Progressive behavioral deficits in DJ-1-deficient mice are associated with normal nigrostriatal function

Loss-of-function mutations in the DJ-1 gene account for an autosomal recessive form of Parkinson's disease (PD). To investigate the physiological functions of DJ-1 in vivo, we generated DJ-1 knockout (DJ-1^−/−) mice. Younger (< 1 year) DJ-1^−/− mice were hypoactive and had mild gait abnormalities. Older DJ-1^−/−, however, showed decreased body weight and grip strength and more severe gait irregularities compared to wild-type littermates. The basal level of extracellular dopamine, evoked dopamine release and dopamine receptor D2 sensitivity appeared normal in the striatum of DJ-1^−/− mice, which was consistent with similar results between DJ-1^−/− and controls in behavioral paradigms specific for the dopaminergic system. An examination of spinal cord, nerve and muscle tissues failed to identify any pathological changes that were consistent with the noted motor deficits. Taken together, our findings suggest that loss of DJ-1 leads to progressive behavioral changes without significant alterations in nigrostriatal dopaminergic and spinal motor systems.     

Clinical and genetic evaluation of DYT1 and DYT6 primary dystonia in China

Background: Dystonia is defined as the presence of sustained involuntary muscle contractions, often leading to abnormal posture and movement. DYT1 is caused by a mutation in the TOR1A gene, whilst mutations in THAP1 gene have been identified as responsible for DYT6. The relative frequency and phenotype differences between DYT1 and DYT6 amongst Chinese primary dystonia patients have not been well‐characterized. Patients and methods: One hundred eleven unrelated Chinese patients with primary dystonia were screened for mutations in TOR1A and THAP1 genes, and correlate this with clinical presentation. Exon 5 of TOR1A and all three exons and exon‐intron conjunctions in THAP1 were screened by direct sequencing. Results: Three subjects were found to have the GAG deletion in the TOR1A gene, and two patients were detected with THAP1 gene mutations/variations (c.224A>T, c.449A>C). The overall mutation frequency was 4.5% in this cohort with TOR1A mutations found in 2.7% and THAP1 mutations found in 1.8%. No mutations were detected in the controls composed of 100 normal Chinese subjects. The clinical presentations of the DYT1 cases included onset in the limbs that could progress to the generalized dystonia within several years but without cranial involvement. Whilst in the DYT6 cases, the onset was cranial or cervical and progresses very slowly. Conclusion: The major clinical differences between DYT1 and DYT6 dystonia in China were the cranial involvement in DYT6 and progress to general dystonia within several years in DYT1.     

Synergistic interaction between an adenosine antagonist and a D1 dopamine agonist on rotational behavior and striatal c-Fos induction in 6-hydroxydopamine-lesioned rats

The interaction between adenosine and D₁ dopamine systems in regulating motor behavior and striatal c-Fos expression was examined in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. These results were compared to the synergistic interaction between D₁ and D₂ dopamine systems in 6-OHDA rats. Coadministration of the adenosine antagonist 3,7-dimethyl-1-propargylxanthine (DMPX: 10 mg/kg) and the D₁ dopamine agonist SKF38393 (0.5 mg/kg) to 6-OHDA-lesioned rats produced significant contralateral rotation and c-Fos expression in the ipsilateral striatum compared to 6-OHDA rats treated with either drug alone. However, the regional pattern of striatal c-Fos activation following treatment of 6-OHDA rats with SKF38393 and DMPX was different from the dorsolateral pattern of striatal c-Fos induction observed after coadministration of D₁ and D₂ dopamine agonists (SKF38393: 0.5 mg/kg+quinpirole: 0.05 mg/kg). These data are consistent with a functional interaction between D₁ dopamine and adenosine systems in the striatum, but suggest that activation of different subsets of striatal neurons underlie the behavioral synergy observed following combined adenosine antagonist-D₁ dopamine agonist and combined D₁ dopamine agonist–D₂ dopamine agonist treatment.     

