Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study.

Ropinirole (ROP) is a dopamine agonist that has been used as therapy for Parkinson's disease. In the present study, we aimed to detect whether gene expression was modulated by ROP in SH-SY5Y cells. SH-SY5Y cell lines were treated with 10 µM ROP for 2 h, after which total RNA was extracted for whole genome analysis. Gene expression profiling revealed that 113 genes were differentially expressed after ROP treatment compared with control cells. Further pathway analysis revealed modulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, with prominent upregulation of PIK3C2B. Moreover, batches of regulated genes, including PIK3C2B, were found to be located on chromosome 1. These findings were validated by quantitative RT-PCR and Western blot analysis. Our study, therefore, revealed that ROP altered gene expression in SH-SY5Y cells, and future investigation of PIK3C2B and other loci on chromosome 1 may provide long-term implications for identifying novel target genes of Parkinson's disease.

Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study

Ropinirole (ROP) is a dopamine agonist that has been used as therapy for Parkinson's disease. In the present study, we aimed to detect whether gene expression was modulated by ROP in SH-SY5Y cells. SH-SY5Y cell lines were treated with 10 µM ROP for 2 h, after which total RNA was extracted for whole genome analysis. Gene expression profiling revealed that 113 genes were differentially expressed after ROP treatment compared with control cells. Further pathway analysis revealed modulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, with prominent upregulation of PIK3C2B. Moreover, batches of regulated genes, including PIK3C2B, were found to be located on chromosome 1. These findings were validated by quantitative RT-PCR and Western blot analysis. Our study, therefore, revealed that ROP altered gene expression in SH-SY5Y cells, and future investigation of PIK3C2B and other loci on chromosome 1 may provide long-term implications for identifying novel target genes of Parkinson's disease.

Examination of the MSX1 gene in patients with Parkinson's disease.

BACKGROUND: Several genetic variants in transcription factor genes have been reported to be associated with Parkinson's disease (PD). The muscle segment homeobox drosophila homolog of 1 gene (MSX1) is a major upstream regulator of the dopaminergic neuronal subtype specification. AIMS OF THE STUDY: To determine whether genetic variation in the coding region of the MSX1 gene plays a role in the etiology of PD. METHODS: We searched for genetic variations in the coding region of the MSX1 gene in 202 patients with PD and 200 normal controls by PCR-single-strand conformation polymorphism (PCR-SSCP) and sequencing. RESULTS: No mutation in the MSX1 gene was identified in our cohort. CONCLUSIONS: Mutations in the coding region of the MSX1 gene play little or no role in the development of PD.

Animal models of restless legs syndrome.

Restless legs syndrome (RLS) is a common disease with prevalence up to 10% in the general population. It is mostly a subjective condition, making animal models intrinsically difficult. General increased activity (urge to move) and limb movements consistent with periodic limb movements of sleep, seen in most patients with RLS, are currently our best behavioral markers. Our best understanding of human RLS demonstrates reduced central nervous system (CNS) iron stores and dysfunction of dopaminergic systems, which most likely involves the spinal cord. Based upon this knowledge, animal manipulations, including destruction of the A11 diencephalic-spinal tract and iron deprivation, have resulted in animal behavior consistent with RLS. Dopamine receptor type 3 knockout mice also show general increased activity. Pharmacologic blockade of dopamine receptors in rodents has also caused movements resembling periodic limb movements of sleep in older rodents but not in younger animals. More sophisticated animal modeling is needed to facilitate our understanding of RLS.

Examination of the SLITRK1 gene in Caucasian patients with Tourette syndrome.

OBJECTIVE: To determine whether variants in the Slit and Trk-like 1 gene (SLITRK1) are present in American Caucasian population of patients with Tourette syndrome (TS). METHODS: We sequenced the 3'-untranslated region for var321 and the whole coding region in the SLITRK1 gene in 82 Caucasian patients with TS from North America. RESULTS: None of the 82 samples from patients with TS showed the non-coding sequence variant (var321). Only one patient with familial TS was heterozygous for a novel 708C > T (Ile236Ile) nucleotide variant. CONCLUSIONS: The var321 and mutation(s) in the coding region of the SLITRK1 gene probably are a rare cause of TS in a Caucasian population; therefore, genetic heterogeneity of TS should be considered. Tests designed to detect variant(s) in the SLITRK1 gene probably will not have a diagnostic utility in clinical practice.

