Juvenile Parkinsonism Research Articles

Involvement of peripheral nervous system in juvenile Parkinson's disease.

We evaluated, by using electrophysiological techniques, 29 patients with juvenile Parkinson's disease (JP), who had no known causes or clinical signs of neuropathy. Electromyographic evidence of chronic partial denervation with reinnervation was observed in nine patients (34.6%). Abnormalities of motor conduction in the common peroneal nerve were present in four (13.8%), Sural sensory conduction in nine (31.9%) and sympathetic skin response (SSR) in eleven (37.9%) patients. The symptoms of dysautonomia correlated poorly with changes in SSR. These abnormalities were independent of age at onset, duration or severity of the disease and antiparkinsonian drugs used. This study suggests that the peripheral nervous system is involved in JP in more than 50% of patients. The commonly observed symptoms of dysautonomia in Parkinson's disease may have a peripheral origin.

SPG11 spastic paraplegia

Autosomal recessive hereditary spastic paraplegia (AR HSP) with thin corpus callosum (TCC) is a rare neurodegenerative disorder often caused by mutations in the gene encoding for spatacsin at the SPG11 locus on chromosome 15q. The disease is characterized by progressive spastic paraparesis and mental retardation which occur during the first two decades of life and frequently with peripheral neuropathy. Brain magnetic resonance imaging (MRI) reveals typical TCC with periventricular white matter changes. We describe two patients, of Turkish descent, from the same consanguineous family and affected with SPG11 in association with unusual early-onset parkinsonism. Parkinsonism occurred during the very early stages of SPG11 in both patients, being in one the inaugural symptom of the disease presented as a resting tremor with akinesia, rigidity and expressing an initial moderate levodopa-response that progressively weakened. The second patient presented a resting tremor with mild akinesia and no levodopa-response. Both patients were affected with progressive spastic paraparesis which had initially occurred at 15 and 12 years of age, respectively, in association with mild mental retardation and an axonal polyneuropathy. TCC with periventricular white matter changes (PWMC) was evident by MRI and (123)I-ioflupane SPECT was abnormal. Genetic analysis detected for both patients a new c.704_705delAT, p.H235RfsX12 homozygous mutation in SPG11. This report provides evidence that parkinsonism may initiate SPG11-linked HSP TCC and that SPG11 may cause juvenile parkinsonism.

Comparación del desempeño neuropsicológico según la edad de inicio en sujetos con enfermedad de Parkinson y parkinsonismo

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, and it is characterised by tremor, bradykinesia, rigidity and postural instability. The most frequent cognitive disorder is executive dysfunction, although global deficits associated to late onset of the disease have also been reported. To describe and to compare cognitive performance in three groups with PD and one with Parkinsonism. A neurological and neuropsychological evaluation was carried out on 175 patients with idiopathic PD and Parkinsonism. The data analysis was performed by comparing the results of the tests carried out on the four groups: three with PD (age of onset: juvenile, adult and late) and one with Parkinsonism, while controlling for age, schooling and time of progression. In the juvenile PD group, alterations were observed in the number of intrusions in verbal memory; in the adult PD and late PD groups, there were alterations in time in continuous visual execution. These differences disappeared when the groups were compared to each other while also controlling for age. The Parkinsonism group obtained results that were lower than those of all the groups with PD for most of the cognitive and functional variables. Idiopathic PD would not be the cause of multiple cognitive impairment, but of a specific alteration, mainly involving the speed of processing and information recall. Age of onset would not be a decisive factor in the degree of impairment of cognitive functioning; important cognitive impairment was only present in the group with Parkinsonism.

Parkin gene therapy

The search for genetic mutations responsible for familial forms of Parkinson's disease (PD) has identified several genes and loci. Mutations in the parkin gene are linked to autosomal recessive juvenile parkinsonism. The genetic forms of PD are uncommon, but gene therapy targeting α-synuclein, parkin, or other pathways may be also applicable for idiopathic PD. Intriguingly, several studies suggest that parkin gene therapy could be useful in a subset of PD patients with mutations in the α-synuclein gene. Furthermore, if parkin overexpression can correct neuronal degeneration in the substantia nigra and striatum, it might be a potentially effective therapy for α-synucleinopathy.

