MAB364 Sigma-AldrichAnti-MAP2A, 2B, 2C Antibody, clone HM-2

Mixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons.To identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons.We performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology.Transcriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons.Our results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

The potential effects of alternative splicing of death-domain expressing genes and neuronal death have not been determined in Alzheimer's disease (AD). We analyzed DENN/MADD/IG20 (DMI), the complex of four splice variants. IG20 is known to be involved in cell death and the DENN/MADD splice variant (DM-SV) in cell survival in non-neural systems. DENN/MADD (DM) and DENN/MADD splice variant 2 were also included. Using SH-SY5Y human neuroblastoma cultures exposed to high concentrations of oligomeric Aβ peptides (oAβ) as a model for neuronal death, there was initially an increased ratio of DM-SV to IG20 (DM-SV/IG20) and knockdown of DMI SVs including DM-SV with antisense DNA then increased cell death. Cultures transfected with small interfering RNAs (siRNAs) specific to subsets of DMI SVs but sparing DM-SV increased the DM-SV/IG20 ratio resulting in a reduction of cell death in the presence of oAβ. Effects on cell survival of DM and DM SV2, the other two SVs expressed in the CNS, are less clear. Compared to normal controls, alternative splicing changes in the CNS of AD patients during disease progression resulted in altered ratios of all of the SVs in a pattern over an extended time that mirrored that of the cultures, and coincided with the accumulation of endogenous, dimeric Aβ (dAβ). Thus, DM-SV may be required for neuronal survival by protecting against oAβ neurotoxicity, and IG20 may contribute to selective neuronal vulnerability in AD.

The mammalian olfactory cortex is a complex structure located along the rostro-caudal extension of the ventrolateral prosencephalon, which is divided into several anatomically and functionally distinct areas: the anterior olfactory nucleus, piriform cortex, olfactory tubercle, amygdaloid olfactory nuclei, and the more caudal entorhinal cortex. Multiple forebrain progenitor domains contribute to the cellular diversity of the olfactory cortex, which is invaded simultaneously by cells originating in distinct germinal areas in the dorsal and ventral forebrain. Using a combination of dye labeling techniques, we identified two novel areas that contribute cells to the developing olfactory cortices, the septum and the ventral pallium, from which cells migrate along a radial and then a tangential path. We characterized these cell populations by comparing their expression of calretinin, calbindin, reelin and Tbr1 with that of other olfactory cell populations.

In conventional MR imaging, it is often difficult to delineate the heterogeneous structure of gliomas. Proton magnetic resonance spectroscopic imaging ((1)H-MRSI) is a noninvasive tool for investigating the spatial distribution of metabolic changes in brain lesions. The aim of this study was to assess the improvements in delineation of gliomas based on segmentation of metabolic changes measured with (1)H-MRSI.

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer's disease mouse model.Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.

A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25muM for 1-7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult-derived neuronal systems, especially for traumatic brain injury (TBI). Copyright 2010 Elsevier B.V. All rights reserved.

2,5-Hexanedione (HD) intoxication is associated with axon atrophy that might be responsible for the characteristic gait abnormalities, hindlimb skeletal muscle weakness and other neurological deficits that accompany neurotoxicity. Although previous mechanistic research focused on neurofilament triplet proteins (NFL, NFM, NFH), other cytoskeletal targets are possible. Therefore, to identify potential non-NF protein targets, we characterized the effects of HD on protein-protein interactions in cosedimentation assays using microtubules and NFs prepared from spinal cord of rats intoxicated at different daily dose rates (175 and 400 mg/kg/day). Results indicate that HD did not alter the presence of alpha- or beta-tubulins in these preparations, nor were changes noted in the distribution of either anterograde (KIF1A, KIF3, KIF5) or retrograde (dynein) molecular motors. The cosedimentation of dynactin, a dynein-associated protein, also was not affected. Immunoblot analysis of microtubule-associated proteins (MAPs) in microtubule preparations revealed substantial reductions (45-80%) in MAP1A, MAP1B heavy chain, MAP2, and tau regardless of HD dose rate. MAP1B light chain content was not altered. Finally, HD intoxication did not influence native NF protein content in either preparation. As per previous research, microtubule and NF preparations were enriched in high-molecular weight NF species. However, these NF derivatives were common to both HD and control samples, suggesting a lack of pathognomonic relevance. These data indicate that, although motor proteins were not affected, HD selectively impaired MAP-microtubule binding, presumably through adduction of lysine residues that mediate such interactions. Given their critical role in cytoskeletal physiology, MAPs could represent a relevant target for the induction of gamma-diketone axonopathy.

