Function Of Dopaminergic Neurons Research Articles

DJ-1 deficient mice demonstrate similar vulnerability to pathogenic Ala53Thr human -syn toxicity

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. A pathological hallmark of PD is the presence of intraneuronal inclusions composed of fibrillized alpha-synuclein (alpha-syn) in affected brain regions. Mutations in the gene, PARK7, which encodes DJ-1, can cause autosomal recessive early-onset PD. Although DJ-1 has been shown to be involved in diverse biological processes, several in vitro studies suggest that it can inhibit the formation and protect against the effects of alpha-syn aggregation. We previously established and characterized transgenic mice expressing pathogenic Ala53Thr human alpha-syn (M83 mice) that develop extensive alpha-syn pathologies in the neuroaxis resulting in severe motor impairments and eventual fatality. In the current study, we have crossbred M83 mice on a DJ-1 null background (M83-DJnull mice) in efforts to determine the effects of the lack of DJ-1 in these mice. Animals were assessed and compared for survival rate, distribution of alpha-syn inclusions, biochemical properties of alpha-syn protein, demise and function of nigral dopaminergic neurons, and extent of gliosis in the neuroaxis. M83 and M83-DJnull mice displayed a similar onset of disease and pathological changes, and none of the analyses to assess for changes in pathogenesis revealed any significant differences between M83 and M83-DJnull mice. These findings suggest that DJ-1 may not function to directly modulate alpha-syn nor does DJ-1 appear to play a role in protecting against the deleterious effects of expressing pathogenic Ala53Thr alpha-syn in vivo. It is possible that alpha-syn and DJ-1 mutations may lead to PD via independent mechanisms.

Reduction of L-Type Amino Acid Transporter 1 mRNA Expression in Brain Capillaries in a Mouse Model of Parkinson's Disease

The blood-brain barrier (BBB) expresses transporters that influence both dopaminergic neuronal function and drug therapy for Parkinson's disease (PD). The purpose of the present study was to clarify changes of transporter mRNA expression at the BBB in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of PD, in order to understand the pathophysiological role of BBB transport function in PD. At 7 d after MPTP treatment, mice showed a motor deficit and a loss of dopaminergic neurons. At the same time, L-type amino acid transporter 1 (LAT1) mRNA expression in the brain capillary fraction of the MPTP-treated mice was significantly reduced by 62.6% compared with saline-treated mice, while no significant change was observed in the expression of glucose transporter 1, creatine transporter 1, taurine transporter, organic cation transporter 2, serotonin transporter, norepinephrine transporter and dopamine transporter. LAT1 mRNA expression in whole brain was not affected at 1, 3 and 5 d after the treatment, but was reduced by 46.3% at 7 d. LAT1 mediates the transport of large neutral amino acids, including tyrosine, as well as the PD-therapeutic drug levodopa, across the BBB. Our findings indicate that decreased LAT1 expression at the BBB in PD patients may adversely affect amino acid supply from the circulating blood and levodopa distribution into the brain.

Caffeine and a selective adenosine A2A receptor antagonist induce sensitization and cross-sensitization behavior associated with increased striatal dopamine in mice

