Neuronal localization of the tyrosine-specific protein kinase p62c-yes in rat basal ganglia

The cellular localization of the tyrosine-specific protein kinase p62^c-yes , the product of the proto-oncogene c-yes, has been examined in the striatonigral neurons which interconnect the rat neostriatum and substantia nigra. Although p62^c-yes was more enriched in the neostriatum than in the substantia nigra, excitotoxin-induced necrosis of nerve cells in the neostriatum led to 50–60% decreases of p62^c-yes both in the lesioned neostriatum and in the ipsilateral substantia nigra. Hence, the p62^c-yes tyrosine kinase is present both in the cell body region and in the axonal and nerve terminal region of the striatonigral neurons. This localization indicates that the enzyme may be involved in both presynaptic and postsynaptic functions in mammalian forebrain neurons.Special issue dedicated to Dr. Paul Greengard.     

Globus Pallidus Externus Neurons Expressing parvalbumin Interconnect the Subthalamic Nucleus and Striatal Interneurons

The globus pallidus externus (GP) is a nucleus of the basal ganglia (BG), containing GABAergic projection neurons that arborize widely throughout the BG, thalamus and cortex. Ongoing work seeks to map axonal projection patterns from GP cell types, as defined by their electrophysiological and molecular properties. Here we use transgenic mice and recombinant viruses to characterize parvalbumin expressing (PV⁺) GP neurons within the BG circuit. We confirm that PV⁺ neurons 1) make up ~40% of the GP neurons 2) exhibit fast-firing spontaneous activity and 3) provide the major axonal arborization to the STN and substantia nigra reticulata/compacta (SNr/c). PV⁺ neurons also innervate the striatum. Retrograde labeling identifies ~17% of pallidostriatal neurons as PV⁺, at least a subset of which also innervate the STN and SNr. Optogenetic experiments in acute brain slices demonstrate that the PV⁺ pallidostriatal axons make potent inhibitory synapses on low threshold spiking (LTS) and fast-spiking interneurons (FS) in the striatum, but rarely on spiny projection neurons (SPNs). Thus PV⁺ GP neurons are synaptically positioned to directly coordinate activity between BG input nuclei, the striatum and STN, and thalamic-output from the SNr.     

Dopaminergic neurons in explants of substantia nigra in culture

Explants of substantia nigra and corpus striatum obtained from newborn rats were maintained in tissue culture for up to six days. Explants of substantia nigra exhibited a net increase in the ability to take up H³-dopamine, a process associated with the dopaminergic neurons; in contrast, the explants of corpus striatum showed a rapid loss in this ability to accumulate H³-dopamine. After three days in culture, the specific activity of tyrosine hydroxylase and monoamine oxidase had decreased 50% in explants of substantia nigra. A medium including fetal calf serum and chick embryo extracts was necessary for the increase in H³-dopamine uptake, and nerve growth factor had an inhibitory effect. Histofluorescent examination of nigral explants cultured for three days indicated morphologically normal dopaminergic neurons.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

DARPP-32 and Phosphatase Inhibitor-1, Two Structurally Related Inhibitors of Protein Phosphatase-1, Are Both Present in Striatonigral Neurons

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of Mr = 32,000) and phosphatase inhibitor-1, two previously characterized inhibitors of protein phosphatase-1, were identified in both the neostriatum and the substantia nigra. Phosphatase inhibitor-1 was partially purified from bovine caudate nucleus and found to be distinct from DARPP-32 in some of its biochemical properties. The neuronal localization of DARPP-32 and phosphatase inhibitor-1 within the rat neostriatum and substantia nigra was investigated by studying the effects of kainic acid. Injection into the neostriatum of kainic acid, which destroys striatonigral neurons and striatonigral fibers, decreased the amounts of DARPP-32 and phosphatase inhibitor-1 to the same extent, both in the lesioned neostriatum and in the ipsilateral substantia nigra. The specific activity of protein phosphatase-1 in the neostriatum was unaffected by kainic acid. The results indicate that, in rat brain, DARPP-32 and phosphatase inhibitor-1 are both present in striatal neurons and in striatonigral fibers, and that they probably coexist in at least a subpopulation of striatonigral neurons. In contrast, protein phosphatase-1 does not appear to be enriched in any specific neuronal subpopulation in the neostriatum.     

