Emmanuel N. Pothos

Leptin Regulation of the Mesoaccumbens Dopamine Pathway

Leptin is an adipose-derived hormone that acts on hypothalamic leptin receptors to regulate energy balance. Leptin receptors are also expressed in extrahypothalamic sites including the ventral tegmental area (VTA), critical to brain reward circuitry. We report that leptin targets DA and GABA neurons of the VTA, inducing phosphorylation of signal-transducer-and-activator-of-transcription-3 (STAT3). Retrograde tracing combined with pSTAT3 immunohistochemistry show leptin-responsive VTA neurons projecting to nucleus accumbens (NAc). Assessing leptin function in the VTA, we showed that ob/ob mice had diminished locomotor response to amphetamine and lacked locomotor sensitization to repeated amphetamine injections, both defects reversed by leptin infusion. Electrically stimulated DA release from NAc shell terminals was markedly reduced in ob/ob slice preparations, and NAc DA levels and TH expression were lower. These data define a role for leptin in mesoaccumbens DA signaling and indicate that the mesoaccumbens DA pathway, critical to integrating motivated behavior, responds to this adipose-derived signal.

Nigrostriatal Dopaminergic Deficits and Hypokinesia Caused by Inactivation of the Familial Parkinsonism-Linked Gene DJ-1

The manifestations of Parkinsons disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.

Impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice

Parkinsons disease (PD) is characterized by the selective vulnerability of the nigrostriatal dopaminergic circuit. Recently, loss-of-function mutations in the PTEN-induced kinase 1 (PINK1) gene have been linked to early-onset PD. How PINK1 deficiency causes dopaminergic dysfunction and degeneration in PD patients is unknown. Here, we investigate the physiological role of PINK1 in the nigrostriatal dopaminergic circuit through the generation and multidisciplinary analysis of PINK1−/− mutant mice. We found that numbers of dopaminergic neurons and levels of striatal dopamine (DA) and DA receptors are unchanged in PINK1−/− mice. Amperometric recordings, however, revealed decreases in evoked DA release in striatal slices and reductions in the quantal size and release frequency of catecholamine in dissociated chromaffin cells. Intracellular recordings of striatal medium spiny neurons, the major dopaminergic target, showed specific impairments of corticostriatal long-term potentiation and long-term depression in PINK1−/− mice. Consistent with a decrease in evoked DA release, these striatal plasticity impairments could be rescued by either DA receptor agonists or agents that increase DA release, such as amphetamine or l-dopa. These results reveal a critical role for PINK1 in DA release and striatal synaptic plasticity in the nigrostriatal circuit and suggest that altered dopaminergic physiology may be a pathogenic precursor to nigrostriatal degeneration.

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.

R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice

Dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinsons disease (PD). The importance of the R1441 residue in the pathogenesis is highlighted by the identification of three distinct missense mutations. To investigate the pathogenic mechanism underlying LRRK2 dysfunction, we generated a knockin (KI) mouse in which the R1441C mutation is expressed under the control of the endogenous regulatory elements. Homozygous R1441C KI mice appear grossly normal and exhibit no dopaminergic (DA) neurodegeneration or alterations in steady-state levels of striatal dopamine up to 2 years of age. However, these KI mice show reductions in amphetamine (AMPH)-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells. The introduction of the R1441C mutation also impairs dopamine D2 receptor function, as suggested by decreased responses of KI mice in locomotor activity to the inhibitory effect of a D2 receptor agonist, quinpirole. Furthermore, the firing of nigral neurons in R1441C KI mice show reduced sensitivity to suppression induced by quinpirole, dopamine, or AMPH. Together, our data suggest that the R1441C mutation in LRRK2 impairs stimulated dopamine neurotransmission and D2 receptor function, which may represent pathogenic precursors preceding dopaminergic degeneration in PD brains.

Restricted eating with weight loss selectively decreases extracellular dopamine in the nucleus accumbens and alters dopamine response to amphetamine, morphine, and food intake

Weight loss is known to alter food intake and drug self-administration, but the neural basis of this is unknown. Therefore, we studied effects of weight loss on neurochemistry of a brain mechanism involved in behavior reinforcement. In rats reduced 20-30% below normal weight, basal extracellular dopamine (DA) in the nucleus accumbens (NAC) decreased up to 50% (p < 0.01), as measured by in vivo microdialysis. No such change was observed in dorsal striatum (STR) or medial prefrontal cortex. In underweight rats, systemic amphetamine (1.5 mg/kg i.p.) transiently restored extracellular DA, but only to basal normal levels. Morphine (20 mg/kg i.p.) or a meal also increased DA, but the percent increase was significantly smaller in underweight than normal weight animals. Amphetamine infused locally by reverse dialysis in the NAC increased extracellular DA more in underweight animals than controls, suggesting that DA had accumulated in the presynaptic terminals. This was confirmed by finding significantly more DA in homogenized NAC micropunches of underweight rats. Receptor counts in micropunches and quantitative receptor autoradiography showed 3H- SCH23390 and 3H-spiperone D1- and D2-type binding in the NAC, STR, frontal cortex and hypothalamus did not change significantly. Locomotor activity was depressed suggesting that low DA release in the NAC may be related to energy conservation during weight loss. Low extracellular DA may also underlie the increase in food and drug intake typically observed in underweight animals and humans when they attempt to restore extracellular DA levels by natural or artificial means.

Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance

Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRb(Nts) neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRb(Nts) neurons with local orexin (OX) neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRb(Nts) neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.

Regulation of Quantal Size by Presynaptic Mechanisms

Quantal size is often modeled as invariant, although it is now well established that the number of transmitter molecules released per synaptic vesicle during exocytosis can be modulated in central and peripheral synapses. In this review, we suggest why presynaptically altered quantal size would be important at social synapses that provide extrasynaptic neurotransmitter. Current techniques used to measure quantal size are reviewed with particular attention to amperometry, the first approach to provide direct measurement of the number of molecules and kinetics of presynaptic quantal release, and to CNS dopamine neuronal terminals. The known interventions that alter quantal size at the presynaptic locus are reviewed and categorized as (1) alteration of transvesicular free energy gradients, (2) modulation of vesicle transmitter transporter activity, (3) modulation of fusion pore kinetics, (4) altered transmitter degranulation, and (5) changes in synaptic vesicle volume. Modulation of the number of molecules released per quantum underlies mechanisms of drug action of L-DOPA and the amphetamines, and seems likely to be involved in both normal synaptic modification and disease states. Statistical analysis for examining quantal size and data presentation is discussed. We include detailed information on performing nonparametric resampling statistical analysis, the Kolmogorov-Smirnov test for two populations, and random walk simulations using spreadsheet programs.

Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats

The association between dietary obesity and mesolimbic systems that regulate hedonic aspects of feeding is currently unresolved. In the present study, we examined differences in baseline and stimulated central dopamine levels in obesity‐prone (OP) and obesity‐resistant (OR) rats. OP rats were hyperphagic and showed a 20% weight gain over OR rats at wk 15 of age, when fed a standard chow diet. This phenotype was associated with a 50% reduction in basal extracellular dopamine, as measured by a microdialysis probe in the nucleus accumbens, a projection site of the mesolimbic dopamine system that has been implicated in food reward. Similar defects were also observed in younger animals (4 wk old). In electrophysiology studies, electrically evoked dopamine release in slice preparations was significantly attenuated in OP rats, not only in the nucleus accumbens but also in additional terminal sites of dopamine neurons such as the accumbens shell, dorsal striatum, and medial prefrontal cortex, suggesting that there may be a widespread dysfunction in mechanisms regulating dopamine release in this obesity model. Moreover, dopamine impairment in OP rats was apparent at birth and associated with changes in expression of several factors regulating dopamine synthesis and release: vesicular monoamine transporter‐2, tyrosine hydroxylase, dopamine transporter, and dopamine receptor‐2 short‐form. Taken together, these results suggest that an attenuated central dopamine system would reduce the hedonic response associated with feeding and induce compensatory hyperphagia, leading to obesity.—Geiger, B. M., Behr, G. G., Frank, L. E., Caldera‐Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. Evidence for defective mesolimbic dopamine exocytosis in obesity‐prone rats. FASEB J. 22, 2740–2746 (2008)

DJ-1 Transcriptionally Up-regulates the Human Tyrosine Hydroxylase by Inhibiting the Sumoylation of Pyrimidine Tract-binding Protein-associated Splicing Factor

Loss-of-function mutations in DJ-1 cause a subset of familial Parkinson disease (PD). However, the mechanism underlying the selective vulnerability in dopaminergic pathway due to the inactivation of DJ-1 is unclear. Previously, we have reported that DJ-1 is a neuroprotective transcriptional co-activator interacting with the transcriptional co-repressor pyrimidine tract-binding protein-associated splicing factor (PSF). Here we show that DJ-1 and PSF bind and regulate the human tyrosine hydroxylase (TH) promoter. Inactivation of DJ-1 by small interference RNA (siRNA) results in decreased TH expression and l-DOPA production in human dopaminergic cell lines. Consistent with its role as a transcriptional regulator, DJ-1 specifically suppresses the global SUMO-1 modification. High molecular weight sumoylated protein species, including PSF, accumulate in the lymphoblast cells from the patients carrying pathogenic DJ-1 mutations. DJ-1 elevates the TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Furthermore, siRNA silencing of DJ-1 decreases the acetylation of TH promoter-bound histones, and histone deacetylase inhibitors restore the DJ-1 siRNA-induced repression of TH. Therefore, our results suggest DJ-1 as a regulator of protein sumoylation and directly link the loss of DJ-1 expression and transcriptional dysfunction to impaired dopamine synthesis.