BackgroundCaffeine, a nonselective adenosine A1 and A2A receptor antagonist, is the most widely used psychoactive substance in the world. Evidence demonstrates that caffeine and selective adenosine A2A antagonists interact with the neuronal systems involved in drug reinforcement, locomotor sensitization, and therapeutic effect in Parkinson's disease (PD). Evidence also indicates that low doses of caffeine and a selective adenosine A2A antagonist SCH58261 elicit locomotor stimulation whereas high doses of these drugs exert locomotor inhibition. Since these behavioral and therapeutic effects are mediated by the mesolimbic and nigrostriatal dopaminergic pathways which project to the striatum, we hypothesize that low doses of caffeine and SCH58261 may modulate the functions of dopaminergic neurons in the striatum.MethodsIn this study, we evaluated the neuroadaptations in the striatum by using reverse-phase high performance liquid chromatography (HPLC) to quantitate the concentrations of striatal dopamine and its metabolites, dihydroxylphenylacetic acid (DOPAC) and homovanilic acid (HVA), and using immunoblotting to measure the level of phosphorylation of tyrosine hydroxylase (TH) at Ser31, following chronic caffeine and SCH58261 sensitization in mice. Moreover, to validate further that the behavior sensitization of caffeine is through antagonism at the adenosine A2A receptor, we also evaluate whether chronic pretreatment with a selective adenosine A2A antagonist SCH58261 or a selective adenosine A1 antagonist DPCPX can sensitize the locomotor stimulating effects of caffeine.ResultsChronic treatments with low dose caffeine (10 mg/kg) or SCH58261 (2 mg/kg) increased the concentrations of dopamine, DOPAC and HVA, concomitant with increased TH phosphorylation at Ser31 and consequently enhanced TH activity in the striatal tissues in both caffeine- and SCH58261-sensitized mice. In addition, chronic caffeine or SCH58261 administration induced locomotor sensitization, and locomotor cross-sensitization to caffeine was observed following chronic treatment of mice with SCH58261 but not with DPCPX.ConclusionsOur study demonstrated that low dosages of caffeine and a selective adenosine A2A antagonist SCH58261 elicited locomotor sensitization and cross-sensitization, which were associated with elevated dopamine concentration and TH phosphorylation at Ser31 in the striatum. Blockade of adenosine A2A receptor may play an important role in the striatal neuroadaptations observed in the caffeine-sensitized and SCH58261-sensitized mice.

A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.

Reciprocal modulation of eye‐blink and pinna‐flexion components of startle during reward anticipation

Because expectancies play a central role in current theories of dopaminergic neuron function, it is important to develop measures of reward anticipation processes. In the present study, reflexogenic bursts of white noise were presented to 39 healthy young adults as they awaited rewards and punishments in a gambling-like task. The rewards were small pieces of chocolate; the punishments, segments of bitter-tasting banana peel. Consistent with prior research on affective valence, postauricular reflexes were larger prior to rewards than punishments, whereas the reverse was true for acoustic blink reflexes. We theorized that potentiation of the postauricular reflex prior to consuming appetizing food is related to the priming of ear-retraction musculature during nursing in our remote ancestors.

MicroRNA regulation of neuron-like differentiation of adipose tissue-derived stem cells

We have reported previously that adipose tissue-derived stem cells (ADSC) could be induced by isobutylmethylxanthine (IBMX) to differentiate into neuron-like cells and such differentiation was mediated by insulin-like growth factor I (IGF-I) signaling. In the present study we show that both IBMX and IGF-I upregulated neural markers β-III-tubulin and paired-like homeobox transcription factor (Pitx3) in ADSC. Because Pitx3 is important for the maturation and function of dopaminergic neurons and has been shown to be regulated by microRNA miR-133b, we also examined the effects of IBMX and IGF-I on miR-133b expression. The results show that both IBMX and IGF-I upregulated miR-133b expression. When miR-133b was overexpressed in ADSC through transfection of a synthetic microRNA mimic, it caused a downregulation of β-III-tubulin as evidenced by immunofluorescence staining and western blot analysis. Overexpression of miR-133b also downregulated Pitx3 and IGF-1 receptor (IGF-IR), and all such downregulation occurred at the protein but not the RNA level. The apparent posttranscriptional regulation was subsequently found to be exerted through a potential miR-133b target in the 3′-untranslated region of IGF-IR, as evidenced by luciferase assay. Thus, IGF-I signaling and miR-133b co-regulate potential neural differentiation of ADSC through a feedback mechanism, in which IGF-I upregulates miR-133b while miR-133b in turn downregulates IGF-IR.

Neurotoxic conversion of β-synuclein: a novel approach to generate a transgenic mouse model of synucleinopathies?