Habenular modulation of dynorphinergic systems in rat ventral mesencephalon

Bilateral electrolytic lesion of the striatonigral pathways (which convey massive afferents to the substantia nigra) caused a marked lowering of alpha-neo-endorphin (alpha-Neo) and dynorphin A(1-8) [Dyn A(1-8)] levels in the substantia nigra without affecting the alpha-Neo content in the ventral tegmental area. Moreover, unilateral infusion of the axon sparing neurotoxin ibotenate into the striatum, but not into the substantia nigra, decrease these two opioid peptides in the substantia nigra on the side ipsilateral to the lesion, failing to modify the alpha-Neo levels in the ventral tegmental area. Bilateral electrolytic lesion of the habenula augmented alpha-Neo content in the substantia nigra and ventral tegmental area at 8-30 days postlesion without affecting the nigral Dyn A(1-8). These results add further support to the view that alpha-Neo- and Dyn A(1-8)-containing neurons projecting to the substantia nigra originate in the striatum and descend through striatonigral pathways. The present data provide evidence that the habenula may participate in the regulation of the activity of alpha-Neo-immunoreactive neurons in the substantia nigra and ventral tegmental area.     

Regulation of GDF-15, a distant TGF-β superfamily member,in a mouse model of cerebral ischemia

GDF-15 is a novel distant member of the TGF-β superfamily and is widely distributed in the brain and peripheral nervous system. We have previously reported that GDF-15 is a potent neurotrophic factor for lesioned dopaminergic neurons in the substantia nigra, and that GDF-15-deficient mice show progressive postnatal losses of motor and sensory neurons. We have now investigated the regulation of GDF-15 mRNA and immunoreactivity in the murine hippocampal formation and selected cortical areas following an ischemic lesion by occlusion of the middle cerebral artery (MCAO). MCAO prominently upregulates GDF-15 mRNA in the hippocampus and parietal cortex at 3 h and 24 h after lesion. GDF-15 immunoreactivity, which is hardly detectable in the unlesioned brain, is drastically upregulated in neurons identified by double-staining with NeuN. NeuN staining reveals that most, if not all, neurons in the granular layer of the dentate gyrus and pyramidal layers of the cornu ammonis become GDF-15-immunoreactive. Moderate induction of GDF-15 immunoreactivity has been observed in a small number of microglial cells identified by labeling with tomato lectin, whereas astroglial cells remain GDF-15-negative after MCAO. Comparative analysis of the size of the infarcted area after MCAO in GDF-15 wild-type and knockout mice has failed to reveal significant differences. Together, our data substantiate the notion that GDF-15 is prominently upregulated in the lesioned brain and might be involved in orchestrating post-lesional responses other than the trophic support of neurons.     

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B‐GFP transgenic mice

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B‐GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (?540 to +31). Using the fresh brain sections of F1B‐GFP transgenic mice, we found that the F1B‐GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B‐GFP⁺ cells existed in the brains of F1B‐GFP transgenic mice. We demonstrated that one population of F1B‐GFP⁺ cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B‐GFP⁺ cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B‐GFP⁺ cells and tyrosine hydroxylase indicated that a subpopulation of F1B‐GFP⁺‐neuronal cells was dopaminergic neurons. Importantly, these F1B‐GFP⁺/TH⁺ cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B‐GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 232–248, 2015     

Nigral and Striatal Comparative Study of the Neurotoxic Action of 1-Methyl-4-Phenylpyridinium Ion: Involvement of Dopamine Uptake System

Abstract: Microdialysis was used in a comparative study of the neurotoxic action of MPP⁺ in the absence or presence of nomifensine (20 µM) in the striatum and substantia nigra. Three different concentrations of MPP⁺ (1, 2.5, and 5 mM) were perfused for 15 min at 24 (day 1) and 48 h (day 2) after surgery. The dopamine basal value in the striatum was ～17 fmol/min. Nomifensine (20 µM) stimulated dopamine release to ～170 fmol/min. The increase of dopamine extracellular output in the striatum after MPP⁺ perfusion on day 1 was independent of the concentration of MPP⁺ perfused and of the absence or presence of nomifensine (20 µM), being ～2,500 fmol/min. The dopamine basal value in the substantia nigra was below the detection limit of our HPLC equipment. Nomifensine (20 µM) stimulated dopamine release to ～6.3 fmol/min. The increase of dopamine extracellular output in the substantia nigra was MPP⁺ dose-dependent (1 mM, 75 fmol/min; 2.5 mM, 150 fmol/min; and 5 mM, 250 fmol/min) and independent of the presence or absence of nomifensine. On day 2, the presence of nomifensine on day 1 produced a total protection against MPP⁺ (1 mM) perfusion in the striatum, which was not observed against MPP⁺ (5 mM). MPP⁺ (1 mM) did not produce any neurotoxic action in the substantia in the absence or presence of nomifensine. The MPP⁺ (2.5 mM) effect on dopamine extracellular output in the absence of nomifensine (20 µM) in the substantia nigra on day 2 was similar to that of MPP⁺ (1 mM) in the striatum. The presence of nomifensine (20 µM) partially prevented the neurotoxic effect of MPP⁺ (2.5 mM) on dopaminergic cell bodies/dendrites in the substantia nigra. The MPP⁺ (5 mM) effect on dopamine extracellular output was similar in both structures studied in the absence or presence of nomifensine on day 2. These results suggest that terminals in the striatum are more sensitive to the neurotoxicity of MPP⁺ than cell bodies/dendrites in the substantia nigra.     

Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways

Background

Parkinson’s disease is the second most common neurodegenerative disorders after Alzheimer’s disease. The main cause of the disease is the massive degeneration of dopaminergic neurons in the substantia nigra. Neuronal apoptosis and neuroinflammation are thought to be the key contributors to the neuronal degeneration.

Results

Both CATH.a cells and ICR mice were treated with 1-methyl-4-phenylpyridin (MPP⁺) to induce neurotoxicity in vitro and in vivo. Western blotting and immunohistochemistry were also used to analyse neurotoxicity, neuroinflammation and aberrant neurogenesis in vivo. The experiment in CATH.a cells showed that the treatment of MPP⁺ impaired intake of cell membrane and activated caspase system, suggesting that the neurotoxic mechanisms of MPP⁺ might include both necrosis and apoptosis. Pretreatment of lithospermic acid might prevent these toxicities. Lithospermic acid possesses specific inhibitory effect on caspase 3. In mitochondria, MPP⁺ caused mitochondrial depolarization and induced endoplasmic reticulum stress via increasing expression of chaperone protein, GRP-78. All the effects mentioned above were reduced by lithospermic acid. In animal model, the immunohistochemistry of mice brain sections revealed that MPP⁺ decreased the amount of dopaminergic neurons, enhanced microglia activation, promoted astrogliosis in both substantia nigra and hippocampus, and MPP⁺ provoked the aberrant neurogenesis in hippocampus. Lithospermic acid significantly attenuates all of these effects induced by MPP⁺.

Conclusions

Lithospermic acid is a potential candidate drug for the novel therapeutic intervention on Parkinson’s disease.     

The Protective Effect of Human Umbilical Cord Blood CD34+ Cells and Estradiol against Focal Cerebral Ischemia in Female Ovariectomized Rat: Cerebral MR Imaging and Immunohistochemical Study

Human umbilical cord blood derived CD34⁺ stem cells are reported to mediate therapeutic effects in stroke animal models. Estrogen was known to protect against ischemic injury. The present study wished to investigate whether the protective effect of CD34⁺ cells against ischemic injury can be reinforced with complemental estradiol treatment in female ovariectomized rat and its possible mechanism. Experiment 1 was to determine the best optimal timing of CD34⁺ cell treatment for the neuroprotective effect after 60-min middle cerebral artery occlusion (MCAO). Experiment 2 was to evaluate the adjuvant effect of 17β-estradiol on CD34⁺ cell neuroprotection after MCAO. Experiment 1 showed intravenous infusion with CD34⁺ cells before MCAO (pre-treatment) caused less infarction size than those infused after MCAO (post-treatment) on 7T magnetic resonance T2-weighted images. Experiment 2 revealed infarction size was most significantly reduced after CD34⁺ + estradiol pre-treatment. When compared with no treatment group, CD34⁺ + estradiol pre-treatment showed significantly less ADC reduction at 2 h and 2 d, less CBF reduction at 2 h and less hyperperfusion at 2 d. The immunoreactivity of c-Fos, c-Jun and GFAP was attenuated, and BDNF showed significant recovery from 2 h to 2 d after MCAO, especially after CD34⁺ + estradiol pre-treatment. The present study suggests pre-treatment with CD34⁺ cells with complemental estradiol can be most protective against ischemic injury, which may act through stabilization of cerebral hemodynamics and normalization of the expressions of immediate early genes and BDNF.     

Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP⁺ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP⁺ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.     

BK Channels Reveal Novel Phosphate Sensitivity in SNr Neurons

Whether large conductance Ca²⁺-activated potassium (BK) channels are present in the substantia nigra pars reticulata (SNr) is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca²⁺]_i) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the K_ATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with K_ATP channels under ischemic stresses.     

Increased acoplasmic transport of H3-dopamine in nigro-neostriatal neurons after reserpine

Recent evidence suggests that dopamine can undergo axoplasmic transport in nigro-neostriatal neurons by binding to amine storage granules. In the present experiments it was demonstrated that reserpine pretreatment (10 mg/kg) 24 hours before stereotaxic injections of ³H-DOPA or ³H-dopamine into the substantia nigra increases the amount of ³H-dopamine transported to the neostriatum by about 300 percent. The activity recovered from the substantia nigra was significantly reduced by reserpine pretreatment however. Stereotaxic injection of ¹⁴C-leucine into the substantia nigra indicated that neither fast nor slow axoplasmic transport of protein was influenced by reserpine pretreatment in these same neurons. The increased transport of dopamine appears therefore to be due to a relatively selective action of reserpine. The results suggest that reserpine either (i) increases the binding of dopamine to newly synthesized amine storage granules, (ii) increases the number of newly synthesized amine storage granules, or (iii) accelerates the rate of transport of amine storage granules. In addition, the results support the view that reserpine can increase the membrane permeability of adrenergic neurons to the outward movement of catecholamines.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

