Alpha-synuclein Knockout Mice Research Articles

Early Effects of Alpha-Synuclein Depletion by Pan-Neuronal Inactivation of Encoding Gene on Electroencephalogram Coherence between Different Brain Regions in Mice.

We analyzed electroencephalogram (EEG) coherence ("functional connectivity") between the cortex (MC), putamen (Pt), and dopamine-producing brain regions (ventral tegmental area, VTA, and substantia nigra, SN) in two groups of two-month-old male mice. We compared EEG coherences in the conditional knockout Sncaflox/flox mice with those in their genetic background (C57Bl6J) one, two, and three months after chronic (for five days) intraperitoneal injections of TAM or the vehicle (corn oil). The EEG coherences in the TAM-treated group were compared with those in the alpha-synuclein knockout mice. A significant suppression of EEG coherence in the TAM-treated mice versus the vehicle group was observed in all inter-structural relations, with the exception of MC-VTA at one and three months and VTA-SN at two months after the injections. Suppressive changes in EEG coherence were observed in the alpha-synuclein knockout mice as well; the changes were similar to those in TAM-treated mice three months after treatment. our data demonstrate a combined time-dependent suppressive effect induced by TAM on intracerebral EEG coherence.

Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called “Lewy pathology” (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in the diseased brain. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brainstem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling, we showed that PSER129 positive punctate structures occur within inactive (i.e., c-fos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antibody recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR-identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in the mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies, either the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because of the natural tendency to accumulate PSER129.

Domain and cell type-specific immunolocalisation of voltage-gated potassium channels in the mouse striatum

Diverse classes of voltage-gated potassium channels (Kv) are integral to the variety of electrical activity patterns that distinguish different classes of neurons in the brain. A feature of their heterogenous expression patterns is the highly precise manner in which specific cell types target their location within functionally specialised sub-cellular domains. Although Kv expression profiles in cortical brain regions are widely reported, their immunolocalisation in sub-cortical areas such as the striatum, and in associated diseases such as Parkinson’s disease (PD), remain less well described. Therefore, the broad aims of this study were to provide a high resolution immunolocalisation analysis of various Kv subtypes within the mouse striatum and assess their potential plasticity in a model of PD. Immunohistochemistry and confocal microscopy revealed that immunoreactivity for Kv1.1, 1.2 and 1.4 overlapped to varying degrees with excitatory and inhibitory axonal marker proteins suggesting these Kv subtypes are targeted to axons innervating striatal medium spiny neurons (MSNs). Immunoreactivity for Kv1.3 strongly overlapped with signal for mitochondrial marker proteins in MSN somata and dendrites. Kv1.5 immunoreactivity was expressed in parvalbumin-immunopositive neurons whereas Kv1.6 was located in cells immunopositive for microglia. Signal for Kv2.1 was concentrated on the somatic and proximal dendritic plasma membrane of MSNs, whilst immunoreactivity for Kv4.2 was targeted to their distal dendritic regions. Finally, striatal Kv2.1 expression, at both the mRNA and protein levels, was decreased in alpha-synuclein overexpressing mice, yet increased in alpha-synuclein knockout mice, compared to wild-type counterparts. The data indicate a variety of Kv expression patterns that are distinctive to the striatum and susceptible to pathology that mirrors PD. Furthermore, these findings advance our understanding of the molecular diversity of various striatal cell types, and potentially have implications for the homeostatic changes of MSN excitability during associated medical conditions such as PD.

Alpha-synuclein supports type 1 interferon signalling in neurons and brain tissue.

The protein alpha-synuclein is predominantly expressed in neurons and is associated with neurodegenerative diseases like Parkinson's disease and dementia with Lewy bodies. However, the normal function of alpha-synuclein in neurons is not clearly defined. We have previously shown that mice lacking alpha-synuclein expression exhibit markedly increased viral growth in the brain, increased mortality and increased neuronal cell death, implicating alpha-synuclein in the neuronal innate immune response. To investigate the mechanism of alpha-synuclein-induced immune responses to viral infections in the brain, we challenged alpha-synuclein knockout mice and human alpha-synuclein knockout dopaminergic neurons with RNA virus infection and discovered that alpha-synuclein is required for neuronal expression of interferon-stimulated genes. Furthermore, human alpha-synuclein knockout neurons treated with type 1 interferon failed to induce a broad range of interferon stimulated genes, implying that alpha-synuclein interacts with type 1 interferon signalling. We next found that alpha-synuclein accumulates in the nucleus of interferon-treated human neurons after interferon treatment and we demonstrated that interferon-mediated phosphorylation of STAT2 is dependent on alpha-synuclein expression in human neurons. Next, we found that activated STAT2 co-localizes with alpha-synuclein following type 1 interferon stimulation in neurons. Finally, we found that brain tissue from patients with viral encephalitis expresses increased levels of phospho-serine129 alpha-synuclein in neurons. Taken together, our results show that alpha-synuclein expression supports neuron-specific interferon responses by localizing to the nucleus, supporting STAT2 activation, co-localizing with phosphorylated STAT2 in neurons and supporting expression of interferon-stimulated genes. These data provide a novel mechanism that links interferon activation and alpha-synuclein function in neurons.

Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Treadmill exercise intervention improves gait and postural control in alpha-synuclein mouse models without inducing cerebral autophagy

Gait and postural control dysfunction are prototypical symptoms compromising quality of life for patients with Parkinson’s disease (PD). Hallmarks of cellular pathology are dopaminergic degeneration and accumulation of the cytosolic protein alpha-synuclein, linked to impaired autophagy-lysosome pathway (ALP) clearance. Physical exercise improves gait in PD patients and motor function in rodent lesion models. Moreover, exercise is considered neuroprotective and ALP induction has been reported, e.g. in human skeletal muscle, rodent peripheral and cerebral tissues. A combined analysis of how distinct exercise paradigms affect motor and central biochemical aspects of PD could maximize benefits for patients. Here we examine the effect of 4 weeks treadmill exercise intervention in 7–8 month non-lesioned mice on a) distinct gait categories, b) ALP activity, c) dopaminergic and alpha-synuclein homeostasis. The study includes wild type, alpha-synuclein knockout, and mice exclusively expressing human alpha-synuclein. Parameters of gait regularity and stability, activity, and dynamic postural control during unforced walk, were assessed by an automated system (CatWalk XT). At baseline, alpha-synuclein mouse models exhibited irregular and less active gait, with impaired dynamic postural control, compared to wild type mice. Treadmill exercise particularly improved speed and stride length, while increasing dual diagonal versus three-paw body support in both the alpha-synuclein knockout and transgenic mice. Biochemical analyses showed higher striatal tyrosine hydroxylase immuno-reactivity and reduced higher-order alpha-synuclein species in the cerebral cortex. However, no significant cerebral ALP induction was measured. In summary, treadmill exercise improved gait activity and postural stability, and promoted dopaminergic and alpha-synuclein homeostasis, without robustly inducing cerebral ALP.

Genetic inactivation of alpha-synuclein affects embryonic development of dopaminergic neurons of the substantia nigra, but not the ventral tegmental area, in mouse brain.

Lesion of the dopaminergic neurons of the nigrostriatal system is a key feature of Parkinson’s disease (PD). Alpha-synuclein is a protein that is a major component of Lewy bodies, histopathological hallmarks of PD, and is involved in regulation of dopamine (DA) neurotransmission. Previous studies of knockout mice have shown that inactivation of alpha-synuclein gene can lead to the reduction in number of DA neurons in the substantia nigra (SN). DA neurons of the SN are known to be the most affected in PD patients whereas DA neurons of neighboring ventral tegmental area (VTA) are much less susceptible to degeneration. Here we have studied the dynamics of changes in TH-positive cell numbers in the SN and VTA during a critical period of their embryonic development in alpha-synuclein knockout mice. This precise study of DA neurons during development of the SN revealed that not only is the number of DA neurons reduced by the end of the period of ontogenic selection, but that the way these neurons are formed is altered in alpha-synuclein knockout mice. At the same time, DA neurons in the VTA are not affected. Alpha-synuclein exerts a modulating effect on the formation of DA neurons in the SN and has no effect on the formation of DA neurons in VTA, the structure that is much less susceptible to degeneration in a brain with PD, suggesting a potential role of alpha-synuclein in the development of the population of DA neurons in substantia nigra.

Abstract 454: Mechanism of Alpha-Synuclein Inhibition of Platelet Granule Exocytosis

