Selective dopaminergic vulnerability in Parkinson's disease: new insights into the role of DAT.

One of the hallmarks of Parkinson's disease (PD) is the progressive loss of dopaminergic neurons and associated dopamine depletion. Several mechanisms, previously considered in isolation, have been proposed to contribute to the pathophysiology of dopaminergic degeneration: dopamine oxidation-mediated neurotoxicity, high dopamine transporter (DAT) expression density per neuron, and autophagy-lysosome pathway (ALP) dysfunction. However, the interrelationships among these mechanisms remained unclear. Our recent research bridges this gap, recognizing autophagy as a novel dopamine homeostasis regulator, unifying these concepts. I propose that autophagy modulates dopamine reuptake by selectively degrading DAT. In PD, ALP dysfunction could increase DAT density per neuron, and enhance dopamine reuptake, oxidation, and neurotoxicity, potentially contributing to the progressive loss of dopaminergic neurons. This integrated understanding may provide a more comprehensive view of aspects of PD pathophysiology and opens new avenues for therapeutic interventions.

A high-affinity cocaine binding site associated with the brain acid soluble protein 1.

Cocaine exerts its stimulant effect by inhibiting dopamine (DA) reuptake, leading to increased dopamine signaling. This action is thought to reflect the binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share cocaine's behavioral actions. Further, recent reports show more potent actions of the drug, implying the existence of a high-affinity receptor for cocaine. We now report high-affinity binding of cocaine associated with the brain acid soluble protein 1 (BASP1) with a dissociation constant (Kd) of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depleting BASP1 in the nucleus accumbens but not the dorsal striatum diminishes locomotor stimulation in mice. Our findings imply that BASP1 is a pharmacologically relevant receptor for cocaine.

D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain.

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.

Cocaine-induced locomotor stimulation involves autophagic degradation of the dopamine transporter.

Cocaine exerts its stimulant effect by inhibiting dopamine reuptake leading to increased dopamine signaling. This action is thought to reflect binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share the behavioral actions of cocaine. We previously showed that toxic levels of cocaine induce autophagic neuronal cell death. Here, we show that subnanomolar concentrations of cocaine elicit neural autophagy in vitro and in vivo. Autophagy inhibitors reduce the locomotor stimulant effect of cocaine in mice. Cocaine-induced autophagy degrades transporters for dopamine but not serotonin in the nucleus accumbens. Autophagy inhibition impairs cocaine conditioned place preference in mice. Our findings indicate that autophagic degradation of DAT modulates behavioral actions of cocaine.

Real-time, functional intra-operative localization of rat cavernous nerve network using near-infrared cyanine voltage-sensitive dye imaging.

Despite current progress achieved in the surgical technique of radical prostatectomy, post-operative complications such as erectile dysfunction and urinary incontinence persist at high incidence rates. In this paper, we present a methodology for functional intra-operative localization of the cavernous nerve (CN) network for nerve-sparing radical prostatectomy using near-infrared cyanine voltage-sensitive dye (VSD) imaging, which visualizes membrane potential variations in the CN and its branches (CNB) in real time. As a proof-of-concept experiment, we demonstrate a functioning complex nerve network in response to electrical stimulation of the CN, which was clearly differentiated from surrounding tissues in an in vivo rat prostate model. Stimulation of an erection was confirmed by correlative intracavernosal pressure (ICP) monitoring. Within 10 minutes, we performed trans-fascial staining of the CN by direct VSD administration. Our findings suggest the applicability of VSD imaging for real-time, functional imaging guidance during nerve-sparing radical prostatectomy.

Transcranial photoacoustic imaging of NMDA-evoked focal circuit dynamics in the rat hippocampus.

OBJECTIVE: We report the transcranial functional photoacoustic (fPA) neuroimaging of N-methyl-D-aspartate (NMDA) evoked neural activity in the rat hippocampus. Concurrent quantitative electroencephalography (qEEG) and microdialysis were used to record real-time circuit dynamics and excitatory neurotransmitter concentrations, respectively. APPROACH: We hypothesized that location-specific fPA voltage-sensitive dye (VSD) contrast would identify neural activity changes in the hippocampus which correlate with NMDA-evoked excitatory neurotransmission. MAIN RESULTS: Transcranial fPA VSD imaging at the contralateral side of the microdialysis probe provided NMDA-evoked VSD responses with positive correlation to extracellular glutamate concentration changes. qEEG validated a wide range of glutamatergic excitation, which culminated in focal seizure activity after a high NMDA dose. We conclude that transcranial fPA VSD imaging can distinguish focal glutamate loads in the rat hippocampus, based on the VSD redistribution mechanism which is sensitive to the electrophysiologic membrane potential. SIGNIFICANCE: Our results suggest the future utility of this emerging technology in both laboratory and clinical sciences as an innovative functional neuroimaging modality.

Transcranial Recording of Electrophysiological Neural Activity in the Rodent Brain in vivo Using Functional Photoacoustic Imaging of Near-Infrared Voltage-Sensitive Dye.

