PLCγ1 in dopamine neurons critically regulates striatal dopamine release via VMAT2 and synapsin III.

Dopamine neurons are essential for voluntary movement, reward learning, and motivation, and their dysfunction is closely linked to various psychological and neurodegenerative diseases. Hence, understanding the detailed signaling mechanisms that functionally modulate dopamine neurons is crucial for the development of better therapeutic strategies against dopamine-related disorders. Phospholipase Cγ1 (PLCγ1) is a key enzyme in intracellular signaling that regulates diverse neuronal functions in the brain. It was proposed that PLCγ1 is implicated in the development of dopaminergic neurons, while the physiological function of PLCγ1 remains to be determined. In this study, we investigated the physiological role of PLCγ1, one of the key effector enzymes in intracellular signaling, in regulating dopaminergic function in vivo. We found that cell type-specific deletion of PLCγ1 does not adversely affect the development and cellular morphology of midbrain dopamine neurons but does facilitate dopamine release from dopaminergic axon terminals in the striatum. The enhancement of dopamine release was accompanied by increased colocalization of vesicular monoamine transporter 2 (VMAT2) at dopaminergic axon terminals. Notably, dopamine neuron-specific knockout of PLCγ1 also led to heightened expression and colocalization of synapsin III, which controls the trafficking of synaptic vesicles. Furthermore, the knockdown of VMAT2 and synapsin III in dopamine neurons resulted in a significant attenuation of dopamine release, while this attenuation was less severe in PLCγ1 cKO mice. Our findings suggest that PLCγ1 in dopamine neurons could critically modulate dopamine release at axon terminals by directly or indirectly interacting with synaptic machinery, including VMAT2 and synapsin III.

Adaptor protein-3 produces synaptic vesicles that release phasic dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Dopaminergic cell protection and alleviation of neuropsychiatric disease symptoms by VMAT2 expression through the class I HDAC inhibitor TC-H 106.

The importance of vesicular monoamine transporter 2 (VMAT2) in dopamine regulation, which is considered crucial for neuropsychiatric disorders, is currently being studied. Moreover, the development of disease treatments using histone deacetylase (HDAC) inhibitors (HDACi) is actively progressing in various fields. Recently, research on the possibility of regulating neuropsychiatric disorders has been conducted. In this study, we evaluated whether VMAT2 expression increased by an HDACi can fine-tune neuropsychotic behavior, such as attention deficit hyperactivity disorder (ADHD) and protect against the cell toxicity through oxidized dopamine. First, approximately 300 candidate HDACi compounds were added to the SH-SY5Y dopaminergic cell line to identify the possible changes in the VMAT2 expression levels, which were measured using quantitative polymerase chain reaction. The results demonstrated, that treatment with pimelic diphenylamide 106 (TC-H 106), a class I HDACi, increased VMAT2 expression in both the SH-SY5Y cells and mouse brain. The increased VMAT2 expression induced by TC-H 106 alleviated the cytotoxicity attributed to 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+ ) and free dopamine treatment. Moreover, dopamine concentrations, both intracellularly and in the synaptosomes, were significantly elevated by increased VMAT2 expression. These results suggest that dopamine concentration regulation by VMAT2 expression induced by TC-H 106 could alter several related behavioral aspects that was confirmed by attenuation of hyperactivity and impulsivity, which were major characteristics of animal model showing ADHD-like behaviors. These results indicate that HDACi-increased VMAT2 expression offers sufficient protections against dopaminergic cell death induced by oxidative stress. Thus, the epigenetic approach could be considered as therapeutic candidate for neuropsychiatric disease regulation.

Single-cell transcriptome analysis of NEUROG3+ cells during pancreatic endocrine differentiation with small molecules.

