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The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy, and the limited resolution of fluorescence microscopy makes it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution.

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress. DOI: http://dx.doi.org/10.7554/eLife.19850.001, eLife digest Our skeleton plays a vital role in giving shape and structure to our body, it also allows us to make coordinated movements. Similarly, each cell contains a microscopic network of structures and supports called the cytoskeleton that helps cells to adopt specific shapes and is crucial for them to move around. Unlike our skeleton, which is relatively unchanging, the cytoskeleton of each cell constantly changes and adapts to the specific needs of the cell. One part of the cytoskeleton is a dense, flexible meshwork of fibers called the cortex that lies just beneath the surface of the cell. The cortex is constructed using a protein called actin, and many of these proteins join together to form each fiber. When cells need to adapt rapidly to an injury or other sudden changes in their environment they activate a so-called stress response. This response often begins with a rapid increase in the amount of calcium ions inside a cell, which can then trigger changes in actin organization. However, it is not clear how cells under stress are able to globally remodel their actin cytoskeleton without compromising stability and integrity of the cortex. Wales, Schuberth, Aufschnaiter et al. used a range of mammalian cells to investigate how actin responds to stress signals. All cells responded to the resulting influx of calcium ions by deconstructing large parts of the actin cortex and simultaneously forming actin filaments near the center of the cell. Wales, Schuberth, Aufschnaiter et al. termed this response calcium-mediated actin reset (CaAR), as it lasted for only a few minutes before the actin cortex reformed. The experiments show that a protein called INF2 controls CaAR by rapidly removing actin from the cortex and forming new filaments near a cell compartment called the endoplasmic reticulum. CaAR allows cells to rapidly and drastically alter the cortex in response to stress. The experiments also show that this sudden shift in actin can change the activity of certain genes, leading to longer-term effects on the cell. The findings of Wales, Schuberth, Aufschnaiter et al. suggest that calcium ions globally regulate the actin cytoskeleton and hence cell shape and movement under stress. This could be relevant for many important processes and conditions such as wound healing, inflammation and cancer. A future challenge will be to understand the role of CaAR in these processes. DOI: http://dx.doi.org/10.7554/eLife.19850.002

Objective Whereas the contribution of α-synuclein to neurodegeneration in Parkinson disease is well accepted, the putative impact of its close homologue, β-synuclein, is enigmatic. β-Synuclein is widely expressed throughout the central nervous system, as is α-synuclein, but the physiological functions of both proteins remain unknown. Recent findings have supported the view that β-synuclein can act as an ameliorating regulator of α-synuclein–induced neurotoxicity, having neuroprotective rather than neurodegenerative capabilities, and being nonaggregating due to the absence of most of the aggregation-promoting NAC domain. However, a mutation of β-synuclein linked to dementia with Lewy bodies rendered the protein neurotoxic in transgenic mice, and fibrillation of β-synuclein has been demonstrated in vitro. Methods Neurotoxicity and aggregation properties of α-, β-, and γ-synuclein were comparatively elucidated in the rat nigro-striatal projection and in cultured neurons. Results Supporting the hypothesis that β-synuclein can act as a neurodegeneration-inducing factor, we demonstrated that wild-type β-synuclein is neurotoxic for cultured primary neurons. Furthermore, β-synuclein formed proteinase K–resistant aggregates in dopaminergic neurons in vivo, leading to pronounced and progressive neurodegeneration in rats. Expression of β-synuclein caused mitochondrial fragmentation, but this fragmentation did not render mitochondria nonfunctional in terms of ion handling and respiration even at late stages of neurodegeneration. A comparison of the neurodegenerative effects induced by α-, β-, and γ-synuclein revealed that β-synuclein was eventually as neurotoxic as α-synuclein for nigral dopaminergic neurons, whereas γ-synuclein proved to be nontoxic and had very low aggregation propensity. Interpretation Our results suggest that the role of β-synuclein as a putative modulator of neuropathology in aggregopathies like Parkinson disease and dementia with Lewy bodies needs to be revisited. Ann Neurol 2013;74:109–118

The prognosis of neurodegenerative disorders is clinically challenging due to the inexistence of established biomarkers for predicting disease progression. Here, we performed an exploratory cross-sectional, case-control study aimed at determining whether gene expression differences in peripheral blood may be used as a signature of Parkinson's disease (PD) progression, thereby shedding light into potential molecular mechanisms underlying disease development. We compared transcriptional profiles in the blood from 34 PD patients who developed postural instability within ten years with those of 33 patients who did not develop postural instability within this time frame. Our study identified200 differentially expressed genes between the two groups. The expression of several of the genes identified was previously found deregulated in animal models of PD and in PD patients. Relevant genes were selected for validation by real-time PCR in a subset of patients. The genes validated were linked to nucleic acid metabolism, mitochondria, immune response and intracellular-transport. Interestingly, we also found deregulation of these genes in a dopaminergic cell model of PD, a simple paradigm that can now be used to further dissect the role of these molecular players on dopaminergic cell loss. Altogether, our study provides preliminary evidence that expression changes in specific groups of genes and pathways, detected in peripheral blood samples, may be correlated with differential PD progression. Our exploratory study suggests that peripheral gene expression profiling may prove valuable for assisting in prediction of PD prognosis, and identifies novel culprits possibly involved in dopaminergic cell death. Given the exploratory nature of our study, further investigations using independent, well-characterized cohorts will be essential in order to validate our candidates as predictors of PD prognosis and to definitively confirm the value of gene expression analysis in aiding patient stratification and therapeutic intervention.