Projections of diencephalic dopamine neurons into the spinal cord in mice.

The aim of this study is to investigate the pathway of diencephalic dopaminergic (DA) neuronal innervating into the spinal cord in mice, the pathway is postulated relevant to clinical restless legs syndrome (RLS). Tyrosine hydroxylase (TH) immunohistochemistry was used to identify the DA neuron. The fluorescent tracer Fluoro-Gold (FG) was stereotaxically injected into the T10-L5 spinal cord of CBL57 mice (n=20) seven days before the animals were sacrificed. The diencephalic sections were stained with TH antibody and the FG tracer present in the diencephalic DA neurons were examined under fluoresce microscope. The average number of total DA neurons per side in A11, A12, A13 and A14 was 66+/-8, 221+/-12, 350+/-17 and 254+/-21 respectively. After being injected into the spinal cord, FG reached the DA neurons within the A10 and A11 groups, but didn't target to any other DA neuron groups including the A8 and A9 groups in substantia nigra (SN). The diencephalic A11 DA neurons possessed long axons extending over several segments and possibly traversing the entire length of the spinal cord. It is the first time to report A10 and A11 DA neuron projections into the spinal cord in mice.

The role of Nurr1 in the development of dopaminergic neurons and Parkinson's disease.

Nurr1, a transcription factor belonging to the orphan nuclear receptor superfamily, is critical in the development and maintenance of the dopaminergic system and as such it may have role in the pathogenesis of Parkinson' disease (PD). Human Nurr1 gene has been mapped to chromosome 2q22-23 and Nurr1 protein is predominantly expressed in central dopaminergic neurons. Nurr1 interacts with other factors critical for the survival of mensencephalic dopaminergic neurons and it appears to regulate the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and l-aromatic amino acid decarboxylase (AADC), all of which are important in the synthesis and storage of dopamine. Experimental studies in Nurr1 knock-out mice indicate that Nurr1 deficiency results in impaired dopaminergic function and increased vulnerability of those midbrain dopaminergic neurons that degenerate in PD. Decreased Nurr1 expression is found in the autopsied PD midbrains, particularly in neurons containing Lewy bodies, as well as in peripheral lymphocytes of patients with parkinsonian disorders. Several variants in Nurr1 gene have been reported in association with PD. All these studies suggest that Nurr1 is not only essential in the development of mensencephalic dopaminergic neurons and maintenance of their functions, but it may also play a role in the pathogenesis of PD.

G309D and W437OPA PINK1 mutations in Caucasian Parkinson's disease patients.

OBJECTIVE: To determine whether the G309D and W437OPA mutations in PINK1 gene are present in American Caucasian population of patients with Parkinson's disease (PD). METHODS: We searched for the G309D and W437OPA mutation by sequencing the regions of interest in the PINK1 gene in 237 unrelated Caucasian patients. RESULTS: None of the 237 samples showed the G309D or W437OPA mutations. CONCLUSIONS: The G309D and W437OPA mutations in PINK1 gene probably do not represent common causes of familial or sporadic PD in a Caucasian population.

Minocycline: neuroprotective mechanisms in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by cardinal features of tremor, bradykinesia, rigidity and postural instability. In addition to the motor symptoms patients experience cognitive decline eventually resulting in severe disability. Pathologically PD is characterized by neurodegeneration in the substantia nigra pars compacta (SNc) with intracytoplasmic inclusions known as Lewy bodies. In addition to the SNc there is neurodegeneration in other areas including cerebral cortex, raphe nuclei, locus ceruleus, nucleus basalis of meynert, cranial nerves and autonomic nervous system. Recent evidence supports the role of inflammation in Parkinson's disease. Apoptosis has been shown to be one of the pathways of cell death in PD. Minocycline, a tetracycline derivative is a caspase inhibitor, and also inhibits the inducible nitric oxide synthase which are important for apoptotic cell death. Furthermore, Minocycline has been shown to block microglial activation of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned parkinsonism animal models and protect against nigrostriatal dopaminergic neurodegeneration. In this review, we present the current experimental evidence for the potential use of tetracycline derivative, minocycline, as a neuroprotective agent in PD.

Association of homozygous 7048G7049 variant in the intron six of Nurr1 gene with Parkinson's disease.