Abnormal cellular translocation of alpha-internexin in spinal motor neurons of Dystonia musculorum mice

Dystonia musculorum (dt) is a mutant mouse with hereditary neuropathy where the dysfunction is mainly found in the dorsal root ganglia (DRG) neurons but not in the spinal motor neurons. However, the accumulation of intermediate filament (IF) proteins in the swelling axons of spinal motor neurons could be found in dt/dt mice. In order to understand the pathological role of neuronal IFs in the swelling axons of spinal motor neurons from dt/dt mice, we extensively examined the distribution of neuronal IF proteins. By immunofluorescence staining, our results indicated that alpha-internexin was a major component in the swelling axon and showed abnormal translocation in the nuclei of spinal motor neurons in dt/dt mice. This abnormal translocation of alpha-internexin in the nuclei of spinal motor neurons was also confirmed by Western blotting and immunoelectron microscopy. Instead of the 10-nm filamentous structure, a diffuse immunopositive pattern of alpha-internexin was observed in the nucleus of spinal motor neurons in dt/dt mutants. We further examined the cell death of spinal motor neurons by TUNEL assay, and no TUNEL-positive cells could be identified from spinal motor neurons in dt/dt mice. From these observations we suggest that abnormal accumulation of neuronal IFs in the swelling axons and abnormal translocation of alpha-internexin in the nuclei of the spinal motor neurons from dt/dt mice may not directly cause cell death of the spinal motor neurons.     

Blunted brain metabolic response to ketamine in mice lacking D1A dopamine receptors

The interaction of glutamatergic and dopamine neurotransmission is thought to have relevance to both the pathophysiology and pharmacotherapy of schizophrenia. For example, subanesthetic doses of the N-methyl- -aspartate receptor (NMDA-R) antagonist ketamine induce schizophrenia-like behavioral effects in humans and both behavioral and brain metabolic activation in rodents. Blockade of NMDA-R results in dopamine release, and antipsychotic drugs that block dopamine neurotransmission decrease NMDA-R antagonist-induced behavioral activation. The involvement of dopamine receptors in brain metabolic activation induced by ketamine is, however, unknown. The present study used D_1A knockout mice to determine the role of dopamine D_1A receptors in the effects of subanesthetic doses of ketamine on both behavioral responses and on alterations in regional [¹⁴C]2-deoxyglucose (2-DG) uptake. There was less ketamine-induced behavioral activation in D_1A knockout mice than in wild-type mice. In wild-type mice, ketamine (30 mg/kg) induced dramatic increases in 2-DG uptake in limbic cortical regions, hippocampal formation, nucleus accumbens, basolateral amygdala, and caudal parts of the substantia nigra pars reticulata. D_1A knockout mice exhibited blunted metabolic activation in response to ketamine in a neuroanatomically specific manner. The selective D₁ antagonist, SCH23390 (0.3 mg/kg), inhibited both ketamine-induced brain metabolic activation and behavioral responses in the wild-type mice, with a similar neuroanatomical specificity observed in the D_1A knockout mice. Thus, the neuroanatomically selective role that D_1A receptors play in ketamine-induced behavior and regional brain metabolic activation in mice provides a useful model for further studies of how the D_1A receptor function may be altered in schizophrenia.     

l-Dopa stimulates c-fos expression in dopamine denervated striatum by combined activation of D-1 and D-2 receptors

Administration of l-dopa to unilaterally 6-hydroxydopamine-lesioned rats, activates the early gene c-fos in the lesioned caudate-putamen. D-1 receptor blockade by SCH 23390, preventedl-dopa-induced Fos-like immunoreactivity in the whole caudate-putamen, while D-2 receptor blockade by raclopride reduced Fos-like immunoreactivity only in the dorso-lateral portion. The results suggest thatl-dopa induces c-fos primarily through an activation of D-1 receptors, while D-2 receptor stimulation plays a facilitatory influence on D-1 mediated c-fos expression.     