Distribution of zinc in the substantia nigra of rats treated with 6-hydroxydopamine

To study the relationship between tissue accumulation of Zinc (Zn) and neurodegeneration in the nigrostriatal dopaminergic pathway,⁶⁵Zn distribution in this pathway was examined after unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rats. When⁶⁵ZnCl₂ was intravenously injected 4 days after treatment with 6-OHDA,⁶⁵Zn was concentrated in the ipsilateral substantia nigra 6 days after⁶⁵Zn injection. On the other hand, 19 d after treatment with 6-OHDA,⁶⁵Zn distribution in the ipsilateral substantia nigra was decreased to the level of the contralateral one. When NH₄ ⁹⁹TcO₄, which cannot go through the blood-brain barrier, was injected into rats 4 d after treatment with 6-OHDA,⁹⁹Tc was concentrated in the ipsilateral substantia nigra 30 min after⁹⁹Tc injection, but no longer detectable 6 d after injection. These results suggest that Zn is necessary for a repair process called replacement gliosis after the death of neurons and that excess Zn does not accumulate in the lesion after completion of the gliosis.     

Tat-Fused Recombinant Human SAG Prevents Dopaminergic Neurodegeneration in a MPTP-Induced Parkinson’s Disease Model

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson’s disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP⁺) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP⁺ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.     

Somatodendritic dopamine release: recent mechanistic insights

Dopamine (DA) is a key transmitter in motor, reward and cogitative pathways, with DA dysfunction implicated in disorders including Parkinson''s disease and addiction. Located in midbrain, DA neurons of the substantia nigra pars compacta project via the medial forebrain bundle to the dorsal striatum (caudate putamen), and DA neurons in the adjacent ventral tegmental area project to the ventral striatum (nucleus accumbens) and prefrontal cortex. In addition to classical vesicular release from axons, midbrain DA neurons exhibit DA release from their cell bodies and dendrites. Somatodendritic DA release leads to activation of D2 DA autoreceptors on DA neurons that inhibit their firing via G-protein-coupled inwardly rectifying K⁺ channels. This helps determine patterns of DA signalling at distant axonal release sites. Somatodendritically released DA also acts via volume transmission to extrasynaptic receptors that modulate local transmitter release and neuronal activity in the midbrain. Thus, somatodendritic release is a pivotal intrinsic feature of DA neurons that must be well defined in order to fully understand the physiology and pathophysiology of DA pathways. Here, we review recent mechanistic aspects of somatodendritic DA release, with particular emphasis on the Ca²⁺ dependence of release and the potential role of exocytotic proteins.     

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons: RELEVANCE TO THE PATHOGENESIS OF PARKINSON DISEASE*

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP⁺) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP⁺-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP⁺-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.     

The neurotoxicity of DOPAL: behavioral and stereological evidence for its role in Parkinson disease pathogenesis

Background

The etiology of Parkinson disease (PD) has yet to be fully elucidated. We examined the consequences of injections of 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of dopamine, into the substantia nigra of rats on motor behavior and neuronal survival.

Methods/Principal Findings

A total of 800 nl/rat of DOPAL (1 µg/200 nl) was injected stereotaxically into the substantia nigra over three sites while control animals received similar injections of phosphate buffered saline. Rotational behavior of these rats was analyzed, optical density of striatal tyrosine hydroxylase was calculated, and unbiased stereological counts of the substantia nigra were made. The rats showed significant rotational asymmetry ipsilateral to the lesion, supporting disruption of dopaminergic nigrostriatal projections. Such disruption was verified since the density of striatal tyrosine hydroxylase decreased significantly (p<0.001) on the side ipsilateral to the DOPAL injections when compared to the non-injected side. Stereological counts of neurons stained for Nissl in pars compacta of the substantia nigra significantly decreased (p<0.001) from control values, while counts of those in pars reticulata were unchanged after DOPAL injections. Counts of neurons immunostained for tyrosine hydroxylase also showed a significant (p = 0.032) loss of dopaminergic neurons. In spite of significant loss of dopaminergic neurons, DOPAL injections did not induce significant glial reaction in the substantia nigra.

Conclusions

The present study provides the first in vivo quantification of substantia nigra pars compacta neuronal loss after injection of the endogenous toxin DOPAL. The results demonstrate that injections of DOPAL selectively kills SN DA neurons, suggests loss of striatal DA terminals, spares non-dopaminergic neurons of the pars reticulata, and triggers a behavioral phenotype (rotational asymmetry) consistent with other PD animal models. This study supports the “catecholaldehyde hypothesis” as an important link for the etiology of sporadic PD.