The pathogenesis of many neurodegenerative disorders arises in association with the misfolding and accumulation of a wide variety of proteins. Much emphasis has been placed on understanding the nature of these protein accumulations, including their composition, the process by which they are formed and the physiological impact they impose at cellular and, ultimately, organismal levels. Alpha-synuclein (ASYN) is the major component of protein inclusions known as Lewy bodies and Lewy neurites, which are the typical pathological hallmarks in disorders referred to as synucleinopathies. In addition, mutations or multiplications in the gene encoding for ASYN have also been shown to cause familial cases of PD, the most common synucleinopathy. Although the precise function of ASYN remains unclear, it appears to be involved in a vast array of cellular processes. Here, we review, in depth, a spectrum of cellular and molecular mechanisms that have been implicated in synucleinopathies. Importantly, detailed understanding of the biology/pathobiology of ASYN may enable the development of novel avenues for diagnosis and/or therapeutic intervention in synucleinopathies.

We have assessed the impact of a-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of a-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction., Instituto de Investigaciones Bioquímicas de La Plata

Abstract 1750 Background: Polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF) are both clonal disorders of CD34+ hematopoietic stem and progenitor cells (HSPC) that undergo inappropriate expansion. The pathological CD34+ clones from both disorders are found at increased levels in the peripheral blood of patients, indicating that the interactions between these CD34+ cells and tissue HSPC niches are different from normal HSPC. Other studies have implicated deficiencies in the CXCR4 signaling axis as a possible reason for the abnormal trafficking of HSPCs in PV and CIMF. We set out to functionally test the importance of CXCR4 for the interactions of HSPCs collected from peripheral blood of patients with PV and CIMF with the bone marrow and other tissue microenvironments. Methods: We purified CD34+ cells from peripheral blood of patients with PV or CIMF, labeled them with fluorescent trackers, and engrafted them into unmanipulated SCID mice. Using video rate, scanning confocal microscopy we tracked CD34+ cell interactions with bone marrow, spleen and liver. To functionally test the role of CXCR4 in the homing of patient CD34+ cells to different microenvironments, we treated CD34+ cells with a blocking antibody prior to engraftment. Results: Flow cytometry revealed that PV CD34+ cells had significantly lower surface expression of CXCR4 than control HSPCs (PV mean: 32.93% positive, range: 14.8–55.1; Control mean: 58.85% positive, range: 33.5–78.7; p value = 0.018) while CIMF samples had scattered CXCR4 expression (mean 61.2% positive range: 0.6–95.4) PV CD34+ cells migrated toward SDF-1 in migration assays, demonstrating that although they had lower surface CXCR4 expression, they were still able to respond to signals from the environment through this receptor. In vivo imaging of mice after engraftment revealed that PV and CIMF CD34+ cells had decreased BM homing compared to control HSPCs. However, CD34+ cells from PV and CIMF patients homed to similar areas of bone marrow vasculature as control cells. Treating CD34+ cells with a blocking antibody against CXCR4 before engraftment significantly reduced the number of PV and CIMF cells that homed to the bone marrow, indicating that their bone marrow homing still relied on CXCR4. Interestingly, there were an increased number of PV and CMF cells that homed to the spleen at baseline compared to control HSPC, suggesting that a distinct homing mechanism favored PV and CMF cell trafficking to the spleen over the BM. The number of cells that homed to the spleen increased upon antibody blockade of CXCR4, indicating that splenic homing of CD34+ cells did not depend on CXCR4/SDF-1 interactions. Conclusion: CD34+ HSPC from PV and CIMF patients and controls homed to similar areas of the BM, although PV and CIMF cells had significantly decreased BM homing. Conversely, PV and CIMF CD34+ cells homed to the spleen in significantly higher numbers than control cells. PV, CIMF and control CD34+ cells were all dependent on CXCR4 for maximal BM homing. Splenic homing was not CXCR4 dependent, however, and PV and CIMF cells homed to the spleen in higher numbers when BM homing was inhibited by CXCR4 blockade. Taken together, we conclude that unique signaling mechanisms regulate trafficking of CD34+ cells to the BM vs. the splenic microenvironments. PV and CIMF CD34+ cells have an abnormal response to these tissue signals, resulting in their preferential homing to the spleen. Molecularly targeted therapies with the Jak2 inhibitors significantly decrease splenomegaly in CIMF patients, although they do not reverse the BM disease process. Recent evidence in a mouse model suggests that the BM is in fact a sanctuary site for disease during treatment with these agents. Whether PV and CIMF CD34+ cells in patients can escape apoptosis during treatment by trafficking to more favorable microenvironments is unknown. Our study suggests that an increased understanding of the mechanisms that these cells use to engage different tissue niches could aid the treatment of these diseases. Disclosures: No relevant conflicts of interest to declare.