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Pathological aggregates of alpha-synuclein are the common hallmarks of synucleinopathies, including Parkinson's disease. There is currently no disease-modifying therapy approved for neurodegenerative synucleinopathies. The induction of macroautophagy by small compounds may be a strategy to reduce the cellular alpha-synuclein burden and to confer neuroprotection. Therefore, in the present study, we investigated a broad spectrum of druggable molecular signaling pathways reported to induce macroautophagy in human cells and compared their protective efficacy against alpha-synuclein-induced toxicity in cultured human postmitotic dopaminergic neurons. Several compounds affecting different pathways were able to activate macroautophagy. All compounds that activated autophagy also protected against alpha-synuclein-induced toxicity. The compounds with the lowest effective concentrations were PI-103, L-690,330, and NF 449, making them particularly interesting for further investigations, including in vivo models. Our findings demonstrate that activation of macroautophagy, as a neuroprotective approach in synucleinopathies, is accessible to pharmacotherapy. Moreover, pharmacological activation of macroautophagy via diverse signaling pathways is effective to protect human dopaminergic neurons against alpha-synuclein-induced toxicity.

Mutations within Leucine-rich repeat kinase 2 (LRRK2) are associated with late-onset Parkinson’s disease. The physiological function of LRRK2 and molecular mechanism underlying the pathogenic role of LRRK2 mutations remain uncertain. Here, we investigated the role of LRRK2 in intracellular signal transduction. We find that deficiency of Lrrk2 in rodents affects insulin-dependent translocation of glucose transporter type 4 (GLUT4). This deficit is restored during aging by prolonged insulin-dependent activation of protein kinase B (PKB, Akt) and Akt substrate of 160 kDa (AS160), and is compensated by elevated basal expression of GLUT4 on the cell surface. Furthermore, we find a crucial role of Rab10 phosphorylation by LRRK2 for efficient insulin signal transduction. Translating our findings into human cell lines, we find comparable molecular alterations in fibroblasts from Parkinson’s patients with the known pathogenic G2019S LRRK2 mutation. Our results highlight the role of LRRK2 in insulin-dependent signalling with potential therapeutic implications.

Summary As mammals evolved with exposure to particular diets, naturally abundant compounds may have become part of the set of environmental co-determinants that shaped brain structure and function. Here we investigated whether bioactive factors found in apples directly affect hippocampal neurogenesis in the adult mouse. We found that quercetin, the most abundant flavanol in apple peel, was anti-proliferative at high concentrations but pro-neurogenic at low concentrations. This was confirmed in vivo, with intraperitoneally delivered quercetin promoting survival and neuronal differentiation, without affecting proliferation. Using a bioassay-guided fractionation approach we also identified additional pro-neurogenic compounds in apple flesh that were not related to flavonoids. We found that 3,5-dihydroxybenzoic acid significantly increased neural precursor cell proliferation and neurogenesis. This work shows that both flavonoids and 3,5-dihydroxybenzoic acid are pro-neurogenic, not only by activating precursor cell proliferation but also by promoting cell-cycle exit, cellular survival, and neuronal differentiation., Graphical Abstract, Highlights • Quercetin promotes hippocampal precursor cell survival and differentiation • Quercetin induces endogenous antioxidants and the AKT pathway • Apple juice supplementation has no effect on adult neurogenesis or learning • 3,5-Dihydroxybenzoic acid increases precursor cell proliferation and neurogenesis, In this article, Kempermann and colleagues show that quercetin, the most abundant flavonoid in apple peel, promotes hippocampal neurogenesis in the adult mouse brain. They also identify 3,5-dihydroxybenzoic acid as another pro-neurogenic compound found in apple flesh.