Norepinephrine loss selectively enhances chronic nigrostriatal dopamine depletion in mice and rats

In this study we investigated whether a selective pattern of norepinephrine loss potentiates methamphetamine-induced striatal dopamine depletion in rats. We also evaluated whether chronic norepinephrine depletion reduces the threshold dose of methamphetamine necessary to induce long-lasting striatal dopamine loss in mice and in rats. Pre-treatment with the selective noradrenergic neurotoxin DSP-4 (50 mg/kg, i.p.) in mice and in rats significantly enhanced methamphetamine-induced striatal dopamine depletion. Administration of a low dose of methamphetamine (1 × 5 mg/kg and 3× 5 mg/kg, respectively, i.p., at 2-h interval) to C57B1/6N mice and Sprague-Dawley rats did not decrease striatal dopamine levels when injected alone but produced a significant decrease in striatal dopamine when given to rodents carrying a long-lasting norepinephrine depletion previously induced by DSP-4. Our results suggest that norepinephrine loss might both enhance neurotoxic damage and decrease the threshold for neurotoxicity to nigrostriatal dopaminergic neurons in different animal species.     

Mechanism study of Aconitum-induced neurotoxicity in PC12 cells: Involvement of dopamine release and oxidative damage

The Aconitum has been widely used as an important component in traditional Chinese medicine. However, it can cause neurotoxicity, and the mechanism has not been fully elucidated. The present study aimed to investigate the potential dopaminergic neurotoxicity of Aconitum and its mechanism. We found that Aconitum significantly evoked dopamine release from cultured PC12 cells and from the nucleus accubens of mice. These results show that Aconitum can promptly trigger dopamine release both in vitro and in vivo. Aconitum exposure induced reactive oxygen species formation with the decrease of superoxide dismutase and glutathione peroxidase. Moreover, PC12 cells proliferation was inhibited and apoptotic death was detected after Aconitum treatment, but this effect could be attenuated by antioxidants. These findings suggest that Aconitum can damage PC12 cells through oxidative stress mechanism. In conclusion, our results indicate that Aconitum can evoke dopamine release from dopaminergic neurons; excessive extracellular of dopamine can then create stresses on cellular antioxidant systems and induce neuron apoptosis.     

Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans

Synaptotagmin 1, encoded by the snt-1 gene in Caenorhabditis elegans, is a major synaptic vesicle protein containing two Ca(2+)-binding (C2) domains. Alternative splicing gives rise to two synaptotagmin 1 isoforms, designated SNT-1A and SNT-1B, which differ in amino acid sequence in the third, fourth, and fifth beta-strands of the second C2 domain (C2B). We report here that expression of either SNT-1 isoform under control of a strong pan-neural promoter fully rescues the snt-1 null phenotype. Furthermore, C-terminal fusions of either isoform with GFP are trafficked properly to synapses and are fully functional, unlike synaptotagmin 1Colon, two colonsGFP fusions in mice. Analysis of isoform expression with genomic GFP reporter constructs revealed that the SNT-1A and-1B isoforms are differentially expressed and localized in the C. elegans nervous system. We also report molecular, behavioral, and immunocytochemical analyses of twenty snt-1 mutations. One of these mutations, md259, specifically disrupts expression of the SNT-1A isoform and has defects in a subset of synaptotagmin 1-mediated behaviors. A second mutation, md220, is an in-frame 9-bp deletion that removes a conserved tri-peptide sequence (VIL) in the second beta-strand of the C2B domain and disrupts the proper intracellular trafficking of synaptotagmin. Site-directed mutagenesis of a functional SNT-1Colon, two colonsGFP fusion protein was used to examine the potential role of the VIL sequence in synaptotagmin trafficking. Although our results suggest the VIL sequence is most likely not a specific targeting motif, the use of SNT-1Colon, two colonsGFP fusions has great potential for investigating synaptotagmin trafficking and localization.     