In addition to the fundamental role of the extracellular glycoprotein Reelin in neuronal development and adult synaptic plasticity, alterations in Reelin-mediated signaling have been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease (AD). In vitro data revealed a biochemical link between Reelin-mediated signaling, Tau phosphorylation, and amyloid precursor protein (APP) processing. To directly address the role of Reelin in amyloid-beta plaque and Tau pathology in vivo, we crossed heterozygous Reelin knock-out mice (reeler) with transgenic AD mice to investigate the temporal and spatial AD-like neuropathology. We demonstrate that a reduction in Reelin expression results in enhanced amyloidogenic APP processing, as indicated by the precocious production of amyloid-beta peptides, the significant increase in number and size of amyloid-beta plaques, as well as age-related aggravation of plaque pathology in double mutant compared with single AD mutant mice of both sexes. Numerous amyloid-beta plaques accumulate in the hippocampal formation and neocortex of double mutants, precisely in layers with strongest Reelin expression and highest accumulation of Reelin plaques in aged wild-type mice. Moreover, concentric accumulations of phosphorylated Tau-positive neurons around amyloid-beta plaques were evident in 15-month-old double versus single mutant mice. Silver stainings indicated the presence of neurofibrillary tangles, selectively associated with amyloid-beta plaques and dystrophic neurites in the entorhinal cortex and hippocampus. Our findings suggest that age-related Reelin aggregation and concomitant reduction in Reelin-mediated signaling play a proximal role in synaptic dysfunction associated with amyloid-beta deposition, sufficient to enhance Tau phosphorylation and tangle formation in the hippocampal formation in aged Reelin-deficient transgenic AD mice.

During cerebral cortex development, different cell populations migrate tangentially through the preplate, traveling from their site of origin toward their final positions. One of the earliest populations formed, the Cajal-Retzius (C-R) cells, is mainly generated in different cortical hem (CH) domains, and they migrate along established and parallel routes to cover the whole cortical mantle. In this study, we present evidence that the phenotype of -Retzius cells, as well as some of their migratory characteristics, is specified in the area where the cells are generated. Nevertheless, when implanted ectopically, these cells can follow new migratory routes, indicating that locally provided genetic cues along the migratory path nonautonomously influence the position of these cells emanating from different portions of the CH. This was witnessed by performing CH implants of tissue expressing fluorescent tracers in live whole embryos. In the same way, tracer injections into the hem of Small eye mutant mice were particularly informative since the lack of Pax6 affects some guidance factors in the migratory environment. As a result, in these animals, the C-R cell population is disorganized, and it forms 1 day late, showing certain differences in gene expression that might help explain these disruptions.

Besides its critical role during neurodevelopment, the extracellular glycoprotein reelin is also a pivotal regulator of adult synaptic function and plasticity, and altered reelin-mediated signalling has been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease. We have recently discovered, in aged rodents and non-human primates, a pronounced decline in reelin-positive interneurons and concomitant accumulation of reelin in extracellular amyloid-like deposits, both being associated with episodic-like memory impairments. Here, we report that these age-related neuropathological changes in hippocampus, entorhinal and piriform cortices of aged wild-type mice are accompanied by abnormal axonal varicosities and altered expression profiles of calcium-binding proteins in plaque-dense areas, as well as a significant reduction in the number of parvalbumin-positive gamma-aminobutyric acid (GABA)ergic projection neurons in basal forebrain areas, including medial septum (MS), ventral and horizontal diagonal Band of Broca (VDB/HDB) and substantia innominata (SI), compared with young subjects. In addition, a significant reduction in the number of choline acetyltransferase-positive cholinergic projection neurons was evident in the HDB/SI area but not in the MS of aged compared with young wild-type mice. No reelin-deposits were found in these basal forebrain regions. Our findings suggest that the elevated reelin plaque load in the projection areas of afferent subcortical GABAergic and cholinergic neurons including hippocampus, entorhinal and piriform cortices affects the axonal integrity and survival of these neurons, potentially contributing to the cognitive impairments observed in aged wild-type mice.,