Many groups have generated alpha-synuclein (alpha-syn) transgenic (tg) mice as a rodent model for human synucleinopathies, including Parkinson's disease and dementia with Lewy bodies (DLB). Indeed, some of the lines displayed limited evidence of neurodegeneration, such as alpha-syn deposits, compromised function of dopaminergic neurons, fibrillization of alpha-syn, and astrogliosis. However, none of them fully replicate the pathological features of synucleinopathies. To better understand the pathogenesis of the synucleinopathies and to develop new therapeutic strategies, improvement of the current version of alpha-syn tg mice may be required. We predict that beta-synuclein (beta-syn), the homologue of alpha-syn, might be a key molecule for this purpose. Although beta-syn is a neuroprotective molecule counteracting the alpha-syn pathology in tg mice, it was previously shown that both beta-syn and gamma-synuclein were associated with axonal pathology in the hippocampus of sporadic cases of Parkinson's disease and DLB. Furthermore, two missense mutations (P123H and V70M) of beta-syn were recently identified in DLB. These mutants of beta-syn were prone to aggregate in vitro and overexpression of these mutant beta-syn proteins in neuroblastoma cells resulted in enhanced lysosomal pathology. Taken together, these results suggest that a toxic gain of function of beta-syn might be involved in the pathogenesis of synucleinopathies. In this context, it is of considerable interest to determine if mutant beta-syn-overexpressing tg mice could exhibit neuropathological features distinct from those in conventional alpha-syn tg mice. Furthermore, it is expected that a bigenic mouse model for mutant beta-syn/alpha-syn might be characterized by a more accelerated phenotype of synucleinopathies.

Injection of HGF Plasmid cDNA to Prevent Manifestation of Parkinson Disease: A Preclinical Study using a Primate Model

Parkinson disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons. Current therapies provide symptomatic treatment only and thus fail to prevent the process of neurodegeneration. Recently, it has been reported that neurotrophic factors could support the survival and enhance the function of dopaminergic neurons. Thus, gene therapy using neurotrophic factors has become the center of interest. From this viewpoint, we focused on hepatocyte growth factor (HGF) as a novel neurotrophic and angiogenic growth factor. In this study, we examined the effects of over-expression of HGF on clinical symptoms induced in a monkey model of PD, aiming toward human gene therapy. Stereotaxic transfection of naked plasmid DNA encoding human HGF cDNA into the striatum resulted in significant prevention of behavioral, immunohistochemical, HPLC and PET scan findings. Overall, the present study demonstrated that over-expression of HGF prevented neuronal death in a PD primate model, providing a potential novel therapy for PD.

Conditional transgenic mice expressing C-terminally truncated human α-synuclein (αSyn119) exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons

BackgroundMissense mutations and multiplications of the α-synuclein gene cause autosomal dominant familial Parkinson's disease (PD). α-Synuclein protein is also a major component of Lewy bodies, the hallmark pathological inclusions of PD. Therefore, α-synuclein plays an important role in the pathogenesis of familial and sporadic PD. To model α-synuclein-linked disease in vivo, transgenic mouse models have been developed that express wild-type or mutant human α-synuclein from a variety of neuronal-selective heterologous promoter elements. These models exhibit a variety of behavioral and neuropathological features resembling some aspects of PD. However, an important deficiency of these models is the observed lack of robust or progressive nigrostriatal dopaminergic neuronal degeneration that is characteristic of PD.ResultsWe have developed conditional α-synuclein transgenic mice that can express A53T, E46K or C-terminally truncated (1–119) human α-synuclein pathological variants from the endogenous murine ROSA26 promoter in a Cre recombinase-dependent manner. Using these mice, we have evaluated the expression of these α-synuclein variants on the integrity and viability of nigral dopaminergic neurons with age. Expression of A53T α-synuclein or truncated αSyn119 selectively in nigrostriatal pathway dopaminergic neurons for up to 12 months fails to precipitate dopaminergic neuronal loss in these mice. However, αSyn119 expression in nigral dopaminergic neurons for up to 12 months causes a marked reduction in the levels of striatal dopamine and its metabolites together with other subtle neurochemical alterations.ConclusionWe have developed and evaluated novel conditional α-synuclein transgenic mice with transgene expression directed selectively to nigrostriatal dopaminergic neurons as a potential new mouse model of PD. Our data support the pathophysiological relevance of C-terminally truncated α-synuclein species in vivo. The expression of αSyn119 in the mouse nigrostriatal dopaminergic pathway may provide a useful model of striatal dopamine depletion and could potentially provide a presymptomatic model of PD perhaps representative of the earliest derangements in dopaminergic neuronal function observed prior to neuronal loss. These conditional α-synuclein transgenic mice provide novel tools for evaluating and dissecting the age-related effects of α-synuclein pathological variants on the function of the nigrostriatal dopaminergic pathway or other specific neuronal populations.

