Α-syn Mice Research Articles

Bimolecular Fluorescence Complementation of Alpha-synuclein Demonstrates its Oligomerization with Dopaminergic Phenotype in Mice

Alpha-synuclein (αSyn) is encoded by the first causal gene identified in Parkinson's disease (PD) and is the main component of Lewy bodies, a pathological hallmark of PD. aSyn-based animal models have contributed to our understanding of PD pathophysiology and to the development of therapeutics. Overexpression of human wildtype αSyn by viral vectors in rodents recapitulates the loss of dopaminergic neurons from the substantia nigra, another defining pathological feature of the disease. The development of a rat model exhibiting bimolecular fluorescence complementation (BiFC) of αSyn by recombinant adeno-associated virus facilitates detection of the toxic αSyn oligomers species. We report here neurochemical, neuropathological and behavioral characterization of BiFC of αSyn in mice. Overexpression and oligomerization of αSyn through BiFC is detected by conjugated fluorescence. Reduced striatal dopamine and loss of nigral dopaminergic neurons are accompanied neuroinflammation and abnormal motor activities. Our mouse model may provide a valuable tool to study the role of αSyn in PD and to explore therapeutic approaches.

The interaction between calcineurin and α-synuclein is regulated by calcium and calmodulin

Calcineurin (CN) is a protein phosphatase and widely distributed in eukaryotes, with an extremely high level of expression in mammalian brain. Alpha-synuclein (α-syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. In our present study, we explored the interactions between CN and α-syn in vitro. Based on the data from microscale thermophoresis, GST pull-down assays, and co-immunoprecipitation, we found that CN binds α-syn. Furthermore, this interaction is mediated by calcium/calmodulin (Ca2+/CaM) signaling. Additionally, thapsigargin (TG) triggered an increase in CN activity and α-syn aggregation in HEK293 cells stably transfected with α-syn. Our previous study in vivo suggest that overexpression of α-syn in transgenic mice significantly promoted CN activity and subsequent nuclear translocation of nuclear factor of activated T-cells (NFAT) in the midbrain dopaminergic (mDA) neurons. These in vivo and in vitro studies have been complementary with each other, representing the changes in the CN-dependent pathway affected by overexpression of α-syn.

Peripheral monocyte entry is required for alpha-Synuclein induced inflammation and Neurodegeneration in a model of Parkinson disease

Accumulation of alpha-synuclein (α-syn) in the central nervous system (CNS) is a core feature of Parkinson disease (PD) that leads to activation of the innate immune system, production of inflammatory cytokines and chemokines, and subsequent neurodegeneration. Here, we used heterozygous reporter knock-in mice in which the first exons of the fractalkine receptor (CX3CR1) and of the C-C chemokine receptor type 2 (CCR2) are replaced with fluorescent reporters to study the role of resident microglia (CX3CR1+) and infiltrating peripheral monocytes (CCR2+), respectively, in the CNS. We used an α-syn mouse model induced by viral over-expression of α-syn. We find that in vivo, expression of full-length human α-syn induces robust infiltration of pro-inflammatory CCR2+ peripheral monocytes into the substantia nigra. Genetic deletion of CCR2 prevents α-syn induced monocyte entry, attenuates MHCII expression and blocks the subsequent degeneration of dopaminergic neurons. These results demonstrate that extravasation of pro-inflammatory peripheral monocytes into the CNS plays a key role in neurodegeneration in this model of PD synucleinopathy, and suggest that peripheral monocytes may be a target of neuroprotective therapies for human PD.

Distinct Parameters in the EEG of the PLP α-SYN Mouse Model for Multiple System Atrophy Reinforce Face Validity.

Multiple system atrophy (MSA) is a neurodegenerative movement disorder characterized by parkinsonian symptoms and cerebellar symptoms. Sleep disturbances also play a crucial role in MSA. One of the most convincing animal models in MSA research is the PLP α-SYN model, but to date no studies on sleep disturbances in this mouse model, frequently found in MSA patients are available. We identified spectral shifts within the EEG of the model, strikingly resembling results of clinical studies. We also characterized muscle activity during REM sleep, which is one of the key symptoms in REM sleep behavioral disorder. Spectral shifts and REM sleep-linked muscle activity were age dependent, supporting Face Validity of the PLP α-SYN model. We also strongly suggest our findings to be critically evaluated for Predictive Validity in future studies. Currently, research on MSA lacks potential compounds attenuating or curing MSA. Future drugs must prove its potential in animal models, for this our study provides potential biomarkers.

