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The pan ROCK inhibitor fasudil acts as a vasodilator and has been used as a medication post cerebral stroke for the past 27 years in Japan and China. More recently, on the basis of the involvement of ROCK inhibition on synaptic function, neuronal survival and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models.To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration we performed a global gene expression analysis on the brains of Alzheimer’s disease model mice treated with fasudil via peripheral i.p injection. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in postmortem neurodegenerative disease brains. The results are most striking in terms of pathway enrichment analysis where pathways regulated in Alzheimer’s disease and by fasudil treatment are overwhelmingly regulated in opposite directions.Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity.

Polymorphisms associated with BIN1 (bridging integrator 1) confer the second greatest risk for developing late-onset Alzheimer’s disease. The biological consequences of this genetic variation are not fully understood; however, BIN1 is a binding partner for tau. Tau is normally a highly soluble cytoplasmic protein, but in Alzheimer’s disease, tau is abnormally phosphorylated and accumulates at synapses to exert synaptotoxicity. The purpose of this study was to determine whether alterations in BIN1 and tau in Alzheimer’s disease promote the damaging redistribution of tau to synapses, as a mechanism by which BIN1 polymorphisms may increase the risk of developing Alzheimer’s disease. We show that BIN1 is lost from the cytoplasmic fraction of Alzheimer’s disease cortex, and this is accompanied by the progressive mislocalization of phosphorylated tau to synapses. We confirmed proline 216 in tau as critical for tau interaction with the BIN1-SH3 domain and showed that the phosphorylation of tau disrupts this binding, suggesting that tau phosphorylation in Alzheimer’s disease disrupts tau–BIN1 associations. Moreover, we show that BIN1 knockdown in rat primary neurons to mimic BIN1 loss in Alzheimer’s disease brain causes the damaging accumulation of phosphorylated tau at synapses and alterations in dendritic spine morphology. We also observed reduced release of tau from neurons upon BIN1 silencing, suggesting that BIN1 loss disrupts the function of extracellular tau. Together, these data indicate that polymorphisms associated with BIN1 that reduce BIN1 protein levels in the brain likely act synergistically with increased tau phosphorylation to increase the risk of Alzheimer’s disease by disrupting cytoplasmic tau–BIN1 interactions, promoting the damaging mis-sorting of phosphorylated tau to synapses to alter synapse structure and reducing the release of physiological forms of tau to disrupt tau function.

BackgroundPost-translational modifications of tau modify its interaction with binding partners and cause tau mislocalisation and altered tau function in Alzheimer’s disease (AD). The AD risk gene BIN1, is a binding partner for tau, however the mechanism by which BIN1 influences tau function is not fully understood. We hypothesised that BIN1 modulates AD risk by causing damaging tau mis-sorting to the synapse.MethodsTau and BIN1 levels, distribution and interactions were assessed in post-mortem control and AD brain and in primary neurons. In primary neurons, tau was further examined using structured illumination microscopy and immunoblotting following BIN1 knockdown, BIN1-tau interactions were examined using proximity ligation assays and tau release from neurons was measured by sensitive sandwich ELISA.ResultsProline 216 in tau was identified as critical for tau interaction with the BIN1-SH3 domain, and tau phosphorylation at serine/threonine residues disrupted this interaction. Subcellular fractionation showed that BIN1 is lost from the cytoplasm of AD brain and this correlated with the mislocalisation of phosphorylated tau to synapses. Mimicking BIN1 loss in AD by knockdown of the protein in primary neurons altered the structure of dendritic spines, caused phosphorylated tau to mis-sort to synapses and reduced the physiological release of predominantly dephosphorylated tau.ConclusionsThese data suggest that BIN1 loss in AD allows phosphorylated tau to be mis-sorted to synapses which likely alters the integrity of the post-synapse, alongside reducing the functionally important release of physiological forms of tau.

The muscarinic M$_{2}$ receptor is a prominent member of the GPCR family and strongly involved in heart diseases. Recently published experimental work explored the cellular response to iperoxo-induced M$_{2}$ receptor stimulation in Chinese hamster ovary (CHO) cells. To better understand these responses, we modelled and analysed the muscarinic M$_{2}$ receptor-dependent signalling pathway combined with relevant secondary messenger molecules using mass action. In our literature-based joint signalling and secondary messenger model, all binding and phosphorylation events are explicitly taken into account in order to enable subsequent stoichiometric matrix analysis. We propose constraint flux sampling (CFS) as a method to characterize the expected shift of the steady state reaction flux distribution due to the known amount of cAMP production and PDE4 activation. CFS correctly predicts an experimentally observable influence on the cytoskeleton structure (marked by actin and tubulin) and in consequence a change of the optical density of cells. In a second step, we use CFS to simulate the effect of knock-out experiments within our biological system, and thus to rank the influence of individual molecules on the observed change of the optical cell density. In particular, we confirm the relevance of the protein RGS14, which is supported by current literature. A combination of CFS with Elementary Flux Mode analysis enabled us to determine the possible underlying mechanism. Our analysis suggests that mathematical tools developed for metabolic network analysis can also be applied to mixed secondary messenger and signalling models. This could be very helpful to perform model checking with little effort and to generate hypotheses for further research if parameters are not known.

