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Age-related dopamine (DA) neuron loss is a primary feature of Parkinson's disease. However, it remains unclear whether similar biological processes occur during healthy aging, albeit to a lesser degree. We therefore determined whether midbrain DA neurons degenerate during aging in mice and humans. In mice, we identified no changes in midbrain neuron numbers throughout aging. Despite this, we found age-related decreases in midbrain mRNA expression of tyrosine hydroxylase (Th), the rate limiting enzyme of DA synthesis. Among midbrain glutamatergic cells, we similarly identified age-related declines in vesicular glutamate transporter 2 (Vglut2) mRNA expression. In co-transmitting Th +/Vglut2 + neurons, Th and Vglut2 transcripts decreased with aging. Importantly, striatal Th and Vglut2 protein expression remained unchanged. In translating our findings to humans, we found no midbrain neurodegeneration during aging and identified age-related decreases in TH and VGLUT2 mRNA expression similar to mouse. Unlike mice, we discovered diminished density of striatal TH+ dopaminergic terminals in aged human subjects. However, TH and VGLUT2 protein expression were unchanged in the remaining striatal boutons. Finally, in contrast to Th and Vglut2 mRNA, expression of most ribosomal genes in Th + neurons was either maintained or even upregulated during aging. This suggests a homeostatic mechanism where age-related declines in transcriptional efficiency are overcome by ongoing ribosomal translation. Overall, we demonstrate species-conserved transcriptional effects of aging in midbrain dopaminergic and glutamatergic neurons that are not accompanied by marked cell death or lower striatal protein expression. This opens the door to novel therapeutic approaches to maintain neurotransmission and bolster neuronal resilience.
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Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.
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Amphetamine (AMPH) is a psychostimulant that is commonly abused. The stimulant properties of AMPH are associated with its ability to increase dopamine (DA) neurotransmission. This increase is promoted by nonvesicular DA release mediated by reversal of DA transporter (DAT) function. Syntaxin 1 (Stx1) is a SNARE protein that is phosphorylated at Ser14 by casein kinase II. We show that Stx1 phosphorylation is critical for AMPH-induced nonvesicular DA release and, in Drosophila melanogaster, regulates the expression of AMPH-induced preference and sexual motivation. Our molecular dynamics simulations of the DAT/Stx1 complex demonstrate that phosphorylation of these proteins is pivotal for DAT to dwell in a DA releasing state. This state is characterized by the breakdown of two key salt bridges within the DAT intracellular gate, causing the opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that we hypothesize underlies nonvesicular DA release.
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Dopamina , Sintaxina 1 , Animales , Anfetamina/farmacología , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Drosophila melanogaster/metabolismo , Fosforilación , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
The aim of this paper is to summarize ultrastructural evidence for glutamatergic dysregulation in several linked regions in postmortem schizophrenia brain. Following a brief summary of glutamate circuitry and how synapses are identified at the electron microscopic (EM) level, we will review EM pathology in the cortex and basal ganglia. We will include the effects of antipsychotic drugs and the relation of treatment response. We will discuss how these findings support or confirm other postmortem findings as well as imaging results. Briefly, synaptic and mitochondrial density in anterior cingulate cortex was decreased in schizophrenia, versus normal controls (NCs), in a selective layer specific pattern. In dorsal striatum, increases in excitatory synaptic density were detected in caudate matrix, a compartment associated with cognitive and motor function, and in the putamen patches, a region associated with limbic function and in the core of the nucleus accumbens. Patients who were treatment resistant or untreated had significantly elevated numbers of excitatory synapses in limbic striatal areas in comparison to NCs and responders. Protein levels of vGLUT2, found in subcortical glutamatergic neurons, were increased in the nucleus accumbens in schizophrenia. At the EM level, schizophrenia subjects had an increase in density of excitatory synapses in several areas of the basal ganglia. In the substantia nigra, the protein levels of vGLUT2 were elevated in untreated patients compared to NCs. The density of inhibitory synapses was decreased in schizophrenia versus NCs. In schizophrenia, glutamatergic synapses are differentially affected depending on the brain region, treatment status, and treatment response.