The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent, potentially irreversible adverse effect of cytotoxic chemotherapy often leading to a reduction or discontinuation of treatment which negatively impacts patients' prognosis. To date, however, neither predictive biomarkers nor preventive treatments for CIPN are available, which is partially due to a lack of suitable experimental models. We therefore aimed to evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for CIPN. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to axonal blebbing and a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not observed for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. Paclitaxel treatment effects were less pronounced after 24 h but prominent when treatment was applied for 72 h. Global transcriptome analyses performed at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways. We further evaluated if known neuroprotective strategies can be reproduced in iPSC-DSN and observed protective effects of lithium replicating findings from rodent dorsal root ganglia cells. Comparing sensory neurons derived from two different healthy donors, we found preliminary evidence that these cell lines react differentially to neurotoxic drugs as expected from the variable presentation of CIPN in patients. In conclusion, iPSC-DSN are a promising platform to study the pathogenesis of CIPN and to evaluate neuroprotective treatment strategies. In the future, the application of patient-specific iPSC-DSN could open new avenues for personalized medicine with individual risk prediction, choice of chemotherapeutic compounds and preventive treatments.

Superparamagnetic iron oxide nanoparticles (SPIO-NPs) have great potential to be used in different pharmaceutical applications, due to their unique and versatile physical and chemical properties. The aim of this study was to quantify in vitro cytotoxicity of dextran 70,000-coated SPIO-NPs labelled/unlabelled with rhodamine 123, in C6 glioma cells and primary hippocampal neural cells. In addition, we analyzed the in vitro and in vivo cellular uptake of labelled SPIO-NPs. The nanoparticles, with average size of 10&ndash, 50 nm and polydispersity index of 0.37, were synthesized using Massart&rsquo, s co-precipitation method. The concentration-dependent cytotoxicity was quantified by using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Intracellular localization of SPIO-NPs was detected by confocal laser microscopy. In vivo confocal neuroimaging (ICON) was performed on male Wistar rats after intravitreal injection followed by ex vivo retina whole mount analysis. When used for in vitro testing concentrations in the range of diagnostic and therapeutic dosages, SPIO-NPs proved to be non-cytotoxic on C6 glioma cells for up to 24 h incubation time. The hippocampal cell culture also did not show impaired viability at low doses after 24 h incubation. Our results indicate that our dextran-coated SPIO-NPs have the potential for in vivo drug delivery applications.

Dietary supplements containing plant materials of Annonaceae species ( Annona muricata L., A. squamosa L., A. mucosa JACQ., A. squamosa × cherimola Mabb.) were extracted by hot, pressurized ethyl acetate and analyzed for their effect in vitro on Lund human mesencephalic neurons. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell death was determined by lactate dehydrogenase levels. Three supplements strongly decreased the cell viability at extract concentrations of 1 µg/mL, of which 1 decreased cell viability at 0.1 µg/µL. Also, strong neuronal toxicities of these supplements were found. Cell death was observed at concentrations of 10 µg/mL. The degree of toxicity was comparable to the ones found in Annonaceous fruit extracts. Two fruit pulps of Annonaceae ( A. muricata and A. squamosa) showed a reduction in cell viability at lower concentrations. The fruit pulp extract of A. muricata revealed the strongest neurotoxic effect, with 67% cell death at a concentration of 1 µg/mL. A high reduction in cell viability coupled with pronounced cell death was found at 0.1 µg/mL for an Annonaceous seed extract. These results demonstrate that the intake of dietary supplements containing plant material from Annonaceae may be hazardous to health in terms of neurotoxicity.

Background: Anti-aggregation drugs play an important role in therapeutic approaches for Alzheimer’s disease. We have previously developed a number of compounds that are able to inhibit the pathological aggregation of Tau protein. One common obstacle to application is the limited penetration across the plasma membranes into cells, where Tau aggregation occurs in the cytosol. We used an inducible N2a cell line which expresses the repeat domain of tau and develops tau aggregates. Objective: Several peptide-polymer conjugates were synthesized to enhance the uptake of compounds into cells and thus to improve their biomedical application. The aim of this study was to test whether the peptide-inhibitor complexes still retain their inhibitory activity on Tau aggregation. Method: We screened peptide sequences with high binding capacity to a subset of aggregation inhibitors and identified them by fluorescence microscopy and MALDI MS/MS with regard to drug solubility and effective complexion. To explore whether the synthesized complexes can influence the aggregation propensity of Tau we performed in vitro and cellular assays. The effect on toxicity was investigated by measuring apoptosis markers. Results/Conclusion: The tested peptide-compound complexes show no decrease in the total Tau levels but decreased ratios of soluble to pelletable Tau species. This indicates a conversion of insoluble Tau oligomers into soluble forms which appear to be less toxic than the insoluble ones, as seen by a decrease of apoptotic cells. Thus the peptide-compound complexes have a higher potency than the compounds alone due to improved bioavailability of the drug.

Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved., α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions, Authors were supported by: H.V.M. (Fundação para a Ciência e Tecnologia (FCT), Portugal SFRH/BPD/64702/ 2009 and SFRH/BPD/109347/2015; EU FP7 project MEFOPA), L.M.A.O (FCT - SFRH/BD/23604/2005; CIRM-BMFB joint grant, 315050 AZ0101-31P6855), R.M.O. and T.S. (FCT SFRH/BPD/41416/2007; SFRH/ BPD/31209/2006); W.X. (Deutsche Forschungsgemeinschaft, SFB539/A3); C.B. and F.G. (Parkinson’s UK and the Medical Research Council, UK). S.E. is supported by Israel Academy of Sciences, Rappaport Family Institute for Research in the Medical Sciences, The Allen and Jewel Prince Center for Neurodegenerative Disorders of the Brain. T.F.O. (EMBO Installation Grant; Marie Curie IRG, Neurofold; DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain; I.C.M. (FCT SFRH/BPD/74287/2010; Investigador FCT IF/00772/ 2013). This work was supported by: FCT PTDC/SAUNEU/105215/2008, PTDC/QUI/73430/2006, PTDC/SAUENB/117013/2010, PTDC/NEU-OSD/5644/2014; EU FP7 project MEFOPA; CIRM-BMFB joint grant (315050 AZ0101-31P6855); Max Planck Society; and European Union (NEURASYNC PITNGA-2009-238316).

Due to its high nitrogen content, melamine was deliberately added to raw milk for increasing the apparent protein content. Previous studies showed that melamine-induced apoptosis and oxidative damage on PC12 cells and rats' hippocampus. Several evidences suggested that vitamin antioxidant reduced oxidative stress and improved organic function. Whether treatments with antioxidant vitamins C or E, otherwise combination of them can attenuate oxidative stress after melamine administration remains to be elucidated. In this study, the reversible effects of vitamin antioxidants was investigated on melamine-induced neurotoxicity in cultured PC12 cells, an in vitro model of neuronal cells. When comparing vitamin C and E, the combination of both statistically increased PC12 cells viability. The results further showed that vitamin complex has effectively reduced the formation of reaction oxygen species, decreased the level of malondialdehyde, and elevated the activities of antioxidative enzymes. Hoechst 33342 staining and flow cytometric analysis of apoptosis showed that vitamin combination treatment effectively prevented PC12 cells from this melamine-induced apoptosis. It revealed the apoptotic nuclear features of the melamine-induced cell death. Additionally, a combination treatment of vitamins effectively inhibited apoptosis via blocking the increased activation of caspase-3. In summary, the vitamin E and C combination treatment could rescue PC12 cells from the injury induced by melamine through the downregulation of oxidative stress and prevention of melamine-induced apoptosis.

Introduction Tau-mediated toxicity in Alzheimer's disease is thought to operate through low-n oligomers, rather than filamentous aggregates. However, the nature of oligomers and pathways of toxicity are poorly understood. Therefore, we investigated structural and functional aspects of highly purified oligomers of a pro-aggregant tau species. Methods Purified oligomers of the tau repeat domain were characterized by biophysical and structural methods. Functional aspects were investigated by cellular assays ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of cell viability, lactate dehydrogenase release assay [for cell toxicity], reactive oxygen species production, and calcium assay), combined with analysis of neuronal dendritic spines exposed to oligomers. Results Purified low-n oligomers are roughly globular, with sizes around 1.6 to 5.4 nm, exhibit an altered conformation, but do not have substantial β-structure. Treatment of primary neurons with oligomers impairs spine morphology and density, accompanied by increased reactive oxygen species and intracellular calcium, but without affecting cell viability (by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of cell viability and lactate dehydrogenase release assay [for cell toxicity]). Discussion Tau oligomers are toxic to synapses but not lethal to cells.