MPTP-induced vulnerability of dopamine neurons in A53T α-synuclein overexpressed mice with the potential involvement of DJ-1 downregulation.

Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the α-synuclein (α-syn) gene. Mutant α-syn expression increases the vulnerability of neurons to exogenous insults. In this study, we developed a new PD model in the transgenic mice expressing mutant hemizygous (hemi) or homozygous (homo) A53T α-synuclein (α-syn Tg) and their wildtype (WT) littermates by treatment with sub-toxic (10 mg/kg, i.p., daily for 5 days) or toxic (30 mg/kg, i.p., daily for 5 days) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tyrosine hydroxylase and Bcl-2 levels were reduced in the α-syn Tg but not WT mice by sub-toxic MPTP injection. In the adhesive removal test, time to remove paper was significantly increased only in the homo α-syn Tg mice. In the challenging beam test, the hemi and homo α-syn Tg mice spent significantly longer time to traverse as compared to that of WT group. In order to find out responsible proteins related with vulnerability of mutant α-syn expressed neurons, DJ-1 and ubiquitin enzyme expressions were examined. In the SN, DJ-1 and ubiquitin conjugating enzyme, UBE2N, levels were significantly decreased in the α-syn Tg mice. Moreover, A53T α-syn overexpression decreased DJ-1 expression in SH-SY5Y cells. These findings suggest that the vulnerability to oxidative injury such as MPTP of A53T α-syn mice can be explained by downregulation of DJ-1.

Molecular and Biological Compatibility with Host Alpha-Synuclein Influences Fibril Pathogenicity

The accumulation and propagation of misfolded α-synuclein (α-Syn) is a central feature of Parkinson's disease and other synucleinopathies. Molecular compatibility between a fibrillar seed and its native protein state is a major determinant of amyloid self-replication. We show that cross-seeded aggregation of human (Hu) and mouse (Ms) α-Syn is bidirectionally restricted. Although fibrils formed by Hu-Ms-α-Syn chimeric mutants can overcome this inhibition in cell-free systems, sequence homology poorly predicts their efficiency in inducing α-Syn pathology in primary neurons or after intracerebral injection into wild-type mice. Chimeric α-Syn fibrils demonstrate enhanced or reduced pathogenicities compared with wild-type Hu- or Ms-α-Syn fibrils. Furthermore, α-Syn mutants induced to polymerize by fibrillar seeds inherit the functional properties of their template, suggesting that transferable pathogenic and non-pathogenic states likely influence the initial engagement between exogenous α-Syn seeds and endogenous neuronal α-Syn. Thus, transmission of synucleinopathies is regulated by biological processes in addition to molecular compatibility.

Alpha-synuclein modulates retinal iron homeostasis by facilitating the uptake of transferrin-bound iron: Implications for visual manifestations of Parkinson's disease

Aggregation of α-synuclein (α-syn) in neurons of the substantia nigra is diagnostic of Parkinson's disease (PD), a neuro-motor disorder with prominent visual symptoms. Here, we demonstrate that α-syn, the principal protein involved in the pathogenesis of PD, is expressed widely in the neuroretina, and facilitates the uptake of transferrin-bound iron (Tf-Fe) by retinal pigment epithelial (RPE) cells that form the outer blood-retinal barrier. Absence of α-syn in knock-out mice (α-syn−/−) resulted in down-regulation of ferritin in the neuroretina, indicating depletion of cellular iron stores. A similar phenotype of iron deficiency was observed in the spleen, femur, and brain tissue of α-syn−/− mice, organs that utilize mainly Tf-Fe for their metabolic needs. The liver and kidney, organs that take up significant amounts of non-Tf-bound iron (NTBI), showed minimal change. Evaluation of the underlying mechanism in the human RPE47 cell line suggested a prominent role of α-syn in the uptake of Tf-Fe by modulating the endocytosis and recycling of transferrin (Tf)/transferrin-receptor (TfR) complex. Down-regulation of α-syn in RPE cells by RNAi resulted in the accumulation of Tf/TfR complex in common recycling endosomes (CREs), indicating disruption of recycling to the plasma membrane. Over-expression of exogenous α-syn in RPE cells, on the other hand, up-regulated ferritin and TfR expression. Interestingly, exposure to exogenous iron increased membrane association and co-localization of α-syn with TfR, supporting its role in iron uptake by the Tf/TfR complex. Together with our observations indicating basolateral expression of α-syn and TfR on RPE cells in vivo, this study reveals a novel function of α-syn in the uptake of Tf-Fe by the neuroretina. It is likely that retinal iron dyshomeostasis due to impaired or altered function of α-syn contributes to the visual symptoms associated with PD.

