MAB318 Sigma-AldrichAnti-Tyrosine Hydroxylase Antibody, clone LNC1

Multiple convergent lines of evidence implicate both α-synuclein (encoded by SCNA) and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson's disease (PD). Occupational exposure to the mitochondrial complex I inhibitor rotenone increases PD risk; rotenone-exposed rats show systemic mitochondrial defects but develop specific neuropathology, including α-synuclein aggregation and degeneration of substantia nigra dopaminergic neurons. Here, we inhibited expression of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus-mediated delivery of a short hairpin RNA (shRNA) targeting the endogenous rat Snca transcript. Knockdown of α-synuclein by ~35% did not affect motor function or cause degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-exposed rats, progressive motor deficits were substantially attenuated contralateral to α-synuclein knockdown. Correspondingly, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites, and their striatal terminals was decreased ipsilateral to α-synuclein knockdown. These data show that α-synuclein knockdown is neuroprotective in the rotenone model of PD and indicate that endogenous α-synuclein contributes to the specific vulnerability of dopaminergic neurons to systemic mitochondrial inhibition. Our findings are consistent with a model in which genetic variants influencing α-synuclein expression modulate cellular susceptibility to environmental exposures in PD patients. shRNA targeting the SNCA transcript should be further evaluated as a possible neuroprotective therapy in PD.

Alpha-synuclein is a key protein in the pathogenesis of Parkinson's disease. Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson's disease, probably through a loss-of-function mechanism. However, the molecular mechanism by which loss of parkin function leads to the development of the disease and the role of alpha-synuclein in parkin-associated Parkinson's disease is still not elucidated. Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach. In this study, we investigated the effect of loss of parkin on alpha-synuclein neuropathology and toxicity in adult rodent brain using viral vectors. Therefore, we overexpressed human wild type alpha-synuclein in the substantia nigra of parkin knockout and wild type mice using two different doses of recombinant adeno-associated viral vectors.No difference was observed in nigral dopaminergic cell loss between the parkin knockout mice and wild type mice up to 16 weeks after viral vector injection. However, the level of alpha-synuclein phosphorylated at serine residue 129 in the substantia nigra was significantly increased in the parkin knockout mice compared to the wild type mice while the total expression level of alpha-synuclein was similar in both groups. The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line.These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons as well as the presence of proteinaceous inclusions named Lewy bodies. α-synuclein (α-syn) is a major constituent of Lewy bodies, and the first disease-causing protein characterized in PD. Several α-syn-based animal models of PD have been developed to investigate the pathophysiology of PD, but none of them recapitulate the full picture of the disease. Ageing is the most compelling and major risk factor for developing PD but its impact on α-syn toxicity remains however unexplored. In this study, we developed and exploited a recombinant adeno-associated viral (AAV) vector of serotype 9 overexpressing mutated α-syn to elucidate the influence of ageing on the dynamics of PD-related neurodegeneration associated with α-syn pathology in different mammalian species.Identical AAV pseudotype 2/9 vectors carrying the DNA for human mutant p.A53T α-syn were injected into the substantia nigra to induce neurodegeneration and synucleinopathy in mice, rats and monkeys. Rats were used first to validate the ability of this serotype to replicate α-syn pathology and second to investigate the relationship between the kinetics of α-syn-induced nigrostriatal degeneration and the progressive onset of motor dysfunctions, strikingly reminiscent of the impairments observed in PD patients. In mice, AAV2/9-hα-syn injection into the substantia nigra was associated with accumulation of α-syn and phosphorylated hα-syn, regardless of mouse strain. However, phenotypic mutants with either accelerated senescence or resistance to senescence did not display differential susceptibility to hα-syn overexpression. Of note, p-α-syn levels correlated with nigrostriatal degeneration in mice. In monkeys, hα-syn-induced degeneration of the nigrostriatal pathway was not affected by the age of the animals. Unlike mice, monkeys did not exhibit correlations between levels of phosphorylated α-syn and neurodegeneration.In conclusion, AAV2/9-mediated hα-syn induces robust nigrostriatal neurodegeneration in mice, rats and monkeys, allowing translational comparisons among species. Ageing, however, neither exacerbated nigrostriatal neurodegeneration nor α-syn pathology per se. Our unprecedented multi-species investigation thus favours the multiple-hit hypothesis for PD wherein ageing would merely be an aggravating, additive, factor superimposed upon an independent disease process.

