Selective Enhancement of Dopamine Release in the Ventral Pallidum of Methamphetamine-Sensitized Mice.

Drugs of abuse induce sensitization, which is defined as enhanced response to additional drug following a period of withdrawal. Sensitization occurs in both humans and animal models of drug reinforcement and contributes substantially to the addictive nature of drugs of abuse, because it is thought to represent enhanced motivational wanting for drug. The ventral pallidum, a key member of the reward pathway, contributes to behaviors associated with reward, such as sensitization. Dopamine inputs to the ventral pallidum have not been directly characterized. Here we provide anatomical, neurochemical, and behavioral evidence demonstrating that dopamine terminals in the ventral pallidum contribute to reward in mice. We report subregional differences in dopamine release, measured by ex vivo fast-scan cyclic voltammetry: rostral ventral pallidum exhibits increased dopamine release and uptake compared with caudal ventral pallidum, which is correlated with tissue expression of dopaminergic proteins. We then subjected mice to a methamphetamine-sensitization protocol to investigate the contribution of dopaminergic projections to the region in reward related behavior. Methamphetamine-sensitized animals displayed a 508% and 307% increase in baseline dopamine release in the rostral and caudal ventral pallidum, respectively. Augmented dopamine release in the rostral ventral pallidum was significantly correlated with sensitized locomotor activity. Moreover, this presynaptic dopaminergic plasticity occurred only in the ventral pallidum and not in the ventral or dorsal striatum, suggesting that dopamine release in the ventral pallidum may be integrally important to drug-induced sensitization.

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.

Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo.

Disruption of neurotransmitter vesicle dynamics (transport, capacity, release) has been implicated in a variety of neurodegenerative and neuropsychiatric conditions. Here, we report a novel mouse model of enhanced vesicular function via bacterial artificial chromosome (BAC)-mediated overexpression of the vesicular monoamine transporter 2 (VMAT2; Slc18a2). A twofold increase in vesicular transport enhances the vesicular capacity for dopamine (56%), dopamine vesicle volume (33%), and basal tissue dopamine levels (21%) in the mouse striatum. The elevated vesicular capacity leads to an increase in stimulated dopamine release (84%) and extracellular dopamine levels (44%). VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and increased basal locomotor activity (41%). Finally, these mice exhibit significant protection from neurotoxic insult by the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as measured by reduced dopamine terminal damage and substantia nigra pars compacta cell loss. The increased release of dopamine and neuroprotection from MPTP toxicity in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in Parkinson disease.

Reduced vesicular storage of catecholamines causes progressive degeneration in the locus ceruleus.

Parkinson's disease (PD) is the most common neurodegenerative motor disease. Pathologically, PD is characterized by Lewy body deposition and subsequent death of dopamine neurons in the substantia nigra pars compacta. PD also consistently features degeneration of the locus ceruleus, the main source of norepinephrine in the central nervous system. We have previously reported a mouse model of dopaminergic neurodegeneration based on reduced expression of the vesicular monoamine transporter (VMAT2 LO). To determine if reduced vesicular storage can also cause noradrenergic degeneration, we examined indices of damage to the catecholaminergic systems in brain and cardiac tissue of VMAT2 LO mice. At two months of age, neurochemical analyses revealed substantial reductions in striatal dopamine (94%), cortical dopamine (57%) and norepinephrine (54%), as well as cardiac norepinephrine (97%). These losses were accompanied by increased conversion of dopamine and norepinephrine to their deaminated metabolites. VMAT2 LO mice exhibited loss of noradrenergic innervation in the cortex, as determined by norepinephrine transporter immunoreactivity and (3)H-nisoxetine binding. Using unbiased stereological techniques, we observed progressive degeneration in the locus ceruleus that preceded degeneration of the substantia nigra pars compacta. In contrast, the ventral tegmental area, which is spared in human PD, remained unaffected. The coordinate loss of dopamine and norepinephrine neurons in VMAT2 LO mice parallels the pattern of neurodegeneration that occurs in human PD, and demonstrates that insufficient catecholamine storage can cause spontaneous degeneration in susceptible neurons, underscoring cytosolic catecholamine catabolism as a determinant of neuronal susceptibility in PD. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'.

Targeting protein tyrosine phosphatase SHP2 for the treatment of PTPN11-associated malignancies.