Variable neurologic phenotype in a GEFS+ family with a novel mutation in SCN1A

PurposeTo describe the spectrum of clinical disease in a mutliplex family with an autosomal dominant form of generalized epilepsy with febrile seizures plus (GEFS+) and determine its genetic etiology.MethodsMedical and family history was obtained on 11 clinically affected individuals and their relatives across three generations through medical chart review and home visits. A candidate gene approach including haplotype analysis and direct sequencing was used.ResultsAn epilepsy-associated haplotype was identified on 2q24. Direct sequencing of the entire SCN1A gene identified seven sequence variants. However, only one of these, c.1162 T > C, was not found in population controls. This transition in exon 8 of SCN1A predicts a substitution (Y388H) of a highly conserved tyrosine residue in the loop between transmembrane segments S5 and S6 of the sodium channel protein (Na_v1.1). Clinical features in mutation carriers of this novel missense mutation were highly variable, ranging from febrile seizures to severe refractory epilepsy.ConclusionA novel missense mutation in the pore-forming region of the sodium channel gene SCN1A causes GEFS+ with a variable phenotype that includes mood and anxiety disorders, as well as ataxia, expanding the GEFS+ spectrum to include neuropsychiatric disease.     

Time course of MPTP-induced degeneration of the nigrostriatal dopamine system in C57 BL/6 mice

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a parkinsonism-inducing dopamine (DA) neurotoxin most effective in primates. MPTP also causes a degeneration of both perikarya and axon terminals of the nigrostriatal DA neurons in C57 BL/6 mice. The time courses of the changes in tyrosine hydroxylase immunoreactive objects, endogenous DA concentrations and specifically bound 3H-mazindol as markers of the integrity of DA neurons were studied in substantia nigra and striatum of adult C57 BL/6 mice, after systemic treatment with MPTP or intranigral injections of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA). A rapid decrease in the three parameters studied was found in the substantia nigra during the first 2 days after MPTP-treatment while the MPTP-induced effects in the striatum were more protracted and maximal reduction was observed 7 days after MPTP. A basically similar pattern was found when studying the 6-OHDA-induced anterograde degeneration of the nigrostriatal system. These results indicate that in C57 BL/6 mice, MPTP primarily destroys the DAergic perikarya with a subsequent anterograde degeneration of the striatal axon terminals, although a limited rapid destruction of some striatal terminals cannot be excluded.     

Extrapyramidal side effects during chronic combined dopamine D1 and D2 antagonist treatment in Cebus apella monkeys

Previous studies in non-human primates have shown that tolerance to dystonia occurs during chronic dopamine D1 (D1) but not D2 antagonism and induction/aggravation of oral dyskinesia (TD) during D2 but not D1 antagonism. We were therefore interested in determining the effects of combined chronic D1 + D2 antagonism on dystonia and dyskinesia. To this intent, 8 male Cebus apella monkeys were treated 10 weeks with gradually increasing doses of D1 antagonist (NNC 112) + a D2 antagonist (raclopride), followed by 2 weeks of treatment with the D2 antagonist alone. Due to previous neuroleptic exposure, 5 monkeys had TD and all were sensitized to dystonia. During the combined antagonist treatment, tolerance to dystonia occurred; the tolerance disappearing upon discontinuation of the D1 antagonist and continuation of the D2 antagonist alone. Parallel to these results, improvement of TD was seen during the combined antagonist treatment with worsening during the D2 antagonist alone. Both the combined antagonists and the D2 antagonist alone resulted in moderate/severe bradykinesia, with no tolerance. These findings indicate that supplementation of traditional D2 antagonism with a D1 antagonist would lessen the risk of dystonia and allow alleviation of preexisting TD, though parkinsonian side effects might still occur. The findings further indicate that separate dopaminergic mechanisms control dystonia/dyskinesia and parkinsonism. Received: 18 February 1999 / Accepted: 11 June 1999     

Involvement of the cerebellar adenosine A1 receptor in cannabinoid-induced motor incoordination in the acute and tolerant state in mice