OBJECTIVE: To determine whether the Nurr1 gene, which is critical for the development and maintenance of nigral dopaminergic neurons, is a risk factor associated with PD. BACKGROUND: The Nurrl gene is highly expressed in the dopaminergic neurons in the midbrain. Knockout of the gene results in agenesis of nigral dopaminergic neurons and heterozygous knockout mice increases 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. METHODS: This study included 105 patients with familial PD (fPD) and 120 patients with sporadic PD (sPD) and 221 age-matched healthy control subjects. The polymorphisms and mutations of the Nurr1 gene in patients with PD were initially examined by heteroduplex analysis and sequencing analysis from PCR-amplified Nurr1 gene fragments. A polymorphism in the BseRI restriction site was identified, and a relatively large-scale analysis then was conducted by three independent investigators who were blinded to the clinical status of the subjects. RESULTS: A homozygous 7048G7049 polymorphism was found in intron 6 of the Nurr1 gene, which was significantly higher in fPD (10/105; 9.5%) and in sPD (5/120; 4.2%) compared with healthy control subjects (2/221; 0.9%). The mean age and the SD at onset of these homozygote patients with PD was 52 +/- 15 years for fPD and 46 +/- 7 years for sPD. The clinical features of these homozygote patients with PD did not differ from those of typical PD. CONCLUSIONS: The homozygote polymorphism of 7048G7049 in intron 6 of the Nurr1 gene is associated with typical PD.

Are dopamine receptor agonists neuroprotective in Parkinson's disease?

Dopamine receptor agonists are playing an increasingly important role in the treatment of not only patients with advanced Parkinson's disease and those with levodopa-induced motor fluctuations, but also in the early treatment of the disease. This shift has been largely due to the demonstrated levodopa-sparing effect of dopamine agonists and their putative neuroprotective effect, with evidence for the latter being based largely on experimental in vitro and in vivo studies. In this article we review the evidence for neuroprotection by the dopamine agonists pramipexole, ropinirole, pergolide, bromocriptine and apomorphine in cell cultures and animal models of injury to the substantia nigra. Most of the studies suggest that dopamine agonists may have neuroprotective effects via direct scavenging of free radicals or increasing the activities of radical-scavenging enzymes, and enhancing neurotrophic activity. However, the finding that pramipexole can normalise mitochondrial membrane potential and inhibit activity of caspase-3 in cytoplasmic hybrid cells derived from mitochondrial DNA of patients with nonfamilial Alzheimer's disease suggests an even broader implication for the neuroprotective role of dopamine agonists. Although the clinical evidence for neuroprotection by dopamine agonists is still limited, the preliminary results from several ongoing clinical trials are promising. Several longitudinal studies are currently in progress designed to demonstrate a delay or slowing of progression of Parkinson's disease using various surrogate markers of neuronal degeneration such as 18F-levodopa positron emission tomography and 123I beta-CIT (carbomethoxy-beta-4-iodophenyl-nortropane) single positron emission computed tomography. The results of these experimental and clinical studies will improve our understanding of the action of dopamine agonists and provide critical information needed for planning future therapeutic strategies for Parkinson's disease and related neurodegenerative disorders.

Antioxidant property of pramipexole independent of dopamine receptor activation in neuroprotection.

Pramipexole and several other dopamine agonists have been recently demonstrated to have neuroprotective effects in vitro and in vivo. We report here that pramipexole can protect DAergic cell line MES 23.5 against dopamine (DA), 6-hydroxydopamine (6-OHDA), and hydrogen peroxide (H2O2)-induced cytotoxicity in vitro possibly through antioxidant effects, and such neuroprotection could not be blocked by selective D2 or D3 antagonists. Incubation with pramipexole (5-20 microM) in MES 23.5 cell cultures increased cellular levels of glutathione (GSH), and elevated glutathione peroxidase (GSH-Px) and catalase activities, but only marginally enhanced SOD activity. Pretreatment with D2 or D3 antagonists did not block the stimulating effects of pramipexole on the cellular levels of GSH, and on the activities of GSH-Px and catalase in MES 23.5 cells. These results indicate that pramipexole's neuroprotective effects are likely associated with its antioxidant property independent of D2/D3 receptor activation.

Stereotaxic injection of IgG from patients with Alzheimer disease initiates injury of cholinergic neurons of the basal forebrain.