A potential strategy to alleviate the aggregation of intrinsically disordered proteins (IDPs) is to maintain the native functional state of the protein by small molecule binding. However, the targeting of the native state of IDPs by small molecules has been challenging due to their heterogeneous conformational ensembles. To tackle this challenge, we applied a high-throughput chemical microarray surface plasmon resonance imaging screen to detect the binding between small molecules and monomeric full-length Tau, a protein linked with the onset of a range of Tauopathies. The screen identified a novel set of drug-like fragment and lead-like compounds that bound to Tau. We verified that the majority of these hit compounds reduced the aggregation of different Tau constructs in vitro and in N2a cells. These results demonstrate that Tau is a viable receptor of drug-like small molecules. The drug discovery approach that we present can be applied to other IDPs linked to other misfolding diseases such as Alzheimer's and Parkinson's diseases.

Epigallo-catechin-3-gallate (EGCG), found in the leaves of Camellia sinensis (green tea), has antioxidant- and scavenger-functions and acts neuroprotectively. It has been publicized as anti-aging remedy but data on potential cellular mechanisms are scarce. Recent studies claimed that EGCG specifically promotes neural precursor cell proliferation in the dentate gyrus of C57Bl/6 mice, without changes at the level of immature and mature new neurons. We here analyzed the effects of EGCG on adult hippocampal neurogenesis in male Balb/C mice and saw a different pattern. Two weeks of treatment with EGCG (0, 0.625, 1.25, 2.5, 5 and 10mg/kg) showed a dose-response curve that peaked at 2.5mg/kg of EGCG with significantly increased cell survival without affecting cell proliferation but decreasing apoptotic cells. Also, EGCG increased the population of doublecortin-(DCX)-expressing cells that comprises the late intermediate progenitor cells (type-2b and -3) as well as immature neurons. After EGCG treatment, the young DCX-positive neurons showed more elaborated dendritic trees. EGCG also significantly increased net neurogenesis in the adult hippocampus and increased the hippocampal levels of phospho-Akt. Ex vivo, EGCG exerted a direct effect on survival and neuronal differentiation of adult hippocampal precursor cells, which was absent, when PI3K, a protein upstream of Akt, was blocked. Our results thus support a pro-survival and a pro-neurogenic role of EGCG. In the context of the conflicting published results, however, potential genetic modifiers must be assumed. These might help to explain the overall variability of study results with EGCG. Our data do indicate, however, that natural compounds such as EGCG can in principle modulate brain plasticity.

The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.

Current cell replacement therapies in Parkinson's disease (PD) are limited by low survival of transplanted cell and lacking regeneration of neuronal circuitries. Therefore, bioartificial cell carriers and growth/differentiation factors are applied to improve the integration of transplants and maximize newly generated and/or residual dopaminergic function. In this work, biohybrid poly(ethylene glycol) (starPEG)-heparin hydrogels releasing fibroblast growth factor 2 (FGF-2) and glial-derived neurotrophic factor (GDNF) were used to trigger dopaminergic tissue formation by primary murine midbrain cells in vitro. Matrix-delivered FGF-2 enhanced cell viability while release of GDNF had a pro-neuronal/dopaminergic effect. Combined delivery of both factors from the glycosaminoglycan-based matrices resulted in a tremendous improvement in survival and maturation capacity of dopaminergic neurons as obvious from tyrosine hydroxylase expression and neurite outgrowth. The reported data demonstrate that glycosaminoglycan-based hydrogels can facilitate the administration of neurotrophic factors and are therefore instrumental in potential future treatments of PD.

