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1.  Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease 
Brain  2013;136(8):2419-2431.
The pace of nigrostriatal degeneration, both with regards to striatal denervation and loss of melanin and tyrosine hydroxylase-positive neurons, is poorly understood especially early in the Parkinson’s disease process. This study investigated the extent of nigrostriatal degeneration in patients with Parkinson’s disease at different disease durations from time of diagnosis. Brains of patients with Parkinson’s disease (n = 28) with post-diagnostic intervals of 1–27 years and normal elderly control subjects (n = 9) were examined. Sections of the post-commissural putamen and substantia nigra pars compacta were processed for tyrosine hydroxylase and dopamine transporter immunohistochemistry. The post-commissural putamen was selected due to tissue availability and the fact that dopamine loss in this region is associated with motor disability in Parkinson’s disease. Quantitative assessments of putaminal dopaminergic fibre density and stereological estimates of the number of melanin-containing and tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta (both in total and in subregions) were performed by blinded investigators in cases where suitable material was available (n = 17). Dopaminergic markers in the dorsal putamen showed a modest loss at 1 year after diagnosis in the single case available for study. There was variable (moderate to marked) loss, at 3 years. At 4 years post-diagnosis and thereafter, there was virtually complete loss of staining in the dorsal putamen with only an occasional abnormal dopaminergic fibre detected. In the substantia nigra pars compacta, there was a 50–90% loss of tyrosine hydroxylase-positive neurons from the earliest time points studied with only marginal additional loss thereafter. There was only a ∼10% loss of melanized neurons in the one case evaluated 1 year post-diagnosis, and variable (30 to 60%) loss during the first several years post-diagnosis with more gradual and subtle loss in the second decade. At all time points, there were more melanin-containing than tyrosine hydroxylase-positive cells. Loss of dopaminergic markers in the dorsal putamen occurs rapidly and is virtually complete by 4 years post-diagnosis. Loss of melanized nigral neurons lags behind the loss of dopamine markers. These findings have important implications for understanding the nature of Parkinson’s disease neurodegeneration and for studies of putative neuroprotective/restorative therapies.
doi:10.1093/brain/awt192
PMCID: PMC3722357  PMID: 23884810
Parkinsons disease; human brain; morphometry; substantia nigra; neuroscience
2.  Nigrostriatal alterations in bone morphogenetic protein receptor II dominant negative mice 
Summary
Background
We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson’s disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway.
Methods
Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection.
Results
BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice.
Conclusions
Endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA.
PMCID: PMC2572854  PMID: 18642641
Bone morphogenetic protein; BMPRII; methamphetamine; apoptosis
3.  Non-motor symptoms of Parkinson’s disease revealed in an animal model with reduced monoamine storage capacity 
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, culminating in severe motor symptoms, including: resting tremor, rigidity, bradykinesia, and postural instability. In addition to motor deficits, there are a variety of non-motor symptoms associated with PD. These symptoms generally precede the onset of motor symptoms, sometimes by years, and include anosmia, problems with gastrointestinal motility, sleep disturbances, sympathetic denervation, anxiety, and depression. Previously, we have shown that mice with a 95% genetic reduction in vesicular monoamine transporter expression (VMAT2-deficient, VMAT2 LO) display progressive loss of striatal dopamine, L-DOPA responsive motor deficits, α-synuclein accumulation, and nigral dopaminergic cell loss. We hypothesized that since these animals exhibit deficits in other monoamine systems (norepinephrine, serotonin), which are known to regulate some of these behaviors that the VMAT2-deficient mice may display some of the non-motor symptoms associated with PD. Here we report that the VMAT2-deficient mice demonstrate progressive deficits in olfactory discrimination, delayed gastric emptying, altered sleep latency, anxiety-like behavior, and age-dependent depressive behavior. These results suggest that the VMAT2-deficient mice may be a useful model of the non-motor symptoms of PD. Furthermore, monoamine dysfunction may contribute to many of the non-motor symptoms of PD and interventions aimed at restoring monoamine function may be beneficial in treating the disease.
doi:10.1523/JNEUROSCI.1495-09.2009
PMCID: PMC2813143  PMID: 19553450
VMAT2; dopamine; norepinephrine; serotonin; depression; olfaction
4.  The Neurotoxicity of DOPAL: Behavioral and Stereological Evidence for Its Role in Parkinson Disease Pathogenesis 
PLoS ONE  2010;5(12):e15251.
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.
doi:10.1371/journal.pone.0015251
PMCID: PMC3001493  PMID: 21179455
5.  CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice 
Cell transplantation  2011;21(6):1213-1223.
Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered protein, which belongs to the evolutionarily conserved CDNF/MANF family of neurotrophic factors. CDNF has been shown to promote the survival of midbrain dopamine neurons in vivo. The degeneration of dopamine neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -treatment is well characterized and efficacy in this model is considered a standard criterion for development of parkinsonian therapies. MPTP is a neurotoxin, which produces parkinsonian symptoms in humans, and in C57/Bl6 mice. To date, there are no reports about the effects of CDNF on dopamine neuron survival or function in the MPTP rodent model, a critical gap. Therefore, we studied whether CDNF has neuroprotective and neurorestorative properties for the nigrostriatal dopamine system after MPTP injections in C57/Bl6 mice. We found that bilateral striatal CDNF injections, given 20-h before MPTP, improved horizontal and vertical motor behavior. CDNF pre-treatment increased tyrosine hydroxylase (TH)-immunoreactivity in the striatum and in the substantia nigra pars reticulata (SNpr), as well as the number of TH-positive cells in substantia nigra pars compacta (SNpc). Post-treatment with CDNF, given 1 week after MPTP injections, increased horizontal and vertical behavior of mice, as well as dopamine fiber densities in the striatum and the number of TH-positive cells in SNpc. CDNF did not alter any of the analyzed dopaminergic biomarkers or locomotor behavior in MPTP-untreated animals. We conclude that striatal CDNF administration is both neuroprotective and neurorestorative for the TH-positive cells in the nigrostriatal dopamine system in the MPTP model, which supports the development of CDNF-based treatment for Parkinson’s disease.
doi:10.3727/096368911X600948
PMCID: PMC3753365  PMID: 21943517
CDNF; MANF; MPTP; Parkinson’s disease
6.  Analysis of inflammation-related nigral degeneration and locomotor function in DJ-1−/− mice 
Background
Complex interactions involving genetic susceptibility and environmental factors are thought to underlie the pathogenesis of Parkinson’s disease (PD). Although the role of inflammatory processes in modulating risk for development of PD has yet to be fully understood, prospective studies suggest that chronic use of NSAIDs reduce the incidence of PD. Loss-of-function mutations in the DJ-1 gene cause a rare form of familial PD with an autosomal recessive pattern of inheritance; however, DJ-1−/− mice do not display nigrostriatal pathway degeneration, suggesting that additional factors such as inflammation may be needed to induce neurodegeneration on the background of DJ-1 gene mutations. Neuroinflammation causes oxidative stress and, based on evidence that DJ-1 plays a protective role against oxidative stress, we investigated whether DJ-1−/− mice display increased vulnerability to inflammation-induced nigral degeneration.
Methods
We exposed adult wild-type and DJ-1−/− mice to repeated intranasal administration of soluble TNF (inTNF) or repeated intraperitoneal injections of low-dose lipopolysaccharide (LPS) or saline vehicle. We measured locomotor performance using a variety of behavior tasks, striatal dopamine (DA) content by HPLC, DA neuron (TH+ cells) and total neuron (NeuN+ cells) number in the substantia nigra pars compacta and ventral tegmental area by unbiased stereology, number of Iba1-positive microglia, and mRNA levels of inflammatory and oxidative stress genes by quantitative PCR in the midbrain, cortex and isolated peritoneal macrophages of DJ-1−/− and wild-type mice.
Results
We found that chronic LPS injections induced similar neuroinflammatory responses in the midbrains of DJ-1−/− mice and wild-type mice and neither group developed locomotor deficits or nigral degeneration. inTNF administration did not appear to induce neuroinflammatory responses in LPS-treated wild-type or DJ-1−/− mice. The lack of vulnerability to inflammation-induced nigral degeneration was not due to enhanced anti-oxidant gene responses in the midbrains of DJ-1−/− mice which, in fact, displayed a blunted response relative to that of wild-type mice. Peripheral macrophages from wild-type and DJ-1−/− mice displayed similar basal and LPS-induced inflammatory and oxidative stress markers in vitro.
Conclusions
Our studies indicate that DJ-1−/− mice do not display increased vulnerability to inflammation-related nigral degeneration in contrast to what has been reported for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. We conclude that either DJ-1 does not have a critical role in protecting DA neurons against inflammation-induced oxidative stress and/or there is compensatory gene expression in the midbrain of DJ-1−/− mice that renders them resistant to the cytotoxic effects triggered by chronic peripheral inflammation.
doi:10.1186/1742-2094-10-50
PMCID: PMC3769147  PMID: 23622116
DJ-1; Dopamine; LPS; Macrophage; Neuroinflammation; Neurodegeneration; Oxidative stress; TNF
7.  JNK Inhibition Protects Dopamine Neurons and Provides Behavioral Improvement in a Rat 6-hydroxydopamine Model of Parkinson’s Disease 
ACS chemical neuroscience  2011;2(4):207-212.
