Erik Floor

Increased Protein Oxidation in Human Substantia Nigra Pars Compacta in Comparison with Basal Ganglia and Prefrontal Cortex Measured with an Improved Dinitrophenylhydrazine Assay

Abstract: The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinsons disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4‐dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size‐dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinsons disease.

CAPS (Mammalian UNC-31) Protein Localizes to Membranes Involved in Dense-Core Vesicle Exocytosis

CAPS is a neural/endocrine-specific protein discovered as a cytosolic factor required for Ca2+-activated dense-core vesicle (DCV) exocytosis in permeable neuroendocrine cells. We report that CAPS is also a membrane-associated, peripherally bound protein in brain homogenates that localizes Selectively to plasma membranes and to DCVs but not to small clear synaptic vesicles (SVs). CAPS exhibits high affinity and saturable binding to DCVs by interaction with bilayer phospholipids. Specific CAPS antibodies inhibit Ca2+-activated norepinephrine release from lysed synaptosomes that contain membrane-associated CAPS, indicating that membrane-bound CAPS is essential for neural DCV exocytosis. CAPS is a functional component of the exocytotic machinery that localizes selectively to DCVs, and it may confer distinct regulatory features on neuropeptide and biogenic amine transmitter secretion.

Role of Synaptic Vesicle Proton Gradient and Protein Phosphorylation on ATP-mediated Activation of Membrane-associated Brain Glutamate Decarboxylase

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464–6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5′-[β,γ-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanidem-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of32P from [γ-32P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking γ-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.

Hydrogen Peroxide Inhibits the Vacuolar H+‐ATPase in Brain Synaptic Vesicles at Micromolar Concentrations

Abstract: Hydrogen peroxide (H2O2) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H2O2 on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H+‐ATPase (V‐ATPase) in the vesicle membrane. We report here that the V‐ATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H2O2. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H2O2‐induced inhibition of the V‐ATPase, and ATP or GTP partially protect the ATPase from inhibition by H2O2. These and other results suggest that the mechanism of inhibition of the V‐ATPase by H2O2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V‐ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down‐regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.

Amphetamine releases dopamine from synaptic vesicles by dual mechanisms

Low concentrations of D-amphetamine (AMPH) released [3H]dopamine (DA) from purified, rat brain synaptic vesicles with exponential kinetics and a half-time of 4 min. Slight alkalinization of the vesicles by AMPH seemed unable to account for this release. Instead, it is likely that AMPH partially inhibits DA uptake after which DA escapes by an uptake-independent pathway. At high concentrations the proton gradient was severely reduced by AMPH and DA was released with a half-time of < 1 min. Loss of the proton gradient was sufficient to account for this DA release. These results show that low concentrations of AMPH release DA from synaptic vesicles and suggest that release occurs by different mechanisms at low and high AMPH concentrations.

Synaptin/synaptophysin, p65 and SV2: their presence in adrenal chromaffin granules and sympathetic large dense core vesicles

The subcellular distribution of three proteins of synaptic vesicles (synaptin/synaptophysin, p65 and SV2) was determined in bovine adrenal medulla and sympathetic nerve axons. In adrenals most p65 and SV2 is confined to chromaffin granules. Part of synaptin/synaptophysin is apparently also present in these organelles, but a considerable portion is found in a light vesicle which does not contain significant concentrations of typical markers of chromaffin granules (cytochrome b-561, dopamine beta-hydroxylase or the amine carrier). An analogous finding was obtained for sympathetic axons. The large dense core vesicles contain most p65 and also SV2 but only a smaller portion of synaptin/synaptophysin. A lighter vesicle containing this latter antigen and some SV2 has also been found. These results establish that in adrenal medulla and sympathetic axons three typical antigens of synaptic vesicles are not restricted to light vesicles. Apparently, a varying part of these antigens is found in chromaffin granules and large dense core vesicles. On the other hand, the light vesicles do not contain significant concentrations of functional antigens of chromaffin granules. Thus, the biogenesis of small presynaptic vesicles which contain all three antigens as well as functional components like the amine carrier is likely to involve considerable membrane sorting.

Enrichment of Presenilin 1 Peptides in Neuronal Large Dense‐Core and Somatodendritic Clathrin‐Coated Vesicles

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N‐terminal and a C‐terminal fragment, both membrane‐associated. More than 40 presenilin 1 mutations have been linked to early‐onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild‐type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear‐core synaptic vesicles, the large dense‐core vesicles, and the somatodendritic and nerve terminal clathrin‐coated vesicles. Both the N‐ and C‐terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense‐core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense‐core vesicle‐mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin‐coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear‐core synaptic vesicle or in the nerve terminal clathrin‐coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full‐length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full‐length protein may not have a vesicular function.

E3 Ubiquitin-Protein Ligase Activity of Parkin Is Dependent on Cooperative Interaction of RING Finger (TRIAD) Elements

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinsons disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity.

Dynamic storage of glutamate in rat brain synaptic vesicles

Storage of [3H]glutamate accumulated by highly purified synaptic vesicles from brain was characterized. [3H]Glutamate was lost with single exponential kinetics with a time constant of minutes after synaptic vesicles were diluted into medium that allowed uptake to continue but that contained unlabeled glutamate in place of [3H]glutamate. This [3H]glutamate efflux occurred at similar rates in media containing 50 and 500 microM glutamate, which suggests that it did not depend on the rate of glutamate transport and was independent of the external and internal glutamate concentrations. All efflux was blocked at 0 degrees C. These results imply that glutamate stored in synaptic vesicles turns over with a half-time of minutes, even during active uptake under physiological conditions.

A one-carbon modification of protein lysine associated with elevated oxidative stress in human substantia nigra.

We describe for the first time a naturally occurring lysine modification that is converted to methyllysine by reduction with sodium borohydride. This modification is ∼1.7 times as abundant in soluble proteins from human substantia nigra pars compacta as in proteins from other brain regions, possibly as a result of elevated oxidative stress in the nigra. Proteins from cultured PC12 cells exposed to oxidative stress conditions also contain elevated levels of this lysine modification. The abundance of the naturally occurring modification is roughly 0.08 nmoles/mg protein in either unstressed brain or PC12 cells. Modification levels remain stable in isolated proteins incubated for 2 h at 37°C in pH 7 buffer. We propose that the endogenous modification is the lysine Schiff base, ε‐N‐methylenelysine, and that lysine modifications may result from a reaction with formaldehyde in vivo. Rat brain contains ∼60 nmoles/g wet weight of formaldehyde, which probably includes both free and reversibly bound forms. Adding ∼35 µm HCHO to PC12 cell growth medium introduces methylenelysine modifications in cell proteins and impairs cell viability. The existence of this post‐translational modification suggests new mechanisms of oxidative stress that may contribute to tissue degeneration, including loss of nigral dopamine neurons during normal aging and in Parkinsons disease.