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Antipsicóticos , Esquizofrenia , Humanos , Antipsicóticos/uso terapéutico , Sinapsis/metabolismo , Cuerpo Estriado/metabolismo , PutamenRESUMEN
Background: Altered dopamine (DA) signaling has been associated with autism spectrum disorder (ASD), a neurodevelopmental condition estimated to impact 1 in 54 children in the United States. There is growing evidence for alterations in both gastrointestinal function and oral microbiome composition in ASD. Recent work suggests that rare variants of the SLC6A3 gene encoding the DA transporter (DAT) identified in individuals with ASD result in structural and functional changes to the DAT. One such recently identified de novo mutation is a threonine to methionine substitution at position 356 of the DAT (DAT T356M). The DAT T356M variant is associated with ASD-like phenotypes in mice homozygous for the mutation (DAT T356M+/+), including social deficits, hyperactivity, and impaired DA signaling. Here, we determine the impact of this altered DA signaling as it relates to altered oral microbiota, and metabolic and gastrointestinal dysfunction. Methods: In the DAT T356M+/+ mouse, we determine the oral microbiota composition, metabolic function, and gastrointestinal (GI) function. We examined oral microbiota by 16S RNA sequencing. We measured metabolic function by examining glucose tolerance and we probed gastrointestinal parameters by measuring fecal dimensions and weight. Results: In the DAT T356M+/+ mouse, we evaluate how altered DA signaling relates to metabolic dysfunction and altered oral microbiota. We demonstrate that male DAT T356M+/+ mice weigh less (Wild type (WT) = 26.48 ± 0.6405 g, DAT T356M+/+ = 24.14 ± 0.4083 g) and have decreased body fat (WT = 14.89 ± 0.6206%, DAT T356M+/+ = 12.72 ± 0.4160%). These mice display improved glucose handling (WT = 32.60 ± 0.3298 kcal/g, DAT T356M+/+ = 36.97 ± 0.4910 kcal/g), and an altered oral microbiota. We found a significant decrease in Fusobacterium abundance. The abundance of Fusobacterium was associated with improved glucose handling and decreased body fat. Conclusions: Our findings provide new insights into how DAT dysfunction may alter gastrointestinal function, composition of the oral microbiota, and metabolism. Our data suggest that impaired DA signaling in ASD is associated with a number of metabolic and gastrointestinal changes which are common in individuals with ASD.
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Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Drosophila melanogaster , Trastornos Distónicos/genética , Enfermedad de Parkinson/genética , Trastornos Psicomotores/genética , Animales , Cloroquina/farmacología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/deficiencia , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Trastornos Distónicos/tratamiento farmacológico , Vuelo Animal/efectos de los fármacos , Células HEK293 , Humanos , Estructura Molecular , Mutación Missense , Enfermedad de Parkinson/tratamiento farmacológico , Trastornos Psicomotores/tratamiento farmacológicoRESUMEN
Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.
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Anfetamina , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Anfetamina/farmacología , Animales , Sitios de Unión , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Drosophila melanogaster , FosfatidilinositolesRESUMEN
Objectives: The substantia nigra (SN) receives glutamatergic and GABAergic inputs that regulate dopaminergic neuronal activity. Imaging studies have shown hyperactivity of the SN in schizophrenia (SZ) patients. We examined neurochemically defined inputs to the SN, synaptic density, and neuromelanin content that might contribute to or reflect this hyperexcitability.Methods: Glutamatergic axon terminals were identified by the immunohistochemical localisation of vGLUT1 and vGLUT2; GABA inputs were identified by the immunohistochemical localisation of GAD67. Neuromelanin granules are visible in unstained sections and thus were assessed in unstained sections. Optical densitometry was measured to assess the density of vGLUT1, vGLUT2 or GAD67 immunolabelled axon terminals and neuromelanin granules. Electron microscopy was used to quantify synaptic and mitochondrial density.Results: Compared to controls, SZ subjects had nonsignificant trends toward a decrease in vGLUT1, and an increase in both vGLUT2 and GAD67. vGLUT1 was negatively correlated with GAD67 in normal controls (NCs) and positively correlated in SZ subjects. A correlation of coefficient analysis showed a significant difference between the negative correlation in NCs and the positive correlation in SZ subjects. Frequency histograms showed the distribution of neuromelanin density was different in SZ subjects compared to NCs. Synaptic density data showed a decrease in inhibitory synapses in SZ subjects. Mitochondrial density was normal in SZ subjects.Conclusions: Synaptic density alterations and the lack of a positive correlation between GAD67 and vGLUT1 could contribute to hyperactivity in the SN.