Cannabinoids are known to impair motor function in humans and laboratory animals. We have demonstrated an accentuation of cannabinoid (CP55,940)-induced motor incoordination in mice by the adenosine A₁ receptor-selective agonist N⁶-cyclohexyladenosine (CHA) (4 ng) using an intracerebellar (ICB) microinjection method. This effect was mediated by the A₁ receptor because pre-treatment with ICB 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 ng), an adenosine A₁ receptor selective antagonist, completely abolished the accentuation. Furthermore, ICB pre-treatment with DPCPX (100 ng) before ICB CP55,940 (15 μg) attenuated the motor incoordination suggesting a modulation by an endogenous adenosine A₁ system. ICB microinjection of CHA or DPCPX prior to ICB vehicle had no effect on normal motor coordination. ICB microinjection of dipyridamole (25 μg), an adenosine transport inhibitor, significantly accentuated the motor incoordination by ICB CP55,940 (15 μg), providing further support for the involvement of endogenous adenosine in the action of CP55,940. Tolerance to the motor incoordinating effect of ICB CP55,940 was demonstrated following 3 days of i.p. CP55,940 (0.1, 1 or 2 mg/kg every 12 or 24 h; total of six or three injections, respectively). Interestingly, animals which exhibited tolerance to ICB CP55,940 also demonstrated tolerance to the accentuating effect of ICB CHA suggesting cross-tolerance between adenosine agonists and cannabinoids. Cross-tolerance was also demonstrated following 3 days of i.p. CHA (0.25 or 1 mg/kg every 24 h; total of three injections) as further evidence of the modulatory role of the cerebellar adenosine system in the acute manifestation of CP55,940-induced motor incoordination. The involvement of cerebellar adenosine and the A₁ receptor in cannabinoid actions is circumstantially supported by previous evidence that CB₁ receptors and A₁ receptors are both localized on cerebellar granule cell parallel fiber terminals and basket cell neurons where they serve to inhibit the release of neurotransmitters.     

Systematic TOR1A non-c.907_909delGAG variant analysis in isolated dystonia and controls

BackgroundAn increasing number of rare, functionally relevant non-c.907_909delGAG (non-ΔGAG) variants in TOR1A have been recognized, associated with phenotypic expressions different from classic DYT1 childhood-onset generalized dystonia. Only recently, DYT1 genotype-phenotype correlations have been proposed, awaiting further elucidation in independent cohorts.MethodsWe screened the entire coding sequence and the 5′-UTR region of TOR1A for rare non-ΔGAG sequence variants in a large series of 940 individuals with various forms of isolated dystonia as well as in 376 ancestry-matched controls. The frequency of rare, predicted deleterious non-ΔGAG TOR1A variants was assessed in the European sample of the Exome Aggregation Consortium (ExAC) dataset.ResultsIn the case cohort, we identified a rare 5′-UTR variant (c.-39G > T), a rare splice-region variant (c.445-8T > C), as well as one novel (p.Ile231Asn) and two rare (p.Ala163Val, p.Thr321Met) missense variants, each in a single patient with adult-onset focal/segmental isolated dystonia. Of these variants, only p.Thr321Met qualified as possibly disease-related according to variant interpretation criteria. One novel, predicted deleterious missense substitution (p.Asn208Ser) was detected in the control cohort. Among European ExAC individuals, the carrier rate of rare, predicted deleterious non-ΔGAG variants was 0.4%.ConclusionsOur study does not allow the establishment of genotype-specific clinical correlations for DYT1. Further large-scale genetic screening accompanied by comprehensive segregation and functional studies is required to conclusively define the contribution of TOR1A whole-gene variation to the pathogenesis of isolated dystonia.     

Effects of MK-801 on D1 dopamine receptor-mediated immediate early gene expression in the dopamine-depleted striatum

Previous work indicates that intrastriatal administration of MK-801 does not completely block D1 agonist-induced gene expression in dopamine-depleted rats. The present study examined the effects of systemic MK-801 on such gene expression. A low dose of MK-801 did not affect induction of c-fos or zif268. A high dose completely blocked induction of c-fos, but only slightly suppressed zif268. The data suggest that NMDA receptor activity may not always be necessary for D1-induced gene expression.     

