MAP2 Immunostaining Research Articles

SRGN amplifies microglia-mediated neuroinflammation and exacerbates ischemic brain injury

BackgroundMicroglia is the major contributor of post-stroke neuroinflammation cascade and the crucial cellular target for the treatment of ischemic stroke. Currently, the endogenous mechanism underlying microglial activation following ischemic stroke remains elusive. Serglycin (SRGN) is a proteoglycan expressed in immune cells. Up to now, the role of SRGN on microglial activation and ischemic stroke is largely unexplored.MethodsSrgn knockout (KO), Cd44-KO and wild-type (WT) mice were subjected to middle cerebral artery occlusion (MCAO) to mimic ischemic stroke. Exogenous SRGN supplementation was achieved by stereotactic injection of recombinant mouse SRGN (rSRGN). Cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Neurological functions were evaluated by the modified neurological severity score (mNSS) and grip strength. Microglial activation was detected by Iba1 immunostaining, morphological analysis and cytokines’ production. Neuronal death was examined by MAP2 immunostaining and FJB staining.ResultsThe expression of SRGN and its receptor CD44 was significantly elevated in the ischemic mouse brains, especially in microglia. In addition, lipopolysaccharide (LPS) induced SRGN upregulation in microglia in vitro. rSRGN worsened ischemic brain injury in mice and amplified post-stroke neuroinflammation, while gene knockout of Srgn exerted reverse impacts. rSRGN promoted microglial proinflammatory activation both in vivo and in vitro, whereas Srgn-deficiency alleviated microglia-mediated inflammatory response. Moreover, the genetic deletion of Cd44 partially rescued rSRGN-induced excessed neuroinflammation and ischemic brain injury in mice. Mechanistically, SRGN boosted the activation of NF-κB signal, and increased glycolysis in microglia.ConclusionSRGN acts as a novel therapeutic target in microglia-boosted proinflammatory response following ischemic stroke.

Genetic deletion of Krüppel-like factor 11 aggravates traumatic brain injury

BackgroundThe long-term functional recovery of traumatic brain injury (TBI) is hampered by pathological events, such as parenchymal neuroinflammation, neuronal death, and white matter injury. Krüppel-like transcription factor 11 (KLF 11) belongs to the zinc finger family of transcription factors and actively participates in various pathophysiological processes in neurological disorders. Up to now, the role and molecular mechanisms of KLF11 in regulating the pathogenesis of brain trauma is poorly understood.MethodsKLF11 knockout (KO) and wild-type (WT) mice were subjected to experimental TBI, and sensorimotor and cognitive functions were evaluated by rotarod, adhesive tape removal, foot fault, water maze, and passive avoidance tests. Brain tissue loss/neuronal death was examined by MAP2 and NeuN immunostaining, and Cresyl violet staining. White matter injury was assessed by Luxol fast blue staining, and also MBP/SMI32 and Caspr/Nav1.6 immunostaining. Activation of cerebral glial cells and infiltration of blood-borne immune cells were detected by GFAP, Iba-1/CD16/32, Iba-1/CD206, Ly-6B, and F4/80 immunostaining. Brian parenchymal inflammatory cytokines were measured with inflammatory array kits.ResultsGenetic deletion of KLF11 worsened brain trauma-induced sensorimotor and cognitive deficits, brain tissue loss and neuronal death, and white matter injury in mice. KLF11 genetic deficiency in mice also accelerated post-trauma astrocytic activation, promoted microglial polarization to a pro-inflammatory phenotype, and increased the infiltration of peripheral neutrophils and macrophages into the brain parenchyma. Mechanistically, loss-of-KLF11 function was found to directly increase the expression of pro-inflammatory cytokines in the brains of TBI mice.ConclusionKLF11 acts as a novel protective factor in TBI. KLF11 genetic deficiency in mice aggravated the neuroinflammatory responses, grey and white matter injury, and impaired long-term sensorimotor and cognitive recovery. Elucidating the functional importance of KLF11 in TBI may lead us to discover novel pharmacological targets for the development of effective therapies against brain trauma.