CONTEXT: The participation of an immune/inflammatory process in the pathomechanism of sporadic Alzheimer disease (AD) has been suggested by evidence for activated microglia and the potential therapeutic benefit of anti-inflammatory medication. OBJECTIVE: To define a possible role for IgG in the immune/inflammatory process of AD in humans, we assayed the ability of IgG samples from patients with AD to target the injury to cholinergic neurons in rat basal forebrain in vivo. DESIGN: IgG purified from the serum or plasma from patients with AD and patients with other neurological disease who were used as control (DC) patients was injected stereotaxically into the medial septum of adult rats. Four weeks later coronal sections of the whole medial septum-diagonal bands of Broca region were immunostained for choline acetyltransferase (ChAT) to identify cholinergic neuronal cells. SETTING: University medical centers. PATIENTS: Blood samples were collected from 8 patients with probable and definite AD and from 6 age-matched DC patients. MAIN OUTCOME MEASURE: Detection of changes in the number of ChAT immunopositive cell profiles in sections and statistical evaluation. RESULTS: Four weeks after the injections, IgG samples from patients with AD significantly reduced the number of ChAT-immunostained cell profiles in the whole medial septum-diagonal bands of Broca region compared with IgGs from DC patients. Neither DC IgGs nor saline solution significantly decreased the number of ChAT-immunopositive neuronal cell profiles. CONCLUSION: Data document that IgG from patients with AD can target a stereotaxically induced immune/inflammatory injury to cholinergic neurons in the rat basal forebrain in vivo.

Clinical correlates of 6-hydroxydopamine injections into A11 dopaminergic neurons in rats: a possible model for restless legs syndrome.

Pursuant to the clinical suspicion that restless legs syndrome (RLS) may involve dopaminergic diencephalic spinal neurons (A11), we performed stereotaxic bilateral 6-hydroxydopamine (6-OHDA) lesions into the A11 nucleus to observe for any behavioral correlates similar to this clinical condition. Pathologic examination demonstrated a 54% reduction in A11 tyrosine hydroxylase staining cells in rats injected with 6-OHDA versus sham treatment. Multiple blindly rated 90-120-minute video epochs demonstrated an increased average number of standing episodes (14.4+/-11.7 versus 7.3+/-5.5 episodes/epoch) and increased total standing time (38.9+/-20.5 versus 25.3+/-12.2 minutes/epoch) but similar total sleep time in four lesioned rats when compared with two sham rats. Treatment of the lesioned rats with intramuscular pramipexole subsequently resulted in fewer standing episodes (4.4+/-3.3 versus 14.4+/-11.7 episodes/epoch) and less total standing time (20.9+/-12.3 versus 38.9+/-20.5 minutes/epoch) when compared with untreated lesioned rats. Despite a large number of observations, the small number of lesioned animals precluded formal statistical analysis. These behaviors are consistent, although not specific, with what would be expected in an animal model of RLS.

Protective role of heme oxygenase-1 in oxidative stress-induced neuronal injury.

Heme oxygenase-1 (HO-1) is a stress protein induced in response to a variety of oxidative challenges. After treatment of the hybrid septal cells SN 56 with beta-amyloid peptide (beta-AP1-40) or hydrogen peroxide (H2O2), we detected high levels of reactive oxygen species, accompanied by a significant elevation in HO-1 expression. Levels of HO-1 increased and then decreased following cell loss. Pretreatment of SN 56 cells with HO-1 antisense oligonucleotides dramatically decreased the immunoreactivity of HO-1 and significantly enhanced the cytotoxicity of beta-AP1-40 and H2O2. In contrast, pretreatment with hemin, an HO-1 inducer, increased the expression of HO-1 and decreased the beta-AP1-40- and H2O2-induced cytotoxicity. These findings support the importance of HO-1 in protecting neurons against oxidative stress-induced injury.

Effects of cerebrospinal fluid from patients with Parkinson disease on dopaminergic cells.

