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Background Platelet activation may be a central mediator of a chain of events leading to microinfarcts, leakage of thrombin and fibrin through the blood‐brain barrier and chronic neuroinflammation that typify Alzheimer’s Disease (AD). The platelet thrombin receptor Protease Activated Receptor 4 (PAR4) is responsible for platelet activation and amplification of thrombin generation. Thrombin cleaves fibrinogen to fibrin, and pathologic fibrin deposition in the cerebral microvasculature is itself a risk factor for Alzheimer’s disease. Fibrin interaction with vascular amyloid β (Aβ) leads to degradation‐resistant blood clots. This process initiates inflammation both in the vessel and adjacent parenchyma. Inflammation in general has been reported to turn on expression of PAR4 in endothelial cells not typically expressing PAR4. Method 1) Data were acquired from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP) to study the correlation of the PAR4 gene expression or methylation with AD diagnosis and longitudinal cognitive decline. 2) 5xFAD amyloid model mice were crossed with PAR4KO mice to test for pathology and markers of inflammation. Result 1) PAR4 gene F2RL3 mRNA was elevated in AD cases and was associated with worse retrospective longitudinal cognitive performance. We also report a significant association of F2RL3 epigenetic demethylation with cognitive decline. 2) 5xFAD mice exhibit increased PAR4 protein expression on vascular endothelial cells. 5xFAD mice exhibit increased vascular fibrin deposits compared to WT mice, and this fibrin deposition is reduced in 5xFAD/PAR4KO compared to 5xFAD. Conclusion In a human study the PAR4 gene F2RL3 mRNA expression is associated with multiple AD‐relevant outcomes and its encoded product, PAR4, may play a role in disease pathogenesis. PAR4 is a platelet receptor that functions upstream of fibrin deposition in 5xFAD amyloid mice, leading to misexpression in the microvasculature. Fibrin deposits have been reported to be inflammatory in 5xFAD and future work will focus on teasing apart the contribution of PAR4 mediated fibrin deposits in inflammation and microglial activation.

Platelet activation may be a central mediator of a chain of events leading to microinfarcts, leakage of thrombin and fibrin through the blood-brain barrier and chronic neuroinflammation that typify Alzheimer's Disease (AD). The platelet thrombin receptor Protease Activated Receptor 4 (PAR4) is responsible for platelet activation and amplification of thrombin generation. Thrombin cleaves fibrinogen to fibrin, and pathologic fibrin deposition in the cerebral microvasculature is itself a risk factor for Alzheimer's disease. Fibrin interaction with vascular amyloid β (Aβ) leads to degradation-resistant blood clots. This process initiates inflammation both in the vessel and adjacent parenchyma. Inflammation in general has been reported to turn on expression of PAR4 in endothelial cells not typically expressing PAR4. 1) Data were acquired from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP) to study the correlation of the PAR4 gene expression or methylation with AD diagnosis and longitudinal cognitive decline. 2) 5xFAD amyloid model mice were crossed with PAR4KO mice to test for pathology and markers of inflammation. 1) PAR4 gene F2RL3 mRNA was elevated in AD cases and was associated with worse retrospective longitudinal cognitive performance. We also report a significant association of F2RL3 epigenetic demethylation with cognitive decline. 2) 5xFAD mice exhibit increased PAR4 protein expression on vascular endothelial cells. 5xFAD mice exhibit increased vascular fibrin deposits compared to WT mice, and this fibrin deposition is reduced in 5xFAD/PAR4KO compared to 5xFAD. In a human study the PAR4 gene F2RL3 mRNA expression is associated with multiple AD-relevant outcomes and its encoded product, PAR4, may play a role in disease pathogenesis. PAR4 is a platelet receptor that functions upstream of fibrin deposition in 5xFAD amyloid mice, leading to misexpression in the microvasculature. Fibrin deposits have been reported to be inflammatory in 5xFAD and future work will focus on teasing apart the contribution of PAR4 mediated fibrin deposits in inflammation and microglial activation.

