Region-specific tauopathy and synucleinopathy in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease

<p>Abstract</p> <p>Background</p> <p>α-synuclein [α-Syn]-mediated activation of GSK-3β leading to increases in hyperphosphorylated Tau has been shown by us to occur in striata of Parkinson's diseased [PD] patients and in animal models of PD. In Alzheimer's disease, tauopathy exists in several brain regions; however, the pattern of distribution of tauopathy in other brain regions of PD or in animal models of PD is not known. The current studies were undertaken to analyze the distribution of tauopathy in different brain regions in a widely used mouse model of PD, the α-Syn overexpressing mouse.</p> <p>Results</p> <p>High levels of α-Syn levels were seen in the brain stem, with a much smaller increase in the frontal cortex; neither cerebellum nor hippocampus showed any overexpression of α-Syn. Elevated levels of p-Tau, hyperphosphorylated at Ser202, Ser262 and Ser396/404, were seen in brain stem, with lower levels seen in hippocampus. In both frontal cortex and cerebellum, increases were seen only in p-Ser396/404 Tau, but not in p-Ser202 and p-Ser262. p-GSK-3β levels were not elevated in any of the brain regions, although total GSK-3β was elevated in brain stem. p-p38MAPK levels were unchanged in all brain regions examined, while p-ERK levels were elevated in brain stem, hippocampus and cerebellum, but not the frontal cortex. p-JNK levels were increased in brain stem and cerebellum but not in the frontal cortex or hippocampus. Elevated levels of free tubulin, indicating microtubule destabilization, were seen only in the brain stem.</p> <p>Conclusion</p> <p>Our combined data suggest that in this animal model of PD, tauopathy, along with microtubule destabilization, exists primarily in the brain stem and striatum, which are also the two major brain regions known to express high levels of α-Syn and undergo the highest levels of degeneration in human PD. Thus, tauopathy in PD may have a very restricted pattern of distribution.</p

Loss of the Intellectual Disability and Autism Gene Cc2d1a and Its Homolog Cc2d1b Differentially Affect Spatial Memory, Anxiety, and Hyperactivity

Hundreds of genes are mutated in non-syndromic intellectual disability (ID) and autism spectrum disorder (ASD), with each gene often involved in only a handful of cases. Such heterogeneity can be daunting, but rare recessive loss of function (LOF) mutations can be a good starting point to provide insight into the mechanisms of neurodevelopmental disease. Biallelic LOF mutations in the signaling scaffold CC2D1Acause a rare form of autosomal recessive ID, sometimes associated with ASD and seizures. In parallel, we recently reported that Cc2d1a-deficient mice present with cognitive and social deficits, hyperactivity and anxiety. In Drosophila, loss of the only ortholog of Cc2d1a, lgd, is embryonically lethal, while in vertebrates, Cc2d1a has a homolog Cc2d1b which appears to be compensating, indicating that Cc2d1a and Cc2d1b have a redundant function in humans and mice. Here, we generate an allelic series of Cc2d1a and Cc2d1b LOF to determine the relative role of these genes during behavioral development. We generated Cc2d1b knockout (KO), Cc2d1a/1b double heterozygous and double KO mice, then performed behavioral studies to analyze learning and memory, social interactions, anxiety, and hyperactivity. We found that Cc2d1a and Cc2d1b have partially overlapping roles. Overall, loss of Cc2d1b is less severe than loss of Cc2d1a, only leading to cognitive deficits, while Cc2d1a/1b double heterozygous animals are similar to Cc2d1a-deficient mice. These results will help us better understand the deficits in individuals with CC2D1A mutations, suggesting that recessive CC2D1B mutations and trans-heterozygous CC2D1A and CC2D1B mutations could also contribute to the genetics of ID

Coalescent-based species delimitation in the sand lizards of the Liolaemus wiegmannii complex (Squamata: Liolaemidae)