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.

Reinforcement learning in the brain

A wealth of research focuses on the decision-making processes that animals and humans employ when selecting actions in the face of reward and punishment. Initially such work stemmed from psychological investigations of conditioned behavior, and explanations of these in terms of computational models. Increasingly, analysis at the computational level has drawn on ideas from reinforcement learning, which provide a normative framework within which decision-making can be analyzed. More recently, the fruits of these extensive lines of research have made contact with investigations into the neural basis of decision making. Converging evidence now links reinforcement learning to specific neural substrates, assigning them precise computational roles. Specifically, electrophysiological recordings in behaving animals and functional imaging of human decision-making have revealed in the brain the existence of a key reinforcement learning signal, the temporal difference reward prediction error. Here, we first introduce the formal reinforcement learning framework. We then review the multiple lines of evidence linking reinforcement learning to the function of dopaminergic neurons in the mammalian midbrain and to more recent data from human imaging experiments. We further extend the discussion to aspects of learning not associated with phasic dopamine signals, such as learning of goal-directed responding that may not be dopamine-dependent, and learning about the vigor (or rate) with which actions should be performed that has been linked to tonic aspects of dopaminergic signaling. We end with a brief discussion of some of the limitations of the reinforcement learning framework, highlighting questions for future research.

Molecular and cellular basis of small- and intermediate-conductance, calcium-activated potassium channel function in the brain

.Small conductance calcium-activated potassium (SK or KCa2) channels link intracellular calcium transients to membrane potential changes. SK channel subtypes present different pharmacology and distribution in the nervous system. The selective blocker apamin, SK enhancers and mice lacking specific SK channel subunits have revealed multifaceted functions of these channels in neurons, glia and cerebral blood vessels. SK channels regulate neuronal firing by contributing to the afterhyperpolarization following action potentials and mediating IAHP, and partake in a calcium-mediated feedback loop with NMDA receptors, controlling the threshold for induction of hippocampal long-term potentiation. The function of distinct SK channel subtypes in different neurons often results from their specific coupling to different calcium sources. The prominent role of SK channels in the modulation of excitability and synaptic function of limbic, dopaminergic and cerebellar neurons hints at their possible involvement in neuronal dysfunction, either as part of the causal mechanism or as potential therapeutic targets.

PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons.

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.

Effect of apoptotic proteins on function of rat vasopressin-and dopaminergic hypothalamic neurons

To study character of effect of apoptosis signal proteins on activities of neurosecretory cells and neurons of rat hypothalamus, pharmacologic inhibitors of proapoptotic protein p53 Pifithrin-alpha and antiapoptotic protein Bcl-2 HA14-1 were injected into the hypothalamus. Activation of vasopressinergic neurosecretory cells at administration of the blocker Bcl-2 HA14-1 was shown: there were observed an increase of vasopressin mRNA in neurons of hypothalamus supraoptical and paraventricular nuclei, a decrease of the immunoreactive vasopressin content in posterior pituitary, and reduction of diuresis. Inactivation of p53 inhibited release of vasopressin from hypothalamus cell bodies, which is indicated by an elevated content of immunoreactive vasopressin in neurosecretory cell bodies with its unchanged synthesis, a decrease of the neurohormone content in the posterior pituitary, and an increase of diuresis rate. Activation of vasopressinergic neurons of the suprachiasmatic nucleus was also shown. Administration of the blocker Bcl-2 has been revealed to decrease functional activity both of dopaminergic neurons (Zona Incerta) and of dopaminergic neurosecretory cells (arcuate nucleus), in which a decrease of the tyrosine hydroxylase content was observed. The p53 inactivation also led to a decrease of activity of dopaminergic neurosecretory cells of arcuate nucleus, whereas activity of the proteins Zone Incerta did not change. Thus, it has been shown that a change of the apoptotic protein content in vasopressinergic and dopaminergic neurons and neurosecretory cells leads to a change of their functional activity, the character and possibly mechanisms of effects of apoptotic proteins on activities of vasopressin- and dopaminergic cells being different.

