rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants. Conclusions In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7

A53T-alpha-synuclein overexpression impairs dopamine signaling and striatal synaptic plasticity in old mice

BACKGROUND: Parkinson's disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice. CONCLUSIONS/SIGNIFICANCE: Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches

Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

The toxicity of α-synuclein invivo is not well understood. Rockenstein etal. describe an α-synuclein transgenic model expressing the E57K mutant that forms stable oligomers. They show that oligomers accumulate at synapses and that the mutation interferes with synaptic vesicles and is associated with behavioural deficits and neurodegeneratio

A53T-Alpha-Synuclein Overexpression Impairs Dopamine Signaling and Striatal Synaptic Plasticity in Old Mice

Parkinson's disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons.Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice.Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches

Dynamic physiological alpha-synuclein S129 phosphorylation is driven by neuronal activity

In Parkinson’s disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA−/− neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker

Analyzes of expression pattern and neuropathology of conditional mouse models of Parkinson's Disease

Das alpha-Synuclein Protein spielt in der Pathogenese neurodegenerativer Erkrankungen, einschließlich der Parkinson-Erkrankung eine wichtige Rolle. Die Parkinson-Krankheit basiert auf progredienten neuropathologischen Abänderungen, die zu motorischen Störungen führen, denen oftmals Riechstörung und kognitive Einbussen vorausgehen. Es ist nicht bekannt, ob der Verlauf der Neuropathologie gestoppt oder umgekehrt werden kann. Um zu klären, ob alpha-Synuclein diese Abänderungen induziert, wurde von uns ein auf dem tet-System basierendes konditionales Mausmodell generiert. Mäuse mit hoher alpha-Synuclein-Expression in unterschiedlichen Hirnarealen entwickelten eine nigrale und hippokampale Neuropathologie, einschließlich einer verminderten Neurogenese und Neurodegeneration, die in einer progredienten motorischen Schwäche und Einbussen im Langzeitgedächtnis resultierten. Das Abschalten der alpha-Synuclein-Expression führte zu einem Stopp der motorischen Symptomatik, führte jedoch nicht zur Reversibilität der Symptome. Mäuse, die das mutierte (A30P) alpha-Synuclein im Riechhirn exprimierten, zeigten eine Reduktion von Monoaminen in dieser Region. In einem durchgeführten Geruchsstest zeigten diese Mäuse eine verminderte Angstreaktion und ein erhöhtes Explorationsverhalten; letzteres könnte ein Hauptmerkmal für eine Depression sein. Die Daten legen nahe, dass in frühen Stadien der Parkinson-Krankheit Therapeutika bzw. behandelnde Strategien, die gegen alpha-Synuclein involvierte pathologische Zellwege gerichtet sind, vorteilhaft für den Krankheitsverlauf sind.Alpha-synuclein has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson’s disease (PD). PD is based on progressive neuropathological alterations leading to motor abnormalities, that are frequently predated by olfactory dysfunction and often accompanied by cognitive decline in later stages of the disease. Whether the neurodegenerative process might be halted or even reversed is presently unknown. In order to explore whether alpha-synuclein induces these alterations we generated conditional mouse models by using the tet-regulatable system. Mice expressing high levels of human wildtype alpha-synuclein in several brain regions developed nigral and hippocampal neuropathology, including reduced neurogenesis and neurodegeneration, leading to progressive motor decline and impaired long-term memory. Turning off transgene expression in aged mice halted progression of motor symptoms but did not reverse the symptoms. Mice expressing the mutated (A30P) alpha-synuclein limited to the olfactory bulb showed a reduction of monoamines in this region. In a conducted TMT-smell test these mice also revealed an impaired anxiety reaction and an increased exploratory behavior; the latter might be a depressive core symptom. Our data suggest that approaches targeting alpha-syn induced pathological pathways might be of benefit in early disease stages

Global Gene Expression Analysis in a Mouse Model for Norrie Disease: Late Involvement of Photoreceptor Cells

PURPOSE. Mutations in the NDP gene give rise to a variety of eye diseases, including classic Norrie disease (ND), X-linked exudative vitreoretinopathy (EVRX), retinal telangiectasis (Coats disease), and advanced retinopathy of prematurity (ROP). The gene product is a cystine-knot–containing extracellular signaling molecule of unknown function. In the current study, gene expression was determined in a mouse model of ND, to unravel disease-associated mechanisms at the molecular level. METHODS. Gene transcription in the eyes of 2-year-old Ndp knockout mice was compared with that in the eyes of age-matched wild-type control animals, by means of cDNA subtraction and microarrays. Clones (n = 3072) from the cDNA subtraction libraries were spotted onto glass slides and hybridized with fluorescently labeled RNA-derived targets. More than 230 differentially expressed clones were sequenced, and their expression patterns were verified by virtual Northern blot analysis. RESULTS. Numerous gene transcripts that are absent or downregulated in the eye of Ndp knockout mice are photoreceptor cell specific. In younger Ndp knockout mice (up to 1 year old), however, all these transcripts were found to be expressed at normal levels. CONCLUSIONS. The identification of numerous photoreceptor cell–specific transcripts with a reduced expression in 2-year-old, but not in young, Ndp knockout mice indicates that normal gene expression in these light-sensitive cells of mutant mice is established and maintained over a long period and that rods and cones are affected relatively late in the mouse model of ND. Obviously, the absence of the Ndp gene product is not compatible with long-term survival of photoreceptor cells in the mouse