Coalescent-based algorithms coupled with the access to genome-wide data have become powerful tools forassessing questions on recent or rapid diversification, as well as delineating species boundaries in the absence of reciprocal monophyly. In southern South America, the diversification of Liolaemus lizards during the Pleistocene is well documented and has been attributed to the climatic changes that characterized this recent period of time. Past climatic changes had harsh effects at extreme latitudes, including Patagonia, but habitat changes at intermediate latitudes of South America have also been recorded, including expansion of sand fields over northern Patagonia and Pampas). In this work, we apply a coalescent-based approach to study the diversification of the Liolaemus wiegmannii species complex, a morphologically conservative clade that inhabits sandy soils across northwest and south-central Argentina, and the south shores of Uruguay. Using four standard sequence markers (mitochondrial DNA and three nuclear loci) along with ddRADseq data we inferred species limits and a time calibrated species tree for the L. wiegmannii complex in order to evaluate the influence of Quaternary sand expansion/retraction cycles on diversification. We also evaluated the evolutionary independence of the recently described L. gardeli and inferred its phylogenetic position relative to L. wiegmannii. We find strong evidence for six allopatric candidate species within L. wiegmannii, which diversified during the Pleistocene. The Great Patagonian Glaciation (∼1 million years before present) likely split the species complex into two main groups: one composed of lineages associated with sub-Andean sedimentary formations, and the other mostly related to sand fields in the Pampas and northern Patagonia. We hypothesize that early speciation within L. wiegmannii was influenced by the expansion of sand dunes throughout central Argentina and Pampas. Finally, L. gardeli is supported as a distinct lineage nested within the L. wiegmannii complex.Fil: Villamil, Joaquín. Universidad de la República. Facultad de Ciencias; UruguayFil: Avila, Luciano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico para el Estudio de los Ecosistemas Continentales; ArgentinaFil: Morando, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico para el Estudio de los Ecosistemas Continentales; ArgentinaFil: Sites, Jack W.. University Brigham Young; Estados UnidosFil: Leaché, Adam D.. University of Washington; Estados UnidosFil: Maneyro, Raúl. Universidad de la República. Facultad de Ciencias; UruguayFil: Camargo Bentaberry, Arley. Universidad de la República; Urugua

Tauopathic Changes in the Striatum of A53T α-Synuclein Mutant Mouse Model of Parkinson's Disease

Tauopathic pathways lead to degenerative changes in Alzheimer's disease and there is evidence that they are also involved in the neurodegenerative pathology of Parkinson's disease [PD]. We have examined tauopathic changes in striatum of the α-synuclein (α-Syn) A53T mutant mouse. Elevated levels of α-Syn were observed in striatum of the adult A53T α-Syn mice. This was accompanied by increases in hyperphosphorylated Tau [p-Tau], phosphorylated at Ser202, Ser262 and Ser396/404, which are the same toxic sites also seen in Alzheimer's disease. There was an increase in active p-GSK-3β, hyperphosphorylated at Tyr216, a major and primary kinase known to phosphorylate Tau at multiple sites. The sites of hyperphosphorylation of Tau in the A53T mutant mice were similar to those seen in post-mortem striata from PD patients, attesting to their pathophysiological relevance. Increases in p-Tau were not due to alterations on protein phosphatases in either A53T mice or in human PD, suggesting lack of involvement of these proteins in tauopathy. Extraction of striata with Triton X-100 showed large increases in oligomeric forms of α-Syn suggesting that α-Syn had formed aggregates the mutant mice. In addition, increased levels of p-GSK-3β and pSer396/404 were also found associated with aggregated α-Syn. Differential solubilization to measure protein binding to cytoskeletal proteins demonstrated that p-Tau in the A53T mutant mouse were unbound to cytoskeletal proteins, consistent with dissociation of p-Tau from the microtubules upon hyperphosphorylation. Interestingly, α-Syn remained tightly bound to the cytoskeleton, while p-GSK-3β was seen in the cytoskeleton-free fractions. Immunohistochemical studies showed that α-Syn, pSer396/404 Tau and p-GSK-3β co-localized with one another and was aggregated and accumulated into large inclusion bodies, leading to cell death of Substantia nigral neurons. Together, these data demonstrate an elevated state of tauopathy in striata of the A53T α-Syn mutant mice, suggesting that tauopathy is a common feature of synucleinopathies

