shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation,Secretion and Toxicity

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.     

Analysis of chromatin structure in mouse preimplantation embryos by fluorescent recovery after photobleaching

Zygotes are totipotent cells that have the ability to differentiate into all cell types. It is believed that this ability is lost gradually and differentiation occurs along with the progression of preimplantation development. Here, we hypothesized that the loose chromatin structure is involved in the totipotency of one-cell stage embryos and that the change from loose to tight chromatin structure is associated with the loss of totipotency. To address this hypothesis, we investigated the mobility of eGFP-tagged histone H2B (eGFP-H2B), which is an index for the looseness of chromatin, during preimplantation development based on fluorescent recovery after photobleaching (FRAP) analysis. The highest mobility of eGFP-H2B was observed in pronuclei in 1-cell stage embryos and mobility gradually decreased during preimplantation development. The decrease in mobility between the 1- and 2-cell stages depended on DNA synthesis in 2-cell stage embryos. In nuclear transferred embryos, chromatin in the pseudopronuclei loosened to a level comparable to the pronuclei in 1-cell stage embryos. These results indicated that the mobility of eGFP-H2B is negatively correlated with the degree of differentiation of preimplantation embryos. Therefore, we suggest that highly loosened chromatin is involved in totipotency of 1-cell embryos and the loss of looseness is associated with differentiation during preimplantation development.     

Battling Alzheimer’s Disease: Targeting SUMOylation-Mediated Pathways

SUMO (small ubiquitin-like modifier) conjugation is a critically important control process in all eukaryotic cells, because it acts as a biochemical switch and regulates the function of hundreds of proteins in many different pathways. Although the diverse functional consequences and molecular targets of SUMOylation remain largely unknown, SUMOylation is becoming increasingly implicated in the pathophysiology of Alzheimer’s disease (AD). Apart from the central SUMO-modified disease-associated proteins, such as amyloid precursor protein, amyloid β, and tau, SUMOylation also regulates several other processes underlying AD. These are involved in inflammation, mitochondrial dynamics, synaptic transmission and plasticity, as well as in protective responses to cell stress. Herein, we review current reports on the involvement of SUMOylation in AD, and present an overview of potential SUMO targets and pathways underlying AD pathogenesis.     

Control of Adult Neurogenesis by Short-Range Morphogenic-Signaling Molecules

Adult neurogenesis is dynamically regulated by a tangled web of local signals emanating from the neural stem cell (NSC) microenvironment. Both soluble and membrane-bound niche factors have been identified as determinants of adult neurogenesis, including morphogens. Here, we review our current understanding of the role and mechanisms of short-range morphogen ligands from the Wnt, Notch, Sonic hedgehog, and bone morphogenetic protein (BMP) families in the regulation of adult neurogenesis. These morphogens are ideally suited to fine-tune stem-cell behavior, progenitor expansion, and differentiation, thereby influencing all stages of the neurogenesis process. We discuss cross talk between their signaling pathways and highlight findings of embryonic development that provide a relevant context for understanding neurogenesis in the adult brain. We also review emerging examples showing that the web of morphogens is in fact tightly linked to the regulation of neurogenesis by diverse physiologic processes.Neurogenesis in the adult mammalian brain is dynamically regulated by a number of genetic and epigenetic intrinsic factors as well as by extrinsic cues (Ninkovic and Götz 2007; Ma et al. 2010; Faigle and Song 2013). Among the latter, local signals emanating from the neural stem cell (NSC) microenvironment are thought to play a prominent modulatory role. This microenvironment, often referred to as the NSC or neurogenic “niche,” is viewed as a complex entity composed of stem and precursor cells, the surrounding mature cell types, cell-to-cell interactions, the extracellular matrix, the basal lamina, and secreted factors (Doetsch 2003). The principal mature cellular constituents of the adult NSC niches are parenchymal astroglial cells, the vasculature, microglia, and ependymal cells, all of which secrete a variety of molecules that mainly control stem-cell behavior, but also influence other stages of the adult neurogenesis process (Basak and Taylor 2009; Mu et al. 2010; Ihrie and Alvarez-Buylla 2011).As opposed to the majority of adult brain regions, the subventricular zone (SVZ) and the dentate gyrus (DG) subgranular zone (SGZ) niches are instructive milieus that allow NSC proliferation while promoting the specification and differentiation of new neurons. The relevance of the SVZ and SGZ microenvironments in adult neurogenesis was first evidenced by heterotopic transplantation experiments showing that precursor cells from a neurogenic niche, such as the SVZ, differentiate into glial cells and not into neurons when grafted to nonneurogenic areas of the brain (Seidenfaden et al. 2006). In contrast, SVZ or spinal cord precursor cells generated neurons when transplanted to a neurogenic region, such as the hippocampal DG (Suhonen et al. 1996; Shihabuddin et al. 2000). Although other in vivo studies have shown that SVZ-derived precursors maintain a certain degree of region-specific potential that is not respecified on transplantation to ectopic sites (Merkle et al. 2007), most studies suggest that local cues in the neurogenic brain niches are key for neuronal differentiation to occur. On the other hand, combined transplantation of both NSCs and niche cells to nonneurogenic areas, or expression of niche factors at the site where NSCs are grafted, promotes neuronal differentiation (Lim et al. 2000, Jiao and Chen 2008). Thus, it has progressively become apparent that extrinsic signals produced by niche cells enable the adult neurogenic program to proceed.More recently, transgenic and virus-based approaches allowing cell type- and temporal-specific manipulation of gene expression in the niches have provided great insights into the identity of the extrinsic signals regulating neurogenesis in vivo and into the molecular mechanisms elicited by those signals. Several soluble and membrane-bound factors have been identified as determinants of SVZ and SGZ neurogenesis, including morphogens, growth factors, neurotrophins, and neurotransmitters. Among these determinants, morphogens are ideally suited to fine-tune the sophisticated processes of stem-cell activation, progenitor expansion, and differentiation required for proper adult neurogenesis. Morphogens are defined as signaling molecules that pattern developing tissues in a concentration-dependent manner (Ashe and Briscoe 2006; Rogers and Schier 2011). They mostly operate in long-range gradients created by synthesis and diffusion of the morphogen proteins from a source and clearance during their flux by diverse mechanisms, such as immobilization, degradation, or endocytosis. Additional molecules that act as anti- or promorphogens further refine their activity. It is important to note that, although morphogens are graded signals, the response they elicit is not graded. Small differences in the concentration of a morphogen can trigger sharp thresholds in the expression of target genes. In addition, morphogens can also act at short range. Lipidation and low-affinity interactions with extracellular matrix components confine the movement of some morphogen proteins and promote effective morphogen–receptor interactions at the cell surface. Cells exposed locally to different morphogen doses respond by adopting different fates and, in this way, a morphogen can assign positional information to cells within a structure or territory, such as a stem-cell niche, and provoke different niche responses or outputs depending on the context (Ashe and Briscoe 2006; Rogers and Schier 2011).Here, we review our current understanding of the role and mechanisms of short-range niche morphogens, including ligands from the Wnt, Notch, Sonic hedgehog, and bone morphogenetic protein (BMP) families, in the regulation of adult neurogenesis. We discuss cross talk between their signaling pathways and intersection with other signaling pathways operating in the niches. We also highlight findings and emerging principles of embryonic development that provide a relevant context for understanding the growing field of adult neurogenesis.