Alison I. Bernstein

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

Significance Several therapeutic strategies have been used to enhance monoamine neurotransmitter signaling. However, many of these interventions have deleterious side effects or lose effectiveness due to off-target actions and system feedback. These undesirable consequences likely occur because of temporal dysregulation of neurotransmitter release and uptake. We demonstrate that increasing vesicular packaging enhances dopamine neurotransmission without this signaling disruption. Mice with elevated vesicular monoamine transporter display increased dopamine release, improved outcomes on anxiety and depressive behaviors, enhanced locomotion, and protection from a Parkinson disease-related neurotoxic insult. The malleable nature of the dopamine vesicle suggests that interventions aimed at enhancing vesicle filling may be of therapeutic benefit. 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.

A locus affecting obesity in human chromosome region 10p12

Aims/hypothesis. Obesity is a complex trait influenced by multiple genes. We evaluated linkage in three regions of human chromosome 10 previously linked to obesity-related phenotypes. Methods. We conducted non-parametric linkage analysis of obesity-related phenotypes in cohorts of 170 European-American and 43 African-American families having extremely obese and normal weight subjects. Results. We found support for linkage of an obesity phenotype (BMI ≥ 27 kg/m2) in both cohorts, as well as in a combined analysis (European-American cohort, Z = 1.90, p = 0.03; African-American cohort, Z = 2.25, p = 0.014; combined cohort, Z = 2.55, p = 0.005). Conclusion/interpretation. These results confirm previous reports of linkage in French and German families. The consistency of results across these four cohorts supports the localization of a quantitative trait locus influencing obesity to human chromosome region 10p12. [Diabetologia (2001) 44: 363–366]

Fungal-derived semiochemical 1-octen-3-ol disrupts dopamine packaging and causes neurodegeneration

Significance Poor air quality from fungal growth in water-damaged, moldy buildings/residences is correlated with a negative impact on human health. The volatile organic compound 1-octen-3-ol is commonly emitted by molds and is responsible for much of the distinctive moldy odor associated with fungal colonization. Using a Drosophila model, we demonstrate via genetic, biochemical, and immunological studies that 1-octen-3-ol causes dopamine neuron degeneration through disruption of dopamine handling. These data demonstrate that 1-octen-3-ol exerts toxicity via disruption of dopamine homeostasis and may represent a naturally occurring environmental agent involved in parkinsonism. Moreover, it provides possible insights into reported movement disorders associated with human exposure to fungi and their volatile organic compounds. Parkinson disease (PD) is the most common movement disorder and, although the exact causes are unknown, recent epidemiological and experimental studies indicate that several environmental agents may be significant risk factors. To date, these suspected environmental risk factors have been man-made chemicals. In this report, we demonstrate via genetic, biochemical, and immunological studies that the common volatile fungal semiochemical 1-octen-3-ol reduces dopamine levels and causes dopamine neuron degeneration in Drosophila melanogaster. Overexpression of the vesicular monoamine transporter (VMAT) rescued the dopamine toxicity and neurodegeneration, whereas mutations decreasing VMAT and tyrosine hydroxylase exacerbated toxicity. Furthermore, 1-octen-3-ol also inhibited uptake of dopamine in human cell lines expressing the human plasma membrane dopamine transporter (DAT) and human VMAT ortholog, VMAT2. These data demonstrate that 1-octen-3-ol exerts toxicity via disruption of dopamine homeostasis and may represent a naturally occurring environmental agent involved in parkinsonism.

5-hydroxymethylcytosine: A new player in brain disorders?

5-Hydroxymethylcytosine (5 hmC), a novel modified cytosine, is oxidized from 5-methylcytosine (5 mC) by the ten-eleven translocation (Tet) protein family. The specific distribution of 5 hmC in mammalian brain and its roles in gene regulation suggest that 5 hmC is important in brain development. 5 hmC may also contribute to the mechanisms underlying neurological diseases. Here, we summarize the current knowledge of 5 hmC, with an emphasis on its roles in neurodevelopmental and neurodegenerative disorders.

The vesicular monoamine transporter 2: an underexplored pharmacological target.

Active transport of neurotransmitters into synaptic vesicles is required for their subsequent exocytotic release. In the monoamine system, this process is carried out by the vesicular monoamine transporters (VMAT1 and VMAT2). These proteins are responsible for vesicular packaging of dopamine, norepinephrine, serotonin, and histamine. These proteins are essential for proper neuronal function; however, compared to their plasma membrane counterparts, there are few drugs available that target these vesicular proteins. This is partly due to the added complexity of crossing the plasma membrane, but also to the technical difficulty of assaying for vesicular uptake in high throughput. Until recently, reagents to enable high throughput screening for function of these vesicular neurotransmitter transporters have not been available. Fortunately, novel compounds and methods are now making such screening possible; thus, a renewed focus on these transporters as potential targets is timely and necessary.

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.

Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease

Significance Here we describe a role for the synaptic vesicle glycoprotein 2C (SV2C) in dopamine neurotransmission and Parkinson disease (PD). SV2C is expressed on the vesicles of dopamine-producing neurons, and genetic deletion of SV2C causes a reduction in synaptic release of dopamine. The reduced dopamine release is associated with a decrease in motor activity. SV2C is suspected of mediating the neuroprotective effects of nicotine, and we show an ablated neurochemical response to nicotine in SV2C-knockout mice. Last, we demonstrate that SV2C expression is specifically disrupted in mice that express mutated α-synuclein and in humans with PD. Together, these data establish SV2C as an important mediator of dopamine homeostasis and a potential contributor to PD pathogenesis. Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

A fluorescent-based assay for live cell, spatially resolved assessment of vesicular monoamine transporter 2-mediated neurotransmitter transport

The vesicular monoamine transporter 2 (VMAT2; Slc18a2) packages monoamines into synaptic vesicles. Monoamine homeostasis is highly regulated and dysfunction may play a role in Parkinsons disease, Huntingtons disease, drug addiction, and neuropsychiatric disorders. The primary function of VMAT2 is to sequester monoamine neurotransmitters into vesicles for subsequent release; it also sequesters toxicants away from cytosolic sites of action. Identification of compounds that modify the action of VMAT2 may be useful as therapeutic agents for preventing or reversing monoamine-related toxicity. Current methods for measuring VMAT2 function are unable to assess uptake in intact cells. Here, we adapted the Neurotransmitter Uptake Assay (Molecular Devices) to develop a measure of VMAT2 function in live whole cells. This assay contains a fluorescent compound, which is transported into cells by the plasma membrane monoamine transporters and has been marketed as a rapid, high-throughput, plate reader based assay for function of these plasma membrane transporters. We demonstrate a modified version of this assay that can be used to visualize and measure transport into vesicles by VMAT2. HEK293 cell lines stably expressing the dopamine transporter and a mCherry-VMAT2 fusion protein were generated. Confocal microscopy confirmed that the fluorescent compound is transported into mCherry-positive compartments. Furthermore, the VMAT2-specific inhibitor tetrabenazine (TBZ) blocks uptake into the mCherry-positive compartment. Confocal images can be analyzed to generate a measure of VMAT2 activity. In summary, we demonstrate a method for spatially resolved analysis of VMAT2-mediated uptake in live intact cells.

MPP+-induces PUMA- and p53-dependent, but ATF3-independent cell death.

Parkinsons disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and depletion of striatal dopamine (DA), leading to a range of motor symptoms, including resting tremor, rigidity, bradykinesia and postural abnormalities. The neurotoxin (MPTP) and its active metabolite, 1-methyl-4-phenylpyridinium (MPP(+)), cause dopaminergic cell loss in a variety of animal species and produce symptoms similar to those seen in PD. Our lab has shown that MPP(+) activates cell stress pathways, including the unfolded protein response (UPR) in mouse primary mesencephalic cultures. The BH3-only protein, PUMA (p53 upregulated mediator of apoptosis), has been shown to be activated in response to many cellular stresses, including endoplasmic reticulum (ER) stress and UPR, and to induce cell death. Therefore, we hypothesized that PUMA may mediate MPP(+) toxicity. To test this hypothesis, we compared the response of primary mesencephalic cultures from wild-type and PUMA deficient (-/-) mice to MPP(+). We also utilized cultures from p53 -/- and activating transcription factor 3 (ATF3) -/- mice to further elucidate the pathways involved. These studies revealed that PUMA and p53, but not ATF3, are required for MPP(+)-induced cell death, suggesting that UPR activation is parallel to the induction of MPP(+)-induced cell death.

Vesicular Monoamine Transporter 2 (VMAT2) Level Regulates MPTP Vulnerability and Clearance of Excess Dopamine in Mouse Striatal Terminals

The vesicular monoamine transporter 2 (VMAT2) packages neurotransmitters for release during neurotransmission and sequesters toxicants into vesicles to prevent neuronal damage. In mice, low VMAT2 levels causes catecholaminergic cell loss and behaviors resembling Parkinsons disease, while high levels of VMAT2 increase dopamine release and protect against dopaminergic toxicants. However, comparisons across these VMAT2 mouse genotypes were impossible due to the differing genetic background strains of the animals. Following back-crossing to a C57BL/6 line, we confirmed that mice with approximately 95% lower VMAT2 levels compared with wild-type (VMAT2-LO) display significantly reduced vesicular uptake, progressive dopaminergic terminal loss with aging, and exacerbated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. Conversely, VMAT2-overexpressing mice (VMAT2-HI) are protected from the loss of striatal terminals following MPTP treatment. We also provide evidence that enhanced vesicular filling in the VMAT2-HI mice modifies the handling of newly synthesized dopamine, indicated by changes in indirect measures of extracellular dopamine clearance. These results confirm the role of VMAT2 in the protection of vulnerable nigrostriatal dopamine neurons and may also provide new insight into the side effects of L-DOPA treatments in Parkinsons disease.