While photoreceptor loss is the most devastating result of inherited retinal degenerations such as retinitis pigmentosa, inner retinal neurons also undergo significant alteration. Detailing these changes has become important as many vision restorative therapies target the remaining neurons. In this study, the rd1-Fos-Tau-LacZ (rd1-FTL) mouse model was used to explore inner retinal change at a late stage of retinal degeneration, after the loss of photoreceptor nuclei. The rd1-FTL model carries a mutation in the phosphodiesterase gene, Pde6b, and an axonally targeted transgenic beta galactosidase reporter system under the control of the c-fos promoter. Retinae of transgenic rd1-FTL mice and control FTL animals aged 2-12 months were processed for indirect fluorescence immunocytochemistry. At 2 months of age, a time when the majority of photoreceptor nuclei are lost, there was negligible c-fos reporter (FTL) expression, however, from 4 months, reporter expression was observed to increase within subpopulations of amacrine and ganglion cells within the central retina. These areas of inner retinal FTL expression coincided with regions that contained aberrant Müller cells. Specifically, these cells exhibited reduced glutamine synthetase and Kir4.1 immunolabelling, whilst showing evidence of proliferative gliosis (increased cyclinD1 and glial fibrillary acidic protein expression). These changes were limited to distinct regions where cone photoreceptor terminals were absent. Overall, these results highlight that distinct areas of the rd1-FTL central retina undergo significant glial alterations after cone photoreceptor loss. These areas coincide with up-regulation of the c-fos reporter in the inner retina, which may represent a change in neuronal function/plasticity. The rd1-FTL mouse is a useful model system to probe changes that occur in the inner retina at later stages of retinal degeneration.

Prevention of relapses is a major challenge in treating anxiety disorders. Fear reinstatement can cause relapse in spite of successful fear reduction through extinction-based exposure therapy. By utilising a contextual fear-conditioning task in mice, we found that reinstatement was accompanied by decreased c-Fos expression in the infralimbic cortex (IL) with reduction of synaptic input and enhanced c-Fos expression in the medial subdivision of the central nucleus of the amygdala (CeM). Moreover, we found that IL dopamine plays a key role in reinstatement. A reinstatement-inducing reminder shock induced c-Fos expression in the IL-projecting dopaminergic neurons in the ventral tegmental area, and the blocking of IL D1 signalling prevented reduction of synaptic input, CeM c-Fos expression, and fear reinstatement. These findings demonstrate that a dopamine-dependent inactivation of extinction circuits underlies fear reinstatement and may explain the comorbidity of substance use disorders and anxiety disorders.

Cardiac sympathetic denervation is found in various cardiac pathologies; however, its relationship with myocardial injury has not been thoroughly investigated.Twenty-four rats were assigned to the normal control group (NC), sympathectomy control group (SC), and a sympathectomy plus mecobalamin group (SM). Sympathectomy was induced by injection of 6-OHDA, after which, the destruction and distribution of sympathetic and vagal nerve in the left ventricle (LV) myocardial tissue were determined by immunofluorescence and ELISA. Heart rate variability (HRV), ECG and echocardiography, and assays for myocardial enzymes in serum before and after sympathectomy were examined. Morphologic changes in the LV by HE staining and transmission electron microscope were used to estimate levels of myocardial injury and concentrations of inflammatory cytokines were used to reflect the inflammatory reaction.Injection of 6-OHDA decreased NE (933.1 ± 179 ng/L for SC vs. 3418.1± 443.6 ng/L for NC, P less than 0.01) and increased NGF (479.4± 56.5 ng/mL for SC vs. 315.85 ± 28.6 ng/mL for NC, P less than 0.01) concentrations. TH expression was reduced, while ChAT expression showed no change. Sympathectomy caused decreased HRV and abnormal ECG and echocardiography results, and histopathologic examinations showed myocardial injury and increased collagen deposition as well as inflammatory cell infiltration in the cardiac tissue of rats in the SC and SM groups. However, all pathologic changes in the SM group were less severe compared to those in the SC group.Chemical sympathectomy with administration of 6-OHDA caused dysregulation of the cardiac autonomic nervous system and myocardial injuries. Mecobalamin alleviated inflammatory and myocardial damage by protecting myocardial sympathetic nerves.