R(+)-8-OH-DPAT, a selective 5-HT1A receptor agonist, attenuated amphetamine-induced dopamine synthesis in rat striatum, but not nucleus accumbens or medial prefrontal cortex

R(+)-8-OH-DPAT (0.05, but not 0.025, 0.1, 1 mg/kg), a 5-HT_1A receptor agonist, decreased l-3,4-dihydroxyphenylalanine (DOPA) accumulation in rat striatum following NSD-1015, an l-aromatic amino acid decarboxylase inhibitor. Amphetamine (1 mg/kg) increased striatal DOPA accumulation, an effect attenuated by R(+)-8-OH-DPAT (0.05 mg/kg). However, both amphetamine (1 mg/kg) and R(+)-8-OH-DPAT (0.05 mg/kg) decreased cortical DOPA accumulation; there were no additional decreases from their combination. Neither amphetamine (1 mg/kg), R(+)-8-OH-DPAT (0.05 mg/kg), or the combination, significantly affected DOPA accumulation in the nucleus accumbens. The significance of and possible mechanisms for these findings are discussed.     

Prolonged fear responses in mice lacking dopamine D1 receptor

Dopamine is an important neurotransmitter involved in learning and memory including emotional memory. The involvement of dopamine in conditioned fear has been widely documented. However, little is known about the molecular mechanisms that underlie contextual fear conditioning and memory consolidation. To address this issue, we used dopamine D₁-deficient mice (D₁−/−) and their wild-type (D₁+/+) and heterozygote (D₁+/−) siblings to assess aversive learning and memory. We quantified two different aspects of fear responses to an environment where the mice have previously received unsignaled footshocks. Using one-trial step-through passive avoidance and conditioned freezing paradigms, mice were conditioned to receive mild inescapable footshocks then tested for acquisition, retention and extinction of conditioned fear responses 5 min after and up to 45–90 days post-training. No differences were observed among any of the genotypes in the acquisition of passive avoidance response or fear-induced freezing behavior. However, with extended testing, D₁−/− mice exhibited prolonged retention and delayed extinction of conditioned fear responses in both tasks, suggesting that D₁−/− mice are capable of acquiring aversive learning normally. These findings demonstrate that the dopamine D₁ receptor is not important for acquisition or consolidation of aversive learning and memory but has an important role in modulating the extinction of fear memory.     

The role of dopamine in the maintenance and breakdown of D1/D2 synergism

The neurochemical factors involved in the maintenance and breakdown of dopamine D₁/D₂ receptor synergism were investigated by giving rats various pharmacological treatments that diminish the ability of dopamine to interact with its D₁ and/or D₂ receptors. Following these treatments, rats were observed for the expression of stereotyped motor behavior in response to independent stimulation of D₁ or D₂ receptors. Independent D₂-mediated responses were observed: (a) 2 h after the last of three daily reserpine (1 mg/kg) injections, (b) 48 h after bilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathways, (c) 24 h after a concentrated 48-h regimen (one injection/6 h) of eticlopride (0.5 mg/kg) or eticlopride + SCH 23390 (0.5 mg each), and (d) 2 h after a concentrated 48-h regimen (one injection/6 h) of α-methyl-p-tyrosine (αMPT; 100 mg/kg), but not after control treatments or a concentrated regimen of SCH 23390 alone. By contrast, independent D₁-mediated responses were observed only after three daily reserpine injections or 48 h after bilateral 6-OHDA lesions. Independent D₁-mediated stereotypy was not observed under control conditions or following a concentrated 48-h regimen of (a) SCH 23390 or eticlopride (0.5 mg/kg each) alone or in combination, (b) a high dose of SCH 23390 (1.0 mg/kg), (c) αMPT (100 mg/kg), or (d) αMPT (100 mg/kg)+SCH 23390 (1.0 mg/kg). Reserpine, bilateral 6-OHDA, and αMPT treatments produced striatal dopamine depletions of 96%, 92%, and 71%, respectively. These data indicate that the breakdown in D₁/D₂ synergism consists of two components: (a) D₁ independence from the controlling influence of D₂ receptors, and (b) D₂ independence from the controlling influence of D₁ receptors. The interaction of synaptic DA with its D₂ receptors plays a major role in determining whether these receptors can function independently of D₁ receptors, whereas reduced DA-D₁ activity alone appears insufficient to elicit D₁ independence.