Abstract 21: Genetic Deletion Of MicroRNA-15a/16-1 In Pericytes Stimulates Cerebral Angiogenesis And Promotes Long-term Functional Recovery After Ischemic Stroke

Introduction: Post-stroke cerebral angiogenesis actively participates in tissue repair and plays a vital role in the long-term functional recovery in stroke patients. Accumulating evidence shows that certain microRNAs (miRs) regulate cerebral angiogenesis after CNS disorders. We have demonstrated that endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis by enhancing activity of pro-angiogenic factors and their receptors. Here we further investigate the effects of pericyte-derived miR-15a/16-1 on post-ischemic cerebral angiogenesis and outcomes. Methods: Inducible pericyte-specific miR-15a/16-1 cKO mice and WT littermate controls were subjected to 1h MCAO and 28d reperfusion. Neurobehavioral outcomes were determined by the foot fault, rotarod, adhesive tape removal, and Morris water maze tests. Brain atrophy was measured by MAP2 immunostaining. Cerebral blood flow (CBF) was monitored by laser speckle imaging. Brain capillary density, functional microvessels, and neurons were examined by CD31/BrdU, tomato lectin/BrdU, and NeuN/BrdU double immunostaining, respectively. In vitro angiogenesis assays, including BrdU cell proliferation, scratch assay, and capillary tube formation were analyzed in mBMEC cutures treated with conditional medium from primary mouse vascular pericytes (mBVPs) with lentivirus-mediated miR-15a/16-1 knockdown. Pro-angiogenic factors were detected by qPCR and western blotting. Results: Pericytes-miR-15a/16-1 cKO mice exhibit improved sensorimotor and cognitive outcomes, increased CBF recovery, and reduced brain atrophy compared to WT controls. The number of newly-generated cerebral microvessels, functional microvessels, and neurons in the peri-infarct brain regions of Pericyte-miR-15a/16-1 cKO mice are higher than WT controls. Treatment of conditional medium from miR-15a/16-1 silencing pericytes significantly increases endothelial cell proliferation, migration, and tube formation, respectively. Mechanistically, lentiviral silencing of miR-15a/16-1 in primary mBVPs remarkably enhances the expression of both FGF2 and VEGFA. Conclusions: Our findings suggest that pericytic miR-15a/16-1 negatively regulates post-stroke cerebral angiogenesis.

Corrigendum: Cocaine Exposure Results in Formation of Dendritic Varicosity in Rat Primary Hippocampal Neurons

The authors have recently noticed that the representative image of MAP2 immunostaining in the control group in Figure 3A was incorrectly chosen. This error was inadvertently caused during the assembly of the images due to our negligence in the process of reformatting our manuscript. Original correct Figure 3A has now been included as shown below. Please note that this amendment does not affect the description, interpretation, statistical analyses, or the original conclusions of the manuscript. The authors apologize for the mistake and any confusion this may have caused.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Abstract 112: Genetic Deletion of Endothelial microRNA-15a/16-1 Promotes Cerebral Angiogenesis and Neurological Recovery in Ischemic Stroke Through Src Signaling Pathway

Introduction: Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. Recently, we demonstrated that endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis and improves long-term neurological recovery by increasing protein levels of VEGFA, FGF2, and their receptors VEGFR2 and FGFR1. Here, we further investigated the downstream signaling pathways of these pro-angiogenic factors by using a potent Src family inhibitor, saracatinib (AZD0530), in endothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice after ischemic stroke. Hypothesis: Pharmacological inhibition of Src signaling cascade using AZD0530 diminishes enhanced post-stroke angiogenesis and long-term neurological recovery induced by the genetic deletion of miR-15a/16-1 in the endothelium. Methods: EC-miR-15a/16-1 cKO and WT littermate controls were subjected to 1h MCAO and 28d reperfusion. AZD0530 (20 mg/kg) was administered daily to both genotypes at 3-21d after MCAO by oral gavage. Cognitive outcomes were determined by Morris water maze tests. Brain atrophy was measured by MAP2 immunostaining. Cerebral blood flow (CBF) was monitored by laser speckle imaging. Brain capillary density and functional vessels were examined by CD31/BrdU and tomato lectin/BrdU double-immunostaining, respectively. Src signaling pathway-related molecules were analyzed by western blotting. Results: Treatment with AZD0530 exacerbates cognitive impairments and brain atrophy in EC-miR-15a/16-1 cKO mice following MCAO. AZD0530 also attenuates the long-term CBF recovery, correlated with inhibiting the generation of newly formed microvessels and functional vessels in the peri-infarct brain regions in EC-miR-15a/16-1 cKO mice after cerebral ischemia. Moreover, EC-targeted deletion of miR-15a/16-1 upregulates the Src signaling pathway, while AZD0530 blocks the Src signaling pathway by downregulating p-Src and its downstream mediators (p-Stat3, p-Akt, p-FAK, p-p38 MAPK) in ischemic brains. Conclusions: Endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis and improves long-term neurological recovery via Src signaling pathway.