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster , the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

Dopamine transporter deficiency syndrome (DTDS) is a primary neurotransmitter disorder caused by loss-of-function mutations in SLC6A3, which encodes the dopamine transporter (DAT). The syndrome is characterised by progressive infantile-onset dystonia-parkinsonism with raised dopamine metabolite homovanillic acid in the cerebrospinal fluid. There are no disease modifying therapies for this life-limiting disorder. The aims of this study were to evaluate the clinical disease spectrum and develop a novel gene therapy approach for DTDS. Patients aged 1–36 years with childhood-onset dystonia-parkinsonism and suggestive neurotransmitter profile had SLC6A3 sequenced. In-vitro functional studies were undertaken for identified missense mutations. We specifically phenotyped DAT knockout (–/–) mice motor behaviour as a model of DTDS and developed a preclinical viral gene therapy construct. We evaluated effects of intracranial delivery of DAT gene therapy in neonatal DAT–/– mice as a proof of concept study. We identified ten new patients harbouring seven novel missense mutations, and found a novel atypical phenotype of juvenile parkinsonism. In-vitro functional characterisation revealed multifactorial disease mechanisms, including abnormal DAT trafficking, glycosylation, and impaired substrate recognition and uptake function. The DAT–/– mouse clearly recapitulated many features of human disease, including reduced survival, early hyperkinesia with later parkinsonism, raised homovanillic acid concentrations, and neurodegeneration. We have evaluated viral gene therapy approaches to target dopaminergic neurons and subsequently delivered DAT via adeno-associated virus type 9 to neonatal DAT–/– mice, with improved survival rates and motor phenotype. We report an expanding disease spectrum in DTDS, in which the clinical presentation mimics both cerebral palsy and juvenile parkinsonism. Genotype–phenotype correlation is evident, with in-vitro functional studies demonstrating greater residual DAT function in later-onset milder forms of disease. The preclinical study of viral gene therapy in neonatal mice is promising and will facilitate the longer term aim towards clinical translation for this untreatable disorder. Medical Research Council Clinical Research Training Fellowship, Great Ormond Street Hospital Children's Charity.

Though much is known about the various physiological functions of each GPCR and the specificity of Gα subunits, the specificity of Gβγ activated by a given GPCR and activating each effector in vivo is not known. Previously, we identified different Gβ and Gγ subunits interacting specifically with α2a-adrenergic receptors (α2aAR). In this study, we examined its in vivo specificity to the soluble NSF attachment proteins (SNARE) complex in adrenergic (auto-α2aAR) and non-adrenergic (hetero-α2aAR) neurons. We applied a quantitative targeted multiple reaction monitoring proteomic analysis of Gβ and Gγ subunits bound to the SNARE complex, and found only a subset of Gβ and Gγ bound. Without stimulation of auto-α2aAR, Gβ1 and Gγ3 interacted with the SNARE complex. When auto-α2aAR were activated, Gβ1, Gβ2, and Gγ3 were found. Further understanding of in vivo Gβγ specificity to its effectors provides new insights into the multiplicity of genes for Gβ and Gγ. Specific Gβγ dimers interact with the SNARE complex following presynaptic α2aAR activation in both adrenergic and non-adrenergic neurons.

Abstract Dopaminergic dysfunction is central to movement disorders and mental diseases. The dopamine transporter (DAT) is essential for the regulation of extracellular dopamine but the genetic and mechanistic link between DAT function and dopamine-related pathologies remains elusive. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here we identify a novel coding DAT variant, DAT-K619N, in a patient with early-onset parkinsonism and comorbid neuropsychiatric disease and in 22 individuals from exome-sequenced samples of neuropsychiatric patients. The variant localizes to the critical C-terminal PDZ-binding motif of DAT and causes reduced uptake capacity, decreased surface expression, and accelerated turnover of DAT in vitro. In vivo, we demonstrate that expression of DAT-K619N in mice and dropsophila imposes impairments in dopamine transmission with accompanying changes in dopamine-directed behaviors. Importantly, both cellular studies and viral overexpression of DAT-K619N in mice show that DAT-K619N has a dominant-negative effect which collectively implies that a single dominant-negative genetic DAT variant can confer risk for neuropsychiatric disease and neurodegenerative early-onset parkinsonism. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵* On behaf of iPSYCH researchers