Neuroprotection by Epigenetic Modulation in a Transgenic Model of Multiple System Atrophy.

Similar to Parkinson disease, multiple system atrophy (MSA) presents neuropathologically with nigral neuronal loss; however, the hallmark intracellular α-synuclein (αSyn) accumulation in MSA affects typically oligodendrocytes to form glial cytoplasmic inclusions. The underlying pathogenic mechanisms remain unclear. As MSA is predominantly sporadic, epigenetic mechanisms may play a role. We tested the effects of the pan-histone deacetylase inhibitor (HDACi) sodium phenylbutyrate in aged mice overexpressing αSyn under the control of the proteolipid protein promoter (PLP–αSyn) designed to model MSA and characterized by αSyn accumulation in oligodendrocytes and nigral neurodegeneration. HDACi improved motor behavior and survival of nigral neurons in PLP–αSyn mice. Furthermore, HDACi reduced the density of oligodendroglial αSyn aggregates, which correlated with the survival of nigral neurons in PLP–αSyn mice. For the first time, we suggest a role of HDACi in the pathogenesis of MSA-like neurodegeneration and support the future development of selective HDACi for MSA therapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s13311-016-0447-1) contains supplementary material, which is available to authorized users.

Osmotherapy With Hypertonic Saline Attenuates Global Cerebral Edema Following Experimental Cardiac Arrest via Perivascular Pool of Aquaporin-4.

We tested the hypothesis that osmotherapy with hypertonic saline attenuates cerebral edema following experimental cardiac arrest and cardiopulmonary resuscitation by exerting its effect via the perivascular pool of aquaporin-4. We used mice with targeted disruption of the gene encoding α-syntrophin (α-Syn) that demonstrate diminished perivascular aquaporin-4 pool but retain the non-endfoot and ependymal pools. Laboratory animal study. University animal research laboratory. Isoflurane-anesthetized adult male wild-type C57B/6 or α-Syn mice were subjected to cardiac arrest/cardiopulmonary resuscitation and treated with either a continuous IV infusion of 0.9% saline or various concentrations of hypertonic saline. Serum osmolality, regional brain water content, blood-brain barrier disruption, and aquaporin-4 protein expression were determined at 24 hours after cardiac arrest/cardiopulmonary resuscitation. Hypertonic saline (7.5%) treatment significantly attenuated water content in the caudoputamen complex and cortex compared with 0.9% saline treatment in wild-type mice subjected to cardiac arrest/cardiopulmonary resuscitation. In contrast, in α-Syn mice subjected to cardiac arrest/cardiopulmonary resuscitation, 7.5% hypertonic saline treatment did not attenuate water content. Treatment with 7.5% hypertonic saline attenuated blood-brain barrier disruption at 24 hours following cardiac arrest/cardiopulmonary resuscitation in wild-type mice but not in α-Syn mice. Total aquaporin-4 protein expression was not different between 0.9% saline and hypertonic saline-treated wild-type mice. Following experimental cardiac arrest/cardiopulmonary resuscitation: 1) continuous hypertonic saline therapy maintained to achieve serum osmolality of ≈ 350 mOsm/L is beneficial for the treatment of cerebral edema; 2) perivascular pool of aquaporin-4 plays a critical role in water egress from brain; and 3) hypertonic saline attenuates blood-brain barrier disruption via perivascular aquaporin-4 pool.

Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (α-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced α-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of α-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on α-Syn pathology in vivo in a transgenic mouse model overexpressing human α-Syn bearing the A53T mutation (α-SynA53T mice). Fasudil treatment improved motor and cognitive functions in α-SynA53T mice as determined by CatwalkTM gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of α-Syn pathology in the midbrain of α-SynA53T mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with α-Syn and attenuates α-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0310-y) contains supplementary material, which is available to authorized users.

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector.

In order to study the molecular pathways of Parkinson's disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic α-SYN mouse models develop gradual α-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson's disease.

Conivaptan, a Selective Arginine Vasopressin V1a and V2 Receptor Antagonist Attenuates Global Cerebral Edema Following Experimental Cardiac Arrest via Perivascular Pool of Aquaporin-4.

