Given the complexity and heterogeneity of the genomic architecture underlying schizophrenia, molecular analyses of these patients with defined and large effect-size genomic defects could provide valuable clues. We established human-induced pluripotent stem cells from two schizophrenia patients with the 22q11.2 deletion (two cell lines from each subject, total of four cell lines) and three controls (total of four cell lines). Neurosphere size, neural differentiation efficiency, neurite outgrowth, cellular migration and the neurogenic-to-gliogenic competence ratio were significantly reduced in patient-derived cells. As an underlying mechanism, we focused on the role of DGCR8, a key gene for microRNA (miRNA) processing and mapped in the deleted region. In mice, Dgcr8 hetero-knockout is known to show a similar phenotype of reduced neurosphere size (Ouchi et al., 2013). The miRNA profiling detected reduced expression levels of miRNAs belonging to miR-17/92 cluster and miR-106a/b in the patient-derived neurospheres. Those miRNAs are reported to target p38α, and conformingly the levels of p38α were upregulated in the patient-derived cells. p38α is known to drive gliogenic differentiation. The inhibition of p38 activity by SB203580 in patient-derived neurospheres partially restored neurogenic competence. Furthermore, we detected elevated expression of GFAP, a gliogenic (astrocyte) marker, in postmortem brains from schizophrenia patients without the 22q11.2 deletion, whereas inflammation markers (IL1B and IL6) remained unchanged. In contrast, a neuronal marker, MAP2 expressions were decreased in schizophrenia brains. These results suggest that a dysregulated balance of neurogenic-to-gliogenic competence may underlie neurodevelopmental disorders such as schizophrenia.

Self-propagating β-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Protein segregation contributes to various cellular processes such as polarization, differentiation, and aging. However, the difficulty in global determination of protein segregation hampers our understanding of its mechanisms and physiological roles. Here, by developing a quantitative proteomics technique, we globally monitored segregation of preexisting and newly synthesized proteins during cell division of budding yeast, and identified crucial domains that determine the segregation of cell-peripheral proteins. Remarkably, the proteomic and subsequent microscopic analyses demonstrated that the flow through the bud neck of the proteins that harbor both endoplasmic reticulum (ER) membrane-spanning and plasma membrane (PM)-binding domains is not restricted by the previously suggested ER membrane or PM diffusion barriers but by septin-mediated partitioning of the PM-associated ER (pmaER). Furthermore, the proteomic analysis revealed that although the PM-spanning t-SNARE Sso2 was retained in mother cells, its paralog Sso1 unexpectedly showed symmetric localization. We found that the transport of Sso1 to buds was required for enhancement of polarized cell growth and resistance to cell-wall stress. Taken together, these data resolve long-standing questions about septin-mediated compartmentalization of the cell periphery, and provide new mechanistic insights into the segregation of cell-periphery proteins and their cellular functions.

Amyloids are β-sheet-rich fibrillar protein aggregates characterized by structural properties of self-propagation and strong resistance to detergent and proteinase. Although a number of causative proteins for neurodegenerative disorders are known to undergo amyloid formation, recent studies have revealed that amyloids may also play beneficial roles in cells. Cellular processes that could be regulated by amyloids are diverse and include translational regulation, programmed cell death and protein storage. Yeast prions of Mod5 and Mot3, non-Mendelian extra-chromosomal factors, also show amyloid-like biophysical properties and have recently been shown to confer host cells resistant to environmental stressors. Furthermore, yeast cells actively respond to environmental stress for fitness adaptation to environmental changes by converting soluble yeast prion proteins into their amyloid forms, allowing cells to survive under stress conditions. Therefore, amyloids are not simply the terminal end-products of protein misfolding but a growing body of evidence suggests that they may possess physiological roles by using their self-propagating properties. Here, we present an overview on recent progress of the research on such functional amyloids.