Synuclein modulation of monoamine transporters

AbstractAlthough well-studied in the context of neurodegenerative disease, a clear biological function for the synuclein proteins remains elusive. Emerging data indicate a role for synucleins in monoamine neurotransmitter homeostasis. A key regulatory component of monoamine neurotransmission is re-uptake of neurotransmitter by the dopamine transporter, norepinephrine transporter, and serotonin transporter, which are common drug targets in the treatment of depression and other mood disorders. Through interactions with these transporters, the neuronal cytoskeleton, and pre-synaptic scaffolding proteins, α-synuclein, β-synuclein, and γ-synuclein modulate trafficking, expression and function of monoamine transporters at the cell surface, thus playing a central role in regulating monoamine re-uptake

Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function.

Expression of A53T mutant human alpha-synuclein under the mouse prion promoter is among the most successful transgenic models of Parkinson's disease. Accumulation of A53T alpha-synuclein causes adult mice to develop severe motor impairment resulting in early death at 8-12 months of age. In younger, pre-symptomatic animals, altered motor activity and anxiety-like behaviors have also been reported. These behavioral changes, which precede severe neuropathology, may stem from non-pathological functions of alpha-synuclein, including modulation of monoamine neurotransmission. Our analysis over the adult life-span of motor activity, anxiety-like, and depressive-like behaviors identifies perturbations both before and after the onset of disease. Young A53T mice had increased distribution of the dopamine transporter (DAT) to the membrane that was associated with increased striatal re-uptake function. DAT function decreased with aging, and was associated with neurochemical alterations that included increased expression of beta-synuclein and gamma synuclein. Prior to normalization of dopamine uptake, transient activation of Tau kinases and hyperphosphorylation of Tau in the striatum were also observed. Aged A53T mice had reduced neuron counts in the substantia nigra pars compacta, yet striatal medium spiny neuron dendritic spine density was largely maintained. These findings highlight the involvement of the synuclein family of proteins and phosphorylation of Tau in the response to dopaminergic dysfunction of the nigrostriatal pathway

Tau and Tau kinase antibodies.

a<p>Protein or phosphorylated epitope recognized by antibody. ^bAntisera raised in mouse (Ms) or rabbit (Rbt). ^cAntibody dilutions applied for immunoblots. ^dAntibody provided as a generous gift by Peter Davies.</p

DAT distribution and function.

<p>Expression and distribution of DAT was analyzed by immunoblot on protein from (A) total lysates (TL) or synaptosomal plasma membrane fractions (SPM). Actin or cadherin expression, respectively, were analyzed as loading controls. Representative blot images from each genotype at each age are presented with approximate molecular mass of nearest protein ladder bands indicated (M_r). Band optical density (OD) of (B) TL and (C) SPM DAT relative to loading controls is presented as percent of two month-old WT (mean ± SEM) and was analyzed by two-way ANOVA with Bonferroni post-hoc tests comparing each A53T group to age-matched controls (*p<0.05). (D) Uptake of [³H]-DA into striatal synaptosomes isolated from WT and A53T mice at 2–12 months of age was measured in triplicate from six animals per group and is presented as percent of age-matched WT control (mean ± SEM). Non-specific uptake was determined in the presence of 100 µM indatraline HCl and has been subtracted. Comparisons between WT and A53T α-Syn at each age were made by t-test (*p<0.05, **p<0.01).</p

Regression analysis of synuclein expression.

a<p>Slope calculated from linear regression of immunoblot optical density of the indicated Syns measured at 2-12 months, where 2-month-old mean of respective genotypes is set to 100% and all other values are expressed relative to 2-month-old level (also measured as percents, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060378#pone-0060378-g004" target="_blank">Fig. 4</a>). Slope units are percent change per month of age. ^bStatistical test for non-zero slopes for each regression analysis. Statistically significant p-values (<0.05) are bolded. ^cHuman A53T α-Syn is not present in WT mice.</p