Cerebral edema is a major cause of mortality following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Arginine vasopressin (AVP) and water channel aquaporin-4 (AQP4) have been implicated in the pathogenesis of CA-evoked cerebral edema. In this study, we examined if conivaptan, a V1a and V2 antagonist, attenuates cerebral edema following CA/CPR in wild type (WT) mice as well as mice with targeted disruption of the gene encoding α-syntrophin (α-syn(-/-)) that demonstrate diminished perivascular AQP4 pool. Isoflurane-anesthetized adult male WT C57Bl/6 and α-syn(-/-) mice were subjected to 8 min CA/CPR and treated with either bolus IV injection (0.15 or 0.3 mg/kg) followed by continuous infusion of conivaptan (0.15 mg/kg/day or 0.3 mg/kg/day), or vehicle infusion for 48 h. Serum osmolality, regional brain water content, and blood-brain barrier (BBB) disruption were determined at the end of the experiment. Sham-operated mice in both strains served as controls. Treatment with conivaptan elevated serum osmolality in a dose-dependent manner. In WT mice, conivaptan at 0.3 mg dose significantly attenuated regional water content in the caudoputamen (81.0 ± 0.5 vs. 82.5 ± 0.4% in controls; mean ± SEM) and cortex (78.8 ± 0.2 vs. 79.4 ± 0.2% in controls), while conivaptan at 0.15 mg was not effective. In α-syn(-/-) mice, conivaptan at 0.3 mg dose did not attenuate water content compared with controls. Conivaptan (0.3 mg/kg/day) attenuated post-CA BBB disruption at 48 h in WT mice but not in α-syn(-/-) mice. Continuous IV infusion of conivaptan attenuates cerebral edema and BBB disruption following CA. These effects of conivaptan that are dependent on the presence of perivascular pool of AQP4 appear be mediated via its dual effect on V1 and V2 receptors.

Commentary: Clinical Correlates of Raphe Serotonergic Dysfunction in Early Parkinson's Disease.

Growing and clinical evidence supports the conclusion that Parkinson’s Disease (PD) is a complex multisystem disorder not exclusively affecting the dopaminergic circuits. In fact, the dopaminergic neuronal loss does not cover all the clinical aspects of PD. Therefore, different non-dopaminergic neurotransmitter systems have been invoked as playing a role in the PD clinical picture. Principally, the inefficiency of serotonergic circuitry has been demonstrated in PD animal models, as well as in post-mortem and in vivo human studies. In the recent article published in Brain, Zahi Qamhawi and colleagues interrogated 123I-FP-CIT single-photon emission computed tomography documenting raphe serotonergic dysfunction in a large group of early PD patients compared to a subset of possible PD patients without evidence of dopaminergic deficit (SWEDD) and a population of healthy controls (1). This paper combined an accurate clinical evaluation with a sophisticated neuroimaging protocol and documented in PD patients a mean raphe serotonin (5-HT) transporter availability significantly lower than both SWEDD patients and controls. These findings, achieved in a large cohort of PD patients, enforced previous autoptic examinations documenting serotonergic neurons loss due to Lewy body pathology in the raphe nuclei of PD patients (2). Accordingly, neuroimaging studies have shown the progressive 5-HT transporter availability reduction in the raphe nuclei as PD pathology progresses (3–5). It is well accepted that concentration of 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in CSF reflects the serotonergic metabolism and turnover in the CNS (6, 7). We performed a case-control study investigating CSF levels of 5-HT and 5-HIAA in a cohort of PD patients, after a 3-day dopaminergic therapy withdrawal and in absence of serotonergic agents (8). We demonstrated the significant reduction of CSF 5-HT and 5-HIAA concentrations in PD patients compared to controls. We also found the significant reduction of CSF 5-HT and 5-HIAA in the PD population with respect to Alzheimer’s Disease patients (8), thus highlighting that the impairment of serotonergic system could represent a specific effect of synuclein-mediated neurodegeneration (8). As a matter of fact, novel experimental studies in alpha-synuclein (α-syn) mouse model of PD showed the impairment of serotonergic system owing to the intracellular accumulation of α-syn in serotonergic neurons coupled with the reduction of 5-HT levels in lower brainstem (9). Based on the clinical presentation, Qamhawi and collegues divided PD patients into two subgroups corresponding to patients with and without resting tremor. Remarkably, patients with tremor had lower mean raphe 5-HT transporter availability than patients unaffected by tremor. Since tremor amplitude, constancy, and severity negatively correlated with raphe 5-HT transporter availability in the whole PD cohort, authors suggested that the serotonergic system inefficiency could be responsible for parkinsonian tremor. This finding was consistent with previous studies detecting an association between 5-HT receptor availability in the raphe nuclei and severity of parkinsonian tremor (10). However, in our CSF study, we found no differences in CSF 5-HT and 5-HIAA levels between tremor dominant and non-tremor dominant patients. This discrepancy could be ascribed to the fact that Qamhawi and collegues studied de novo early PD patients, whereas we investigated patients with higher Hoehn and Yahr stage and greater motor disability at the Unified PD Rating Scale-motor section. It could be of interest to evaluate in follow-up studies how the serotonergic transmission damage may influence PD motor symptoms along with the progression of the disease. Concurrently, the reliability of the proposed difference in raphe 5-HT transporter availability found by Qamhawi and coworkers in early PD patients should be also analyzed in more advanced PD patients. Remarkably, Qamhawi and coauthors documented that raphe 5-HT transporter availability did not correlate with PD non-motor symptoms, such as fatigue, depression, and sleep-wake cycle disturbances. In agreement with this finding, we did not identify mutual interplays linking CSF 5-HT and 5-HIAA concentrations to depression, apathy, and sleep disturbances. Although our report sustained Qamhawi and coworkers results, literature proposes controversial data regarding the involvement of serotonergic pathways breakage in the pathophysiology of PD non-motor symptoms (11), thus requiring further investigations. All considering, we suppose that the reduced 5-HT transporter availability, as shown by Qamhawi and collegues, and the 5-HT synthesis, metabolism, and turnover impairment, resulting in reduced CSF levels of 5-HT and 5-HIAA (8) may both contribute to the serotonergic transmission dysfunction evident in PD. Hence, serotonin circuitry inefficiency could represent the fitting partner of the dopaminergic deficit in PD.