The efficiency of inducing human embryonic stem cells into NEUROG3+ pancreatic endocrine cells is a bottleneck in stem cell therapy for diabetes. To understand the cell properties and fate decisions during differentiation, we analyzed the modified induction method using single-cell transcriptome and found that DAPT combined with four factors (4FS): nicotinamide, dexamethasone, forskolin and Alk5 inhibitor II (DAPT + 4FS) increased the expression of NEUROG3 to approximately 34.3%. The increased NEUROG3+ cells were mainly concentrated in Insulin + Glucagon + (INS + GCG+) and SLAC18A1 + Chromogranin A+(SLAC18A1 + CHGA +) populations, indicating that the increased NEUROG3+ cells promoted the differentiation of pancreatic endocrine cells and enterochromaffin-like cells. Single-cell transcriptome analysis provided valuable clues for further screening of pancreatic endocrine cells and differentiation of pancreatic islet cells. The gene set enrichment analysis (GSEA) suggest that we can try to promote the expression of INS + GCG+ population by up-regulating G protein-coupled receptor (GPCR) and mitogen-activated protein kinase signals and down-regulating Wnt, NIK/NF-KappaB and cytokine-mediated signal pathways. We can also try to regulate GPCR signaling through PLCE1, so as to increase the proportion of NEUROG3+ cells in INS+GCG+ populations. To exclude non-pancreatic endocrine cells, ALCAMhigh CD9low could be used as a marker for endocrine populations, and ALCAMhigh CD9lowCDH1low could remove the SLC18A1 + CHGA+ population.

A case report of infantile parkinsonism-dystonia-2 caused by homozygous mutation in the SLC18A2 gene.

Background: The monoamine neurotransmitter disorders are neurometabolic syndromes caused by disturbances in the synthesis, transport and metabolism of the biogenic amines (the catecholamines dopamine, norepinephrine and epinephrine; serotonin), which are increasingly recognized as an expanding group of inherited neurometabolic syndromes.Case Description: A 6-month-old male infant who presented with developmental delay and suspected cerebral palsy was diagnosed with infantile parkinsonism-dystonia-2 (MIM: 618049). The whole-exome sequencing identified a homozygous c.710C > T (p.Pro237His) transition in the monoamine transporter gene SLC18A2, which was due to paternal uniparental disomy (UPD) of chromosome 10p15.3q26.3, resulting in brain dopamine-serotonin vesicular transport disease. Sanger sequencing confirmed that his unaffected father carried the same mutation in the heterozygous state, while his mother did not carry the same mutation. Autosomal recessive gene mutations in SLC18A2 has been identified in three families in different countries. The infant was treated with pramipexole, a dopamine agonist, and the static tremor was better compared with that before treatment, but the movement disorder was not significantly improved.Conclusion: This case confirmed the causal mutation of SLC18A2 gene and brain dopamine-serotonin vesicular transport disease, which suggested the mechanism of UPD homozygous formation, and confirmed that dopamine agonist treatment could improve some symptoms in affected individuals.

Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.

PURPOSE: Brain monoamine vesicular transport disease is an infantile-onset movement disorder that mimics cerebral palsy. In 2013, the homozygous SLC18A2 variant, p.Pro387Leu, was first reported as a cause of this rare disorder, and dopamine agonists were efficient for treating affected individuals from a single large family. To date, only 6 variants have been reported. In this study, we evaluated genotype-phenotype correlations in individuals with biallelic SLC18A2 variants. METHODS: A total of 42 affected individuals with homozygous SLC18A2 variant alleles were identified. We evaluated genotype-phenotype correlations and the missense variants in the affected individuals based on the structural modeling of rat VMAT2 encoded by Slc18a2, with cytoplasm- and lumen-facing conformations. A Caenorhabditis elegans model was created for functional studies. RESULTS: A total of 19 homozygous SLC18A2 variants, including 3 recurrent variants, were identified using exome sequencing. The affected individuals typically showed global developmental delay, hypotonia, dystonia, oculogyric crisis, and autonomic nervous system involvement (temperature dysregulation/sweating, hypersalivation, and gastrointestinal dysmotility). Among the 58 affected individuals described to date, 16 (28%) died before the age of 13 years. Of the 17 patients with p.Pro237His, 9 died, whereas all 14 patients with p.Pro387Leu survived. Although a dopamine agonist mildly improved the disease symptoms in 18 of 21 patients (86%), some affected individuals with p.Ile43Phe and p.Pro387Leu showed milder phenotypes and presented prolonged survival even without treatment. The C. elegans model showed behavioral abnormalities. CONCLUSION: These data expand the phenotypic and genotypic spectra of SLC18A2-related disorders.