Introduction Synapse loss is the structural correlate of the cognitive decline indicative of dementia. In the brains of Alzheimer's disease sufferers, amyloid β (Aβ) peptides aggregate to form senile plaques but as soluble peptides are toxic to synapses. We previously demonstrated that Aβ induces Dickkopf-1 (Dkk1), which in turn activates the Wnt–planar cell polarity (Wnt-PCP) pathway to drive tau pathology and neuronal death. Methods We compared the effects of Aβ and of Dkk1 on synapse morphology and memory impairment while inhibiting or silencing key elements of the Wnt-PCP pathway. Results We demonstrate that Aβ synaptotoxicity is also Dkk1 and Wnt-PCP dependent, mediated by the arm of Wnt-PCP regulating actin cytoskeletal dynamics via Daam1, RhoA and ROCK, and can be blocked by the drug fasudil. Discussion Our data add to the importance of aberrant Wnt signaling in Alzheimer's disease neuropathology and indicate that fasudil could be repurposed as a treatment for the disease., Highlights • Aβ synaptotoxicity is Dickkopf-1 and Wnt-PCP dependent. • The Wnt-PCP pathway drives Aβ-driven synapse loss via RhoA and ROCK. • ROCK inhibitor fasudil blocks Aβ-driven synapse loss and cognitive impairment. • Fasudil should be assessed for repurposing for Alzheimer's disease.

DISC1 is a multifunctional, intracellular scaffold protein. At the cellular level, DISC1 plays a pivotal role in neural progenitor proliferation, migration, and synaptic maturation. Perturbation of the biological pathways involving DISC1 is known to lead to behavioral changes in rodents, which supports a clinical report of a Scottish pedigree in which the majority of family members with disruption of the DISC1 gene manifest depression, schizophrenia, and related mental conditions. The discrepancy between modest evidence in genetics and strong biological support for the role of DISC1 in mental conditions suggests a working hypothesis that regulation of DISC1 at the protein level, such as posttranslational modification, may play a role in the pathology of mental conditions. In this study, we report on the SUMOylation of DISC1. This posttranslational modification occurs on lysine residues where the small ubiquitin-related modifier (SUMO) and its homologs are conjugated to a large number of cellular proteins, which in turn regulates their subcellular distribution and protein stability. By using in silico, biochemical, and cell-biological approaches, we now demonstrate that human DISC1 is SUMOylated at one specific lysine 643 (K643). We also show that this residue is crucial for proper neural progenitor proliferation in the developing cortex.

The failure of remyelination in multiple sclerosis is largely unexplained. Lindner et al. report that glial cells in demyelinating lesions show increased expression of fibroblast growth factor 9 (FGF9). This induces astrocyte-dependent responses that inhibit remyelination and stimulate expression of pro-inflammatory chemokines, supporting a feedback loop that amplifies disease activity., The failure of remyelination in multiple sclerosis is largely unexplained. Lindner et al. report that glial cells in demyelinating lesions show increased expression of fibroblast growth factor 9 (FGF9). This induces astrocyte-dependent responses that inhibit remyelination and stimulate expression of pro-inflammatory chemokines, supporting a feedback loop that amplifies disease activity, Remyelination failure plays an important role in the pathophysiology of multiple sclerosis, but the underlying cellular and molecular mechanisms remain poorly understood. We now report actively demyelinating lesions in patients with multiple sclerosis are associated with increased glial expression of fibroblast growth factor 9 (FGF9), which we demonstrate inhibits myelination and remyelination in vitro. This inhibitory activity is associated with the appearance of multi-branched ‘pre-myelinating’ MBP+/PLP+ oligodendrocytes that interact with axons but fail to assemble myelin sheaths; an oligodendrocyte phenotype described previously in chronically demyelinated multiple sclerosis lesions. This inhibitory activity is not due to a direct effect of FGF9 on cells of the oligodendrocyte lineage but is mediated by factors secreted by astrocytes. Transcriptional profiling and functional validation studies demonstrate that these include effects dependent on increased expression of tissue inhibitor of metalloproteinase-sensitive proteases, enzymes more commonly associated with extracellular matrix remodelling. Further, we found that FGF9 induces expression of Ccl2 and Ccl7, two pro-inflammatory chemokines that contribute to recruitment of microglia and macrophages into multiple sclerosis lesions. These data indicate glial expression of FGF9 can initiate a complex astrocyte-dependent response that contributes to two distinct pathogenic pathways involved in the development of multiple sclerosis lesions. Namely, induction of a pro-inflammatory environment and failure of remyelination; a combination of effects predicted to exacerbate axonal injury and loss in patients.

