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Alzheimer's & Dementia : the Journal of... Apr 2023Lysosomes are degradative organelles that maintain cellular homeostasis and protein quality control. Transcription factor EB (TFEB)-mediated lysosome biogenesis enhances...
INTRODUCTION
Lysosomes are degradative organelles that maintain cellular homeostasis and protein quality control. Transcription factor EB (TFEB)-mediated lysosome biogenesis enhances lysosome-dependent degradation and alleviates neurodegenerative diseases, but the mechanisms underlying TFEB regulation and modification are still poorly understood.
METHODS
By screening novel small-molecule compounds, we identified a group of lysosome-enhancing compounds (LYECs) that promote TFEB activation and lysosome biogenesis.
RESULTS
One of these compounds, LH2-051, significantly inhibited the function of the dopamine transporter (DAT) and subsequently promoted lysosome biogenesis. We uncovered cyclin-dependent kinase 9 (CDK9) as a novel regulator of DAT-mediated lysosome biogenesis and identified six novel CDK9-phosphorylated sites on TFEB. We observed that signal transduction by the DAT-CDK9-TFEB axis occurs on lysosomes. Finally, we found that LH2-051 enhanced the degradation of amyloid beta plaques and improved the memory of amyloid precursor protein (APP)/Presenilin 1 (PS1) mice.
DISCUSSION
We identified the DAT-CDK9-TFEB signaling axis as a novel regulator of lysosome biogenesis. Our study sheds light on the mechanisms of protein quality control under pathophysiological conditions.
Topics: Mice; Animals; Alzheimer Disease; Amyloid beta-Peptides; Dopamine Plasma Membrane Transport Proteins; Amyloid beta-Protein Precursor; Lysosomes; Autophagy
PubMed: 36130073
DOI: 10.1002/alz.12776 -
AJNR. American Journal of Neuroradiology Feb 2015The functional imaging technique most widely used in European clinics to differentiate a true parkinsonian syndrome from vascular parkinsonism, drug-induced changes, or... (Review)
Review
The functional imaging technique most widely used in European clinics to differentiate a true parkinsonian syndrome from vascular parkinsonism, drug-induced changes, or essential tremor is dopamine-transporter SPECT. This technique commonly reports dopamine-transporter function, with decreasing striatal uptake demonstrating increasingly severe disease. The strength of dopamine-transporter SPECT is that nigrostriatal degeneration is observed in both clinically inconclusive parkinsonism and early, even premotor, disease. In this clinical review (Part 2), we present the dopamine-transporter SPECT findings in a variety of neurodegenerative diseases, including multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, and dementia with Lewy bodies. The findings in vascular parkinsonism, drug-induced parkinsonism, and essential tremor are also described. It is hoped that this technique will be the forerunner of a range of routinely used, process-specific ligands that can identify early degenerative disease and subsequently guide disease-modifying interventions.
Topics: Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Essential Tremor; Humans; Lewy Body Disease; Multiple System Atrophy; Parkinsonian Disorders; Supranuclear Palsy, Progressive; Tomography, Emission-Computed, Single-Photon
PubMed: 24924549
DOI: 10.3174/ajnr.A3971 -
The Journal of Biological Chemistry Nov 2015The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the...
The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders.
Topics: Cell Line; Dopamine; Dopamine Plasma Membrane Transport Proteins; Down-Regulation; Endocytosis; Humans; Kinetics; Lipoylation; Protein Kinase C
PubMed: 26424792
DOI: 10.1074/jbc.M115.667055 -
Brain Research Bulletin Dec 2020Synaptic dopamine (DA) concentrations are largely determined by the activities of presynaptic D and D autoreceptors (DR and DR) and DA transporter (DAT). Furthermore,...
Synaptic dopamine (DA) concentrations are largely determined by the activities of presynaptic D and D autoreceptors (DR and DR) and DA transporter (DAT). Furthermore, the activity of DAT is regulated by phosphorylation events and protein interactions that affect its surface expression. Because DA autoreceptors and DAT coordinately maintain synaptic DA homeostasis, we hypothesized that DR might crosstalk with DAT to fine-tune synaptic DA concentrations. To test this hypothesis, we established [H]DA uptake and DAT surface expression assays in hD/rDAT-double-transfected HEK-293 cells or limbic forebrain synaptosomal preparations. Ropinirole, a preferential DR agonist, reduced [H]DA uptake in HEK-hD/rDAT cells in a dose-dependent manner, an effect which could be blocked by the DR/DR antagonist, raclopride. Furthermore, ropinirole also reduced DAT surface expression in limbic forebrain synaptosomes, and this effect could be blocked by raclopride or the internalization inhibitor, concanavalin A. To identify potential mediators of this apparent DR-DAT crosstalk, DAT-associated proteins were co-immunoprecipitated from limbic forebrain synaptosomes after DR activation and identified by MALDI-TOF. From this analysis, the Hsc70 chaperone was identified as a DAT-associated protein. Interestingly, ropinirole induced the association of Hsc70/Hsp70 with DAT, and the Hsc70/Hsp70 inhibitor, apoptozole, prevented the ropinirole-induced reduction of DAT surface expression. Together, these results suggest that DR negatively regulates DAT activity by promoting the association of DAT and Hsc70/Hsp70.