Hyperphosphorylated tau aggregates in the cortex and hippocampus of transgenic mice with mutant human FTDP-17 Tau and lacking the PARK2 gene

Mutations in the PARK2 gene encoding parkin cause autosomal recessive juvenile parkinsonism, but have also been found in patients diagnosed with certain tauopathies. Conversely, mutations in the MAPT gene encoding tau are present in some types of parkinsonism. In order to investigate the possible relationship between these two proteins, we generated a double mutant mouse that is deficient in PARK2 and that over-expresses the hTauVLW transgene, a mutant form of the tau protein present in FTDP-17. Independent deletion of PARK2 or over-expression of the hTauVLW transgene produces mild phenotypic alterations, while a substantial increase in parkin expression is observed in hTauVLW transgenic mice. However, double mutant mice present memory and exploratory deficits, and accumulation of PHF-1 and AT8 hyperphosphorylated tau epitopes in neurons. These phenomena are coupled with reactive astrocytosis, DNA fragmentation, and variable cerebral atrophy. Here, we show that cortical and hippocampal neurons of double mutant mice develop argyrophilic Gallyas-Braak aggregates of phosphorylated tau from 3 months of age. Their number decreases in old animals. Moreover, numerous phosphorylated tau aggregates were identified with the conformation-dependent Alz-50 antibody and the S-Thioflavin staining. Ventral motor nuclei of the spinal cord also present Alz-50, AT8, and PHF1 hyperphosphorylated tau aggregates when parkin is deleted in mice over-expressing the hTauVLW transgene, begining at early ages. Thus, the combination of PARK2 gene deletion with hTauVLW over-expression in mice produces abnormal hyperphosphorylated tau aggregates, similar to those observed in the brain of patients diagnosed with certain tauopathies. In the light of these changes, these mice may help to understand the molecular processes responsible for these diseases, and they may aid the development of new therapeutic strategies to treat neurodegenerative diseases related to tau and parkin proteins.

Significance of the parkin and PINK1gene in Jordanian families with incidences of young-onset and juvenile parkinsonism

BackgroundParkinson's disease is a progressive neurodegenerative disorder, where most cases are sporadic with a late onset. In rare incidences familial forms of early-onset parkinsonism occur, and when recessively inherited, cases are often explained by mutations in either the parkin (PARK2) or PINK1 (PARK6) gene or on exceptional occasions the DJ-1 (PARK7) or ATP13A2 (PARK9) gene. Recessively inherited deletions/duplications and point mutations in the parkin gene are the most common cause of early-onset parkinsonism known so far, but in an increasing number of studies, genetic variations in the serine/threonine kinase domain of the PINK1 gene are found to explain early-onset parkinsonism.MethodsIn this study all families were from a population with a high incidence of consanguinity. We investigated 11 consanguineous families comprising 17 affected with recessively inherited young-onset parkinsonism for mutations both in the parkin and PINK1 gene. Exons and flanking regions were sequenced, and segregation patterns of genetic variation were assessed in members of the respective families. An exon dosage analysis was performed for all exons in both genes.ResultsIn the parkin gene, a three generation family was identified with an exon 4 deletion segregating with disease. Both affected were homozygous for the deletion that segregated on a haplotype that spanned the gene in a haplotype segregation analysis that was performed using additional markers. Exon dosage analysis confirmed the recessive pattern of inheritance with heterozygous deletions segregating in healthy family members. In the PINK1 gene we identified two novel putative pathogenic substitutions, P416R and S419P, located in a conserved motif of the serine/threonine kinase domain. Both substitutions segregated with disease in agreement with a recessive pattern of inheritance within respective families and both were present as homozygous in two affected each. We also discuss common polymorphisms in the two genes found to be co-segregating within families.ConclusionOur results further extend on the involvement of PINK1 mutations in recessive early-onset parkinsonism with clinical features similar to carriers of parkin mutations.