Parkinson’s disease (PD) results from the loss of dopamine neurons located in the substantia nigra pars compacta (SNpc) that project to the striatum. A therapeutic has yet to be identified that halts this neurodegenerative process, and as such, development of a brain penetrant small molecule neuroprotective agent would represent a significant advancement in the treatment of the disease. To fill this void we developed an aminopyrimidine JNK inhibitor (SR-3306) that reduced the loss of dopaminergic cell bodies in the SNpc and their terminals in the striatum produced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway. Administration of SR-3306 [10 mg/kg/day (s.c.) for 14 days] increased the number of tyrosine hydroxylase immunoreactive (TH+) neurons in the SNpc by six-fold and reduced the loss of the TH+ terminals in the striatum relative to the corresponding side of 6-OHDA-lesioned rats that received only vehicle (p<0.05). In addition, SR-3306 [10 mg/kg/day (s.c.) for 14 days] decreased d-amphetamine-induced circling by 87% compared to 6-OHDA-lesioned animals given vehicle. Steady-state brain levels of SR-3306 at day 14 were 347 nM, which was approximately two-fold higher than the cell-based IC50 for this compound. Finally, immunohistochemical staining for phospho-c-jun (p-c-jun) revealed that SR-3306 [10 mg/kg/day (s.c.) for 14 days] produced a 2.3-fold reduction of the number of immunoreactive neurons in the SNpc relative to vehicle treated rats. Collectively, these data suggest that orally bioavailable JNK inhibitors may be useful neuroprotective agents for the treatment of Parkinson’s disease.
doi:10.1021/cn1001107
PMCID: PMC3110072  PMID: 21666838
8.  JNK Inhibition Protects Dopamine Neurons and Provides Behavioral Improvement in a Rat 6-Hydroxydopamine Model of Parkinson’s Disease 
ACS Chemical Neuroscience  2011;2(4):207-212.
Parkinson’s disease (PD) results from the loss of dopamine neurons located in the substantia nigra pars compacta (SNpc) that project to the striatum. A therapeutic has yet to be identified that halts this neurodegenerative process, and as such, development of a brain penetrant small molecule neuroprotective agent would represent a significant advancement in the treatment of the disease. To fill this void, we developed an aminopyrimidine JNK inhibitor (SR-3306) that reduced the loss of dopaminergic cell bodies in the SNpc and their terminals in the striatum produced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway. Administration of SR-3306 [10 mg/kg/day (s.c.) for 14 days] increased the number of tyrosine hydroxylase immunoreactive (TH+) neurons in the SNpc by 6-fold and reduced the loss of the TH+ terminals in the striatum relative to the corresponding side of 6-OHDA-lesioned rats that received only vehicle (p < 0.05). In addition, SR-3306 [10 mg/kg/day (s.c.) for 14 days] decreased d-amphetamine-induced circling by 87% compared to 6-OHDA-lesioned animals given vehicle. Steady-state brain levels of SR-3306 at day 14 were 347 nM, which was approximately 2-fold higher than the cell-based IC50 for this compound. Finally, immunohistochemical staining for phospho-c-jun (p-c-jun) revealed that SR-3306 [10 mg/kg/day (s.c.) for 14 days] produced a 2.3-fold reduction of the number of immunoreactive neurons in the SNpc relative to vehicle treated rats. Collectively, these data suggest that orally bioavailable JNK inhibitors may be useful neuroprotective agents for the treatment of Parkinson’s disease.
doi:10.1021/cn1001107
PMCID: PMC3110072  PMID: 21666838
JNK; 6-OHDA; neuroprotection; Parkinson’s disease
9.  Behavioral and Histopathological Consequences of Paraquat Intoxication in Mice: Effects of α-Synuclein Over-Expression 
Synapse (New York, N.Y.)  2007;61(12):991-1001.
Genetic variability in the α-synuclein gene and long-term exposure to the pesticide paraquat constitute possible risk factors for sporadic Parkinson’s disease. The goal of the present study was to further characterize the effects of paraquat in mice as a model of Parkinson’s disease and to determine whether it acted synergistically with α-synuclein over-expression to cause nigrostriatal cell death or dysfunction. Paraquat (10 mg/kg i.p.) was administered once a week for 3 weeks to mice over-expressing human α-synuclein under the Thy1 promoter and their wild-type littermates. The effect of paraquat on catecholaminergic neurons was reminiscent of that of Parkinson’s disease, with preferential loss of dopaminergic neurons in the ventral tier of the substantia nigra pars compacta and loss of tyrosine hydroxylase staining in the locus coeruleus. α-Synuclein over-expression did not increase paraquat-induced cell loss, and paraquat did not worsen the behavioral deficits observed in the transgenic mice. However, paraquat markedly increased proteinase-K-resistant α-synuclein aggregates in substantia nigra of the transgenic mice. The data further validate the use of paraquat to model Parkinson’s disease in mice and show that although paraquat and α-synuclein over-expression act synergistically to increase protein aggregation in vivo, this interaction does not result in short-term neuroprotection or increased vulnerability of nigrostriatal neurons.
doi:10.1002/syn.20456
PMCID: PMC3097512  PMID: 17879265
pesticide; Parkinson’s disease; substantia nigra; striatum; locus coeruleus; mice
10.  Aged Tgfβ2/Gdnf double-heterozygous mice show no morphological and functional alterations in the nigrostriatal system 
Journal of Neural Transmission  2010;117(6):719-727.