Differential regional effects of methamphetamine on dopamine transport

Multiple high-dose methamphetamine administrations cause long-lasting (> 1 week) deficits in striatal dopaminergic neuronal function. This stimulant likewise causes rapid (within 1 h) and persistent (at least 48 h) decreases in activities of striatal: 1) dopamine transporters, as assessed in synaptosomes; and 2) vesicular monoamine transporter -2 (VMAT-2), as assessed in a non-membrane-associated (referred to herein as cytoplasmic) vesicular subcellular fraction. Importantly, not all brain areas are vulnerable to methamphetamine-induced long-lasting deficits. Similarly, the present study indicates that methamphetamine exerts differential acute effects on monoaminergic transporters according to brain region. In particular, results revealed that in the nucleus accumbens, methamphetamine rapidly, but reversibly (within 24 h), decreased plasmalemmal dopamine transporter function, without effect on plasmalemmal dopamine transporter immunoreactivity. Methamphetamine also rapidly and reversibly (within 48 h) decreased cytoplasmic VMAT-2 function in this region, with relatively little effect on cytoplasmic VMAT-2 immunoreactivity. In contrast, methamphetamine did not alter either dopamine transporter or VMAT-2 activity in the hypothalamus. Noteworthy, the nucleus accumbens and hypothalamus did not display the persistent long-lasting striatal dopamine depletions caused by the stimulant. Taken together, these data suggest that deficits in plasmalemmal and vesicular monoamine transporter activity lasting greater than 24–48 h may be linked to the long-lasting dopaminergic deficits caused by methamphetamine and appear to be region specific.

MicroRNAs and cancer

The Nobel Prize in Medicine and Physiology was awarded to the RNA interference (RNAi) field in 2006 because of the huge therapeutic potential this technique harbours. However, the RNAi technology is based on a natural mechanism that utilizes short noncoding RNA molecules (microRNAs) to regulate gene expression. This paper reviews our current knowledge about microRNAs focusing on their involvement in cancer and their potential as diagnostics and therapeutics.

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons

BackgroundWe initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription.MethodologyDopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined.Principal FindingsA number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.ConclusionsThe present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

Protective Effects of K6PC-5, A Sphingosine Kinase Activator, Against 1-methyl-4-phenylpyridinium-induced Dopaminergic Neuronal Cell Death

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metaolite, regulates multiple cellular responses such as Ca signaling, cellular growth and survival, and differentiation. Sphingosine kinase (SphK) is a key enzyme in modulating the levels of S1P and is emerging as an important regulating enzyme. Here we have investigated whether K6PC-5, a newly synthesized SphK activator, plays a neuroprotective role by activating cell survival systems such as protein kinase C, phosphatidylinositol-3-kinase (PI3K), and acting on the anti-apoptotic and the pro-apoptotic genes in SN4741 dopaminergic cells. 1-methyl-4-phenylpyridinium is a neurotoxin that selectively inhibits the mitochondrial functions of dopaminergic neurons in the substantia nigra pars compacta. In the present study, we found that MPP induced a decrease in SN4741 mouse dopaminergic cell viability. K6PC-5 restored the reduced phospho-PKC and phospho-PI3K activities caused by MPP + toxicity. In addition, gene expression analysis revealed that K6PC-5 prevented both the MPP + induced expression of the pro-apoptotic gene mRNA, Bax, and the decrease of the anti-apoptotic gene mRNA, bcl-w. These results suggest that the neuroprotective mechanism of K6PC-5 against MPP-induced apoptotic cell death includes stimulation of PKC and PI3K, and modulation of cell survival and death genes.