Recent research findings have provided convincing evidence indicating a role for Interleukin-33 (IL-33) signalling pathway in a number of central nervous system (CNS) diseases including multiple sclerosis (MS) and Alzheimer’s disease. However, the exact function of IL-33 molecule within the CNS under normal and pathological conditions is currently unknown. In this study, we have mapped cellular expression of IL-33 and its receptor ST2 by immunohistochemistry in the brain tissues of MS patients and appropriate controls; and investigated the functional significance of these findings in vitro using a myelinating culture system. Our results demonstrate that IL-33 is expressed by neurons, astrocytes and microglia as well as oligodendrocytes, while ST2 is expressed in the lesions by oligodendrocytes and within and around axons. Furthermore, the expression levels and patterns of IL-33 and ST2 in the lesions of acute and chronic MS patient brain samples are enhanced compared with the healthy brain tissues. Finally, our data using rat myelinating co-cultures suggest that IL-33 may play an important role in MS development by inhibiting CNS myelination. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0344-1) contains supplementary material, which is available to authorized users.

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4BY358C mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and β-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4BY358C mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4BY358C mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4BY358C mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4BY358C mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Canavan disease (CD) is identified as a fatal spongiform leukodystrophy caused by missense mutations in the Aspa gene (1,2).

Canavan disease (CD) is a rare leukodystrophy characterized by diffuse spongiform white matter degeneration, dysmyelination and intramyelinic oedema with consequent impairment of psychomotor development and early death. The molecular cause of CD has been identified as being mutations of the gene encoding the enzyme aspartoacylase (ASPA) leading to its functional deficiency. The physiological role of ASPA is to hydrolyse N-acetyl-l-aspartic acid (NAA), producing l-aspartic acid and acetate; as a result, its deficiency leads to abnormally high central nervous system NAA levels. The aim of this article is to review what is currently known regarding the aetiopathogenesis and treatment of CD, with emphasis on the non-genetic therapeutic strategies, both at an experimental and a clinical level, by highlighting: (a) major related hypotheses, (b) the results of the available experimental simulatory approaches, as well as (c) the relevance of the so far examined markers of CD neuropathology. The potential and the limitations of the current non-genetic neuroprotective approaches to the treatment of CD are particularly discussed in the current article, in a context that could be used to direct future experimental and (eventually) clinical work in the field.

Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal 'head' domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1's C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1(D453G) mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1(D453G) mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1(D453G) mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans.

Inflammatory basal ganglia disorders are infectious and autoimmune conditions presenting with movement disorders and psychiatric symptoms. Autoimmune basal ganglia disorders (ABGA) include Sydenham's chorea and recently described basal ganglia encephalitis associated with autoantibody against dopamine D2 receptor. Although autoantibodies are implicated in the pathogenesis of ABGA, the presence of seronegative cases suggests their etiological diversity. To elucidate the immune mechanisms of pediatric ABGA, we retrospectively analyzed clinical and neuroradiological data of patients with acute-onset inflammatory basal ganglia disorders and studied immunological markers in sera and CSF. Fourteen patients (11 males) aged between 8 months to 13 years were enrolled. Initial presentation was preceded by streptococcal infection in 5 cases. Clinical manifestations included chorea (n = 7), dystonia (n = 6), oral dyskinesia (n = 5), psychiatric symptoms (n = 7), and seizures (n = 9). Eleven showed magnetic resonance image signal abnormalities in basal ganglia. Moderate cerebrospinal fluid (CSF) pleocytosis (more than 50 cells per microliter) was found in 4 patients. Cytokines (interferon gamma, interleukin (IL)-6, IL-10, IL-12p70, tumor necrosis factor alpha) and chemokines (CCL2, CCL4, CXCL8, CXCL9, CXCL10) were measured in sera and CSF obtained from 9 patients. They were divided into two groups according to the presence of CSF pleocytosis: pleocytosis-positive (P group, n = 4) and -negative (N group, n = 5). Serum CCL4 level was significantly higher in N group than in P group (P b 0.05) and CSF IL-6 was significantly higher in P group than in N group (P b 0.05). CSF to serum ratio of CXCL9 and CXCL10 were significantly higher in P group than in N group (P b 0.05). Antineuronal antibodies tested by immunocytochemistry using rat primary neuronal culture detected antibodies against dendrite (n = 5), soma (n = 3), and nuclei (n = 5), with similar frequency between two groups. These results suggest that intracerebral inflammation generates chemokine gradient to recruit leukocytes into the brain in P group, whereas inflammation predominantly occurs systemically in N group.