Protein Expression Profile on Differentiation of Bone Marrow Mesenchymal Stem Cells into Retinal Ganglion-Like Cells.

To explore possible approaches to differentiating rat bone marrow mesenchymal stem cells (BMSCs) into retinal ganglion-like cells and to demonstrate the dynamic changes in protein expression profiles of BMSCs throughout the differentiation. BMSCs were isolated from adult rats and cultured in medium conditioned by neonatal rat retinal cells to induce BMSC differentiation into retinal ganglion-like cells. Immunostaining for neurofilament, nestin, Map2, and Thy1.1 was used to follow the differentiation process. Two types of protein arrays were employed to profile the BMSCs, the differentiated retinal ganglion-like cells, and the primary retinal ganglion cells (RGCs) using the Biomarker Wizard System. After 7 days of culture in conditioned medium, cells showing a neural-cell-like modality appeared. The differentiated retinal ganglion-like cells showed that network-like connections were positive for nestin, neurofilament, Map2, and Thy1.1. In total, 16 marker proteins were highly expressed in both retinal ganglion-like cells and RGCs and no obvious expression was observed in BMSCs. Among them, nine proteins were expressed more highly in RGCs than in retinal ganglion-like cells. BMSCs can be induced to differentiate into retinal ganglion-like cells by neonatal rat retinal cells, and the induced cells show protein profiles resembling those of isolated RGCs.

Anti-brain cancer activity of chloroform and hexane extracts of Tinospora cordifolia Miers: an in vitro perspective

Plants have been suggested as safest source of therapeutic agents, with multi targeted mode of action and least side effects. Tinospora cordifolia, commonly known as Guduchi in India, is one of the most highly valued herbs in Ayurvedic medicine. It possesses potential anti-cancer, anti-inflammatory, hepatoprotective, anti-diabetic, immune-stimulatory and various other beneficial activities. The present study was aimed to investigate the differentiation inducing potential of chloroform and hexane extracts of T. cordifolia using U87MG glioblastoma and IMR-32 neuroblastoma cell lines as model system. Chloroform (Chl-TCE) and hexane (Hex-TCE) extracts significantly reduced the rate of proliferation and induced cell differentiation as evidenced by MTT assay and immunostaining for GFAP and MAP-2 in glioblastoma and neuroblastoma, respectively. Further these extracts increased the expression of stress markers HSP70 and Mortalin and induced senescence. Chloroform and hexane extracts also inhibited the migration of U87MG glioblastoma and IMR-32 neuroblastoma as indicated by wound scratch assay and supported by reduced expression of NCAM. Furthermore these extracts are not toxic to normal cells as they showed no inhibitory effects on primary astrocytic and neuronal cultures. The present study suggests that chloroform and hexane extracts of T. cordifolia retard the rate of proliferation, induce differentiation and inhibit migration of human glioblastomas and neuroblastomas, thus may act as potential phytotherapeutic intervention in treatment of neural cancers.

Remote limb ischemic postconditioning promotes motor function recovery in a rat model of ischemic stroke via the up-regulation of endogenous tissue kallikrein.

Remote ischemic conditionings, such as pre- and per-conditioning, are known to provide cardioprotection in animal models of ischemia. However, little is known about the neuroprotection effect of postconditioning after cerebral ischemia. In this study, we aim to evaluate the motor function rescuing effect of remote limb ischemic postconditioning (RIPostC) in a rat model of acute cerebral stroke. Left middle cerebral artery occlusion (MCAO) was performed to generate the rat model of ischemic stroke, followed by daily RIPostC treatment for maximum 21days. The motor function after RIPostC was assessed with foot fault test and balance beam test. Local infarct volume was measured through MRI scanning. Neuronal status was evaluated with Nissl's, HE, and MAP2 immunostaining. Lectin immunostaining was performed to evaluate the microvessel density and area. Daily RIPostC for more than 21days promoted motor function recovery and provided long-lasting neuroprotection after MCAO. Reduced infarct volume, rescued neuronal loss, and enhanced microvessel density and size in the injured areas were observed. In addition, the RIPostC effect was associated with the up-regulation of endogenous tissue kallikrein (TK) level in circulating blood and local ischemic brain regions. A TK receptor antagonist HOE-140 partially reversed RIPostC-induced improvements, indicating the specificity of endogenous TK mediating the neuroprotection effect of RIPostC. Our study demonstrates RIPostC treatment as an effective rehabilitation therapy to provide motor function recovery and alleviate brain impairment in a rat model of acute cerebral ischemia. We also for the first time provide evidence showing that the up-regulation of endogenous TK from remote conditioning regions underlies the observed effects of RIPostC.