Overexpression of α-synuclein simultaneously increases glutamate NMDA receptor phosphorylation and reduces glucocerebrosidase activity

Progressive accumulation of α-synuclein (α-syn)-containing protein aggregates throughout the nervous system is a pathological hallmark of Parkinson’s disease (PD). The mechanisms whereby α-syn exerts neurodegeneration remain to be fully understood. Here we show that overexpression of α-syn in transgenic mice leads to increased phosphorylation of glutamate NMDA receptor (NMDAR) subunits NR1 and NR2B in substantia nigra and striatum as well as reduced glucocerebrosidase (GCase) levels. Similarly, molecular studies performed in mouse N2A cells stably overexpressing human α-syn (α-synN2A) showed that phosphorylation states of the same NMDAR subunits were increased, whereas GCase levels and lysosomal GCase activity were reduced. α-synN2A cells showed an increased sensitivity to neurotoxicity towards 6-hydroxydopamine and NMDA. However, wildtype N2A, but not α-synN2A cells, showed a further reduction in viability when co-incubated with 6-hydroxydopamine and the lysosomal inhibitors NH4Cl and leupeptin, suggesting that α-syn per se perturbs lysosomal functions. NMDA treatment reduced lysosomal GCase activity to the same extent in α-synN2A cells as in wildtype N2A cells, indicating that the α-syn-dependent difference in NMDA neurotoxicity is unrelated to an altered GCase activity. Nevertheless, these data provide molecular evidence that overexpression of α-syn simultaneously induces two potential neurotoxic hits by increasing glutamate NMDA receptor phosphorylation, consistent with increased NMDA receptors functionality, and reducing GCase activity.

Differential calcium alterations in animal models of neurodegenerative disease: Reversal by FK506

Abnormal accumulation of amyloid β (Aβ), α-synuclein (α-syn), and microtubule-associated protein tau (tau) have been implicated in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Pick’s disease (PiD). The mechanisms through which aggregated versions of α-syn, Aβ, and tau may lead to neurodegeneration are not entirely clear, however, there is emerging evidence that neuronal calcium dysregulation is at play. Two-photon microscopy is a powerful tool that can be used to measure in vivo alterations of calcium transients using animal models of neurodegeneration, and when coupled with statistical methods to characterize functional signals, can reveal features that identify and discern between distinct mouse types. We studied four mouse models of neurodegenerative diseases, wild-type (WT) α-syn, E57K α-syn, amyloid precursor protein (APP), and triple-repeat (3R)-Tau and Non-transgenic (tg) littermates using two-photon microscopy. We found that for calcium transients, simple measures such as area under the curve (AUC) and peak width in the 1-Hz whisker pad stimulation paradigm, were significantly increased for WT α-syn, E57K α-syn and APP mice across all cortical depths compared to Non-tg mice. A similar result was found in the 3-Hz paradigm in E57K α-syn mice. Spontaneous calcium transient AUC was significantly higher in WT α-syn mice and lower for APP and 3R Tau mice at 150-μm depth. Going beyond simple measure differences such as group means for AUC, signal peak width, and spontaneous calcium activity counts, we built statistical classifiers to characterize neuronal calcium signals to identify and discern, with quantified measures of confidence, all mouse types. We tested our classifier with FK506, which regulates mitochondrial calcium and found that this drug modulated the WT α-syn calcium transients to such an extent that the classifier easily identified the calcium transients as belonging to Non-tg mice. The coupling of two-photon microscopy data and statistical classifiers serves to effectively create a bioassay where the number of animals and scientific resources can be reduced without compromising the results of the experiment.

Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinson's disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague–Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting that PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time-point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.

Increased 14-3-3 phosphorylation observed in Parkinson's disease reduces neuroprotective potential of 14-3-3 proteins

14-3-3 proteins are key regulators of cell survival. We have previously demonstrated that 14-3-3 levels are decreased in an alpha-synuclein (αsyn) mouse model of Parkinson's disease (PD), and that overexpression of certain 14-3-3 isoforms is protective in several PD models. Here we examine whether changes in 14-3-3 phosphorylation may contribute to the neurodegenerative process in PD. We examine three key 14-3-3 phosphorylation sites that normally regulate 14-3-3 function, including serine 58 (S58), serine 184 (S184), and serine/threonine 232 (S/T232), in several models of PD and in human PD brain. We observed that an increase in S232 phosphorylation is observed in rotenone-treated neuroblastoma cells, in cells overexpressing αsyn, and in human PD brains. Alterations in S58 phosphorylation were less consistent in these models, and we did not observe any phosphorylation changes at S184. Phosphorylation at S232 induced by rotenone is reduced by casein kinase inhibitors, and is not dependent on αsyn. Mutation of the S232 site affected 14-3-3θ's neuroprotective effects against rotenone and 1-methyl-4-phenylpyridinium (MPP+), with the S232D mutant lacking any protective effect compared to wildtype or S232A 14-3-3θ. The S232D mutant partially reduced the ability of 14-3-3θ to inhibit Bax activation in response to rotenone. Based on these findings, we propose that phosphorylation of 14-3-3s at serine 232 contributes to the neurodegenerative process in PD.

Issue Cover (December 2014)

Front cover: GFAP immunostaining of brainstem sections from (Thy1)-h[A30P]-αSyn mice. Lifelong high fat diet led to a significantly premature onset of brainstem astrogliosis (cover) and synucleinopathy (not shown). J. Neurochem. 2014, vol. 131 (6), pp. 848–858. Read the full article on doi: 10.1111/jnc.12813

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.

The regulation of catalase activity by PPAR γ is affected by α ‐synuclein

ObjectiveWhile evidence for oxidative injury is frequently detected in brains of humans affected by Parkinson's disease (PD) and in relevant animal models, there is uncertainty regarding its cause. We tested the potential role of catalase in the oxidative injury that characterizes PD.MethodsUtilizing brains of A53T α-Syn and ntg mice, and cultured cells, we analyzed catalase activity and expression, and performed biochemical analyses of peroxisomal metabolites.ResultsLower catalase expression and lower activity levels were detected in A53T α-Syn brains and α-Syn-expressing cells. The effect on catalase activity was independent of disease progression, represented by mouse age and α-Syn mutation, suggesting a potential physiological function for α-Syn. Notably, catalase activity and expression were unaffected in brains of mice modeling Alzheimer's disease. Moreover, we found that α-Syn expression downregulate the peroxisome proliferator-activated receptor (PPAR)γ, which controls catalase transcription. Importantly, activation of either PPARγ2, PPARα or retinoic X receptor eliminated the inhibiting effect of α-Syn on catalase activity. In addition, activation of these nuclear receptors enhanced the accumulation of soluble α-Syn oligomers, resulting in a positive association between the degree of soluble α-Syn oligomers and catalase activity. Of note, a comprehensive biochemical analysis of specific peroxisomal metabolites indicated no signs of dysfunction in specific peroxisomal activities in brains of A53T α-Syn mice.InterpretationOur results suggest that α-Syn expression may interfere with the complex and overlapping network of nuclear receptors transcription activation. In result, catalase activity is affected through mechanisms involved in the regulation of soluble α-Syn oligomers.