Topics: Animals; Dopamine Agonists; Dopamine Plasma Membrane Transport Proteins; HEK293 Cells; HSC70 Heat-Shock Proteins; Humans; Indoles; Mice; Neurons; Phosphorylation; Prosencephalon; Receptors, Dopamine D3; Synaptosomes
PubMed: 33049353
DOI: 10.1016/j.brainresbull.2020.10.005 -
The Journal of Biological Chemistry Feb 2023Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA...
Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGlu5, drove rapid biphasic DAT membrane trafficking in ex vivo striatal slices, with region-specific differences between ventral and dorsal striata. DAT insertion required D2 DA autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetric studies revealed that DAT trafficking impacts DA clearance. Furthermore, dopaminergic mGlu5 silencing elevated DAT surface expression and abolished motor learning, which was rescued by inhibiting DAT with a subthreshold CE-158 dose. We discovered that presynaptic DAT trafficking is complex, multimodal, and region specific, and for the first time, we identified cell autonomous mechanisms that govern presynaptic DAT tone. Importantly, the findings are consistent with a role for regulated DAT trafficking in DA clearance and motor function.
Topics: Dopamine; Dopamine Plasma Membrane Transport Proteins; Receptors, Presynaptic; Animals; Mice; Corpus Striatum
PubMed: 36640864
DOI: 10.1016/j.jbc.2023.102900 -
Journal of Neurochemistry Oct 2021Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits....
Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits. Evidence suggests that the rapid effects of stimulants that are structurally and mechanistically similar to MCAT on monoamine transporter function may contribute to the abuse liability and/or persistent monoaminergic deficits caused by these agents. Thus, effects of MCAT on 1) striatal dopamine (DA) transporter (DAT); and 2) striatal and hippocampal serotonin transporter (SERT) function, as determined in tissues from adult male rats, were assessed. As reported previously, a single administration of MCAT rapidly (within 1 hr) decreases striatal [ H]DA uptake. Similarly, incubation of rat synaptosomes with MCAT at 37℃ (but not 4˚C) decreased striatal [ H]DA uptake. Incubation with MCAT likewise decreased [ H]5HT but not vesicular [ H]DA uptake. MCAT incubation in vitro was without effect on [ H]DA uptake in striatal synaptosomes prepared from MCAT-treated rats. The decrease in [ H]DA uptake caused by MCAT incubation: (a) reflected a decrease in V , with minimal change in K , and (b) was attenuated by co-incubation with the cell-permeable calcium chelator, N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-1,1'-bis[(acetyloxy)methyl] ester-glycine (BAPTA-AM), as well as the non-selective protein kinase-C (PKC) inhibitors bisindolylmaleimide-1 (BIM-1) and 2-[1-3(Aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (or Bisindolylmaleimide VIII; Ro-31-7549). Taken together, these results suggest that in vitro MCAT incubation may model important aspects of MCAT administration in vivo, and that calcium and PKC contribute to the in vitro effects of MCAT on DAT.
Topics: Animals; Central Nervous System Stimulants; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Male; Propiophenones; Protein Kinase C; Rats; Rats, Sprague-Dawley; Synaptosomes
PubMed: 34320222
DOI: 10.1111/jnc.15483 -
Archives of Toxicology Mar 2016Reserpine is used as an animal model of parkinsonism. We hypothesized that the involuntary movements induced by reserpine in rodents are induced by dopaminergic toxicity...
Reserpine is used as an animal model of parkinsonism. We hypothesized that the involuntary movements induced by reserpine in rodents are induced by dopaminergic toxicity caused by extracellular dopamine accumulation. The present study tested the effects of reserpine on the dopaminergic system in Caenorhabditis elegans. Reserpine was toxic to worms (decreased the survival, food intake, development and changed egg laying and defecation cycles). In addition, reserpine increased the worms' locomotor rate on food and decreased dopamine levels. Morphological evaluations of dopaminergic CEP neurons confirmed neurodegeneration characterized by decreased fluorescence intensity and the number of worms with intact CEP neurons, and increased number of shrunken somas per worm. These effects were unrelated to reserpine's effect on decreased expression of the dopamine transporter, dat-1. Interestingly, the locomotor rate on food and the neurodegenerative parameters fully recovered to basal conditions upon reserpine withdrawal. Furthermore, reserpine decreased survival in vesicular monoamine transporter and dat-1 loss-of-function mutant worms. In addition, worms pre-exposed to dopamine followed by exposure to reserpine had decreased survival. Reserpine activated gst-4, which controls a phase II detoxification enzymes downstream of nuclear factor (erythroid-derived-2)-like 2. Our findings establish that the dopamine transporter, dat-1, plays an important role in reserpine toxicity, likely by increasing extracellular dopamine concentrations.