18F-FDOPA Kinetics in Brain Tumors

L-3,4-Dihydroxy-6-(18)F-fluoro-phenyl-alanine ((18)F-FDOPA) is an amino acid analog used to evaluate presynaptic dopaminergic neuronal function. Evaluation of tumor recurrence in neurooncology is another application. Here, the kinetics of (18)F-FDOPA in brain tumors were investigated. A total of 37 patients underwent 45 studies; 10 had grade IV, 10 had grade III, and 13 had grade II brain tumors; 2 had metastases; and 2 had benign lesions. After (18)F-DOPA was administered at 1.5-5 MBq/kg, dynamic PET images were acquired for 75 min. Images were reconstructed with iterative algorithms, and corrections for attenuation and scatter were applied. Images representing venous structures, the striatum, and tumors were generated with factor analysis, and from these, input and output functions were derived with simple threshold techniques. Compartmental modeling was applied to estimate rate constants. A 2-compartment model was able to describe (18)F-FDOPA kinetics in tumors and the cerebellum but not the striatum. A 3-compartment model with corrections for tissue blood volume, metabolites, and partial volume appeared to be superior for describing (18)F-FDOPA kinetics in tumors and the striatum. A significant correlation was found between influx rate constant K and late uptake (standardized uptake value from 65 to 75 min), whereas the correlation of K with early uptake was weak. High-grade tumors had significantly higher transport rate constant k(1), equilibrium distribution volumes, and influx rate constant K than did low-grade tumors (P < 0.01). Tumor uptake showed a maximum at about 15 min, whereas the striatum typically showed a plateau-shaped curve. Patlak graphical analysis did not provide accurate parameter estimates. Logan graphical analysis yielded reliable estimates of the distribution volume and could separate newly diagnosed high-grade tumors from low-grade tumors. A 2-compartment model was able to describe (18)F-FDOPA kinetics in tumors in a first approximation. A 3-compartment model with corrections for metabolites and partial volume could adequately describe (18)F-FDOPA kinetics in tumors, the striatum, and the cerebellum. This model suggests that (18)F-FDOPA was transported but not trapped in tumors, unlike in the striatum. The shape of the uptake curve appeared to be related to tumor grade. After an early maximum, high-grade tumors had a steep descending branch, whereas low-grade tumors had a slowly declining curve, like that for the cerebellum but on a higher scale.

Inhibition of the leucine-rich repeat protein LINGO-1 enhances survival, structure, and function of dopaminergic neurons in Parkinson's disease models

The nervous system-specific leucine-rich repeat Ig-containing protein LINGO-1 is associated with the Nogo-66 receptor complex and is endowed with a canonical EGF receptor (EGFR)-like tyrosine phosphorylation site. Our studies indicate that LINGO-1 expression is elevated in the substantia nigra of Parkinson's disease (PD) patients compared with age-matched controls and in animal models of PD after neurotoxic lesions. LINGO-1 expression is present in midbrain dopaminergic (DA) neurons in the human and rodent brain. Therefore, the role of LINGO-1 in cell damage responses of DA neurons was examined in vitro and in experimental models of PD induced by either oxidative (6-hydroxydopamine) or mitochondrial (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity. In LINGO-1 knockout mice, DA neuron survival was increased and behavioral abnormalities were reduced compared with WT. This neuroprotection was accompanied by increased Akt phosphorylation (p-Akt). Similar neuroprotective in vivo effects on midbrain DA neurons were obtained in WT mice by blocking LINGO-1 activity using LINGO-1-Fc protein. Neuroprotection and enhanced neurite growth were also demonstrated for midbrain DA neurons in vitro. LINGO-1 antagonists (LINGO-1-Fc, dominant negative LINGO-1, and anti-LINGO-1 antibody) improved DA neuron survival in response to MPP+ in part by mechanisms that involve activation of the EGFR/Akt signaling pathway through a direct inhibition of LINGO-1's binding to EGFR. These results show that inhibitory agents of LINGO-1 activity can protect DA neurons against degeneration and indicate a role for the leucine-rich repeat protein LINGO-1 and related classes of proteins in the pathophysiological responses of midbrain DA neurons in PD.