In this work we investigate the role of CHIP in a new CHIP-mutation related ataxia and the therapeutic potential of trehalose. The patient's fibroblasts with a new form of hereditary ataxia, related to STUB1 gene (CHIP) mutations, and three age and sex-matched controls were treated with epoxomicin and trehalose. The effects on cell death, protein misfolding and proteostasis were evaluated. Recent studies have revealed that mutations in STUB-1 gene lead to a growing list of molecular defects as deregulation of protein quality, inhibition of proteasome, cell death, decreased autophagy and alteration in CHIP and HSP70 levels. In this CHIP-mutant patient fibroblasts the inhibition of proteasome with epoxomicin induced severe pathophysiological age-associated changes, cell death and protein ubiquitination. Additionally, treatment with epoxomicin produced a dose-dependent increase in the number of cleaved caspase-3 positive cells. However, co-treatment with trehalose, a disaccharide of glucose present in a wide variety of organisms and known as a autophagy enhancer, reduced these pathological events. Trehalose application also increased CHIP and HSP70 expression and GSH free radical levels. Furthermore, trehalose augmented macro and chaperone mediated autophagy (CMA), rising the levels of LC3, LAMP2, CD63 and increasing the expression of Beclin-1 and Atg5-Atg12. Trehalose treatment in addition increased the percentage of immunoreactive cells to HSC70 and LAMP2 and reduced the autophagic substrate, p62. Although this is an individual case based on only one patient and the statistical comparisons are not valid between controls and patient, the low variability among controls and the obvious differences with this patient allow us to conclude that trehalose, through its autophagy activation capacity, anti-aggregation properties, anti-oxidative effects and lack of toxicity, could be very promising for the treatment of CHIP-mutation related ataxia, and possibly a wide spectrum of neurodegenerative disorders related to protein disconformation.

Olfactory dysfunction, often preceding the cardinal motor symptoms, such as bradykinesia, rigidity, tremor at rest and postural instability, is frequently reported in Parkinson's disease. This symptom appears to be related to an increased number of dopamine neurons in the periglomerular layer of the olfactory bulb. In animal models of Parkinson's disease, adult neural progenitor cells migrating from the subventricular zone of the lateral ventricle to the olfactory bulb are evidently altered in their survival and progeny. The modulation of neural progenitor cells contributing to the number of dopamine neurons in the periglomerular layer, however, is still poorly understood. In this study, we have investigated the survival and neuronal differentiation of newly generated cells in the olfactory bulb, following treatment with the dopamine precursor l-DOPA and the monoamine oxidase-B inhibitor selegiline in a unilateral, intranigral 6-hydroxydopamine lesion model in mice. Our data show that the number of neural progenitor cells in the subventricular zone is decreased after an intranigral 6-hydroxydopamine lesion, while there is no difference from control in lesioned mice with selegiline or l-DOPA treatment. Selegiline is able to normalize the number of dopamine neurons in the periglomerular layer, while l-DOPA treatment sustains the increased number observed in 6-hydroxydopamine lesioned animals. We conclude that there is a distinct modulation of newly generated dopamine neurons of the olfactory bulb after l-DOPA and selegiline treatment. The differential effects of the two drugs might also play a role in olfactory dysfunction in Parkinson's disease patients.

Neuronal activity provokes myelinating oligodendrocytes to release exosomes by stimulation of ionotropic glutamate receptors, and that once released, these vesicles are internalized by neurons conveying neuroprotection., Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca2+ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity., Author Summary Brain function largely depends on the communication between electrically excitable neurons and surrounding glial cells. Myelinating oligodendrocytes are a type of brain cell that insulate major neuronal processes (axons) and help to sustainably maintain axonal health, which is poorly understood in molecular terms. Several cell types release microvesicles termed exosomes that include genetic information (primarily RNA) and can act as vehicles transferring specific cargo to target cells. Here, we demonstrate that exosomes secreted by oligodendrocytes in response to neuronal signals enter neurons to make their cargo functionally available to the neuronal metabolism. We revealed in cultured cells that exosome release from oligodendrocytes is triggered by the neurotransmitter glutamate through activation of ionotropic glutamate receptors. We also show that glial exosomes are internalized by neurons via an endocytic pathway. By modifying oligodendroglial exosomes with a reporter enzyme, we could demonstrate that the exosome cargo is recovered by target neurons in culture as well as in vivo after injection of exosomes into the mouse brain. Neurons challenged with stressful growth conditions were protected when treated with oligodendroglial exosomes. The study introduces a new concept of reciprocal cell communication in the nervous system and identifies the signal-mediated transfer of exosomes from oligodendrocytes to neurons contributing to the preservation of axonal health.