Inhibition of VMAT2 by ß2-adrenergic agonists, antagonists, and the atypical antipsychotic ziprasidone.

Vesicular monoamine transporter 2 (VMAT2) is responsible for packing monoamine neurotransmitters into synaptic vesicles for storage and subsequent neurotransmission. VMAT2 inhibitors are approved for symptomatic treatment of tardive dyskinesia and Huntington's chorea, but despite being much-studied inhibitors their exact binding site and mechanism behind binding and inhibition of monoamine transport are not known. Here we report the identification of several approved drugs, notably ß2-adrenergic agonists salmeterol, vilanterol and formoterol, ß2-adrenergic antagonist carvedilol and the atypical antipsychotic ziprasidone as inhibitors of rat VMAT2. Further, plausible binding modes of the established VMAT2 inhibitors reserpine and tetrabenazine and hit compounds salmeterol and ziprasidone were identified using molecular dynamics simulations and functional assays using VMAT2 wild-type and mutants. Our findings show VMAT2 as a potential off-target of treatments with several approved drugs in use today and can also provide important first steps in both drug repurposing and therapy development targeting VMAT2 function.

Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks.

Receptors, transporters, and ion channels are important targets for therapy development in neurological diseases, but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on the whole-brain level. We combine CRISPR-Cas9 gene editing with in vivo positron emission tomography (PET) and functional MRI (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene network relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR-Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a tool to investigate the impact of specific genes on brain molecular and functional dynamics, which will help to develop tailored therapies for normalizing brain function.

Sclerosing Paragangliomas: Correlations of Histological Features with Patients' Genotype and Vesicular Monoamine Transporter Expression.

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score ≥ 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas.

Motility phenotype in a zebrafish vmat2 mutant.

In the present study, we characterize a novel zebrafish mutant of solute carrier 18A2 (slc18a2), also known as vesicular monoamine transporter 2 (vmat2), that exhibits a behavioural phenotype partially consistent with human Parkinson´s disease. At six days-post-fertilization, behaviour was analysed and demonstrated that vmat2 homozygous mutant larvae, relative to wild types, show changes in motility in a photomotor assay, altered sleep parameters, and reduced dopamine cell number. Following an abrupt lights-off stimulus mutant larvae initiate larger movements but subsequently inhibit them to a lesser extent in comparison to wild-type larvae. Conversely, during a lights-on period, the mutant larvae are hypomotile. Thigmotaxis, a preference to avoid the centre of a behavioural arena, was increased in homozygotes over heterozygotes and wild types, as was daytime sleep ratio. Furthermore, incubating mutant larvae in pramipexole or L-Dopa partially rescued the motor phenotypes, as did injecting glial cell-derived neurotrophic factor (GDNF) into their brains. This novel vmat2 model represents a tool for high throughput pharmaceutical screens for novel therapeutics, in particular those that increase monoamine transport, and for studies of the function of monoamine transporters.

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish.

AIM: We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. METHODS: A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. RESULTS: The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. CONCLUSION: Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.

Combined Exposure to Metals in Drinking Water Alters the Dopamine System in Mouse Striatum.