Loss of dopaminergic neurons in the substantia nigra pars compacta and the resulting decrease in striatal dopamine levels are the hallmarks of Parkinson’s disease. Tgfβ and Gdnf have been identified as neurotrophic factors for dopaminergic midbrain neurons in vivo and in vitro. Haploinsufficiency for either Tgfβ or Gdnf led to dopaminergic deficits. In this study we therefore analyzed the nigrostriatal system of aged Tgfβ2+/−/Gdnf+/− double-heterozygous mice. Unexpectedly, we found no morphological changes in the nigrostriatal system as compared with age-matched wild-type mice. There were no significant differences in the number of TH-positive midbrain neurons and no changes in the optical density of TH immunoreactivity in striata of Tgfβ2+/−/Gdnf+/− double-heterozygous mice. Moreover, we found no significant differences in the striatal levels of dopamine and its metabolites dihydroxyphenylacetic acid and homovanillic acid. Our results indicate that a combined haploinsufficiency for Tgfβ2 and Gdnf has no impact on the function and the survival of midbrain DA neurons under normal aging conditions.
doi:10.1007/s00702-010-0406-7
PMCID: PMC2879483  PMID: 20458508
Tgfβ2; Gdnf; Dopamine; Nigro-striatal system; Midbrain
11.  Loss of cannabinoid CB1 receptor expression in the 6-hydroxydopamine-induced nigrostriatal terminal lesion model of Parkinson’s disease in the rat 
Brain research bulletin  2010;81(6):543-548.
The endocannabinoid system is emerging as a potential alternative to the dopaminergic system for the treatment of Parkinson’s disease. Like all emerging targets, validation of this system’s potential for treating human Parkinsonism necessitates testing in animal models of the condition. However, if components of the endocannabinoid system are altered by the induction of a Parkinsonian state in animal models, this could have an impact on the interpretation of such preclinical experiments. This study sought to determine if expression of the CB1 subtype of cannabinoid receptor is altered in the two most commonly used rat models of Parkinson’s disease. Parkinsonian lesions were induced by stereotaxic injection of 6-hydroxydopamine into the axons (medial forebrain bundle) or terminals (striatum) of the nigrostriatal pathway. On days 1, 3, 7, 14 and 28 post-lesion, rats were sacrificed and brains were processed for tyrosine hydroxylase and CB1 receptor immunohistochemistry. The CB1 receptor was expressed strongly in the substantia nigra pars reticulata, minimally overlapping with tyrosine hydroxylase immunoreactivity in the pars compacta. Interestingly, while there was little change in CB1 receptor expression following axonal lesion, expression of the receptor was significantly reduced following terminal lesion. Loss of CB1 receptor expression in the pars reticulata correlated significantly with the loss of striatal and nigral volume after terminal lesion indicating this may have been due to 6-hydroxydopamine-induced non-specific damage of striatonigral neurons which are known to express CB1 receptors. Thus, this result has implications for the choice of model and interpretation of studies used to investigate potential cannabinoid-based therapies for Parkinson’s disease as well as striatonigral diseases such as Huntington’s disease and Multiple Systems Atrophy.
doi:10.1016/j.brainresbull.2010.01.009
PMCID: PMC3659808  PMID: 20097273
Parkinson’s disease; Endocannabinoid system; CB1 receptor; 6-Hydroxydopamine; Substantia nigra
12.  Dopamine receptor 3 might be an essential molecule in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity 
BMC Neuroscience  2013;14:76.
Background
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinson’s disease (PD)-like neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) via its oxidized product, 1-methyl-4-phenylpyridinium (MPP+), which is transported by the dopamine (DA) transporter into DA nerve terminals. DA receptor subtype 3 (D3 receptor) participates in neurotransmitter transport, gene regulation in the DA system, physiological accommodation via G protein-coupled superfamily receptors and other physiological processes in the nervous system. This study investigated the possible correlation between D3 receptors and MPTP-induced neurotoxicity. A series of behavioral experiments and histological analyses were conducted in D3 receptor-deficient mice, using an MPTP-induced model of PD.
Results
After the fourth MPTP injection, wild-type animals that received 15 mg/kg per day displayed significant neurotoxin-related bradykinesia. D3 receptor-deficient mice displayed attenuated MPTP-induced locomotor activity changes. Consistent with the behavioral observations, further neurohistological assessment showed that MPTP-induced neuronal damage in the SNpc was reduced in D3 receptor-deficient mice.
Conclusions
Our study indicates that the D3 receptor might be an essential molecule in MPTP-induced PD and provides a new molecular mechanism for MPTP neurotoxicity.
doi:10.1186/1471-2202-14-76
PMCID: PMC3734004  PMID: 23902361
Dopamine receptor 3; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity; Parkinson’s disease
13.  Heptachlor induced nigral dopaminergic neuronal loss and Parkinsonism-like movement deficits in mice 
Epidemiological studies have suggested an association between pesticide exposure and Parkinson's disease. In this study, we examined the neurotoxicity of an organochlorine pesticide, heptachlor, in vitro and in vivo. In cultured SH-SY5Y cells, heptachlor induced mitochondria-mediated apoptosis. When injected into mice intraperitoneally on a subchronic schedule, heptachlor induced selective loss of dopaminergic neurons in the substantia nigra pars compacta. In addition, the heptachlor injection induced gliosis of microglia and astrocytes selectively in the ventral midbrain area. When the general locomotor activities were monitored by open field test, the heptachlor injection did not induce any gross motor dysfunction. However, the compound induced Parkinsonism-like movement deficits when assessed by a gait and a pole test. These results suggest that heptachlor can induce Parkinson's disease-related neurotoxicities in vivo.
doi:10.1038/emm.2014.12
PMCID: PMC3944446  PMID: 24577234
apoptosis; dopaminergic neuron; heptachlor; mitochondria; organochlorine; Parkinson's disease
14.  Nigral stimulation for resistant axial motor impairment in Parkinson’s disease? A randomized controlled trial 
Brain  2013;136(7):2098-2108.