Disaggregation of amyloid brils is a fundamental biological process required for amyloid propagation.However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust, in vitro reconstituted system of yeast prion propagation and found that Hsp104, Ssa1, and Sis1 chaperones are essential for e cient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule uorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to the amyloid. Remarkably, we uncovered two distinct, prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperon dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These ndings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloidassociated neurodegenerative diseases.

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. Here, using yeast and human fibroblasts, we show that cellular senescence, irreversible cell cycle arrest contributing to organismal aging, is the third outcome of PMD. To identify the genes essential for PMD response, we performed a systematic yeast genome-wide screen. The screen identified 48 genes. The top hits in the screen are the endosomal sorting complexes required for transport (ESCRT) genes. Strikingly, the replicative lifespan regulator genes are enriched in our 48 hits, and indeed, PMD limits the replicative lifespan in budding yeast; the ESCRT activator AAA-ATPase VPS4-overexpression extends it. In normal human fibroblasts, PMD induces cellular senescence via p53. Our study demonstrates that PMD limits replicative lifespan in two different eukaryotic cell types and highlights an underappreciated but ubiquitous senescent cell subtype, namely PMD-dependent senescent cells.

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SHP-2 enhanced neurogenesis and suppressed gliogenesis. Blockade of MAP kinase activity inhibited the SHP-2-mediated increase in neurogenesis, indicating that this is the likely pathway used by NS SHP-2. Together, these results indicate that SHP-2 plays a key role in integrating extrinsic signals, and functions to promote neurogenesis at the same time that it inhibits gliogenesis. We are currently investigating whether the neuronal versus glial ratio is altered in a mouse model of Noonan syndrome, with the ultimate goal of determining whether this accounts for the cognitive impairments in NS patients. Cellular mechanisms regulating neural plasticity [S45]Role of proteolytic conversion of proBDNF to mBDNF in synaptic development and plasticity B. LuGene, Cognition and Psychosis (GCAP) Program, NIMH, NIH, USA BDNF has recently emerged as a key regulator for synaptic development and plasticity. In a recent study, we examined the role of BDNF in late-phase LTP (L-LTP). We demonstrate that the extracellular protease tPA converts the precursor proBDNF to the mature BDNF (mBDNF) in the hippocampus, and such conversion is critical for L-LTP. Furthermore, application of mBDNF allows L-LTP to occur when protein synthesis is blocked and converts early-phase LTP to L-LTP, suggesting that mBDNF is the key protein synthesis product responsible for L-LTP expression. Our study has identified tPA/plasmin as an endogenous enzyme system that converts proBDNF to mBDNF in the hippocampus, and provided a mechanistic link between tPA and BDNF in L-LTP.ProBDNF and mBDNF are thought to bind to two distinct receptors: the pan neurotrophin receptor p75 (p75 NTR ) and the TrkB tyrosine kinase, respectively. Previous, studies indicate that mBDNF facilitates LTP through TrkB. We show that the proBDNF, by activating its preferred receptor p75 NTR , selectively enhances the NMDA-dependent form of LTD at the hippocampal synapses. Together with the finding that mBDNF promotes LTP, our results support a Yin-Yang model in which proBDNF promotes LTD whereas mBDNF facilitates LTP. Thus, extracellular cleavage of proBDNF becomes a key step that controls the direction of BDNF regulation.ProBDNF ! mBDNF conversion may also play a role in synapse development. Using a cell culture system in which a single myocyte is innervated by two spinal neurons, proBDNF induces retraction of the less active terminal by activating p75 NTR , whereas mature BDNF (mBDNF) facilitates the stabilization of the active one through TrkB. Muscle activity drives the secretion of proBDNF, which is converted to mBDNF by proteases selectively expressed by the active, presynaptic terminal. These results demonstrate that the activity-dependent conversion of proBDNF to mBDNF is critical for synaptic competition and elimination during development.Brain functions are shaped by sensory experience during early postnatal life. We have found that the critical period for binocular vision is triggered by specific GABAergic connections in the neocortex. Maturation of these interneurons (and hen...