Environmental exposure to arsenic (As), lead (Pb), and cadmium (Cd) frequently occurs; however, data on the specific effects of combined exposure on neurotransmission, specifically dopaminergic neurotransmission, are lacking. In this study, motor coordination and dopamine content, along with the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and dopamine receptors (DRs), were examined in the striatum of adult male mice following exposure to drinking water containing As, Pb, and/or Cd. We found that exposure to a metal mixture impaired motor coordination. After 4 weeks of treatment, a significant decrease in dopamine content and expression of TH, DAT, and VMAT2 was observed in the striatum of metal-mixture-treated mice, compared to the controls or single-metal-exposed groups. However, DRD1 and DRD2 expression did not significantly change with metal treatment. These results suggest that altered dopaminergic neurotransmission by the collective action of metals may contribute to metal-mixture-induced neurobehavioral disorders.

Restoration of Noradrenergic Function in Parkinson's Disease Model Mice.

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine ß-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

Genetic or Toxicant-Induced Disruption of Vesicular Monoamine Storage and Global Metabolic Profiling in Caenorhabditis elegans.

The proper storage and release of monoamines contributes to a wide range of neuronal activity. Here, we examine the effects of altered vesicular monoamine transport in the nematode Caenorhabditis elegans. The gene cat-1 is responsible for the encoding of the vesicular monoamine transporter (VMAT) in C. elegans and is analogous to the mammalian vesicular monoamine transporter 2 (VMAT2). Our laboratory has previously shown that reduced VMAT2 activity confers vulnerability on catecholamine neurons in mice. The purpose of this article was to determine whether this function is conserved and to determine the impact of reduced VMAT activity in C. elegans. Here we show that deletion of cat-1/VMAT increases sensitivity to the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) as measured by enhanced degeneration of dopamine neurons. Reduced cat-1/VMAT also induces changes in dopamine-mediated behaviors. High-resolution mass spectrometry-based metabolomics in the whole organism reveals changes in amino acid metabolism, including tyrosine metabolism in the cat-1/VMAT mutants. Treatment with MPP+ disrupted tryptophan metabolism. Both conditions altered glycerophospholipid metabolism, suggesting a convergent pathway of neuronal dysfunction. Our results demonstrate the evolutionarily conserved nature of monoamine function in C. elegans and further suggest that high-resolution mass spectrometry-based metabolomics can be used in this model to study environmental and genetic contributors to complex human disease.

Targeting barrel field spiny stellate cells using a vesicular monoaminergic transporter 2-Cre mouse line.

Rodent primary somatosensory cortex (S1) is organized in defined layers, where layer IV serves as the main target for thalamocortical projections. Serotoninergic signaling is important for the organization of thalamocortical projections and consequently proper barrel field development in rodents, and the vesicular monoamine transporter 2 (VMAT2) can be detected locally in layer IV S1 cortical neurons in mice as old as P10, but the identity of the Vmat2-expressing neurons is unknown. We here show that Vmat2 mRNA and also Vmat2-Cre recombinase are still expressed in adult mice in a sub-population of the S1 cortical neurons in the barrel field. The Vmat2-Cre cells showed a homogenous intrinsically bursting firing pattern determined by whole-cell patch-clamp, localized radial densely spinous basal dendritic trees and almost exclusively lack of apical dendrite, indicative of layer IV spiny stellate cells. Single cell mRNA sequencing analysis showed that S1 cortical Vmat2-Cre;tdTomato cells express the layer IV marker Rorb and mainly cluster with layer IV neurons, and RNAscope analysis revealed that adult Vmat2-Cre neurons express Vmat2 and vesicular glutamate transporter 1 (Vglut1) and Vglut2 mRNA to a high extent. In conclusion, our analysis shows that cortical Vmat2 expression is mainly confined to layer IV neurons with morphological, electrophysiological and transcriptional characteristics indicative of spiny stellate cells.

Ambient Light Regulates Retinal Dopamine Signaling and Myopia Susceptibility.

