Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically-defined tumor types, coupled with an expanding portfolio of molecularly-targeted therapies, demands flexible and comprehensive approaches to profile clinically significant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative utilizing a comprehensive assay, MSK-IMPACT, through which we have compiled matched tumor and normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel non-coding alterations, and mutational signatures that were shared among common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

Parkinson’s disease [PD], a progressive neurodegenerative disease, results in abnormal accumulation of insoluble alpha-synuclein [α-Syn] in dopaminergic neurons. Here we examined tauopathic changes and the α-Syn/p-GSK-3β/proteasome pathway in postmortem striata and inferior frontal gyri [IFG] from patients with PD and PD with dementia [PDD]. In both PD and PDD, α-Syn levels were high, especially the insoluble form of this protein; in PDD, insoluble α-Syn levels were persistently higher than PD across both brain regions. Levels of p-GSK-3β phosphorylated at Tyr 216, which hyperphosphorylates Tau to produce toxic pathological forms of p-Tau, were higher in striata of both PD and PDD compared to controls, but were unaltered in IFG. While proteasomal activity was unchanged in striatum of PD and PDD, such activity was diminished in the IFG of both PD and PDD. A decrease in 19S subunit of the proteasomes was seen in IFG of PDD, while lower levels of 20S subunits were seen in striatum and IFG of both PD and PDD patients. Parkin levels were similar in PD and PDD, suggesting lack of involvement of this protein. Most interestingly, tauopathic changes were noted only in striatum of PD and PDD, with increased hyperphosphorylation seen at Ser262 and Ser396/404; increases in Ser202 levels were seen only in PD but not in PDD striatum. We were unable to detect any tauopathy in IFG in either PD or PDD despite increased levels of α-Syn, and decreased proteasomal activity, and is probably due to lack of increase in p-GSK-3β in IFG. Unlike Alzheimer’s disease where tauopathy is more globally observed in diverse brain regions, our data demonstrates restricted expression of tauopathy in brains of PD and PDD, probably limited to dopaminergic neurons of the nigrostriatal region.

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.

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.