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization.

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

Ataxin-3 (ATXN3), the disease protein in spinocerebellar ataxia type 3 (SCA3), binds to target gene promoters and modulates transcription by interaction with transcriptional regulators. Here, we show that ATXN3 interacts with the forkhead box O (FOXO) transcription factor FOXO4 and activates the FOXO4-dependent transcription of the manganese superoxide dismutase (SOD2) gene. Upon oxidative stress, ATXN3 and FOXO4 translocate to the nucleus, concomitantly bind to the SOD2 gene promoter and increase the expression of the antioxidant enzyme SOD2. Compared with normal ATXN3, mutant ATXN3 has a reduced capability to activate the FOXO4-mediated SOD2 expression and interferes with binding of FOXO4 to the SOD2 gene promoter. These findings are consistent with a downregulation of SOD2 in pontine brain tissue and lymphoblastoid cell (LC) lines of SCA3 patients. In response to oxidative stress, LCs from SCA3 patients show a specific impairment to upregulate SOD2 expression in correlation with a significantly increased formation of reactive oxygen species and cytotoxicity. The impairment to increase the expression of SOD2 under oxidative stress conditions is associated with a significantly reduced binding of FOXO4 to the SOD2 gene promoter in SCA3-LCs. Finally and consistent with a regulatory role of ATXN3 in SOD2 expression, knockdown of endogenous ATXN3 by RNA interference represses the expression of SOD2. These findings support that ATXN3 plays an important role in regulating the FOXO4-dependent antioxidant stress response via SOD2 and suggest that a decreased antioxidative capacity and increased susceptibility towards oxidative stress contributes to neuronal cell death in SCA3.

Inhibition of pro-survival Bcl-2 family proteins by BH3-only proteins is a key initial step leading to apoptotic cell death. In neurons, investigating cell death pathways is often hampered by the multi-factorial nature of the stress stimuli employed. Here we investigate the action of ABT-737, a small molecule inhibitor which specifically targets the BH3-protein binding domain of pro-survival Bcl-2, Bcl-X L and Bcl-w. ABT-737 produced a time- and concentration-dependent neuronal cell death which displayed the classical hallmarks of apoptosis. Cell death was maximal by around 4 h ABT-737 treatment, and the effect of ABT-737 could be delayed by the broad spectrum caspase inhibitor zVADfmk. Examining, using real-time confocal microscopy, the molecular basis for the onset of response demonstrated recruitment of pro-apoptotic Bax to specific mitochondrial foci, followed by mitochondrial fragmentation. Treatment of neurons with ABT-737 also produced cleavage of Bid, a BH3-only protein known to be a caspase substrate. Interestingly, cleaved Bid translocated to mitochondria but did not colocalise with Bax foci. zVADfmk inhibited Bid cleavage and slowed the rate of fragmentation, suggesting a role for cleaved Bid in the amplification of the apoptotic response. siRNA-mediated knockdown of Bax significantly inhibited ABT-737 induced cell death, whereas knockdown of the BH3-only proteins Bid or Bim had no effect. ABT-737 therefore appears to be a useful tool with which to examine neuronal apoptotic pathways. Our data suggests that caspase-dependent cleavage of Bid may be a downstream amplification event which enhances the rate of mitochondrial fragmentation.

International audience; In this paper we report a new method that permitted for the first time to selectively track a polysaccharide-based hydrogel on bone tissue explants, several weeks after its implantation. The hydrogel, which was developed for bone healing and tissue engineering, was labelled with a ruthenium complex and implanted into rabbit bone defects in order to investigate its in vivo degradation. 1, 2, 3 and 8 weeks after surgery, the bone explants were analyzed by synchrotron X-ray microfluorescence, infrared mapping spectroscopy, scanning electron microscopy, and optical microscopy after histological coloration. The results showed that the labelled polysaccharide-based hydrogel was likely to undergo phagocytosis that seemed to occur from the edge to the center of the implantation site up to at least the 8th week.

The mitochondrial chaperone mortalin was implicated in Parkinson's disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes.