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Optical brain clearing combined with immunolabeling is valuable for analyzing molecular tissue structures, including complex synaptic connectivity. However, the presence of aberrant lipid deposition due to aging and brain disorders poses a challenge for achieving antibody penetration throughout the entire brain volume. Herein, we present an efficient brain-wide immunolabeling method, the immuno-active clearing technique (iACT). The treatment of brain tissues with a zwitterionic detergent, specifically SB3-12, significantly enhanced tissue permeability by effectively mitigating lipid barriers. Notably, Quadrol treatment further refines the methodology by effectively eliminating residual detergents from cleared brain tissues, subsequently amplifying volumetric fluorescence signals. Employing iACT, we uncover disrupted axonal projections within the mesolimbic dopaminergic (DA) circuits in 5xFAD mice. Subsequent characterization of DA neural circuits in 5xFAD mice revealed proximal axonal swelling and misrouting of distal axonal compartments in proximity to amyloid-beta plaques. Importantly, these structural anomalies in DA axons correlate with a marked reduction in DA release within the nucleus accumbens. Collectively, our findings highlight the efficacy of optical volumetric imaging with iACT in resolving intricate structural alterations in deep brain neural circuits. Furthermore, we unveil the compromised integrity of DA pathways, contributing to the underlying neuropathology of Alzheimer's disease. The iACT technique thus holds significant promise as a valuable asset for advancing our understanding of complex neurodegenerative disorders and may pave the way for targeted therapeutic interventions., (© 2023. The Author(s).)

The use of time-lapse live imaging enables us to track the dynamic changes in neurites during their formation. Ex vivo live imaging with acute brain slices provides a more physiological environment than cultured cells. To accomplish this, a certain method of labeling is necessary to visualize and identify neurite morphology. To understand the dynamics of neurite structure at early stages of neurite formation, we describe in this chapter ex vivo live imaging using a confocal microscope at P0 in combination with in utero electroporation (IUE)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mammalian TRPC5 channels are predominantly expressed in the brain, where they increase intracellular Ca 2+ and induce depolarization. Because they augment presynaptic vesicle release, cause persistent neural activity, and show constitutive activity, TRPC5s could play a functional role in late developmental brain events. We used immunohistochemistry to examine TRPC5 in the chick embryo brain between 8 and 20 days of incubation, and provide the first detailed description of their distribution in birds and in the whole brain of any animal species. Stained areas substantially increased between E8 and E16, and staining intensity in many areas peaked at E16, a time when chick brains first show organized patterns of whole-brain metabolic activation like what is seen consistently after hatching. Areas showing cell soma staining match areas showing Trpc5 mRNA or protein in adult rodents (cerebral cortex, hippocampus, amygdala, cerebellar Purkinje cells). Chick embryos show protein staining in the optic tectum, cerebellar nuclei, and several brainstem nuclei; equivalent areas in the Allen Institute mouse maps express Trpc5 mRNA. The strongest cell soma staining was found in a dorsal hypothalamic area (matching a group of parvicellular arginine vasotocin neurons and a pallial amygdalohypothalamic cell corridor) and the vagal motor complex. Purkinje cells showed strong dendritic staining at E20. Unexpectedly, we also describe neurite staining in the septum, several hypothalamic nuclei, and a paramedian raphe area; the strongest neurite staining was in the median eminence. These novel localizations suggest new unexplored TRPC5 functions, and possible roles in late embryonic brain development., (© 2021 Wiley Periodicals LLC.)

Loss-of-function mutations in the human vacuolar protein sorting the 13 homolog A (VPS13A) gene cause Chorea-acanthocytosis (ChAc), with selective degeneration of the striatum as the main neuropathologic feature. Very little is known about the VPS13A expression in the brain. The main objective of this work was to assess, for the first time, the spatiotemporal distribution of VPS13A in the mouse brain. We found VPS13A expression present in neurons already in the embryonic stage, with stable levels until adulthood. VPS13A mRNA and protein distributions were similar in the adult mouse brain. We found a widespread VPS13A distribution, with the strongest expression profiles in the pons, hippocampus, and cerebellum. Interestingly, expression was weak in the basal ganglia. VPS13A staining was positive in glutamatergic, GABAergic, and cholinergic neurons, but rarely in glial cells. At the cellular level, VPS13A was mainly located in the soma and neurites, co-localizing with both the endoplasmic reticulum and mitochondria. However, it was not enriched in dendritic spines or the synaptosomal fraction of cortical neurons. In vivo pharmacological modulation of the glutamatergic, dopaminergic or cholinergic systems did not modulate VPS13A concentration in the hippocampus, cerebral cortex, or striatum. These results indicate that VPS13A has remarkable stability in neuronal cells. Understanding the distinct expression pattern of VPS13A can provide relevant information to unravel pathophysiological hallmarks of ChAc.

The sigma 1 receptor (S1R) is an enigmatic ligand-operated chaperone involved in many important biological processes, and its functions are not fully understood yet. Herein, we developed a novel series of bitopic S1R ligands as versatile tools to investigate binding processes, allosteric modulation, and the oligomerization mechanism. These molecules have been prepared in the enantiopure form and subjected to a preliminary biological evaluation, while in silico investigations helped to rationalize the results. Compound 7 emerged as the first bitopic S1R ligand endowed with low nanomolar affinity ( K i = 2.6 nM) reported thus far. Computational analyses suggested that 7 may stabilize the open conformation of the S1R by simultaneously binding the occluded primary binding site and a peripheral site on the cytosol-exposed surface. These findings pave the way to new S1R ligands with enhanced activity and/or selectivity, which could also be used as probes for the identification of a potential allosteric site.

Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Neurogenesis and functional brain activity require complex associations of inherently programmed secretory elements that are regulated precisely and temporally. Family with sequence similarity 19 A1 (FAM19A1) is a secreted protein primarily expressed in subsets of terminally differentiated neuronal precursor cells and fully mature neurons in specific brain substructures. Several recent studies have demonstrated the importance of FAM19A1 in brain physiology; however, additional information is needed to support its role in neuronal maturation and function. In this study, dendritic spine morphology in Fam19a1 -ablated mice and neurite development during in vitro neurogenesis were examined to understand the putative role of FAM19A1 in neural integrity. Adult Fam19a1 -deficient mice showed low dendritic spine density and maturity with reduced dendrite complexity compared to wild-type (WT) littermates. To further explore the effect of FAM19A1 on neuronal maturation, the neurite outgrowth pattern in primary neurons was analyzed in vitro with and without FAM19A1. In response to FAM19A1, WT primary neurons showed reduced neurite complexity, whereas Fam19a1 -decifient primary neurons exhibited increased neurite arborization, which was reversed by supplementation with recombinant FAM19A1. Together, these findings suggest that FAM19A1 participates in dendritic spine development and neurite arborization.

Environmental risk factors that operate at foetal or neonatal levels increase the vulnerability to schizophrenia, plausibly via stress-immune activation that perturbs the epidermal growth factor (EGF) system, a system critical for neurodevelopment. We investigated potential associations between environmental insults and immune and EGF system changes through a maternal immune activation (MIA) model, using the precocial spiny mice (Acomys cahirinus). After mid-gestation MIA prepubescent offspring showed elevated NF-κB1 protein in nucleus accumbens, decreased EGFR in caudate putamen and a trend for increased PI3K-110δ in ventral hippocampus. Thus, prenatal stress may cause a heightened NF-κB1-mediated immune attenuation of EGF system signalling., (Copyright © 2020. Published by Elsevier B.V.)

The nonreceptor protein tyrosine kinase Fyn and protein Ser/Thr phosphatase 2A (PP2A) are major multifunctional signaling molecules. Deregulation of Fyn and altered PP2A methylation are implicated in cancer and Alzheimer's disease (AD). Here, we tested the hypothesis that the methylation state of PP2A catalytic subunit, which influences PP2A subunit composition and substrate specificity, can affect Fyn regulation and function. Using Neuro-2a (N2a) neuroblastoma cell models, we first show that methylated PP2A holoenzymes containing the Bα subunit coimmunoprecipitate and copurify with Fyn in membrane rafts. PP2A methylation status regulates Fyn distribution and Fyn-dependent neuritogenesis, likely in part by affecting actin dynamics. A methylation-incompetent PP2A mutant fails to interact with Fyn. It perturbs the normal partitioning of Fyn and amyloid precursor protein (APP) in membrane microdomains, which governs Fyn function and APP processing. This correlates with enhanced amyloidogenic cleavage of APP, a hallmark of AD pathogenesis. Conversely, enhanced PP2A methylation promotes the nonamyloidogenic cleavage of APP in a Fyn-dependent manner. Disturbances in one-carbon metabolic pathways that control cellular methylation are associated with AD and cancer. Notably, they induce a parallel loss of membrane-associated methylated PP2A and Fyn enzymes in N2a cells and acute mouse brain slices. One-carbon metabolism also modulates Fyn-dependent process outgrowth in N2a cells. Thus, our findings identify a novel methylation-dependent PP2A/Fyn signaling module. They highlight the underestimated importance of cross talks between essential metabolic pathways and signaling scaffolds that are involved in normal cell homeostasis and currently being targeted for cancer and AD treatment., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Takenouchi-Kosaki syndrome (TKS) is an autosomal dominant congenital syndrome, of which pathogenesis is not well understood. Recently, a heterozygous mutation c.1449T > C/p.(Tyr64Cys) in the CDC42 gene, encoding a Rho family small GTPase, has been demonstrated to contribute to the TKS clinical features, including developmental delay with intellectual disability (ID). However, specific molecular mechanisms underlying the neuronal pathophysiology of TKS remain largely unknown. In this study, biochemical analyses revealed that the mutation moderately activates Cdc42. In utero electroporation-based acute expression of Cdc42-Y64C in ventricular zone progenitor cells in embryonic mice cerebral cortex resulted in migration defects and cluster formation of excitatory neurons. Expression the mutant in primary cultured hippocampal neurons caused impaired axon elongation. These data suggest that the c.1449T > C/p.(Tyr64Cys) mutation causes altered CDC42 function and results in defects in neuronal morphology and migration during brain development, which is likely to be responsible for pathophysiology of psychomotor delay and ID in TKS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Abnormal phosphorylation of microtubule-associated proteins such as tau has been shown to play a role in neurodegenerative disorders. It is hypothesized that oxidative stress-induced aggregates of hyperphosphorylated tau could lead to the microtubule network degradation commonly associated with neurodegeneration. We investigated whether oxidative stress induced tau hyperphosphorylation and focused on neurite degradation using cultured neurons isolated from the embryonic chick brain as a model system. Cells were isolated from the cerebrum, cerebellum, and tectum of 14-day-old chicks, grown separately in culture, and treated with tert-Butyl hydroperoxide (to simulate oxidative stress) for 48 hr. Relative expression and localization of tau or phospho-tau and β-tubulin III in neurites were determined using quantitative immunocytochemistry and confocal microscopy. In untreated cells, tau was tightly colocalized with β-tubulin III. Increasing levels of oxidative stress induced an increase in overall tau expression in neurites of cerebral and tectal but not the cerebellar neurons, coupled with a decrease in phospho-tau expression in tectal but not the cerebral or cerebellar neurons. In addition, oxidative stress induced the degeneration of the distal ends of the neurites and redistribution of phospho-tau toward the neuronal soma in the cerebral but not the tectal and cerebellar neurons. These results suggest that oxidative stress induces changes in tau protein that precede cytoskeletal degradation and neurite retraction. Additionally, there is a differential susceptibility of neuronal subpopulations to oxidative stress, which may offer potential avenues for investigation of the cellular mechanisms underlying the differential manifestations of neurodegenerative disorders in different regions of the brain., (© 2019 Wiley Periodicals, Inc.)

The transforming growth factor β (TGF-β) signaling pathway is evolutionarily conserved and widely used in the animal kingdom to regulate diverse developmental processes. Prior studies have shown that Baboon (Babo), a Drosophila type I TGF-β receptor, plays essential roles in brain development and neural circuit formation. However, the expression pattern for Babo in the developing brain has not been previously reported. We generated a knock-in fly with a human influenza hemagglutinin (HA) tag at the C-terminus of Babo and assessed its localization. Babo::HA was primarily expressed in brain structures enriched with neurites, including the mushroom body lobe and neuropils of the optic lobe, where Babo has been shown to instruct neuronal morphogenesis. Since the babo 3' untranslated region contains a predicted microRNA-34 (miR-34) target sequence, we further tested whether Babo::HA expression was affected by modulating the level of miR-34. We found that Babo was upregulated by mir-34 deletion and downregulated by miR-34 overexpression, confirming that it is indeed a miR-34 target gene. Taken together, our results demonstrate that the babo HA fly permits accurate visualization of endogenous Babo expression during brain development and the construction of functional neural circuits.

Memory deficits affect a large proportion of the human population and are associated with aging and many neurologic, neurodegenerative, and psychiatric diseases. Treatment of this mental disorder has been disappointing because all potential candidates studied thus far have failed to produce consistent effects across various types of memory and have shown limited to no effects on memory deficits. Here, we show that the promotion of neuronal arborization through the expression of the regulator of G-protein signaling 14 of 414 amino acids (RGS14 414 ) not only induced robust enhancement of multiple types of memory but was also sufficient for the recovery of recognition, spatial, and temporal memory, which are kinds of episodic memory that are primarily affected in patients or individuals with memory dysfunction. We observed that a surge in neuronal arborization was mediated by up-regulation of brain-derived neurotrophic factor (BDNF) signaling and that the deletion of BDNF abrogated both neuronal arborization activation and memory enhancement. The activation of BDNF-dependent neuronal arborization generated almost 2-fold increases in synapse numbers in dendrites of pyramidal neurons and in neurites of nonpyramidal neurons. This increase in synaptic connections might have evoked reorganization within neuronal circuits and eventually supported an increase in the activity of such circuits. Thus, in addition to showing the potential of RGS14 414 for rescuing memory deficits, our results suggest that a boost in circuit activity could facilitate memory enhancement and the reversal of memory deficits.-Masmudi-Martín, M., Navarro-Lobato, I., López-Aranda, M. F., Delgado, G., Martín-Montañez, E., Quiros-Ortega, M. E., Carretero-Rey, M., Narváez, L., Garcia-Garrido, M. F., Posadas, S., López-Téllez, J. F., Blanco, E., Jiménez-Recuerda, I., Granados-Durán, P., Paez-Rueda, J., López, J. C., Khan, Z. U. RGS14 414 treatment induces memory enhancement and rescues episodic memory deficits.

The neuronal pathways of the circadian clock in the brain of R. prolixus have been described in detail previously, but there is no information concerning the cells or their pathways which relay either inputs to the clock (e.g. for light entrainment), or outputs from it to driven rhythms. Here, we employ antisera to three neuropeptides (type A allatostatin-7, crustacean cardioactive peptide and FMRFamide), and serotonin in confocal laser scanning immunohistochemistry to analyze the distribution of cell bodies and their projections in relation to the principle circadian clock cells (lateral cells, LNs) for all four neuron types. LNs are revealed following labelling with anti- pigment dispersing factor in double labelled preparations. Regions of potential communication between ramifications of the LNs and each of the four other neuron types is described (identified by close superposition of their neurites in various brain regions), as is their detailed projections within the brain. Neuromodulation is sometimes suggested by close, but not intimate, proximity of varicosities of neurites. We infer that some neuron types comprise input pathways to the LNs, some are outputs to neuroendocrine or behavioral rhythms, and others participate in both input and output pathways, sometimes by the same neuron type but in different locations. For example, one retinula cell in each ommatidium is immunoreactive for allatostatin A; its axon projects to the medulla making superpositions with LNs, as do serotonin cells in the optic lobe, indicating roles of both neuron types in light input (entrainment) to the clock. But in other brain areas, these same types appear to mediate outputs from the clock. The accessory medulla has been widely reported as the principle center of integration in other insects; but we found sparse evidence of this in R. prolixus as it contains few neurites other than those from the clock cells. Rather, the importance of neural pathways involving the medulla and the superior protocerebrum is emphasized. We conclude that there is a vast and complex web of interactions in the brain with the LNs, which potentially receive multiple pathways of inputs and outputs that could drive rhythmicity in a multitude of downstream cells, rendering a host of output pathways rhythmic, notably hormone release from neurosecretory cells and behaviors., (Copyright © 2018. Published by Elsevier Inc.)

Purpose: Neurite orientation dispersion and density imaging (NODDI) is a clinically feasible approach to measure intra-neurite volume fraction (f in ). However, the sophisticated fitting procedure takes several hours. And the NODDI model relied on several questionable assumptions. Recent analytical work demonstrated that f in could be simply calculated from the spherical mean signal (MEANS) averaged over all gradient directions with a more solid theoretical foundation. The current study aims to compare NODDI and MEANS for measuring f in in human brain and investigate the potential of MEANS as a fast approach in clinics., Methods: NODDI f in and MEANS f in were measured and compared on the same dataset. NODDI f in was obtained using the NODDI MATLAB Toolbox. MEANS f in is the product of the spherical mean signal and 2bD/π, where D is the intra-neurite intrinsic diffusivity., Results: NODDI f in and MEANS f in maps are similar. The voxel-by-voxel correlation suggests that NODDI f in and MEANS f in are approximately equivalent to each other., Conclusion: MEANS may have potential to serve a fast and simple approach to estimate f in in clinics., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Radiation induces DNA and protein damage and free radical formation, effectively establishing cellular senescence in a variety of models. We demonstrate the effects of two known pleiotropic drugs following gamma radiation damage in neurosphere/cerebral organoid system based on human embryonic stem cells. mTORC1 repression by rapamycin prior to irradiation, or metabolic activation by minocycline after irradiation, partially rescues neuroepithelium integrity, neurite-growing capacity, ventricle formation and extracellular acidification rate as an integral measure of metabolic output. Cerebral organoid model thus provides valid and robust readouts for radiation studies in a complex 3D setting.

EphA5 and its ligand ephrin-A5 interaction can trigger synaptogenesis during early hippocampus development. We have previously reported that abnormal EphA5 expression can result in synaptogenesis disorder in congenital hypothyroidism (CH) rats. To better understand its precise molecular mechanism, we further analyzed the characteristics of ephrin-A5 expression in the hippocampus of CH rats. Our study revealed that ephrin-A5 expression was downregulated by thyroid hormone deficiency in the developing hippocampus and hippocampal neurons in rats. Thyroxine treatment for hypothyroid hippocampus and triiodothyronine treatment for hypothyroid hippocampal neurons significantly improved ephrin-A5 expression but could not restore its expression to control levels. Hypothyroid hippocampal neurons in-vitro showed synaptogenesis disorder characterized by a reduction in the number and length of neurites. Furthermore, the synaptogenesis-associated molecular expressions of NMDAR-1 (NR1), PSD95 and CaMKII were all downregulated correspondingly. These results suggest that ephrin-A5 expression may be decreased in CH, and abnormal activation of ephrin-A5/EphA5 signaling affects synaptogenesis during brain development. Such findings provide an important basis for exploring the pathogenesis of CH genetically.

A population of descending neurons connect the brain and thoracic motor center, playing a critical role in controlling behavior. We examined the anatomical organization of descending neurons (DNs) in the brain of the silkmoth Bombyx mori. Moth pheromone orientation is a good model to investigate neuronal mechanisms of behavior. Based on mass staining and single-cell staining, we evaluated the anatomical organization of neurite distribution by DNs in the brain. Dense innervation was observed in the posterior-ventral part of the brain called the posterior slope (PS). We analyzed the morphology of DNs innervating the lateral accessory lobe (LAL), which is considered important for moth olfactory behavior. We observed that all LAL DNs also innervate the PS, suggesting the integration of signals from the LAL and PS. We also identified a set of DNs innervating the PS but not the LAL. These DNs were sensitive to the sex pheromone, suggesting a role of the PS in motor control for pheromone processing. Here we discuss the organization of descending pathways for pheromone orientation.

This etiologic model proposes that Alzheimers Disease (AD) arises when an unusually rapid increase in ventricle volume triggers axon stretch that culminates in the physical separation of trans-synaptic proteins. As a result, these proteins, such as neurexin, neuroligin, N-Cadherin and Amyloid Precursor Protein (APP), experience a change in the configuration of their cytoplasmic tail, so that instead of transmitting signals to create and maintain synaptic structure they activate enzymes, and generate molecules, that stimulate neurite growth; for example, the transformation of the N-Cadherin tail dissolves its complex with presenilin and β-catenin triggering activation of glycogen synthase kinase 3 beta (GSK3β) and cytoskeletal disruption. This disruption leads to an increase in pro nerve growth factor (proNGF), a molecule that stimulates neurite growth via the p75 neurotrophin receptor (p75). GSK3β contributes to this growth by increasing microtubule instability through the phosphorylation of tau. Separation of trans-synaptic APPs leads to their cis dimerization and this stimulates production of amyloid beta (Aβ), an autocrine growth factor that interacts with both the p75 and alpha 7 nicotinic acetylcholine receptors. Cis dimerization of APPs may also allow the autophosphorylation of Y682 and T668 in the APP cytoplasmic tail, triggering activation of c-Jun N terminal kinase, Abelson kinase and cyclin dependent kinase 5, all of which play a role in neurite growth. ProNGF, Aβ and the kinase cascades work together to transform synapses into growth cones and stimulate sprouting of neuropil threads in an attempt to reconnect axons and dendrites. Neurofibrillary tangles, located in neural cell soma, consist of neurofilaments and microtubules needed to fuel this renewal of neurite growth. The model suggests that the best way to treat AD is to prevent synaptic separation by identifying individuals experiencing unusually high rates of ventricle growth and reducing this to more normal levels by shunting or some other technique., (Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.)

Frontotemporal Lobar Degeneration (FTLD) encompasses certain related neurodegenerative disorders which alter personality and cognition. Heterogeneous ribonuclear proteins (hnRNPs) maintain RNA metabolism and changes in their function may underpin the pathogenesis of FTLD. Immunostaining for hnRNP E2 was performed on sections of frontal and temporal cortex with hippocampus from 80 patients with FTLD, stratified by pathology into FTLD-tau and FTLD-TDP type A, B and C subtypes, and by genetics into patients with C9orf72 expansions, MAPT or GRN mutations, or those with no known mutation, and on 10 healthy controls. Semi-quantitative analysis assessed hnRNP staining in frontal and temporal cortex, and in dentate gyrus (DG) of hippocampus, in the different pathology and genetic groups. We find that hnRNP E2 immunostaining detects the TDP-43 positive dystrophic neurites (DN) within frontal and temporal cortex, and the neuronal cytoplasmic inclusions (NCI) seen in DG granule cells, characteristic of patients with Semantic Dementia (SD) and type C TDP-43 pathology, but did not detect TDP-43 or tau inclusions in any of the other pathological or genetic variants of FTLD. Double immunofluorescence for hnRNP E2 and TDP-43 showed most TDP-43 immunopositive DN to contain hnRNP E2. Present findings indicate an association between TDP-43 and hnRNP E2 which might underlie the pathogenetic mechanism of this form of FTLD.

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ 5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of G i/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated G i/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα 13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα 13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα 13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα 13 ·R7-RGS complexes. Because Gα 13 /R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ 5 with or without R7BP. We found that neurite retraction evoked by Gα 12/13 -dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα 12/13 but not Gα i/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα 13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rapidly progressive Alzheimer's disease (rpAD) is a particularly aggressive form of Alzheimer's disease, with a median survival time of 7-10 months after diagnosis. Why these patients have such a rapid progression of Alzheimer's disease is currently unknown. To further understand pathological differences between rpAD and typical sporadic Alzheimer's disease (sAD) we used localized proteomics to analyze the protein differences in amyloid plaques in rpAD and sAD. Label-free quantitative LC-MS/MS was performed on amyloid plaques microdissected from rpAD and sAD patients (n = 22 for each patient group) and protein expression differences were quantified. On average, 913 ± 30 (mean ± SEM) proteins were quantified in plaques from each patient and 279 of these proteins were consistently found in plaques from every patient. We found significant differences in protein composition between rpAD and sAD plaques. We found that rpAD plaques contained significantly higher levels of neuronal proteins (p = 0.0017) and significantly lower levels of astrocytic proteins (p = 1.08 × 10 -6 ). Unexpectedly, cumulative protein differences in rpAD plaques did not suggest accelerated typical sAD. Plaques from patients with rpAD were particularly abundant in synaptic proteins, especially those involved in synaptic vesicle release, highlighting the potential importance of synaptic dysfunction in the accelerated development of plaque pathology in rpAD. Combined, our data provide new direct evidence that amyloid plaques do not all have the same protein composition and that the proteomic differences in plaques could provide important insight into the factors that contribute to plaque development. The cumulative protein differences in rpAD plaques suggest rpAD may be a novel subtype of Alzheimer's disease.

The development of neurons and vessels shares striking anatomical and molecular features, and it is presumably orchestrated by an overlapping repertoire of extracellular signals. CNS macrophages have been implicated in various developmental functions, including the morphogenesis of neurons and vessels. However, whether CNS macrophages can coordinately influence neurovascular development and the identity of the signals involved therein is unclear. Here, we demonstrate that activity of the cell surface receptor CD95 regulates neuronal and vascular morphogenesis in the post-natal brain and retina. Furthermore, we identify CNS macrophages as the main source of CD95L, and macrophage-specific deletion thereof reduces both neurovascular complexity and synaptic activity in the brain. CD95L-induced neuronal and vascular growth is mediated through src-family kinase (SFK) and PI3K signaling. Together, our study highlights a coordinated neurovascular development instructed by CNS macrophage-derived CD95L, and it underlines the importance of macrophages for the establishment of the neurovascular network during CNS development., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Familial British dementia (FBD) and familial Danish dementia (FDD) are caused by mutations in the BRI 2 gene. These diseases are characterized clinically by progressive dementia and ataxia and neuropathologically by amyloid deposits and neurofibrillary tangles. Herein, we investigate BRI 2 protein accumulation in FBD, FDD, Alzheimer disease and Gerstmann-Sträussler-Scheinker disease. In FBD and FDD, we observed reduced processing of the mutant BRI 2 pro-protein, which was found accumulating intracellularly in the Golgi of neurons and glial cells. In addition, we observed an accumulation of a mature form of BRI 2 protein in dystrophic neurites, surrounding amyloid cores. Accumulation of BRI 2 was also observed in dystrophic neurites of Alzheimer disease and Gerstmann-Sträussler-Scheinker disease cases. Although it remains to be determined whether intracellular accumulation of BRI 2 may lead to cell damage in these degenerative diseases, our study provides new insights into the role of mutant BRI 2 in the pathogenesis of FBD and FDD and implicates BRI 2 as a potential indicator of neuritic damage in diseases characterized by cerebral amyloid deposition., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is characterized by slow, progressive degeneration of dopaminergic neurons in the substantia nigra. The cause of neuronal death in PD is largely unknown, but several genetic loci, including leucine-rich repeat kinase 2 (LRRK2), have been identified. LRRK2 has guanosine triphosphatase (GTPase) and kinase activities, and mutations in LRRK2 are the major cause of autosomal-dominant familial PD. Histone deacetylases (HDACs) remove acetyl groups from lysine residues on histone tails, promoting transcriptional repression via condensation of chromatin. Here, we demonstrate that LRRK2 binds to and directly phosphorylates HDAC3 at Ser-424, thereby stimulating HDAC activity. Specifically, LRRK2 promoted the deacetylation of Lys-5 and Lys-12 on histone H4, causing repression of gene transcription. Moreover, LRRK2 stimulated nuclear translocation of HDAC3 via the phoshorylation of karyopherin subunit α2 and α6. HDAC3 phosphorylation and its nuclear translocation were increased in response to 6-hydroxydopamine (6-OHDA) treatment. LRRK2 also inhibited myocyte-specific enhancer factor 2D activity, which is required for neuronal survival. LRRK2 ultimately promoted 6-OHDA-induced cell death via positive modulation of HDAC3. These findings suggest that LRRK2 affects epigenetic histone modification and neuronal survival by facilitating HDAC3 activity and regulating its localization., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein-induced neuropathology. In mice expressing a human α-synucleinopathy-associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Its pathological hallmarks are senile plaques (SPs), which contain extracellular deposits of amyloid β (Aβ) protein fibrils and dystrophic neurites (DNs), and neurofibrillary tangles (NFTs) containing hyperphosphorylated tau. Impairment of protein-degradation systems, including the ubiquitin-proteasome and the autophagy-lysosome systems, has been proposed as one of the causes of the accumulation of these aberrant proteins in AD brains. Tom1 (target of Myb1) was originally identified by the induction of its expression by the v-Myb oncogene and is a part of two major protein-degradation systems. The present study was conducted by immunohistochemical and immunofluorescent stainings to show that Tom1 was localized in DNs, perisomatic granules (PSGs), and NFTs in AD brains. Moreover, in DNs, Tom1 colocalized with ubiquitin, lysosomal proteins, and Tom1-related proteins (Tollip and myosin VI), which act in both protein-degradation systems via Tom1. These results indicate that Tom1 plays important roles in protein-degradation systems in AD pathogenesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Alterations in genes that regulate neurodevelopment can lead to cortical malformations, resulting in malfunction during postnatal life. The NF-κB pathway has a key role during neurodevelopment by regulating the maintenance of the neural progenitor cell pool and inhibiting neuronal differentiation. In this study, we evaluated whether mice lacking the NF-κB p50 subunit (KO) present alterations in cortical structure and associated behavioral impairment. We found that, compared with wild type (WT), KO mice at postnatal day 2 present an increase in radial glial cells, an increase in Reelin protein expression levels, in addition to an increase of specific layer thickness. Moreover, adult KO mice display abnormal columnar organization in the somatosensory cortex, a specific decrease in somatostatin- and parvalbumin-expressing interneurons, altered neurite orientation, and a decrease in Synapsin I protein levels. Concerning behavior, KO mice, in addition to an increase in locomotor and exploratory activity, display impairment in social behaviors, with a reduction in social interaction. Finally, we found that risperidone treatment decreased hyperactivity of KO mice, but had no effect on defective social interaction. Altogether, these data add complexity to a growing body of data, suggesting a link between dysregulation of the NF-κB pathway and neurodevelopmental disorders pathogenesis., (© The Author 2016. Published by Oxford University Press.)

Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22-C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypothesis, we found that only LCBs, the substrates common for all of the CerS isoforms, but not ceramides and complex sphingolipids, were restored to the wild-type levels in the Cers2-rescued Cers1 mutant mouse brains. Furthermore, LCBs induced neurite fragmentation in cultured neurons at concentrations corresponding to the elevated levels in the CerS1-deficient brain. The strong association of LCB levels with neuronal survival both in vivo and in vitro suggests high-level accumulation of LCBs is a possible underlying cause of the CerS1 deficiency-induced neuronal death.

In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Bexarotene, a selective agonist for Retinoid X receptors (RXR) improves cognitive deficits and amyloid-β (Aβ) clearance in mice. Here we examine if the effect of bexarotene on RXR cistrome and transcriptomes depend on APOE isoform and Aβ deposition. We found bexarotene increased RXR binding to promoter regions in cortex of APOE3 mice. Rho family GTPases and Wnt signaling pathway were highly enriched in ChIP-seq and RNA-seq datasets and members of those pathways - Lrp1, Lrp5, Sfrp5 and Sema3f were validated. The effect of APOE isoform was compared in APOE3 and APOE4 mice and we found significant overlapping in affected pathways. ChIP-seq using mouse embryonic stem cells and enrichment levels of histone marks H3K4me3 and H3K27me3 revealed that, bexarotene induced epigenetic changes, consistent with increased neuronal differentiation and in correlation with changes in transcription. Comparison of transcriptome in APOE3 and APP/APOE3 mice revealed that amyloid deposition significantly affects the response to bexarotene. In primary neurons, bexarotene ameliorated the damaged dendrite complexity and loss of neurites caused by Aβ42. Finally, we show that the disruption of actin cytoskeleton induced by Aβ42 in vitro was inhibited by bexarotene treatment. Our results suggest a mechanism to establish RXR therapeutic targets with significance in neurodegeneration.

β-Amyloid (Aβ) oligomers may play an important role in the early pathogenesis of Alzheimer's disease: cognitive impairment caused by synaptic dysfunction. Dystrophic neurites surrounding Aβ plaques, another pathological feature of Alzheimer's disease, are plaque-associated neuritic alterations preceding the appearance of synaptic loss. In the present review, we focus on the mechanism of dystrophic neurite formation by Aß oligomers, and discuss the neurotoxic role of Aβ-induced calsyntenin-3 in mediating dystrophic neurite formation., (© 2016 Japan Geriatrics Society.)

Nogo-A is a myelin associated protein and one of the most potent neurite growth inhibitors in the central nervous system. Interference with Nogo-A signaling has thus been investigated as therapeutic target to promote functional recovery in CNS injuries. Still, the finding that Nogo-A presents a fairly ubiquitous expression in many types of neurons in different brain regions, in the eye and even in the inner ear suggests for further functions besides the neurite growth repression. Indeed, a growing number of studies identified a variety of functions including regulation of neuronal stem cells, modulation of microglial activity, inhibition of angiogenesis and interference with memory formation. Aim of the present commentary is to draw attention on these less well-known and sometimes controversial roles of Nogo-A. Furthermore, we are addressing the role of Nogo-A in neuropathological conditions such as ischemic stroke, schizophrenia and neurodegenerative diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The androgen dehydroepiandrosterone (DHEA) has trophic and anti-glucocorticoid actions on brain growth. The adrenal gland of the spiny mouse (Acomys cahirinus) synthesizes DHEA. The aim of this study was to determine whether the brain of this precocial species is also able to produce DHEA de novo during fetal, neonatal and adult life. The expression of P450c17 and cytochrome b5 (Cytb5), the enzyme and accessory protein responsible for the synthesis of DHEA, was determined in fetal, neonatal and adult brains by immunocytochemistry, and P450c17 bioactivity was determined by the conversion of pregnenolone to DHEA. Homogenates of fetal brain produced significantly more DHEA after 48 h in culture (22.46 ± 2.0 ng/mg tissue) than adult brain homogenates (5.04 ± 2.0 ng/mg tissue; p < 0.0001). P450c17 and Cytb5 were co-expressed in fetal neurons but predominantly in oligodendrocytes and white matter tracts in the adult brain. Because DHEA modulates glucocorticoids actions, the expression of the glucocorticoid receptor (GR) was also determined. In the brainstem, medulla, midbrain, and cerebellum, the predominant GR localization changed from neurons in the fetal brain to oligodendrocytes and white matter tracts in the adult brain. The change of expression of P450c17, Cytb5 and GR proteins with cell type, brain region and developmental age indicates that DHEA is an endogenous neurosteroid in this species that may have important trophic and stress-modifying actions during both prenatal and postnatal life., (© 2015 S. Karger AG, Basel.)

Objective: SCA12 is a progressive autosomal-dominant disorder, caused by a CAG/CTG repeat expansion in PPP2R2B on chromosome 5q32, and characterized by tremor, gait ataxia, hyperreflexia, dysmetria, abnormal eye movements, anxiety, depression, and sometimes cognitive impairment. Neuroimaging has demonstrated cerebellar and cortical atrophy. We now present the neuropathology of the first autopsied SCA12 brain and utilize cell models to characterize potential mechanisms of SCA12 neurodegeneration., Methods: A fixed SCA12 brain was examined using gross, microscopic, and immunohistochemical methods. The effect of the repeat expansion on PPP2R2B Bβ1 expression was examined in multiple cell types by transient transfection of constructs containing the PPP2R2B Bβ1 promoter region attached to a luciferase reporter. The neurotoxic effect of PPP2R2B overexpression was examined in transfected rat primary neurons., Results: Neuropathological investigation revealed enlarged ventricles, marked cerebral cortical atrophy and Purkinje cell loss, less-prominent cerebellar and pontine atrophy, and neuronal intranuclear ubiquitin-positive inclusions, consistent with Marinesco bodies, which did not stain for long polyglutamine tracts, alpha-synuclein, tau, or transactive response DNA-binding protein 43. Reporter assays demonstrated that the region of PPP2R2B containing the repeat functions as a promoter, and that promoter activity increases with longer repeat length and is dependent on cell type, repeat sequence, and sequence flanking the repeat. Overexpression of PPP2R2B in primary cortical neurons disrupted normal morphology., Conclusions: SCA12 involves extensive, but selective, neurodegeneration distinct from Alzheimer's disease, synucleinopathies, tauopathies, and glutamine expansion diseases. SCA12 neuropathology may arise from the neurotoxic effect of repeat-expansion-induced overexpression of PPP2R2B., (© 2015 International Parkinson and Movement Disorder Society.)

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

In the central nervous system (CNS), insulin resistance (I/R) can cause defective neurite outgrowth and neuronal cell death, which can eventually lead to cognitive deficits. Recent research has focused on the relationship between I/R and the cognitive impairment caused by dementia, with the goal of developing treatments for dementia. Insulin signal transduction mediated by insulin receptor substrate (IRS-1) has been thoroughly studied in the CNS of patients with I/R. In the present study, we investigated whether the impairment of IRS-1-mediated insulin signaling contributes to neurite outgrowth and neuronal loss, both in mice fed a high-fat diet and in mouse neuroblastoma (Neuro2A) cells. To investigate the changes caused by the inhibition of IRS-1-mediated insulin signaling in the brain, we performed Cresyl Violet staining and immunochemical analysis. To investigate the changes caused by the inhibition of IRS-1-mediated insulin signaling in neuroblastoma cells, we performed Western blot analysis, reverse transcription-PCR, and immunochemical analysis. We show that the deactivation of IRS-1-mediated insulin signaling can inhibit neuronal outgrowth and aggravate neuronal cell death in the insulin-resistant CNS. Thus, IRS-1-mediated insulin signal transduction may be an important factor in the treatment of cognitive decline induced by I/R., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration.

Aims: Alzheimer's disease and the transmissible spongiform encephalopathies or prion diseases accumulate misfolded and aggregated forms of neuronal cell membrane proteins. Distinctive membrane lesions caused by the accumulation of disease-associated prion protein (PrP(d)) are found in prion disease but morphological changes of membranes are not associated with Aβ in Alzheimer's disease. Membrane changes occur in all prion diseases where PrP(d) is attached to cell membranes by a glycosyl-phosphoinositol (GPI) anchor but are absent from transgenic mice expressing anchorless PrP(d). Here we investigate whether GPI membrane attached Aβ may also cause prion-like membrane lesions., Methods: We used immunogold electron microscopy to determine the localization and pathology of Aβ accumulation in groups of transgenic mice expressing anchored or unanchored forms of Aβ or mutated human Alzheimer's precursor protein., Results: GPI attached Aβ did not replicate the membrane lesions of PrP(d). However, as with PrP(d) in prion disease, Aβ peptides derived from each transgenic mouse line initially accumulated on morphologically normal neurite membranes, elicited rapid glial recognition and neurite Aβ was transferred to attenuated microglial and astrocytic processes., Conclusions: GPI attachment of misfolded membrane proteins is insufficient to cause prion-like membrane lesions. Prion disease and murine Aβ amyloidosis both accumulate misfolded monomeric or oligomeric membrane proteins that are recognized by glial processes and acquire such misfolded proteins prior to their accumulation in the extracellular space. In contrast to prion disease where glial cells efficiently endocytose PrP(d) to endolysosomes, activated microglial cells in murine Aβ amyloidosis are not as efficient phagocytes., (© 2014 British Neuropathological Society.)

Growing evidence indicates that microRNAs (miRNAs) are important mediators of brain development and neurite growth. However, the affected signaling mechanisms are not clearly clarified. In the present study, we confirm that miR-29c is expressed during mice brain development and increases neurite outgrowth via decreasing PTEN expression. We first screen the picked-out miR-29c up-regulated in PC12 cells induced by nerve growth factor (NGF). In silico analysis of possible miR-29c targets, VEGFA, MAPK3, PDGFB, and PTEN mRNA are proposed as relatively likely putative binding sites for miR-29c. Subsequently, we detect that miR-29c is involved in brain development and has a negative relationship with the expression of PTEN. Then, using luciferase reporter assay,we demonstrate that miR-29c could directly target to the 3'-UTR of PTEN mRNA and result in down-expression of PTEN. By infecting PC12 cells with lentiviral pLKO-miR-29c or control, we also find that increasing levels of miR-29c markedly increase Akt phosphorylation level, and thus, promote neurite outgrowth of PC12 cells. Together, our results identify that miR-29c is required for mice brain development and modulates neurite outgrowth in PC12 cells via targeting PTEN and has a promising therapeutic target for neural disease.

Alzheimer's disease (AD) is the most common form of dementia and has no effective treatment. Besides the well-known pathologic characteristics, this disease also has a vascular component, and substantial evidence shows increased thrombosis as well as a critical role for fibrin(ogen) in AD. This molecule has been implicated in neuroinflammation, neurovascular damage, blood-brain barrier permeability, vascular amyloid deposition, and memory deficits that are observed in AD. Here, we present evidence demonstrating that fibrin deposition increases in the AD brain and correlates with the degree of pathology. Moreover, we show that fibrin(ogen) is present in areas of dystrophic neurites and that a modest decrease in fibrinogen levels improves neuronal health and ameliorates amyloid pathology in the subiculum of AD mice. Our results further characterize the important role of fibrin(ogen) in this disease and support the design of therapeutic strategies aimed at blocking the interaction between fibrinogen and amyloid-β (Aβ) and/or normalizing the increased thrombosis present in AD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

NGF induces neuronal differentiation by modulating [Ca(2+)]i. However, the role of the three isoforms of the main Ca(2+)-extruding system, the Na(+)/Ca(2+) exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca(2+)]i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca(2+) content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na(+) currents and 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na(+)]i significantly increased in cells overexpressing NCX1.4 as well as ER Ca(2+) content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca(2+)]i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca(2+) content and PI3K signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have previously proposed that specific proteins may form insoluble aggregates as a response to an illness-specific proteostatic dysbalance in a subset of brains from individuals with mental illness, as is the case for other chronic brain conditions. So far, established risk factors DISC1 and dysbindin were seen to specifically aggregate in a subset of such patients, as was a novel schizophrenia-related protein, CRMP1, identified through a condition-specific epitope discovery approach. In this process, antibodies are raised against the pooled insoluble protein fractions (aggregomes) of post mortem brain samples from schizophrenia patients, followed by epitope identification and confirmation using additional techniques. Pursuing this epitope discovery paradigm further, we reveal TRIO binding protein (TRIOBP) to be a major substrate of a monoclonal antibody with a high specificity to brain aggregomes from patients with chronic mental illness. TRIOBP is a gene previously associated with deafness which encodes for several distinct protein species, each involved in actin cytoskeletal dynamics. The 3' splice variant TRIOBP-1 is found to be the antibody substrate and has a high aggregation propensity when over-expressed in neuroblastoma cells, while the major 5' splice variant, TRIOBP-4, does not. Endogenous TRIOBP-1 can also spontaneously aggregate, doing so to a greater extent in cell cultures which are post-mitotic, consistent with aggregated TRIOBP-1 being able to accumulate in the differentiated neurons of the brain. Finally, upon expression in Neuroscreen-1 cells, aggregated TRIOBP-1 affects cell morphology, indicating that TRIOBP-1 aggregates may directly affect cell development, as opposed to simply being a by-product of other processes involved in major mental illness. While further experiments in clinical samples are required to clarify their relevance to chronic mental illness in the general population, TRIOBP-1 aggregates are thus implicated for the first time as a biological element of the neuropathology of a subset of chronic mental illness.

Current neuropathological Alzheimer's disease (AD) criteria from the National Institute on Aging-Alzheimer's Association (NIA-AA) incorporate two staging systems for Aβ pathology, namely the Thal Aβ phase (TAP) and the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) methods. The goal of this study was to compare and contrast results obtained with these two different staging systems for Aβ pathology since this is critical for future correlations of Aβ amyloid imaging data with Aβ neuropathology data based on immunohistochemical detection of Aβ deposits. A total of 123 cases, divided into 82 training and 41 validation cases, with a diagnosis of either unremarkable adult brain (normal) or AD and CERAD scores ranging from none to frequent were included. There was no clear and consistent relationship between CERAD and the TAP Aβ scores with the exception of scores for the highest plaque burdens (i.e., CERAD C3 and TAP A3) in the cases studied here. However, we developed an algorithm that relates CERAD scores to TAP scores with high agreement (94 % in training and 98 % in the validation set). In addition, TAP scores were a better predictor of dementia (sensitivity of 94 % specificity 87.7 %) than CERAD scores (sensitivity of 57 % specificity 100 %). Yet, further research is needed to define strategies to relate CERAD and TAP Aβ plaque scores to compare their utility and for determining the clinical associations of these different amyloid staging systems with aging and AD.

Intracellular Ca(2+) signal is a key regulator of axonal growth during brain development. As transient receptor potential (TRP) channels are permeable to Ca(2+) and mediate numerous brain functions, it is conceivable that many TRP channels would regulate neuronal differentiation. We therefore screened TRP channels that are involved in the regulation of neurite growth. Among the TRP channels, the Trpm2 level was inversely associated with neurite growth. TRPM2 was highly expressed in embryonic brain. Pharmacological perturbation or knockdown of TRPM2 markedly increased the axonal growth, whereas its overexpression inhibited the axonal growth. Addition of ADP ribose, an endogenous activator of TRPM2, to PC12 cells significantly repressed the axonal growth. TRPM2 was actively involved in the neuronal retraction induced by cerebrospinal fluid-rich lysophosphatidic acid (LPA). More importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Therefore, we conclude that TRPM2 mediates the LPA-induced suppression of axonal growth, which provides a long-sought mechanism underlying the effect of LPA on neuronal development.

Brain neurons form synapses throughout the life span. This process is initiated by neuronal depolarization, however the numbers of synapses thus formed depend on brain levels of three key nutrients-uridine, the omega-3 fatty acid DHA, and choline. Given together, these nutrients accelerate formation of synaptic membrane, the major component of synapses. In infants, when synaptogenesis is maximal, relatively large amounts of all three nutrients are provided in bioavailable forms (e.g., uridine in the UMP of mothers' milk and infant formulas). However, in adults the uridine in foods, mostly present at RNA, is not bioavailable, and no food has ever been compelling demonstrated to elevate plasma uridine levels. Moreover, the quantities of DHA and choline in regular foods can be insufficient for raising their blood levels enough to promote optimal synaptogenesis. In Alzheimer's disease (AD) the need for extra quantities of the three nutrients is enhanced, both because their basal plasma levels may be subnormal (reflecting impaired hepatic synthesis), and because especially high brain levels are needed for correcting the disease-related deficiencies in synaptic membrane and synapses.

The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect., (Copyright © 2014 Elsevier B.V. All rights reserved.)