Pael‐R transgenic mice crossed with parkin deficient mice displayed progressive and selective catecholaminergic neuronal loss

Parkin, a ubiquitin ligase, is responsible for autosomal recessive juvenile parkinsonism (AR-JP). We identified parkin-associated endothelin receptor-like receptor (Pael-R) as a substrate of parkin, whose accumulation is thought to induce unfolded protein response (UPR) -mediated cell death, leading to dopaminergic neurodegeneration. To create an animal model of AR-JP, we generated parkin knockout/Pael-R transgenic (parkin-ko/Pael-R-tg) mice. parkin-ko/Pael-R-tg mice exhibited early and progressive loss of dopaminergic as well as noradrenergic neurons without formation of inclusion bodies, recapitulating the pathological features of AR-JP. Evidence of activation of UPR and up-regulation of dopamine and its metabolites were observed throughout the lifetime. Moreover, complex I activity of mitochondria isolated from parkin-ko/Pael-R-tg mice was significantly reduced later in life. These findings suggest that persistent induction of unfolded protein stress underlies chronic progressive catecholaminergic neuronal death, and that dysfunction of mitochondrial complex I and oxidative stress might be involved in the progression of Parkinson's disease. parkin-ko/Pael-R-tg mice represents an AR-JP mouse model displaying chronic and selective loss of catecholaminergic neurons.

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms' distribution pattern and in exons' expression both in wild type controls and dopamine D3 receptor's knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.

Parkin as a tumor suppressor gene for hepatocellular carcinoma

The parkin was first identified as a gene implicated in autosomal recessive juvenile Parkinsonism. Deregulation of the parkin gene, however, has been observed in various human cancers, suggesting that the parkin gene may be important in tumorigenesis. To gain insight into the physiologic role of parkin, we generated parkin-/- mice lacking exon 3 of the parkin gene. We demonstrated here that parkin-/- mice had enhanced hepatocyte proliferation and developed macroscopic hepatic tumors with the characteristics of hepatocellular carcinoma. Microarray analyses revealed that parkin deficiency caused the alteration of gene expression profiles in the liver. Among them, endogenous follistatin is commonly upregulated in both nontumorous and tumorous liver tissues of parkin-deficient mice. Parkin deficiency resulted in suppression of caspase activation and rendered hepatocytes resistant to apoptosis in a follistatin-dependent manner. These results suggested that parkin deficiency caused enhanced hepatocyte proliferation and resistance to apoptosis, resulting in hepatic tumor development, partially through the upregulation of endogenous follistatin. The finding that parkin-deficient mice are susceptible to hepatocarcinogenesis provided the first evidence showing that parkin is indeed a tumor suppressor gene.

HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin

Parkin is the gene product identified as the major cause of autosomal recessive juvenile Parkinsonism (AR-JP). Parkin, a ubiquitin ligase E3, contains a unique ubiquitin-like domain in its N-terminus designated Uld which is assumed to be a interaction domain with the Rpn 10 subunit of 26S proteasome. To elucidate the structural and functional role of Uld in parkin at the atomic level, the X-ray crystal structure of murine Uld was determined and a molecular dynamics simulation of wild Uld and its five mutants (K27N, R33Q, R42P, K48A and V56E) identified from AR-JP patients was performed. Murine Uld consists of two α helices [Ile23–Arg33 (α1) and Val56–Gln57 (α2)] and five β strands [Met1–Phe7 (β1), Tyr11–Asp18 (β2), Leu41–Phe45 (β3), Lys48–Pro51 (β4) and Ser65–Arg72 (β5)] and its overall structure is essentially the same as that of human ubiquitin with a 1.22 Å rmsd for the backbone atoms of residues 1–76; however, the sequential identity and similarity between both molecules are 32% and 63%, respectively. This close resemblance is due to the core structure built by same hydrogen bond formations between and within the backbone chains of α1 and β1/2/5 secondary structure elements and by nearly the same hydrophobic interactions formed between the nonpolar amino acids of their secondary structures. The side chain NηH of Lys27 on the α1 helix was crucial to the stabilization of the spatial orientations of β3 and β4 strands, possible binding region with Rpn 10 subunit, through three hydrogen bonds. The MD simulations showed the K27N and R33Q mutations increase the structural fluctuation of these β strands including the α1 helix. Reversely, the V56E mutant restricted the spatial flexibility at the periphery of the short α2 helix by the interactions between the polar atoms of Glu56 and Ser19 residues. However, a large fluctuation of β4 strand with respect to β5 strand was induced in the R42P mutant, because of the impossibility of forming paired hydrogen bonds of Pro for Arg42 in wild Uld. The X-ray structure showed that the side chains of Asp39, Gln40 and Arg42 at the N-terminal periphery of β3 strand protrude from the molecular surface of Uld and participate in hydrogen bonds with the polar residues of neighboring Ulds. Thus, the MD simulation suggests that the mutation substitution of Pro for Arg42 not only causes the large fluctuation of β3 strand in the Uld but also leads to the loss of the ability of Uld to trap the Rpn 10 subunit. In contrast, the MD simulation of K48A mutant showed little influence on the β3–β4 loop structure, but a large fluctuation of Lys48 side chain, suggesting the importance of flexibility of this side chain for the interaction with the Rpn 10 subunit. The present results would be important in elucidating the impaired proteasomal binding mechanism of parkin in AR-JP.

Memory and exploratory impairment in mice that lack the Park-2 gene and that over-express the human FTDP-17 mutant Tau

While mutations in the Park-2 gene are the most frequent cause of autosomal-recessive juvenile parkinsonism (AR-JP), they are also present in several forms of tauopathies. Conversely, in some forms of parkinsonism, mutations in the tau gene have also been observed. Deletion of the Park-2 gene and over-expression of mutant tau independently produce mild brain alterations in mice. However, the presence of both mutations simultaneously causes a tau neuropathology, involving reactive astrocytosis, neuron loss in the cortex and hippocampus, and lesions in nigrostriatal and motor neurons. Furthermore, mutant tau over-expression in mice produces important memory impairment. When “parkin” function was abolished in young tau transgenic mice, the memory alterations were exaggerated. Moreover, additional exploratory and motor deficits were observed in older mice, causing the memory alterations to be underestimated. Thus, while memory deficits are more severe in young mice they were somehow attenuated by exploratory impairments in ageing mutants. This double mutant animal will serve as a useful experimental tool to investigate the abnormal processing of hyperphosphorylated tau and its relationship to the development of the cognitive deficits associated with certain neurodegenerative diseases.

Immunohistochemical localization of a ubiquitin ligase HRD1 in murine brain

HRD1 is an E3 ubiquitin ligase and plays an important role in endoplasmic reticulum-associated degradation (ERAD). Parkin-associated endothelin receptor-like receptor (Pael-R) is a substrate of the E3 ubiquitin ligase parkin, which has been implicated in ER stress-induced cell death in dopamine neurons in autosomal recessive juvenile parkinsonism (AR-JP). Recently, we demonstrated that endogenous HRD1 interacts with Pael-R, and that HRD1 promotes the degradation of Pael-R and protects cell death caused by the accumulation of Pael-R. Another group recently reported that HRD1 suppresses the toxicity of polyglutamine-expanded huntingtin. However, the topographical localization of HRD1 protein in the brain, especially related to neurodegenerative disease, is unclear. In this study, we used immunohistochemistry to investigate the topographical localization of HRD1 in the brain and demonstrated that HRD1 immunoreactivity was expressed widely in the substantia nigra pars compacta (SNC) containing dopaminergic neurons and was expressed in the cerebral cortex, hippocampus, dentate gyrus, striatum, globus pallidus, and Purkinje cells of the cerebellar cortex. Furthermore, HRD1 immunoreactivity was detected in the neuronal cells but not in the glial cells. These results suggest that HRD1 may play an important role in maintaining higher brain function, including motor function or learning and memory. In addition, HRD1 may have substrates other than Pael-R that are implicated in neurodegenerative disorders.