OBJECTIVE: We aimed to examine the regulation of lipocalin-2 (LCN2) in multiple sclerosis (MS) and its potential functional relevance with regard to myelination and neurodegeneration.METHODS: We determined LCN2 levels in 3 different studies: (1) in CSF and plasma from a case-control study comparing patients with MS (n = 147) with controls (n = 50) and patients with relapsing-remitting MS (n = 75) with patients with progressive MS (n = 72); (2) in CSF and brain tissue microdialysates from a case series of 7 patients with progressive MS; and (3) in CSF at baseline and 60 weeks after natalizumab treatment in a cohort study of 17 patients with progressive MS. Correlation to neurofilament light, a marker of neuroaxonal injury, was tested. The effect of LCN2 on myelination and neurodegeneration was studied in a rat in vitro neuroglial cell coculture model.RESULTS: Intrathecal production of LCN2 was increased predominantly in patients with progressive MS (p < 0.005 vs relapsing-remitting MS) and displayed a positive correlation to neurofilament light (p = 0.005). Levels of LCN2 in brain microdialysates were severalfold higher than in the CSF, suggesting local production in progressive MS. Treatment with natalizumab in progressive MS reduced LCN2 levels an average of 13% (p < 0.0001). LCN2 was found to inhibit remyelination in a dose-dependent manner in vitro.CONCLUSIONS: LCN2 production is predominantly increased in progressive MS. Although this moderate increase does not support the use of LCN2 as a biomarker, the correlation to neurofilament light and the inhibitory effect on remyelination suggest that LCN2 might contribute to neurodegeneration through myelination-dependent pathways.

We report a non-destructive in-line monitoring method developed for Cd diffusion into InP on SACM-APD structure. Photocurrent vs voltage measurement are taken directly via proving diffused diodes on a wafer. We demonstrate that there is linear correlation between punch-through voltages Vpt on the photo I-V curves and diffusion depth measured by SIMS and Polaron profiles. It has been established that Vpt can be extracted easily from I-V curves and used for re-diffusion to approach target depth.

Objective The objective of the study was to determine whether the designation of a procedure as newer leads more patients to choose that procedure over 1 that is designated as older. Study Design Women with stress incontinence read two 1-page descriptions of surgical procedures for treatment of stress incontinence and were asked to indicate which of the 2 surgical procedures they would choose whether they were going to choose surgical treatment. Randomly for half the participants, a rectus fascia sling was described as being a newer procedure and a mesh sling as an older procedure. For the other half of participants, a mesh sling was described as older and a fascia sling as newer. All participants were also asked whether, in general, they considered that newer surgical procedures were better than older surgical procedures and why. Results Forty-eight women of mean age 57 years (range, 33-82) were interviewed. Thirty-two patients (68%) chose the newer procedure, and 35 (74%) chose the fascia procedure, both percentages higher than would be expected by chance. When fascia was presented as being the newer procedure, it was chosen over the older mesh by 22 of 24 patients (92%), whereas when fascia was presented as being the older procedure, it was chosen over the newer mesh by a smaller margin. The great majority of patients (79%) stated that newer procedures are better in general. Conclusions Our results suggest that the use of the words newer or older may overshadow other important information that physicians intend to convey during surgical counseling.

Highlights • PDE4D7 is phosphorylated by PKA in the unique N-terminal region at serine 42. • PDE4D7 phosphorylation at serine 42 negatively regulates PDE4 activity. • Ablation of phosphorylation at serine 42 activates PDE4D7 and this reduces phosphorylation in the UCR1 domain., The cyclic AMP phosphodiesterases type 4 (PDE4s) are expressed in a cell specific manner, with intracellular targeting directed by unique N-terminal anchor domains. All long form PDE4s are phosphorylated and activated by PKA phosphorylation within their upstream conserved region 1 (UCR1). Here, we identify and characterise a novel PKA site (serine 42) within the N-terminal region of PDE4D7, an isoform whose activity is known to be important in prostate cancer progression and ischemic stroke. In contrast to the UCR1 site, PKA phosphorylation of the PDE4D7 N-terminus appears to occur constitutively and inhibits PDE4 activity to allow cAMP signalling under basal conditions.