Alpha-synuclein is only expressed in the brain and platelets. The normal function of alpha-synuclein in the brain is not known, but studies indicate it is an inhibitor of exocytosis in platelets. Platelet granule exocytosis is an important step in stable thrombus formation and its regulatory mechanisms are poorly defined. We therefore sought to determine the mechanism of alpha-synuclein inhibition of exocytosis. Using platelets from alpha-synuclein knockout (syn−/−) mice we first confirmed that syn−/− mice have increased alpha-granule release in response to agonist stimulation as compared to wild-type (WT) control mice. Using a mesenteric intravital microscopy model, we have also found that syn−/− mice have shortened time to thrombus formation as compared to wild-type control mice. The granule exocytosis and thrombosis phenotypic changes are restored to normal in syn null mice made transgenic to express human alpha-synuclein, clearly demonstrating that alpha-synuclein is an inhibitor of exocytosis in platelets. We have also determined the mechanism by which alpha-synuclein inhibits platelet granule exocytosis. Alpha-synuclein interacts with the exocytosis associated molecule Rab3, a modulator of the ATPase molecule N-ethylmaleimide Sensitive Factor (NSF). NSF activity is necessary for exocytosis and is increased by Rab3 interactions. Using platelet lysates, addition of an excess of recombinant alpha-synuclein inhibits NSF and Rab3 co-immunoprecipitation. Furthermore, syn−/− mouse platelets have increased Rab3 immunoprecipitation with NSF as compared to WT controls, that is restored to normal in synuclein Tg mice. Using recombinant tagged alpha-synuclein, NSF, and Rab3 in pull down assays we have confirmed that alpha-synuclein inhibits NSF-Rab3 interactions, and furthermore, decreases NSF ATPase activity. In summary, we now demonstrate using in vitro, ex vivo, and in vivo models that alpha-synuclein is a negative regulator of platelet granule exocytosis by inhibiting NSF-Rab3 association and thus NSF ATPase activity.

Substrate‐mediated enhancement of phosphorylated tyrosine hydroxylase in nigrostriatal dopamine neurons: evidence for a role of α‐synuclein

Tyrosine hydroxylase (TH) protein, phosphorylated at serine-40, serine-31 and serine-19, and enzyme catalytic activity were compared under basal conditions and in activated nigrostriatal dopamine (NSDA) neurons of wild-type and homozygous alpha-synuclein knockout mice. Mice were injected with the D2 antagonist raclopride to stimulate NSDA neuronal activity in the presence or absence of supplemental l-tyrosine. There was no difference in phosphorylated TH levels or TH catalytic activity between wild-type and alpha-synuclein knockout mice under basal conditions or following raclopride-induced acceleration of NSDA activity. In wild-type animals, tyrosine administration potentiated the raclopride-induced increase in phosphorylated TH and enzyme activity. However, tyrosine administration did not enhance phosphorylated TH levels or enzyme catalytic activity in raclopride-stimulated NSDA neurons in alpha-synuclein knockout mice. These findings suggest that alpha-synuclein plays a role in the ability of tyrosine to either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity. The present study supports the hypothesis that alpha-synuclein functions as a molecular chaperone protein that regulates the phosphorylation state of TH in a substrate and activity-dependent manner.

Coenzyme Q<sub>10</sub> Provides Neuroprotection in Iron-Induced Apoptosis in Dopaminergic Neurons

The exact molecular mechanism of progressive loss of neuromelanin containing nigrostriatal dopaminergic neurons in Parkinson's disease (PD) remains unknown, yet evidence suggests that iron might play an important role in PD pathology. In this study we have determined the neuroprotective role of coenzyme Q(10) (CoQ(10)) in ironinduced apoptosis in cultured human dopaminergic (SK-N-SH) neurons, in metallothionein gene- manipulated mice, and in alpha-synuclein knockout (alpha-synko) mice with a primary objective to assess a possible therapeutic and anti-inflammatory potential for CoQ(10) in PD. Iron-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species production, increased metallothionein and glutathione synthesis, caspase- 3 activation, NF-kappaB induction, and decreased Bcl-2 expression, without any significant change in Bax expression. Lower concentrations of FeSO4 (1-10 microM) induced perinuclear aggregation of mitochondria, whereas higher concentrations (100-250 microM) induced CoQ(10) depletion, plasma membrane perforations, mitochondrial damage, and nuclear DNA condensation and fragmentation. FeSO(4)-induced deleterious changes were attenuated by pretreatment with CoQ(10) and by deferoxamine, a potent iron chelator, in SK-N-SH cells. 1-Methyl, 4-phenyl, 1,2,3,6- tetrahydropyridine (MPTP)-induced striatal release of free iron, and NF-kappaB expression were significantly increased; whereas ferritin and melanin synthesis were significantly reduced in the substantia nigra pars compacta (SNpc) of MT(dko) mice as compared with control(wt) mice, MT(trans) mice, and alpha-synko mice. CoQ(10) treatment inhibited MPTP-induced NF-kappaB induction in all of the genotypes. These data suggest that glutathione and metallothionein synthesis might be induced as an attempt to combat iron-induced oxidative stress, whereas exogenous administration of CoQ(10) or of metallothionein induction might provide CoQ(10)-mediated neuroprotection in PD.