David Sulzer

Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinsons disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.

CARGO RECOGNITION FAILURE IS RESPONSIBLE FOR INEFFICIENT AUTOPHAGY IN HUNTINGTON’S DISEASE

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntingtons disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Dopamine-modified α-synuclein blocks chaperone-mediated autophagy

Altered degradation of alpha-synuclein (alpha-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that alpha-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of alpha-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic alpha-syn mutations are rare, alpha-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of alpha-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified alpha-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Amphetamine and other psychostimulants reduce pH gradients in midbrain dopaminergic neurons and chromaffin granules: A mechanism of action

Rewarding properties of psychostimulants result from reduced uptake and/or increased release of dopamine at mesolimbic synapses. As exemplified by cocaine, many psychostimulants act by binding to the dopamine uptake transporter. However, this does not explain the action of other psychostimulants, including amphetamine. As most psychostimulants are weak bases and dopamine uptake into synaptic vesicles uses an interior-acidic pH gradient, we examined the possibility that psychostimulants might inhibit acidification. Pharmacologically relevant concentrations of amphetamine as well as cocaine and phencyclidine rapidly reduced pH gradients in cultured midbrain dopaminergic neurons. To examine direct effects on vesicles, we used chromaffin granules. The three psychostimulants, as well as fenfluramine, imipramine, and tyramine, reduced the pH gradient, resulting in reduced uptake and increased release of neurotransmitter. Inhibition of acidification by psychoactive amines contributes to their pharmacology and may provide a principal molecular mechanism of action of amphetamine.

Interplay between Cytosolic Dopamine, Calcium, and α-Synuclein Causes Selective Death of Substantia Nigra Neurons

The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinsons disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DA(cyt)) in cultured midbrain neurons, we confirm that elevated DA(cyt) and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DA(cyt) provide neuroprotection. L-DOPA increased DA(cyt) in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DA(cyt) was not altered by alpha-synuclein deletion, although dopaminergic neurons lacking alpha-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DA(cyt), and alpha-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets.

Heterosynaptic Dopamine Neurotransmission Selects Sets of Corticostriatal Terminals

Dopamine input to the striatum is required for voluntary motor movement, behavioral reinforcement, and responses to drugs of abuse. It is speculated that these functions are dependent on either excitatory or inhibitory modulation of corticostriatal synapses onto medium spiny neurons (MSNs). While dopamine modulates MSN excitability, a direct presynaptic effect on the corticostriatal input has not been clearly demonstrated. We combined optical monitoring of synaptic vesicle exocytosis from motor area corticostriatal afferents and electrochemical recordings of striatal dopamine release to directly measure effects of dopamine at the level of individual presynaptic terminals. Dopamine released by either electrical stimulation or amphetamine acted via D2 receptors to inhibit the activity of subsets of corticostriatal terminals. Optical and electrophysiological data suggest that heterosynaptic inhibition was enhanced by higher frequency stimulation and was selective for the least active terminals. Thus, dopamine, by filtering less active inputs, appears to reinforce specific sets of corticostriatal synaptic connections.

Loss of mTOR-Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

α-Synuclein Overexpression in PC12 and Chromaffin Cells Impairs Catecholamine Release by Interfering with a Late Step in Exocytosis

α-Synuclein (α-syn), a protein implicated in Parkinsons disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how α-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous α-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant α-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically “docked” vesicles was apparent in the α-syn-overexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) α-syn, as well as chromaffin cells from control and α-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant α-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P α-syn-overexpressing cells. The α-syn-overexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that α-syn overexpression inhibits a vesicle “priming” step, after secretory vesicle trafficking to “docking” sites but before calcium-dependent vesicle membrane fusion.

Vesicular Transport Regulates Monoamine Storage and Release but Is Not Essential for Amphetamine Action

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2-/- mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2-/- animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.