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is a neurodegenerative lysosomal storage disease caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). The PPT1-deficient mouse (Cln1(-/-)) is a useful phenocopy of human INCL. Cln1(-/-) mice display retinal dysfunction, seizures, motor deficits, and die at ~8 months of age. However, little is known about the cognitive and behavioral functions of Cln1(-/-) mice during disease progression. In the present study, younger (~1-2 months of age) Cln1(-/-) mice showed minor deficits in motor/sensorimotor functions while older (~5-6 months of age) Cln1(-/-) mice exhibited more severe impairments, including decreased locomotor activity, inferior cued water maze performance, decreased running wheel ability, and altered auditory cue conditioning. Unexpectedly, certain cognitive functions such as some learning and memory capabilities seemed intact in older Cln1(-/-) mice. Younger and older Cln1(-/-) mice presented with walking initiation defects, gait abnormalities, and slowed movements, which are analogous to some symptoms reported in INCL and parkinsonism. However, there was no evidence of alterations in dopaminergic markers in Cln1(-/-) mice. Results from this study demonstrate quantifiable changes in behavioral functions during progression of murine INCL and suggest that Parkinson-like motor/sensorimotor deficits in Cln1(-/-) mice are not mediated by dopamine deficiency.

We investigated the distribution patterns of the extracellular matrix protein Reelin and of crucial Reelin signaling components in murine midbrain and striatum. The cellular distribution of the Reelin receptors VLDLr and ApoER2, the intracellular downstream mediator Dab1, and the alternative Reelin receptor APP were analyzed at embryonic day 16, at postnatal stage 15 (P15), and in 3-month-old mice. Reelin was expressed intracellularly and extracellularly in midbrain mesencephalic dopaminergic (mDA) neurons of newborns. In the striatum, Calbindin D-28k(+) neurons exhibited Reelin intracellularly at E16 and extracellularly at P15 and 3 months. ApoER2 and VLDLr were expressed in mDA neurons at E16 and P15 and in oligodendrocytes at 3 months, whereas Dab1 and APP immunoreactivity was observed in mDA at all stages analyzed. In the striatum, Calbindin D-28k(+)/GAD67(+) inhibitory neurons expressed VLDLr, ApoER2, and Dab1 at P15, but only Dab1 at E16 and 3 months. APP was always expressed in mouse striatum in which it colocalized with Calbindin D-28k. Our data underline the importance of Reelin signalling during embryonic development and early postnatal maturation of the mesostriatal and mesocorticolimbic system, and suggest that the striatum and not the midbrain is the primary source of Reelin for midbrain neurons. The loss of ApoER2 and VLDLr expression in the mature midbrain and striatum implies that Reelin functions are restricted to migratory events and early postnatal maturation and are dispensable for the maintenance of dopaminergic neurons.

The axon initial segment (AIS) is a specialized structure near the start of the axon that is a site of neuronal plasticity. Changes in activity levels in vitro and in vivo can produce structural AIS changes in excitatory cells that have been linked to alterations in excitability, but these effects have never been described in inhibitory interneurons. In the mammalian olfactory bulb (OB), dopaminergic interneurons are particularly plastic, undergoing constitutive turnover throughout life and regulating tyrosine hydroxylase expression in an activity-dependent manner. Here we used dissociated cultures of rat and mouse OB to show that a subset of bulbar dopaminergic neurons possess an AIS and that these AIS-positive cells are morphologically and functionally distinct from their AIS-negative counterparts. Under baseline conditions, OB dopaminergic AISs were short and located distally along the axon but, in response to chronic 24 h depolarization, lengthened and relocated proximally toward the soma. These activity-dependent changes were in the opposite direction to both those we saw in non-GABAergic OB neurons and those reported previously for excitatory cell types. Inverted AIS plasticity in OB dopaminergic cells was bidirectional, involved all major components of the structure, was dependent on the activity of L-type CaV1 calcium channels but not on the activity of the calcium-activated phosphatase calcineurin, and was opposed by the actions of cyclin-dependent kinase 5. Such distinct forms of AIS plasticity in inhibitory interneurons and excitatory projection neurons may allow considerable flexibility when neuronal networks must adapt to perturbations in their ongoing activity.

Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented. Using electron microcopy, we found that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon segments that are continuous with axon terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change the segregation of the two vesicular types, suggesting the existence of highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2 vesicles make asymmetric synapses, commonly associated with excitatory signaling. Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate can be released from the same axons, but are not normally released at the same site or from the same synaptic vesicles.