN of asymptomatic animals had a slight increase in lipofuscin fluorescence within dopaminergic neurons and a small reduction in the number of dopaminergic cells, while fluorescent intensity of individual neurons was unchanged. The SN of the symptomatic animal displayed a sharp decline in the number of DA neurons along with an increase in autofluorescent pigment granules; these changes were most pronounced in the central and medial regions of the SN. These data suggest that after MPTP the terminals of the nigrostriatal pathway are affected before the cell bodies. In the one symptomatic animal emergence of parkinsonian disability corresponded with a marked loss of DA neurons and DA concentration in the central and medial regions of the SN. In the control monkeys a gradient in the concentration of amines and metabolites was observed within the SN; the lateral region contained the highest and the medial region the lowest concentration.
Immunohistochemical examination of the midbrain of vervet monkeys treated with MPTP revealed a marked loss of dopaminergic neurons in the lateral, but not medial, region of the A8 dopamine (DA) cell group. In the same animals, the number of DA neurons in the substantia nigra was only slightly decreased. Biochemical assessment revealed a marked (greater than 85%) depletion of DA in the striatum. However, in the DA cell body regions significant decreases in DA and homovanillic acid were observed only in the lateral A8 region, and not in the medial A8 region or substantia nigra. These data suggest that those A8 DA neurons which project to the striatum are preferentially vulnerable to MPTP.
Mild footshock stress results in the metabolic activation of the prefrontal cortical dopamine (DA) innervation, but does not augment DA utilization in mesolimbic areas (such as the nucleus accumbens septi, NAS) or the striatum. However, increases in either the intensity or duration of footshock stress increase DA utilization in the subcortical sites. DA afferents to the prefrontal cortex (PFC) hold corticofugal projection neurons under tonic inhibition. Previous data suggest that removal of these corticofugal glutamatergic neurons from tonic DA inhibition results in a transsynaptic alteration in the NAS, such that the DA innervation of the NAS is rendered hyperresponsive to certain perturbations. We therefore examined the effects of stress on subcortical DA systems in rats previously subjected to 6-hydroxydopamine lesions of the PFC DA innervation. Mild footshock stress resulted in an increase in concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the PFC, but not NAS or striatum, of sham-lesioned animals. Footshock resulted in a significant increase in the concentration of DOPAC in the nucleus accumbens of animals sustaining PFC lesions two weeks previously. The PFC lesion did not result in a stress-induced increase in DA release in the striatum. These results suggest that disruption of the PFC DA innervation results in an enhanced responsiveness of the mesolimbic DA innervation to stress. These data may help explain the stress-elicited exacerbation of the psychotic process in schizophrenia.
2,5-Hexanedione (2,5-HD) induces a toxic neuropathy characterized by massive, focal axonal neurofilament (NF) accumulation. Covalent interaction of 2,5-HD with NF protein amines, resulting in pyrrole adduct formation, has been proposed as a critical step in its mechanism. The present study was undertaken to evaluate the hypothesis of selective 2,5-HD/lysine modification, by quantitating in vitro adduction in the NF proteins and in specific polypeptide domains of each protein. Native rat spinal cord NFs were exposed to 0-212.5 mM [14C]2,5-HD for 2-16 h (37 degrees C under argon), followed by removal of non-covalently bound radioactivity. Incorporation of radioactivity and pyrrole formation in NFs increased linearly with 2,5-HD concentration and biphasically with time. SDS-PAGE and fluorography demonstrated prominent labeling of the three NF subunit proteins (H, M, and L), in addition to high-MW, crosslinked material derived from NF-H and -M. Mild chymotryptic cleavage was employed to isolate the carboxyl-terminal 'tail' domains of NF-H and -M, and the pooled amino-terminal NF 'rod' regions, all of which were radiolabeled. Specific activity (mol adduct/mol protein) of adducted NF proteins and polypeptide domains was determined by scintillation counting of electroeluted proteins. Stable binding in the NF-H and -M proteins was 4- to 6-fold higher than in the NF-L protein at all 2,5-HD concentrations, with specific activities of approximately 6.9, 4.7, and 1.3 mol/mol protein, respectively, at 212.5 mM. Approximately 70-80% of NF-H and -M binding was localized to the tail domains. In contrast, NF-L and pooled rod domain adduction did not substantially exceed 1 mol/mol protein. These findings provide the first direct evidence for limited and selective pyrrole adduction in the NF proteins following 2,5-HD exposure.