Minimally-invasive monitoring of electrophysiological neural activities in real-time-that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET-presents a very challenging yet significant task for neuroimaging. In this paper, we present in vivo functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Our normalized time-frequency analysis presented in vivo VSD response in the seizure group significantly distinguishable from those of the control groups at sub-mm spatial resolution. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that the near-infrared fPA VSD imaging is a promising tool for in vivo recording of brain activities through intact scalp, which would pave a way to its future translation in real time human brain imaging.

Histone H2AX promotes neuronal health by controlling mitochondrial homeostasis.

Phosphorylation of histone H2AX is a major contributor to efficient DNA repair. We recently reported neurobehavioral deficits in mice lacking H2AX. Here we establish that this neural failure stems from impairment of mitochondrial function and repression of the mitochondrial biogenesis gene PGC-1α. H2AX loss leads to reduced levels of the major subunits of the mitochondrial respiratory complexes in mouse embryonic fibroblasts and in the striatum, a brain region particularly vulnerable to mitochondrial damage. These defects are substantiated by disruption of the mitochondrial shape in H2AX mutant cells. Ectopic expression of PGC-1α restores mitochondrial oxidative phosphorylation complexes and mitigates cell death. H2AX knockout mice display increased neuronal death in the brain when challenged with 3-nitropronionic acid, which targets mitochondria. This study establishes a role for H2AX in mitochondrial homeostasis associated with neuroprotection.

Antidepressant Actions of Ketamine Mediated by the Mechanistic Target of Rapamycin, Nitric Oxide, and Rheb.

The weeks/months it takes for traditional antidepressants to act pose an obstacle in the management of depression. Ketamine's prompt and sustained antidepressant effects constitute a major advance. Multiple studies implicate glutamatergic signaling to protein synthesis machinery and synapse formation in ketamine's antidepressant effects. Here we review evidence linking ketamine to glutamate receptor subtypes and protein homeostasis. We describe a signaling cascade wherein nitric oxide drives the formation of a ternary protein complex comprised of glyceraldehyde 3-phosphate dehydrogenase, seven in absentia homolog 1, and Ras homolog enriched in brain downstream of the glutamate N-methyl-D-aspartate receptor. Seven in absentia homolog 1 ubiquitylates and degrades Ras homolog enriched in brain leading to inhibition of mechanistic target of rapamycin. Ketamine inhibits this molecular cascade leading to activation of mechanistic target of rapamycin and, in turn, to antidepressant actions.

Neuronal migration is mediated by inositol hexakisphosphate kinase 1 via α-actinin and focal adhesion kinase.

Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report that IP6K1 deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions. IP6K1 deletion disrupts α-actinin's intracellular localization and function. The IP6K1 deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion of IP6K1 abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased in IP6K1 deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] recapitulates the phenotype of IP6K1 deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1.

Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1-dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1-dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.

Cocaine elicits autophagic cytotoxicity via a nitric oxide-GAPDH signaling cascade.

Cocaine exerts its behavioral stimulant effects by facilitating synaptic actions of neurotransmitters such as dopamine and serotonin. It is also neurotoxic and broadly cytotoxic, leading to overdose deaths. We demonstrate that the cytotoxic actions of cocaine reflect selective enhancement of autophagy, a process that physiologically degrades metabolites and cellular organelles, and that uncontrolled autophagy can also lead to cell death. In brain cultures, cocaine markedly increases levels of LC3-II and depletes p62, both actions characteristic of autophagy. By contrast, cocaine fails to stimulate cell death processes reflecting parthanatos, monitored by cleavage of poly(ADP ribose)polymerase-1 (PARP-1), or necroptosis, assessed by levels of phosphorylated mixed lineage kinase domain-like protein. Pharmacologic inhibition of autophagy protects neurons against cocaine-induced cell death. On the other hand, inhibition of parthanatos, necroptosis, or apoptosis did not change cocaine cytotoxicity. Depletion of ATG5 or beclin-1, major mediators of autophagy, prevents cocaine-induced cell death. By contrast, depleting caspase-3, whose cleavage reflects apoptosis, fails to alter cocaine cytotoxicity, and cocaine does not alter caspase-3 cleavage. Moreover, depleting PARP-1 or RIPK1, key mediators of parthanatos and necroptosis, respectively, did not prevent cocaine-induced cell death. Autophagic actions of cocaine are mediated by the nitric oxide-glyceraldehyde-3-phosphate dehydrogenase signaling pathway. Thus, cocaine-associated autophagy is abolished by depleting GAPDH via shRNA; by the drug CGP3466B, which prevents GAPDH nitrosylation; and by mutating cysteine-150 of GAPDH, its site of nitrosylation. Treatments that selectively influence cocaine-associated autophagy may afford therapeutic benefit.

Human GAPDH Is a Target of Aspirin's Primary Metabolite Salicylic Acid and Its Derivatives.

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA's multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson's drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death.

Huntington's disease: Neural dysfunction linked to inositol polyphosphate multikinase.

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD.