Gait and balance disturbances typically emerge in advanced Parkinson’s disease with generally limited response to dopaminergic medication and subthalamic nucleus deep brain stimulation. Therefore, advanced programming with interleaved pulses was put forward to introduce concomittant nigral stimulation on caudal contacts of a subthalamic lead. Here, we hypothesized that the combined stimulation of subthalamic nucleus and substantia nigra pars reticulata improves axial symptoms compared with standard subthalamic nucleus stimulation. Twelve patients were enrolled in this 2 × 2 cross-over double-blind randomized controlled clinical trial and both the safety and efficacy of combined subthalamic nucleus and substantia nigra pars reticulata stimulation were evaluated compared with standard subthalamic nucleus stimulation. The primary outcome measure was the change of a broad-scaled cumulative axial Unified Parkinson’s Disease Rating Scale score (Scale II items 13–15, Scale III items 27–31) at ‘3-week follow-up’. Secondary outcome measures specifically addressed freezing of gait, balance, quality of life, non-motor symptoms and neuropsychiatric symptoms. For the primary outcome measure no statistically significant improvement was observed for combined subthalamic nucleus and substantia nigra pars reticulata stimulation at the ‘3-week follow-up’. The secondary endpoints, however, revealed that the combined stimulation of subthalamic nucleus and substantia nigra pars reticulata might specifically improve freezing of gait, whereas balance impairment remained unchanged. The combined stimulation of subthalamic nucleus and substantia nigra pars reticulata was safe, and of note, no clinically relevant neuropsychiatric adverse effect was observed. Patients treated with subthalamic nucleus and substantia nigra pars reticulata stimulation revealed no ‘global’ effect on axial motor domains. However, this study opens the perspective that concomittant stimulation of the substantia nigra pars reticulata possibly improves otherwise resistant freezing of gait and, therefore, highly warrants a subsequent phase III randomized controlled trial.
doi:10.1093/brain/awt122
PMCID: PMC3692032  PMID: 23757762
Parkinson’s disease; DBS; gait; freezing; subthalamic nucleus
15.  Region-specific deficits in dopamine, but not norepinephrine, signaling in a novel A30P α-synuclein BAC transgenic mouse☆ 
Neurobiology of Disease  2014;62(100):193-207.
Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5–10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111 kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca −/−) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca −/− animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca −/− mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca −/− mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD.
Highlights
•SNCA A30P BAC transgenic mice recapitulate endogenous α-synuclein expression pattern•SNCA A30P BAC transgenic mice demonstrate a region specific deficit in evoked DA, but not NE, release.•Expression of A30P or WT α-syn restored the sensitivity of DA neurons to MPTP.•A30P BAC mice had no Lewy body-like aggregation or neuronal loss in SNpc or LC.•Early changes in DA neurotransmission in the absence of aggregation
doi:10.1016/j.nbd.2013.10.005
PMCID: PMC3898275  PMID: 24121116
Parkinson's disease; α-Synuclein; Dopamine; Norepinephrine; Voltammetry; Behavior
16.  Absence of Ret Signaling in Mice Causes Progressive and Late Degeneration of the Nigrostriatal System 
PLoS Biology  2007;5(3):e39.
Support of ageing neurons by endogenous neurotrophic factors such as glial cell line–derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration. GDNF has been tested in clinical trials for the treatment of Parkinson disease (PD), a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. BDNF modulates nigrostriatal functions and rescues DA neurons in PD animal models. The physiological roles of GDNF and BDNF signaling in the adult nigrostriatal DA system are unknown. We generated mice with regionally selective ablations of the genes encoding the receptors for GDNF (Ret) and BDNF (TrkB). We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation. These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.
Author Summary
What does a neuron need to survive? Our body produces its own survival factors for neurons, so-called neurotrophic factors, which have additional roles in neuron differentiation, growth, and function. Declining production of a neurotrophic factor or impaired signal transduction in ageing neurons may contribute to pathological neurodegeneration in humans. Glial cell line–derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) have been suggested as survival factors for midbrain dopaminergic neurons, a group of neurons primarily affected in Parkinson disease.
To investigate the physiological requirements for GDNF and BDNF to establish and maintain an important output pathway of these neurons—the nigrostriatal pathway—in the intact brain, we generated mutant mice with regionally selective ablations of the receptors for these survival factors, Ret (receptor of GDNF and related family members) or TrkB (BDNF receptor). Surprisingly, these mice survive to adulthood and show normal development and maturation of the nigrostriatal system. However, in ageing mice, ablation of Ret leads to a progressive and cell-type–specific loss of substantia nigra pars compacta neurons and their projections into the striatum. Our findings establish Ret and subsequent downstream effectors as critical regulators of long-term maintenance of the nigrostriatal system.