From juvenile parkinsonism to encephalitis lethargica, a new phenotype of post-streptococcal disorders: Case report

We report the case of a 16-year-old boy presented with a mild akinetic-rigid parkinsonism shortly after a post-streptococcal infection. After stopping corticoids, he had a rapid neurological deterioration to a fatal encephalitis lethargica-like syndrome. Serum analysis demonstrated consistently elevated anti-streptolysin-O. This case illustrates a new severe phenotype in the spectrum of the post-streptococcal disorders. This etiology should be considered in the differential diagnosis of a movement disorder with a rapid detrimental evolution.

発症後３０年経過したＰＡＲＫ６の１例−脳機能画像による検討−

We have reported a case of autosomal recessive juvenile parkinsonism PARK6 with a 30-year history. She developed tremor of right lower limb at the age of 23. At the age of 28, she received a clinical diagnosis of early-onset Parkinson's disease. She showed clinical improvements by the treatment with trihexyphenidyl, but symptoms showed slow progression over the subsequent years. L-DOPA therapy was introduced at the age of 42, and five years later, L-DOPA-induced dyskinesia developed. Dystonia, diurnal fluctuation and sleep benefit were absent. She carried a homozygous missense mutation in PINK1 gene, and was diagnosed as PARK6. The brain MRI did not show apparent abnormality. 18F-FDG-positron emission topography (PET) displayed normal uptake in the brain, suggesting normal glucose metabolism. PET imaging with a dopamine D2 receptor ligand 11C-raclopride revealed that postsynaptic 11C-raclopride uptake was normal in the bilateral putamen. After the introduction of pramipexisol, she showed clinical improvements. L-DOPA-induced dyskinesia disappeared with the gradual tapering and withdrawal of L-DOPA. In this PARK6 case, postsynaptic D2 receptors of the nigro-striatal dopaminergic neurons were thought to be maintained despite a long disease history.

Involvement of spinal motor neurons in parkin‐positive autosomal recessive juvenile parkinsonism

We intensively examined the spinal cord of an autosomal recessive juvenile parkinsonism (ARJP) female patient with a homozygous exon 3 deletion in the parkin gene, anticipating a possible involvement of anterior horn neurons. Although the clinical features of the patient were consistent with parkinsonism as a result of parkin mutation, her tendon reflex was abolished in the lower limbs. This feature was in contrast with hyperreflexia, usually found in previous reports of ARJP. Histologically, on the level of the cervical, thoracic, and sacral spinal cord, anterior horn neurons were well preserved and normal. However, the lumbar spinal cord exhibited many swellings of proximal axons (spheroids) and degenerative changes in the somata of the large anterior horn neurons such as central chromatolysis, cystatin C-negative small eosinophilic inclusions, and eosinophilic Lewy body-like inclusions. Ultrastructurally, accumulations of neurofilaments and abnormal structures, such as inclusion bodies similar to skein-like inclusions and disorganized rough endoplasmic reticulum, were observed in the somata and neuronal processes. Lewy body-like inclusions in this study were positively immunostained for both alpha-synuclein and ubiquitin that closely resemble Lewy bodies, but are different from Lewy body-like inclusions negatively immunostained for alpha-synuclein in amyotrophic lateral sclerosis. These findings suggest that eosinophilic inclusions that closely resemble Lewy bodies may be formed in the spinal motor neurons of ARJP patients with parkin mutations and the motor neurons of these patients may be vulnerable to neurodegeneration.

A Disease State Mutation Unfolds the Parkin Ubiquitin-like Domain

E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.