Nitric Oxide-GAPDH Transcriptional Signaling Mediates Behavioral Actions of Cocaine.

Psychotropic actions of cocaine are generally thought to involve its blockade of monoamine transporters leading to increased synaptic levels of monoamines, especially dopamine. Subsequent intracellular events have been less well characterized. We describe a signaling system wherein lower behavioral stimulant doses of cocaine, as well as higher neurotoxic doses, activate a cascade wherein nitric oxide nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to generate a complex with the ubiquitin-E3-ligase Siah1 which translocates to the nucleus. With lower cocaine doses, nuclear GAPDH augments CREB signaling, while at higher doses p53 signaling is enhanced. The drug CGP3466B very potently blocks GAPDH nitrosylation, hindering both signaling cascades and inhibits both behavioral activating and neurotoxic effects of cocaine. This system affords potentially novel approaches to the therapy of cocaine abuse.

MiR-223 regulates the differentiation of immature neurons.

BACKGROUND: Small non-coding microRNA RNA molecules can regulate stem cell function. The role of microRNAs in neural stem/progenitor cells (NS/PCs) differentiation is not entirely clear. METHODS: MiRNA profiling, loss and gain of function studies coupled with dendritic tree development morphometric analysis and calcium influx imaging were utilized to investigate the role of micoRNA-223 in differentiating NS/PCs. RESULTS: MiRNA profiling in human NS/PCs before and after differentiation in vitro reveals modulation of miRNAs following differentiation of NS/PCs. MiR-223, a microRNA well characterized as a hematopoietic-specific miRNA was identified. Cell-autonomous inhibition of miR-223 in the adult mouse dentate gyrus NS/PCs led to a significant increase in immature neurons soma size, dendritic tree total length, branch number per neuron and complexity, while neuronal migration in the dentate gyrus remained unaffected. Overexpression of miR-223 decreased dendritic tree total length, branch number and complexity in neurons differentiated from human embryonic stem cells (hESCs). Inhibition of miR-223 enhanced N-methyl-D-aspartate (NMDA) induced calcium influx in human neurons differentiated from NS/PCs. CONCLUSIONS: Taken together, these findings indicate that miR-223 regulates the differentiation of neurons derived from NS/PCs.

Botch is a γ-glutamyl cyclotransferase that deglycinates and antagonizes Notch.

Botch promotes embryonic neurogenesis by inhibiting the initial S1 furin-like cleavage step of Notch maturation. The biochemical process by which Botch inhibits Notch maturation is not known. Here, we show that Botch has γ-glutamyl cyclotransferase (GGCT) activity that deglycinates Notch, which prevents the S1 furin-like cleavage. Moreover, Notch is monoglycinated on the γ-glutamyl carbon of glutamate 1,669. The deglycinase activity of Botch is required for inhibition of Notch signaling both in vitro and in vivo. When the γ-glutamyl-glycine at position 1,669 of Notch is degylcinated, it is replaced by 5-oxy-proline. These results reveal that Botch regulates Notch signaling through deglycination and identify a posttranslational modification of Notch that plays an important role in neurogenesis.

Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction.

Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

MicroRNA-223 is neuroprotective by targeting glutamate receptors.

Stroke is a major cause of mortality and morbidity worldwide. Extracellular glutamate accumulation leading to overstimulation of the ionotropic glutamate receptors mediates neuronal injury in stroke and in neurodegenerative disorders. Here we show that miR-223 controls the response to neuronal injury by regulating the functional expression of the glutamate receptor subunits GluR2 and NR2B in brain. Overexpression of miR-223 lowers the levels of GluR2 and NR2B by targeting 3'-UTR target sites (TSs) in GluR2 and NR2B, inhibits NMDA-induced calcium influx in hippocampal neurons, and protects the brain from neuronal cell death following transient global ischemia and excitotoxic injury. MiR-223 deficiency results in higher levels of NR2B and GluR2, enhanced NMDA-induced calcium influx, and increased miniature excitatory postsynaptic currents in hippocampal neurons. In addition, the absence of MiR-223 leads to contextual, but not cued memory deficits and increased neuronal cell death following transient global ischemia and excitotoxicity. These data identify miR-223 as a major regulator of the expression of GluR2 and NR2B, and suggest a therapeutic role for miR-223 in stroke and other excitotoxic neuronal disorders.

Botch promotes neurogenesis by antagonizing Notch.

Regulation of self-renewal and differentiation of neural stem cells is still poorly understood. Here we investigate the role of a developmentally expressed protein, Botch, which blocks Notch, in neocortical development. Downregulation of Botch in vivo leads to cellular retention in the ventricular and subventricular zones, whereas overexpression of Botch drives neural stem cells into the intermediate zone and cortical plate. In vitro neurosphere and differentiation assays indicate that Botch regulates neurogenesis by promoting neuronal differentiation. Botch prevents cell surface presentation of Notch by inhibiting the S1 furin-like cleavage of Notch, maintaining Notch in the immature full-length form. Understanding the function of Botch expands our knowledge regarding both the regulation of Notch signaling and the complex signaling mediating neuronal development.