Ret, a receptor for glial cell line-derived neurotrophic factor, selectively regulates long-term maintenance of the nigrostriatal dopaminergic system.
doi:10.1371/journal.pbio.0050039
PMCID: PMC1808500  PMID: 17298183
17.  Cyclooxygenase-2 mediates microglial activation and secondary dopaminergic cell death in the mouse MPTP model of Parkinson's disease 
Background
Accumulating evidence suggests that inflammation plays an important role in the progression of Parkinson's disease (PD). Among many inflammatory factors found in the PD brain, cyclooxygenase (COX), specifically the inducible isoform, COX-2, is believed to be a critical enzyme in the inflammatory response. Induction of COX-2 is also found in an experimental model of PD produced by administration of 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).
Method
COX-2-deficient mice or C57BL/6 mice were treated with MPTP to investigate the effects of COX-2 deficiency or by using various doses of valdecoxib, a specific COX-2 inhibitor, which induces inhibition of COX-2 on dopaminergic neuronal toxicity and locomotor activity impairment. Immunohistochemistry, stereological cell counts, immunoblotting, an automated spontaneous locomotor activity recorder and rotarod behavioral testing apparatus were used to assess microglial activation, cell loss, and behavioral impariments.
Results
MPTP reduced tyrosine hydroxylase (TH)-positive cell counts in the substantia nigra pars compacta (SNpc); total distance traveled, vertical activity, and coordination on a rotarod; and increased microglia activation. Valdecoxib alleviated the microglial activation, the loss of TH-positive cells and the decrease in open field and vertical activity. COX-2 deficiency attenuated MPTP-induced microglial activation, degeneration of TH-positive cells, and loss of coordination.
Conclusion
These results indicate that reducing COX-2 activity can mitigate the secondary and progressive loss of dopaminergic neurons as well as the motor deficits induced by MPTP, possibly by suppression of microglial activation in the SNpc.
doi:10.1186/1742-2094-3-6
PMCID: PMC1440849  PMID: 16566823
18.  Photobiomodulation preserves behaviour and midbrain dopaminergic cells from MPTP toxicity: evidence from two mouse strains 
BMC Neuroscience  2013;14:40.
Background
We have shown previously that near-infrared light (NIr) treatment or photobiomodulation neuroprotects dopaminergic cells in substantia nigra pars compacta (SNc) from degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in Balb/c albino mice, a well-known model for Parkinson’s disease. The present study explores whether NIr treatment offers neuroprotection to these cells in C57BL/6 pigmented mice. In addition, we examine whether NIr influences behavioural activity in both strains after MPTP treatment. We tested for various locomotive parameters in an open-field test, namely velocity, high mobility and immobility.
Results
Balb/c (albino) and C57BL/6 (pigmented) mice received injections of MPTP (total of 50 mg/kg) or saline and NIr treatments (or not) over 48 hours. After each injection and/or NIr treatment, the locomotor activity of the mice was tested. After six days survival, brains were processed for TH (tyrosine hydroxylase) immunochemistry and the number of TH+ cells in the substantia nigra pars compacta (SNc) was estimated using stereology. Results showed higher numbers of TH+ cells in the MPTP-NIr groups of both strains, compared to the MPTP groups, with the protection greater in the Balb/c mice (30% vs 20%). The behavioural tests revealed strain differences also. For Balb/c mice, the MPTP-NIr group showed greater preservation of locomotor activity than the MPTP group. Behavioural preservation was less evident in the C57BL/6 strain however, with little effect of NIr being recorded in the MPTP-treated cases of this strain. Finally, there were differences between the two strains in terms of NIr penetration across the skin and fur. Our measurements indicated that NIr penetration was considerably less in the pigmented C57BL/6, compared to the albino Balb/c mice.
Conclusions
In summary, our results revealed the neuroprotective benefits of NIr treatment after parkinsonian insult at both cellular and behavioural levels and suggest that Balb/c strain, due to greater penetration of NIr through skin and fur, provides a clearer model of protection than the C57BL/6 strain.
doi:10.1186/1471-2202-14-40
PMCID: PMC3616839  PMID: 23531041
Tyrosine hydroxylase; Substantia nigra; Balb/c; C57BL/6; Neuroprotection
19.  Baicalin and deferoxamine alleviate iron accumulation in different brain regions of Parkinson's disease rats★ 
Neural Regeneration Research  2012;7(27):2092-2098.