Topics: Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Defecation; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Female; Gene Expression Regulation; Green Fluorescent Proteins; Ovum; Parkinson Disease; Reserpine
PubMed: 25579234
DOI: 10.1007/s00204-015-1451-7 -
Addiction Biology Jan 2017To compare the effects of heroin and methamphetamine (METH) addiction on dopamine transporters (DATs) in the same dose and duration, we assessed DAT levels in the... (Comparative Study)
Comparative Study
To compare the effects of heroin and methamphetamine (METH) addiction on dopamine transporters (DATs) in the same dose and duration, we assessed DAT levels in the striatum by Tc-TRODAT-1 single-photon emission computed tomography (SPECT) brain images in people with heroin and METH dependence. We recruited 21 healthy human controls, 23 heroin-dependent subjects and 25 METH abusers. The heroin- and METH-dependent subjects exhibited negative urine toxicology after undergoing physiological detoxification. All subjects underwent SPECT brain imaging, and specific tracer uptake ratios (SURs) were assessed bilaterally in the regions of interest. A significant SUR reduction in heroin-dependent subjects and METH-dependent subjects compared with healthy controls was found in the left striatum, right striatum, left caudate nucleus, right caudate nucleus, left putamen and right putamen. There were no significant differences in the heroin group and METH group for the left striatum, right striatum, left caudate nucleus, right caudate nucleus, left putamen and right putamen. The scores of craving, HAMA (Hamilton Anxiety Rating Scale), in heroin abusers were lower than in the METH abusers. Our results show that people with heroin and METH dependence who are currently abstinent had lower DAT levels in the striatum than healthy controls. There were no differences in striatal DAT in heroin and METH users. These results suggest that chronic heroin and METH abuse appears to produce similar effects in striatal DAT in humans. METH users may have more serious craving and anxiety symptoms than heroin users with prolonged abstinence.
Topics: Adult; Amphetamine-Related Disorders; Caudate Nucleus; Chronic Disease; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Female; Heroin; Heroin Dependence; Humans; Male; Methamphetamine; Middle Aged; Putamen; Tomography, Emission-Computed, Single-Photon; Young Adult
PubMed: 26040446
DOI: 10.1111/adb.12271 -
International Journal of Molecular... Nov 2020Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia....
Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (-2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors.
Topics: Animals; Basal Ganglia; Dopamine; Dopamine Plasma Membrane Transport Proteins; Female; Heterozygote; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Psychomotor Agitation; Receptors, Dopamine D3; Synaptic Transmission; Up-Regulation
PubMed: 33153031
DOI: 10.3390/ijms21218216 -
Scientific Reports Oct 2019The aim of this study was to evaluate associations of motor and non-motor symptoms with dopamine transporter binding in prodromal stage of synucleinopathies. We examined...
The aim of this study was to evaluate associations of motor and non-motor symptoms with dopamine transporter binding in prodromal stage of synucleinopathies. We examined 74 patients with idiopathic REM sleep behavior disorder (RBD), which is a prodromal synucleinopathy, and 39 controls using Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), Montreal Cognitive Assessment, University of Pennsylvania Smell Identification Test (UPSIT), Farnsworth-Munsell 100 hue test, orthostatic test, Scales for Outcomes in PD-Autonomic, Beck depression inventory-II, State-Trait Anxiety Inventory, and video-polysomnography. Electromyographic muscle activity during REM sleep was quantified according to Sleep Innsbruck-Barcelona criteria. In 65 patients, dopamine transporter single-photon emission computed tomography (DAT-SPECT) imaging was performed, putaminal binding ratio was calculated and scans were classified as normal, borderline, or abnormal. Compared to controls, RBD patients had significantly more severe scores in all examined tests. Patients with abnormal DAT-SPECT had higher MDS-UPDRS motor score (p = 0.006) and higher prevalence of orthostatic hypotension (p = 0.008). Putaminal binding ratio was positively associated with UPSIT score (p = 0.03) and negatively associated with tonic (p = 0.003) and phasic (p = 0.01) muscle activity during REM sleep. These associations likely reflect simultaneous advancement of underlying pathology in substantia nigra and susceptible brainstem and olfactory nuclei in prodromal synucleinopathy.
Topics: Aged; Case-Control Studies; Dopamine Plasma Membrane Transport Proteins; Female; Humans; Male; Mental Status and Dementia Tests; Middle Aged; Polysomnography; Protein Binding; REM Sleep Behavior Disorder
PubMed: 31664065
DOI: 10.1038/s41598-019-51710-y