Activating mutations in PTPN11 (encoding SHP2), a protein tyrosine phosphatase (PTP) that plays an overall positive role in growth factor and cytokine signaling, are directly associated with the pathogenesis of Noonan syndrome and childhood leukemias. Identification of SHP2-selective inhibitors could lead to the development of new drugs that ultimately serve as treatments for PTPN11-associated diseases. As the catalytic core of SHP2 shares extremely high homology to those of SHP1 and other PTPs that play negative roles in cell signaling, to identify selective inhibitors of SHP2 using computer-aided drug design, we targeted a protein surface pocket that is adjacent to the catalytic site, is predicted to be important for binding to phosphopeptide substrates, and has structural features unique to SHP2. From computationally selected candidate compounds, #220-324 effectively inhibited SHP2 activity with an IC50 of 14 µmol/L. Fluorescence titration experiments confirmed its direct binding to SHP2. This active compound was further verified for its ability to inhibit SHP2-mediated cell signaling and cellular function with minimal off-target effects. Furthermore, mouse myeloid progenitors with the activating mutation (E76K) in PTPN11 and patient leukemic cells with the same mutation were more sensitive to this inhibitor than wild-type cells. This study provides evidence that SHP2 is a "druggable" target for the treatment of PTPN11-associated diseases. As the small-molecule SHP2 inhibitor identified has a simple chemical structure, it represents an ideal lead compound for the development of novel anti-SHP2 drugs. Mol Cancer Ther; 12(9); 1738-48. ©2013 AACR.

Determinants of buildup of the toxic dopamine metabolite DOPAL in Parkinson's disease.

Intra-neuronal metabolism of dopamine (DA) begins with production of 3,4-dihydroxyphenylacetaldehyde (DOPAL),which is toxic. According to the 'catecholaldehyde hypothesis', DOPAL destroys nigrostriatal DA terminals and contributes to the profound putamen DA deficiency that characterizes Parkinson's disease (PD). We tested the feasibility of using post-mortem patterns of putamen tissue catechols to examine contributions of altered activities of the type 2 vesicular monoamine transporter (VMAT2) and aldehyde dehydrogenase(ALDH) to the increased DOPAL levels found in PD. Theoretically, the DA : DOPA concentration ratio indicates vesicular uptake, and the 3,4-dihydroxyphenylacetic acid: DOPAL ratio indicates ALDH activity. We validated these indices in transgenic mice with very low vesicular uptake VMAT2-Lo) or with knockouts of the genes encoding ALDH1A1 and ALDH2 (ALDH1A1,2 KO), applied these indices in PD putamen, and estimated the percent decreases in vesicular uptake and ALDH activity in PD. VMAT2-Lo mice had markedly decreased DA:DOPA (50 vs. 1377, p < 0.0001),and ALDH1A1,2 KO mice had decreased 3,4-dihydroxyphenylacetic acid:DOPAL (1.0 vs. 11.2, p < 0.0001). In PD putamen, vesicular uptake was estimated to be decreased by 89% and ALDH activity by 70%. Elevated DOPAL levels in PD putamen reflect a combination of decreased vesicular uptake of cytosolic DA and decreased DOPAL detoxification by ALDH.

Vesicular integrity in Parkinson's disease.

The defining motor characteristics of Parkinson's disease (PD) are mediated by the neurotransmitter dopamine (DA). Dopamine molecules spend most of their lifespan stored in intracellular vesicles awaiting release and very little time in the extracellular space or the cytosol. Without proper packaging of transmitter and trafficking of vesicles to the active zone, dopamine neurotransmission cannot occur. In the cytosol, dopamine is readily oxidized; excessive cytosolic dopamine oxidation may be pathogenic to nigral neurons in PD. Thus, factors that disrupt vesicular function may impair signaling and increase the vulnerability of dopamine neurons. This review outlines the many mechanisms by which disruption of vesicular function may contribute to the pathogenesis of PD. From direct inhibition of dopamine transport into vesicles by pharmacological or toxicological agents to alterations in vesicle trafficking by PD-related gene products, variations in the proper compartmentalization of dopamine can wreak havoc on a functional dopamine pathway. Findings from patient populations, imaging studies, transgenic models, and mechanistic studies will be presented to document the relationship between impaired vesicular function and vulnerability of the nigrostriatal dopamine system. Given the deleterious effects of impaired vesicular function, strategies aimed at enhancing vesicular function may be beneficial in the treatment of PD.

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ß-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ß-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.

SHP-2 tyrosine phosphatase in human diseases.

SHP-2, a ubiquitously expressed Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTP), plays a critical role in physiology and disease. SHP-2 has been clearly demonstrated to be an important molecule in various cytoplasmic signal transduction pathways. In addition, emerging evidence indicates that SHP-2 may function in the nucleus and in the mitochondria. However, the signaling mechanisms of SHP-2 are not completely understood. Interestingly, genetic mutations in SHP-2 that either enhance or inactivate its catalytic activity have been identified in human diseases with overlapping phenotypes. In light of this hint given by nature, new cell and animal models now provide the opportunity to uncover how this molecule functions in multiple cellular processes, and more importantly, how its known mutations induce human diseases.