Previous studies found that iron accumulates in the substantia nigra of Parkinson’s disease patients. However, it is still unclear whether other brain regions have iron accumulation as well. In this experiment, rats with rotenone-induced Parkinson’s disease were treated by gastric perfusion of baicalin or intraperitoneal injection of deferoxamine. Immunohistochemical staining demonstrated that iron accumulated not only in the substantia nigra pars compacta, but also significantly in the striatum globus pallidus, the dentate gyrus granular layer of the hippocampus, the dentate-interpositus and the facial nucleus of the cerebellum. Both baicalin and deferoxamine, which are iron chelating agents, significantly inhibited iron deposition in these brain areas, and substantially reduced the loss of tyrosine hydroxylase-positive cells. These chelators also reduced iron content in the substantia nigra. In addition to the substantia nigra, iron deposition was observed in other brain regions as well. Both baicalin and deferoxamine significantly inhibited iron accumulation in different brain regions, and had a protective effect on dopaminergic neurons.
doi:10.3969/j.issn.1673-5374.2012.27.002
PMCID: PMC4281409  PMID: 25558221
Parkinson’s disease; rotenone; iron; baicalin; deferoxamine; substantia nigra; striatum; hippocampus; cerebellum; neurodegenerative disease; regeneration; neural regeneration
20.  Brain region specific mitophagy capacity could contribute to selective neuronal vulnerability in Parkinson's disease 
Proteome Science  2011;9:59.
Parkinson's disease (PD) is histologically well defined by its characteristic degeneration of dopaminergic neurons in the substantia nigra pars compacta. Remarkably, divergent PD-related mutations can generate comparable brain region specific pathologies. This indicates that some intrinsic region-specificity respecting differential neuron vulnerability exists, which codetermines the disease progression. To gain insight into the pathomechanism of PD, we investigated protein expression and protein oxidation patterns of three different brain regions in a PD mouse model, the PINK1 knockout mice (PINK1-KO), in comparison to wild type control mice. The dysfunction of PINK1 presumably affects mitochondrial turnover by disturbing mitochondrial autophagic pathways. The three brain regions investigated are the midbrain, which is the location of substantia nigra; striatum, the major efferent region of substantia nigra; and cerebral cortex, which is more distal to PD pathology. In all three regions, mitochondrial proteins responsible for energy metabolism and membrane potential were significantly altered in the PINK1-KO mice, but with very different region specific accents in terms of up/down-regulations. This suggests that disturbed mitophagy presumably induced by PINK1 knockout has heterogeneous impacts on different brain regions. Specifically, the midbrain tissue seems to be most severely hit by defective mitochondrial turnover, whereas cortex and striatum could compensate for mitophagy nonfunction by feedback stimulation of other catabolic programs. In addition, cerebral cortex tissues showed the mildest level of protein oxidation in both PINK1-KO and wild type mice, indicating either a better oxidative protection or less reactive oxygen species (ROS) pressure in this brain region. Ultra-structural histological examination in normal mouse brain revealed higher incidences of mitophagy vacuoles in cerebral cortex than in striatum and substantia nigra. Taken together, the delicate balance between oxidative protection and mitophagy capacity in different brain regions could contribute to brain region-specific pathological patterns in PD.
doi:10.1186/1477-5956-9-59
PMCID: PMC3196908  PMID: 21943346
Parkinson's disease; mitophagy; proteomics; PINK1; 2DE
21.  Parkin Disease: A Clinicopathologic Entity? 
JAMA neurology  2013;70(5):571-579.
Importance
Mutations in the gene encoding parkin (PARK2) are the most common cause of autosomal recessive juvenile-onset and young-onset parkinsonism. The few available detailed neuropathologic reports suggest that homozygous and compound heterozygous parkin mutations are characterized by severe substantia nigra pars compacta neuronal loss.
Objective
To investigate whether parkin -linked parkinsonism is a different clinicopathologic entity to Parkinson disease (PD).
Design, Setting, and Participants
We describe the clinical, genetic, and neuropathologic findings of 5 unrelated cases of parkin disease and compare them with 5 pathologically confirmed PD cases and 4 control subjects. The PD control cases and normal control subjects were matched first for age at death then disease duration (PD only) for comparison.
Results
Presenting signs in the parkin disease cases were hand or leg tremor often combined with dystonia. Mean age at onset was 34 years; all cases were compound heterozygous for mutations of parkin. Freezing of gait, postural deformity, and motor fluctuations were common late features. No patients had any evidence of cognitive impairment or dementia. Neuronal counts in the substantia nigra pars compacta revealed that neuronal loss in the parkin cases was as severe as that seen in PD, but relative preservation of the dorsal tier was seen in comparison with PD (P = .04). Mild neuronal loss was identified in the locus coeruleus and dorsal motor nucleus of the vagus, but not in the nucleus basalis of Meynert, raphe nucleus, or other brain regions. Sparse Lewy bodies were identified in 2 cases (brainstem and cortex).
Conclusions and Relevance
These findings support the notion that parkin disease is characterized by a more restricted morphologic abnormality than is found in PD, with predominantly ventral nigral degeneration and absent or rare Lewy bodies.
doi:10.1001/jamaneurol.2013.172
PMCID: PMC4202385  PMID: 23459986
22.  Environmental neurotoxin-induced progressive model of parkinsonism in rats 
Annals of neurology  2010;68(1):70-80.