Purpose: Exposure to high-intensity or outdoor lighting has been shown to decrease the severity of myopia in both human epidemiological studies and animal models. Currently, it is not fully understood how light interacts with visual signaling to impact myopia. Previous work performed in the mouse retina has demonstrated that functional rod photoreceptors are needed to develop experimentally-induced myopia, alluding to an essential role for rod signaling in refractive development. Methods: To determine whether dim rod-dominated illuminance levels influence myopia susceptibility, we housed male C57BL/6J mice under 12:12 light/dark cycles with scotopic (1.6 × 10-3 candela/m2), mesopic (1.6 × 101 cd/m2), or photopic (4.7 × 103 cd/m2) lighting from post-natal day 23 (P23) to P38. Half the mice received monocular exposure to -10 diopter (D) lens defocus from P28-38. Molecular assays to measure expression and content of DA-related genes and protein were conducted to determine how illuminance and lens defocus alter dopamine (DA) synthesis, storage, uptake, and degradation and affect myopia susceptibility in mice. Results: We found that mice exposed to either scotopic or photopic lighting developed significantly less severe myopic refractive shifts (lens treated eye minus contralateral eye; -1.62 ± 0.37D and -1.74 ± 0.44D, respectively) than mice exposed to mesopic lighting (-3.61 ± 0.50D; P < 0.005). The 3,4-dihydroxyphenylacetic acid /DA ratio, indicating DA activity, was highest under photopic light regardless of lens defocus treatment (controls: 0.09 ± 0.011 pg/mg, lens defocus: 0.08 ± 0.008 pg/mg). Conclusions: Lens defocus interacted with ambient conditions to differentially alter myopia susceptibility and DA-related genes and proteins. Collectively, these results show that scotopic and photopic lighting protect against lens-induced myopia, potentially indicating that a broad range of light levels are important in refractive development.

Improved functional and histochemical outcomes in l-DOPA plus tolcapone treated VMAT2-deficient mice.

Parkinson disease is typically treated with L-3,4-dihydroxyphenylalanine (or levodopa) co-prescribed with concentration stabilizers to prevent undesired motor fluctuations. However, the beneficial role of the chronic combined therapy on disease progression has not been thoroughly explored. We hypothesized that tolcapone, a catechol-O-methyl-transferase inhibitor, co-administered with levodopa may offer beneficial long-term disease-modifying effects through its dopamine stabilization actions. Here, we followed vesicular monoamine transporter 2-deficient and wild-type mice treated twice daily per os with vehicle, levodopa (20 mg/kg), tolcapone (15 mg/kg) or levodopa (12.5 mg/kg) + tolcapone (15 mg/kg) for 17 weeks. We assessed open field, bar test and rotarod performances at baseline and every 4th week thereafter, corresponding to OFF-medication weeks. Finally, we collected coronal sections from the frontal caudate-putamen and determined the reactivity level of dopamine transporter. Vesicular monoamine transporter 2-deficient mice responded positively to chronic levodopa + tolcapone intervention in the bar test during OFF-periods. Neither levodopa nor tolcapone interventions offered significant improvements on their own. Similarly, chronic levodopa + tolcapone intervention was associated with partially rescued dopamine transporter levels, whereas animals treated solely with levodopa or tolcapone did not present this effect. Interestingly, 4-month progression of bar test scores correlated significantly with dopamine-transporter-label density. Overall, we observed a moderate functional and histopathological improvement effect by chronic dopamine replacement when combined with tolcapone in vesicular monoamine transporter 2-deficient mice. Altogether, chronic stabilization of dopamine levels by catechol-O-methyl-transferase inhibition, besides its intended immediate actions, arises as a potential long-term beneficial approach during the progression of Parkinson disease.

VMAT2 Safeguards ß-Cells Against Dopamine Cytotoxicity Under High-Fat Diet-Induced Stress.

