Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients’ fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modelling for drug screening.

Several neurological diseases, includingThe importance of ␣-synuclein to the pathogenesis of Parkinson disease (PD) 4 and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1)(2)(3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4 -6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7-9), which increase levels of ␣-synuclein with no alteration in sequence, also cause PD or DLB.␣-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11,12). Phosphorylation at tyrosine residues has been observed by some investigators (13,14) but not by others (10 -12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress ␣-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of ␣-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate ␣-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an ␣-synuclein kinase. The ability of PLK2 to directly phosphorylate ␣-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates ␣-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brai...

The interaction of amyloid-beta (Aβ) and tau in the pathogenesis of Alzheimer's disease is a subject of intense inquiry, with the bulk of evidence indicating that changes in tau are downstream of Aβ. It has been shown however, that human tau overexpression in amyloid precursor protein transgenic mice increases Aβ plaque deposition. Here, we confirm that human tau increases Aβ levels. To determine if the observed changes in Aβ levels were because of intracellular or extracellular secreted tau (eTau for extracellular tau), we affinity purified secreted tau from Alzheimer's disease patient-derived cortical neuron conditioned media and analyzed it by liquid chromatography-mass spectrometry. We found the extracellular species to be composed predominantly of a series of N-terminal fragments of tau, with no evidence of C-terminal tau fragments. We characterized a subset of high affinity tau antibodies, each capable of engaging and neutralizing eTau. We found that neutralizing eTau reduces Aβ levels in vitro in primary human cortical neurons where exogenously adding eTau increases Aβ levels. In vivo, neutralizing human tau in 2 human tau transgenic models also reduced Aβ levels. We show that the human tau insert sequence is sufficient to cause the observed increase in Aβ levels. Our data furthermore suggest that neuronal hyperactivity may be the mechanism by which this regulation occurs. We show that neuronal hyperactivity regulates both eTau secretion and Aβ production. Electrophysiological analysis shows for the first time that secreted eTau causes neuronal hyperactivity. Its induction of hyperactivity may be the mechanism by which eTau regulates Aβ production. Together with previous findings, these data posit a novel connection between tau and Aβ, suggesting a dynamic mechanism of positive feed forward regulation. Aβ drives the disease pathway through tau, with eTau further increasing Aβ levels, perpetuating a destructive cycle.

We have previously shown that a WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor ␤ (TGF-␤) receptor. In this report, we show that another WD-40 repeat protein, the B␣ subunit of protein phosphatase 2A, associates with the cytoplasmic domain of type I TGF-␤ receptors. This association depends on the kinase activity of the type I receptor, is increased by coexpression of the type II receptor, which is known to phosphorylate and activate the type I receptor, and allows the type I receptor to phosphorylate B␣. Furthermore, B␣ enhances the growth inhibition activity of TGF-␤ in a receptor-dependent manner. Because B␣ has been characterized as a regulator of phosphatase 2A activity, our observations suggest possible functional interactions between the TGF-␤ receptor complex and the regulation of protein phosphatase 2A.Mitogenic stimulation of cells by extracellular factors is often mediated by transmembrane tyrosine kinase receptors or receptors that associate with cytoplasmic tyrosine kinases. The signaling pathways generated by many of these receptors are well characterized (23). In contrast to the tyrosine kinase receptors, the receptor signaling pathways for transforming growth factor ␤ (TGF-␤) and the many TGF-␤-related factors have only recently been characterized (12, 24). TGF-␤ and TGF-␤-related factors are secreted proteins which mediate their activities through transmembrane serine/threonine kinase receptors. Ligand-induced activation of these receptors and signaling leads to potent growth inhibition and gene expression responses. Two type I and two type II receptors form the signaling TGF-␤ receptor complex at the cell surface, in which the type II receptors (T␤RII) are constitutively active and autophosphorylated, and the type I receptors (T␤RI) require phosphorylation by T␤RII for activation (12,24).Several proteins have been shown to associate with TGF-␤ receptors. Smad2 and Smad3, which act as effectors of TGF-␤ signaling, can associate with the receptor complex and are phosphorylated by T␤RI. Once dissociated, they are translocated as a complex with Smad4/DPC4 into the nucleus, where they function as transcriptional activators (11,24,33). Another receptor-associated protein is TRIP-1, which interacts with and is phosphorylated by T␤RII (8) and contains five WD-40 repeats (40). WD-40 repeats are minimally conserved sequences of approximately 40 amino acids that typically end in tryptophan-aspartate (WD) and are thought to mediate protein-protein interactions (40). Since TRIP-1 is largely composed of WD-40 repeats, it is possible that other WD-40 repeat proteins may bind to serine/threonine kinase receptors. The association of WD-40 repeat proteins may then allow them to play a role in signaling by the serine/threonine kinase receptors. WD-40 repeats have been identified in a variety of proteins (40), including the B␣ subunit of the serine/threonine protein phosphatase 2A (PP2A).PP2A is one of the major, albeit poorly understood, serine/ threonine phosphatases which re...

The type I and type II receptors for transforming growth factor-␤ (TGF-␤) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-␤ signaling, we characterized the kinase activity of the type II TGF-␤ receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32 P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-␤ receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-␤ receptor signaling.