Although clinically distinct diseases, tauopathies and synucleinopathies share common genesis and mechanisms, leading to overlapping degenerative changes within neurons. In human postmortem striatum of Parkinson's disease [PD] and PD with dementia, we have recently described elevated levels of tauopathy, indexed as increased hyperphosphorylated Tau [p-Tau]. Here we assessed tauopathy in striatum of a transgenic animal model of PD, overexpressing human α-synuclein under the PDGF promoter. At 11 months of age, large and progressive increases in p-Tau in transgenic mice, hyperphosphorylated at sites reminiscent of Alzheimer's disease, were noted, along with elevated levels of α-synuclein and p-GSK-3β, a major kinase involved in hyperphosphorylation of Tau. Differential Triton X-100 extraction of striata showed the presence of aggregated α-Syn in the Tg mice, along with p-Tau and p-GSK-3β, which was also confirmed through immunohistochemistry. After p-Tau formation, both Tau and MAP1 dissociated from the cytoskeleton, consistent with diminished ability of these cytoskeleton-binding proteins to bind microtubules. Increases in free tubulin and actin were also noted, indicative of cytoskeleton remodeling and destabilization. In vivo magnetic resonance imaging of the transgenic animals showed a reduction in brain volume of transgenic mice indicating substantial atrophy. From immunohistochemical studies, α-synuclein, p-Tau and p-GSK-3β were found to be overexpressed and co-localized in large inclusion bodies, reminiscent of Lewy bodies. The elevated state of tauopathy seen in these PDGF-α-synuclein mice provides further confirmation that Parkinson's may be a tauopathic disease. KeywordsAlzheimer's disease; neurodegeneration; synucleinopathies; tauopathies Sporadic Parkinson's disease [PD] is a progressive neurodegenerative disease of unknown etiology [Pollanen et al, 1993], resulting in loss of motor function and degeneration of dopaminergic neurons [Pollanen et al, 1993;Jakes et al, 1994;Forno, 1996;Spillantini et al, 1998;Corti et al 2005]. Alpha-synuclein [α-Syn], a presynaptic protein, is causal in the genesis of PD [Forno, 1996;Spillantini et al, 1998], and gene duplication and triplication of α-Syn are found in sporadic and early onset forms of PD [Singleton et al, 2003] NIH Public Access Author ManuscriptEur J Neurosci. Author manuscript; available in PMC 2012 May 1. A53T and E46K] in the gene are linked to autosomal dominant familial forms of the disease [Polymeropoulos et al, 1997;Kruger et al, 1998]. Although a soluble protein, under pathological conditions, α-Syn becomes insoluble, self-aggregates and accumulates into intra-neuronal inclusion bodies [Spillantini et al, 1998;Masliah et al, 2000;El-Agnaf et al, 1998;Gosavi et al, 2002]. Similar to α-Syn, Tau is also a highly soluble protein that becomes insoluble by pathological hyperphosphorylation, leading to tauopathies, including Alzheimer's disease (AD), [Dickson et al, 2002;Esper et al, 2007;Gasparini et al, 2007;Wadia & Lang, 2007;Gong & Iqba...

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.

Loss-of-function (LOF) mutations in CC2D1A cause a spectrum of neurodevelopmental disorders, including intellectual disability, autism spectrum disorder, and seizures, identifying a critical role for this gene in cognitive and social development. CC2D1A regulates intracellular signaling processes that are critical for neuronal function, but previous attempts to model the human LOF phenotypes have been prevented by perinatal lethality in Cc2d1a-deficient mice. To overcome this challenge, we generated a floxed Cc2d1a allele for conditional removal of Cc2d1a in the brain using Cre recombinase. While removal of Cc2d1a in neuronal progenitors using Cre expressed from the Nestin promoter still causes death at birth, conditional postnatal removal of Cc2d1a in the forebrain via calcium/calmodulin-dependent protein kinase II-alpha (CamKIIa) promoter-driven Cre generates animals that are viable and fertile with grossly normal anatomy. Analysis of neuronal morphology identified abnormal cortical dendrite organization and a reduction in dendritic spine density. These animals display deficits in neuronal plasticity and in spatial learning and memory that are accompanied by reduced sociability, hyperactivity, anxiety, and excessive grooming. Cc2d1a conditional knockout mice therefore recapitulate features of both cognitive and social impairment caused by human CC2D1A mutation, and represent a model that could provide much needed insights into the developmental mechanisms underlying nonsyndromic neurodevelopmental disorders.

ObjectiveMost neurodegenerative diseases contain hyperphosphorylated Tau [p-Tau]. We examined for the first time epitopes at which Tau is hyperphosphorylated in Parkinson’s disease, dementia with Lewy bodies and Alzheimer’s disease, and also select Tau kinases.MethodsPostmortem frontal cortex from Parkinson’s disease, dementia with Lewy bodies, Alzheimer’s disease and striata from Parkinson’s disease, were analyzed by immunoblots using commercially available antibodies against 20 different phospho-epitopes of Tau. Major Tau kinases were also screened. Results in diseased tissues were compared to nondiseased controls.ResultsIn Alzheimer’s disease, Tau was hyperphosphorylated at all the 20 epitopes of p-Tau. In dementia with Lewy bodies, p-Tau formation occurred at 6 sites sharing 30% overlap with Alzheimer’s disease, while in Parkinson’s frontal cortex, an area which does not degenerate, Tau hyperphosphorylation was seen at just 3 epitopes, indicating 15% overlap with Alzheimer’s disease. In Parkinson’s disease striatum, an area which undergoes considerable neurodegeneration, Tau was hyperphosphorylated at 10 epitopes, sharing 50% overlap with Alzheimer’s disease. Between frontal cortex of Parkinson’s disease and dementia with Lewy bodies, there were only two p-Tau epitopes in common. In striata of Parkinson’s disease, there were 3 clusters of Tau hyperphosphorylated at 3 contiguous sites, while two such clusters were detected in dementia with Lewy bodies; such clusters disrupt axonal transport of mitochondria, cause microtubule remodeling and result in cell death. p-GSK-3β, a major Tau kinase, was activated in all brain regions examined, except in dementia with Lewy bodies. Activation of other Tau kinases was seen in all brain regions, with no clear pattern of activation.InterpretationOur studies suggest that the three neurodegenerative diseases each have a signature-specific profile of p-Tau formation which may be useful in understanding the genesis of the diseases and for the development of a panel of specific biomarkers.

Amplification of a particular DNA fragment from a mixture of organisms by PCR is a common first step in methods of examining microbial community structure. The use of group-specific primers in community DNA profiling applications can provide enhanced sensitivity and phylogenetic detail compared to domain-specific primers. Other uses for group-specific primers include quantitative PCR and library screening. The purpose of the present study was to develop several primer sets targeting commonly occurring and important groups. Primers specific for the 16S ribosomal sequences of Alphaproteobacteria, Betaproteobacteria, Bacilli, Actinobacteria, and Planctomycetes and for parts of both the 18S ribosomal sequence and the internal transcribed spacer region of Basidiomycota were examined. Primers were tested by comparison to sequences in the ARB 2003 database, and chosen primers were further tested by cloning and sequencing from soil community DNA. Eighty-five to 100% of the sequences obtained from clone libraries were found to be placed with the groups intended as targets, demonstrating the specificity of the primers under field conditions. It will be important to reevaluate primers over time because of the continual growth of sequence databases and revision of microbial taxonomy.

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.

SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.

Although 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, alpha-synuclein, beta-synuclein, and gamma-synuclein modulate trafficking, expression and function of monoamine transporters at the cell surface, thus playing a central role in regulating monoamine re-uptake.