Vesicular monoamine transporter 2 (VMAT2) uptakes cytoplasmic monoamines into vesicles for storage. VMAT2 plays a role in modulating insulin release by regulating dopamine levels in the pancreas, although the exact mechanism remains elusive. We found that VMAT2 expression in ß-cells specifically increases under high blood glucose conditions. The islets isolated from ß-cell-specific Vmat2 knockout (ßVmat2KO) mice show elevated insulin secretion levels in response to glucose stimulation. Under prolonged high-fat diet feedings, the ßVmat2KO mice exhibit impaired glucose and insulin tolerance and progressive ß-cell dysfunction. Here we demonstrate VMAT2 uptake of dopamine to protect dopamine from degradation by monoamine oxidase, thereby safeguarding ß-cells from excess reactive oxygen species (ROS) exposure. In the context of high demand for insulin secretion, the absence of VMAT2 leads to elevated ROS in ß-cells, which accelerates ß-cell dedifferentiation and ß-cell loss. Therefore, VMAT2 controls the amount of dopamine in ß-cells, thereby protecting pancreatic ß-cells from excessive oxidative stress.

Differences in the expression of catecholamine-synthesizing enzymes between vesicular monoamine transporter 1- and 2-immunoreactive glomus cells in the rat carotid body.

Vesicular monoamine transporters (VMAT) 1 and 2 are responsible for monoamine transportation into secretary vesicles and are tissue-specifically expressed in central and peripheral monoaminergic tissues, including the carotid body (CB). The aim of the present study was to examine the expression of catecholamine-synthesizing enzymes in VMAT1- and VMAT2-immunoreactive glomus cells in the rat CB using multiple immunolabeling. The expression of VMAT1 and VMAT2 mRNA in the CB was confirmed by RT-PCR. Immunohistochemistry revealed that VMAT1 immunoreactivity was predominant in glomus cells rather than VMAT2 immunoreactivity. Glomus cells with VMAT1 immunoreactivity exhibited weak/negative VMAT2 immunoreactivity, and vice versa. Immunoreactivities for VMAT1 and tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, were co-localized in the same glomus cells and a positive correlation was confirmed between the two immunoreactivities (Spearman's coefficient = 0.82; p <  0.05). Although some glomus cells showed co-localization of VMAT2 and dopamine ß-hydroxylase immunoreactivity, the biosynthetic enzyme for noradrenaline, VMAT2 immunoreactivity appeared to be less associated with both catecholamine-synthesizing enzymes as indicated by a correlation analysis (TH: Spearman's coefficient = 0.38, DBH: Spearman's coefficient = 0.26). These results indicate that heterogeneity on functional role would exist among glomus cells in terms of VMAT isoform and catecholamine-synthesizing enzymes expression.

Vesicular monoamine transporter 2 mediates fear behavior in mice.

A subset of people exposed to a traumatic event develops post-traumatic stress disorder (PTSD), which is associated with dysregulated fear behavior. Genetic variation in SLC18A2, the gene that encodes vesicular monoamine transporter 2 (VMAT2), has been reported to affect risk for the development of PTSD in humans. Here, we use transgenic mice that express either 5% (VMAT2-LO mice) or 200% (VMAT2-HI mice) of wild-type levels of VMAT2 protein. We report that VMAT2-LO mice have reduced VMAT2 protein in the hippocampus and amygdala, impaired monoaminergic vesicular storage capacity in both the striatum and frontal cortex, decreased monoamine metabolite abundance and a greatly reduced capacity to release dopamine upon stimulation. Furthermore, VMAT2-LO mice showed exaggerated cued and contextual fear expression, altered fear habituation, inability to discriminate threat from safety cues, altered startle response compared with wild-type mice and an anxiogenic-like phenotype, but displayed no deficits in social function. By contrast, VMAT2-HI mice exhibited increased VMAT2 protein throughout the brain, higher vesicular storage capacity and greater dopamine release upon stimulation compared with wild-type controls. Behaviorally, VMAT2-HI mice were similar to wild-type mice in most assays, with some evidence of a reduced anxiety-like responses. Together, these data show that presynaptic monoamine function mediates PTSD-like outcomes in our mouse model, and suggest a causal link between reduced VMAT2 expression and fear behavior, consistent with the correlational relationship between VMAT2 genotype and PTSD risk in humans. Targeting this system is a potential strategy for the development of pharmacotherapies for disorders like PTSD.