Objective
Exposure to a number of drugs, chemicals or environmental factors can cause parkinsonism. Epidemiologic evidence supports a causal link between the consumption of flour made from the washed seeds of the plant, Cycas micronesica, by the Chamorro population of Guam and the development of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC).
Methods
We now report that consumption of washed cycad flour pellets by Sprague-Dawley male rats induces progressive parkinsonism.
Results
Cycad-fed rats displayed motor abnormalities after two to three months of feeding such as spontaneous unilateral rotation, shuffling gait and stereotypy. Histological and biochemical examination of brains from cycad-fed rats revealed an initial decrease in the levels of dopamine and its metabolites in the striatum (STR), followed by neurodegeneration of dopaminergic (DAergic) cell bodies in the substantia nigra pars compacta (SNc). α-synuclein (α-syn; proteinase K-resistant) and ubiquitin aggregates were found in the DAergic neurons of the SNc and neurites in the STR. In addition, we identified α-syn aggregates in neurons of the locus coeruleus and cingulate cortex. No loss of motor neurons in the spinal cord was found after chronic consumption of cycad flour. In an organotypic slice culture of the rat substantia nigra and the striatum, an organic extract of cycad causes a selective loss of DA neurons and α-synuclein aggregates in the substantia nigra.
Interpretation
Cycad-fed rats exhibit progressive behavioral, biochemical, and histological hallmarks of parkinsonism, coupled with a lack of fatality.
doi:10.1002/ana.22018
PMCID: PMC2988442  PMID: 20582986
α-synuclein; progressive neurodegeneration; cycad neurotoxins
23.  Forebrain Ischemia Triggers GABAergic System Degeneration in Substantia Nigra at Chronic Stages in Rats 
The long-term consequences of forebrain ischemia include delayed Parkinson's syndrome. This study revealed delayed neurodegeneration in the substantia nigra 8 weeks after 12.5 minutes of global ischemia in rat brain. Following neuronal loss of 30–40% in central and dorsolateral striatum at day 3, neuronal damage in the substantia nigra (SN) was assessed at 4–8 weeks using immunohistochemistry for glutamate decarboxylase 67 (GAD67), vesicular GABA transporter (VGAT), and calretinin (CR). At day 56, the optical density of GAD67-, but not VGAT-, immunoreactivity in substantia nigra pars reticulata (SNR)—significantly decreased. CR-neurons concentrated in substantia nigra pars compacta (SNC) were reduced by 27% from day 3 (n = 5) to day 56 (n = 7, ANOVA, p < .01). Movement coordination was impaired at day 56, as evaluated using beam-walking test (time-to-traverse 5.6 ± 1.2 sec versus 11.8 ± 5.4 sec; sham versus ischemia, p < .05, n = 5, and 7, resp.). Our results demonstrate delayed impairment of the GABAergic system components in SN and associated with movement deficits after global ischemia.
doi:10.1155/2010/506952
PMCID: PMC2957857  PMID: 20981346
24.  Overexpression of parkin in rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity 
Experimental neurology  2013;247:359-372.
Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity.
doi:10.1016/j.expneurol.2013.01.001
PMCID: PMC4321803  PMID: 23313192
methamphetamine; toxicity; parkin; neuroprotection; dopamine; striatum
25.  Reduced vesicular storage of catecholamines causes progressive degeneration in the locus ceruleus 
Neuropharmacology  2013;76(0 0):97-105.
Parkinson’s disease (PD) is the most common neurodegenerative motor disease. Pathologically, PD is characterized by Lewy body deposition and subsequent death of dopamine neurons in the substantia nigra pars compacta. PD also consistently features degeneration of the locus ceruleus, the main source of norepinephrine in the central nervous system. We have previously reported a mouse model of dopaminergic neurodegeneration based on reduced expression of the vesicular monoamine transporter (VMAT2 LO). To determine if reduced vesicular storage can also cause noradrenergic degeneration, we examined indices of damage to the catecholaminergic systems in brain and cardiac tissue of VMAT2 LO mice. At two months of age, neurochemical analyses revealed substantial reductions in striatal dopamine (94%), cortical dopamine (57%) and norepinephrine (54%), as well as cardiac norepinephrine (97%). These losses were accompanied by increased conversion of dopamine and norepinephrine to their deaminated metabolites. VMAT2 LO mice exhibited loss of noradrenergic innervation in the cortex, as determined by norepinephrine transporter immunoreactivity and 3H-nisoxetine binding. Using unbiased stereological techniques, we observed progressive degeneration in the locus ceruleus that preceded degeneration of the substantia nigra pars compacta. In contrast, the ventral tegmental area, which is spared in human PD, remained unaffected. The coordinate loss of dopamine and norepinephrine neurons in VMAT2-LO mice parallels the pattern of neurodegeneration that occurs in human PD, and demonstrates that insufficient catecholamine storage can cause spontaneous degeneration in susceptible neurons, underscoring cytosolic catecholamine catabolism as a determinant of neuronal susceptibility in PD.
doi:10.1016/j.neuropharm.2013.08.033
PMCID: PMC4049095  PMID: 24025942
VMAT2; norepinephrine; Parkinson’s disease; locus ceruleus

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