Overexpression of Septin 4, the Drosophila homologue of human CDCrel‐1, is toxic for dopaminergic neurons

parkin loss-of-function mutations are linked to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that promotes degradation of specific target proteins by the proteasome. It has been proposed that loss of Parkin activity will result in accumulation of its substrates, thus leading to dopaminergic (DA) neuron death. In Drosophila, parkin mutations cause degeneration of a subset of DA neurons in the brain but no Parkin substrates have yet been described. Here we characterized the septin 4 gene, which encodes the Drosophila orthologue of human CDCrel-1, a Parkin substrate. We showed that Septin 4 overexpression causes age-dependent disruption of DA neuron integrity in the dorsomedial cluster, which is suppressed by coexpression of Parkin and enhanced by reducing parkin function. Furthermore, other phenotypes caused by Septin 4 overexpression are also enhanced in a heterozygous parkin mutant background. This indicates that Septin 4 accumulation is toxic for DA neurons and suggests that Septin 4 could be a genuine substrate of Drosophila Parkin. Regarding this, we also showed that both proteins are able to interact physically with each other in vitro, thus supporting this hypothesis.

Comparison of the time courses of selective gene expression and dopaminergic depletion induced by MPP + in MN9D cells

Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons with unknown etiology. MPP + (1-methyl-4-phenylpyridinium ion) is the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like symptoms in humans and animals. MPTP/MPP + produces selective dopaminergic neuronal degeneration, therefore, these agents are commonly used to study the pathogenesis of PD. However, the mechanisms of their toxicity have not been fully elucidated. Recently, we reported in a microarray study using a midbrain-derived dopaminergic neuronal cell line, MN9D, that MPP + induced significant changes in a number of genes known to be associated with the dopaminergic system. In this study, we investigated the expression time courses of six genes using real-time RT-PCR, and compared them with the progressive dopaminergic depletion caused by MPP +. Our data showed that dopamine content was significantly decreased after 0.5 h of MPP + (200 μM) exposure and was completely depleted after 40 h. The expression of Gpr37, which is closely related to the pathogenesis of autosomal recessive juvenile Parkinsonism, was up-regulated after 0.5 h, and stayed up-regulated up to 48 h. Txnip, which is critical to the adjustment of cellular redox status, was down-regulated after 1 h and stayed down-regulated up to 48 h. Ldh1 and Cdo1, which are also involved in oxidative stress, were down-regulated after 16 h and stayed down-regulated up to 48 h. Two pro-apoptotic genes, Egln3 and Bnip3, were down-regulated after 2 and 4 h, and stayed down-regulated up to 48 h. These findings suggested that the time course of expression for multiple genes correlated with the dopaminergic depletion; and MPP +-induced neurotoxicity in MN9D cells could be used as a model to further explore the roles of these and other genes in the pathogenesis and possible treatment of PD.

Modulation of parkin gene expression in noradrenergic neuronal cells

Typical autosomal recessive juvenile Parkinsonism (AR-JP) is resulted from the loss of function mutation in the parkin gene. In an effort to learn more about the cell type-specific functional role of parkin, we used in vitro model such as locus coeruleus (LC) noradrenergic (NA) neuronal progenitor cell line, LC3541. Employing this in vitro model, we revealed that parkin plays a crucial role in phenotypic differentiation of NA neurons. Our results showed that parkin mRNA was expressed during the differentiation of NA neuronal progenitor cell line and that the level of the parkin mRNA was down-regulated by oxidative stress in the NA neuronal cells. Parkin protein overexpression in LC3541 cells induced expression of NA markers (TH, phox2a and DBH). Small interfering RNA (SiRNA) for parkin supressed NA differentiation and DBH expression. Preventing protein kinase A (PKA) activation with PKI attenuated expression of DBH in parkin overexpressed LC3541 cells. These findings suggest that the relative abundance of parkin enhances differentiation of NA phenotypes via a PKA-dependent pathway.