BACKGROUND: The pathogenesis of substantia nigra pars compacta neuronal injury in Parkinson disease (PD) remains unknown. Cerebrospinal fluid (CSF) has been reported to contain factors toxic to dopaminergic neurons. OBJECTIVES: To determine whether the cytotoxic effects of CSF of PD patients are specific for dopaminergic neurons, dependent on prior levodopa therapy, and mediated by the cytokine tumor necrosis factor alpha (TNF-alpha). DESIGN: Specimens of CSF were evaluated in dopaminergic (MES 23.5) and nondopaminergic (N18TG2) cell lines for cytotoxicity by viability assay and by the inhibition of tyrosine hydroxylase. After specificity and time and dose response were established, CSF specimens were assayed in a blinded manner. The TNF-alpha levels in CSF were determined by enzyme-linked immunosorbent assay. The toxicity of TNF-alpha in MES 23.5 cells was determined. SETTING: A university-based research facility. SUBJECTS: There were 4 groups of subjects: normal control subjects (n = 10), control subjects with neurologic disease (n = 8), PD patients treated with levodopa (n = 10), and untreated subjects with PD (n= 20). RESULTS: Specimens of CSF from 15 (50%) of 30 PD patients and 2 (11%) of 18 control subjects were cytotoxic to dopaminergic MES 23.5 cells and were nontoxic to the parental cell line N18TG2. There was no correlation between the degree of PD CSF cytotoxicity, levodopa therapy, or the severity and duration of PD. Terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) for DNA fragmentation suggested the involvement of apoptotic mechanisms. The inhibition of tyrosine hydroxylase was an early effect of cell injury by PD CSF and correlated with the viability assay. The mean TNF-alpha level was 2.6-fold higher in CSF specimens from PD patients than in those of controls. The addition of recombinant human TNF-alpha equivalent to the highest level determined in PD CSF was not cytotoxic to MES 23.5 cultures. CONCLUSIONS: Blinded CSF specimens from PD patients, regardless of therapy, contain factors that cause specific dopaminergic neuronal cell injury. These factors are present in a substantial proportion of CSF specimens from patients with early PD, before the institution of medical therapy. Levels of TNF-alpha are elevated in the CSF of PD patients, but TNF-alpha is not responsible for the cytotoxicity.

Experimental destruction of substantia nigra initiated by Parkinson disease immunoglobulins.

BACKGROUND: Increased levels of free radicals and oxidative stress may contribute to the pathogenesis of substantia nigra (SN) injury in Parkinson disease (PD), but the initiating etiologic factors remain undefined in most cases. OBJECTIVE: To determine the potential importance of immune mechanisms in triggering or amplifying neuronal injury, we assayed serum samples from patients with PD to determine the ability of IgG to initiate relatively specific SN injury in vivo. METHODS: IgG purified from the serum of 5 patients with PD and 10 disease control (DC) patients was injected into the right side of the SN in adult rats. Coronal sections were cut from the whole brain at the level of the stereotaxic injections, stained for tyrosine hydroxylase and with cresyl violet, and cellular profiles were counted in identical brain regions at the injection and contralateral sides. The ratio of cell profile counts of the corresponding injected and uninjected regions was used as an internal standard. RESULTS: Four weeks following injection of IgG, a 50% decrease in tyrosine hydroxylase-positive cellular profiles was noted on the injected sides compared with the contralateral sides of the same animals. Similarly, applied DC IgG caused only an 18% decrease. Cresyl violet staining revealed a 35% decrease in neuronal profiles of PD IgG injected into the SN pars compacta compared with the contralateral uninjected side, whereas DC IgG caused a minimal 10% decrease. Even at 4 weeks after the PD IgG injections, perivascular inflammation and significant microglial infiltration were present near injured SN pars compacta neurons. No cytotoxic effects of PD IgG were noted in choline acetyltransferase-positive neurons after stereotaxic injections into the medial septal region. Absorption of PD IgG with mesencephalic membranes and protein A agarose gel beads removed cytotoxicity, while absorption with liver membranes did not change the cytotoxicity. CONCLUSIONS: Our data suggest that PD IgG can initiate a relatively specific inflammatory destruction of SN pars compacta neurons in vivo and demonstrate the potential relevance of immune mechanisms in PD.

Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons.

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the floor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.

Beta-amyloid-induced neurotoxicity of a hybrid septal cell line associated with increased tau phosphorylation and expression of beta-amyloid precursor protein.

Recent evidence suggests that beta-amyloid peptide (beta-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which beta-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated beta-AP(1-40) treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser(199/202) and Ser396. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted beta-amyloid precursor protein (beta-APP) was markedly elevated. Application of antisense oligonucleotide to beta-APP reduced expression of beta-APP and immunoreactivity of phosphorylated tau. Control peptide beta-AP(1-28) did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated beta-APP. These results suggest that betaAP(1-40)-induced tau phosphorylation may be associated with increased beta-APP expression in degenerated neurons.