Pathology-MRI Correlations in Diffuse Low-Grade Epilepsy Associated Tumors.

It is recognized that IDH mutation negative, low-grade epilepsy associated tumors (LEAT) can show diffuse growth patterns and lack the diagnostic hallmarks of either classical dysembryoplastic neuroepithelial tumors (DNT) or typical ganglioglioma. “Nonspecific or diffuse DNT” and more recently “polymorphous low-grade neuroepithelial tumor of the young” have been terms used for these entities. There are few reports on the MRI recognition of these diffuse glioneuronal tumors (dGNT), which is important in planning the extent of surgical resection. In 27 LEATs T1, T2, FLAIR, and postcontrast T1 MRI were evaluated and the pathology reviewed, including immunostaining for NeuN, CD34, MAP2, and IDH1. Each case was then independently classified by pathology or MRI as simple DNT, complex DNT, or dGNT. There was agreement in 23/27 (85%; Kappa score 0.62; p < 0.01). In 4 cases, there was discrepancy in the diagnosis of simple versus complex DNT but 100% agreement achieved for dGNT. DNT showed significantly more expansion of the cortex, cystic change and ventricle extension than dGNT. dGNT showed significantly more subcortical T2w hyperintensity and focal cortical atrophy which correlated on pathology with CD34 expression, cortical neuronal loss and white matter rarefaction. There was no distinct cortical dysplasia component identified by MRI or pathology in any case. This study highlights that dGNT can be reliably discriminated on MRI from DNT.

Outer brain barriers in rat and human development.

Complex barriers at the brain's surface, particularly in development, are poorly defined. In the adult, arachnoid blood-cerebrospinal fluid (CSF) barrier separates the fenestrated dural vessels from the CSF by means of a cell layer joined by tight junctions. Outer CSF-brain barrier provides diffusion restriction between brain and subarachnoid CSF through an initial radial glial end feet layer covered with a pial surface layer. To further characterize these interfaces we examined embryonic rat brains from E10 to P0 and forebrains from human embryos and fetuses (6–21st weeks post-conception) and adults using immunohistochemistry and confocal microscopy. Antibodies against claudin-11, BLBP, collagen 1, SSEA-4, MAP2, YKL-40, and its receptor IL-13Rα2 and EAAT1 were used to describe morphological characteristics and functional aspects of the outer brain barriers. Claudin-11 was a reliable marker of the arachnoid blood-CSF barrier. Collagen 1 delineated the subarachnoid space and stained pial surface layer. BLBP defined radial glial end feet layer and SSEA-4 and YKL-40 were present in both leptomeningeal cells and end feet layer, which transformed into glial limitans. IL-13Rα2 and EAAT1 were present in the end feet layer illustrating transporter/receptor presence in the outer CSF-brain barrier. MAP2 immunostaining in adult brain outlined the lower border of glia limitans; remnants of end feet were YKL-40 positive in some areas. We propose that outer brain barriers are composed of at least 3 interfaces: blood-CSF barrier across arachnoid barrier cell layer, blood-CSF barrier across pial microvessels, and outer CSF-brain barrier comprising glial end feet layer/pial surface layer.

Intraoperative vascular DIVA surgery reveals angiogenic hotspots in tumor zones of malignant gliomas

Malignant gliomas belong to the most threatening tumor entities and are hallmarked by rapid proliferation, hypervascularization and an invasive growth pattern. The primary obstacle in surgical treatment lies in differentiation between healthy and pathological tissue at the tumor margins, where current visualization methods reach their limits. Here, we report on a novel technique (vascular dual intraoperative visualization approach - vDIVA) enabling visualization of different tumor zones (TZ I–III) on the basis of angiogenic hotspots. We investigated glioblastoma patients who underwent 5-ALA fluorescence-guided surgery with simultaneous intraoperative ICG fluorescence angiography. This vDIVA technique revealed hypervascularized areas which were further histologically investigated. Neuropathological assessments revealed tissue areas at the resection margins corresponding to TZ II, and postoperative CD34- and Map2 immunostaining confirmed these angiogenic hotspots to be occupied by glioma cells. Hence, the vascular architecture in this transitional zone could be well differentiated from both primary tumor bulk and healthy brain parenchyma. These data demonstrate that ICG fluorescence angiography improves state-of-the-art glioma surgery techniques and facilitates the future characterization of polyclonal attributes of malignant gliomas.

Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts

We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O2). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O2 tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O2 conditions compared to 50% under high O2 growth conditions, confirming the beneficial aspect of reprogramming under low O2. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3β and Smad-mediated pathways. Our results showed that lowering the O2 tension at the initiation of reprogramming is a simple and efficient stratergy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

Enhanced neuroregenerative effects by scaffold for the treatment of a rat spinal cord injury with Wnt3a-secreting fibroblasts

Wnt proteins are bifunctional axon guidance molecules, several of which appear to mediate guidance of corticospinal tract axons along the spinal cord. Here, we studied increasing effect on regeneration by Wnt-containing alginate scaffolds on spinal cord injury (SCI). A total of 32 rats were injured at the T7-8 level with an NYU impactor. According to transplantation materials, rats were classified into four groups: a Wnt3a-secreting fibroblast transplantation group (Wnt group, n = 8), a Wnt3a-secreting fibroblast with alginate transplantation group (Wnt + alginate group, n = 8), an alginate transplantation group (alginate group, n = 8), and a contusion-only group (sham group, n = 8). Behavioral tests were performed on the first, second, and third days after injury, and then weekly for 8 weeks. Five of the eight rats from each group were selected for manganese-enhanced magnetic resonance imaging (ME-MRI). Two rats from each group were examined for GAP43 and MAP2 expression using monoclonal and polyclonal primary antibodies, respectively. Seven weeks after transplantation (8 weeks after SCI), Wnt + alginate group rats achieved an average Basso-Beattie-Bresnahan locomotor score of 19.0, which was significantly higher than that of other groups. ME-MRI at 8 weeks after SCI revealed significantly higher relative signal intensities in the Wnt + alginate group. Gap43 and Map2 immunostaining, showed strong positive in the Wnt + alginate group. The Wnt + alginate complex exerted significantly enhanced recovery in a rat SCI model compared to alginate or Wnt3a alone. These results suggest that alginate scaffolds facilitate the regeneration of axon working with Wnt3a protein that promotes regeneration of the injured spinal cord.

Early Postnatal Lesion of the Medial Dorsal Nucleus Leads to Loss of Dendrites and Spines in Adult Prefrontal Cortex

Research suggests that the medial dorsal nucleus (MD) of the thalamus influences pyramidal cell development in the prefrontal cortex (PFC) in an activity-dependent manner. The MD is reciprocally connected to the PFC. Many psychiatric disorders, such as schizophrenia, affect the PFC, and one of the most consistent findings in schizophrenia is a decrease in volume and neuronal number in the MD. Therefore, understanding the role the MD plays in the development of the PFC is important and may help in understanding the progression of psychiatric disorders that have their root in development. Focusing on the interplay between the MD and the PFC, this study examined the hypothesis that the MD plays a role in the dendritic development of pyramidal cells in the PFC. Unilateral electrolytic lesions of the MD in Long-Evans rat pups were made on postnatal day 4 (P4), and the animals developed to P60. We then examined dendritic morphology by examining MAP2 immunostaining and by using Golgi techniques to determine basilar dendrite number and spine density. Additionally, we examined pyramidal cell density in cingulate area 1 (Cg1), prelimbic region, and dorsolateral anterior cortex, which receive afferents from the MD. Thalamic lesions caused a mean MD volume decrease of 12.4% which led to a significant decrease in MAP2 staining in both superficial and deep layers in all 3 cortical areas. The lesions also caused a significant decrease in spine density and in the number of primary and secondary basilar dendrites on superficial and deep layer pyramidal neurons in all 3 regions. No significant difference was observed in pyramidal cell density in any of the regions or layers, but a nonsignificant increase in cell density was observed in 2 regions. Our data are thus consistent with the hypothesis that the MD plays a role in the development of the PFC and, therefore, may be a good model to begin to examine neurodevelopmental disorders such as autism and schizophrenia.

RP58 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Cerebral Cortex

Accumulating evidence suggests that many brain diseases are associated with defects in neuronal migration, suggesting that this step of neurogenesis is critical for brain organization. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here, we identified the zinc-finger transcriptional repressor RP58 as a key regulator of neuronal migration via multipolar-to-bipolar transition. RP58(-/-) neurons exhibited severe defects in the formation of leading processes and never shifted to the locomotion mode. Cre-mediated deletion of RP58 using in utero electroporation in RP58(flox/flox) mice revealed that RP58 functions in cell-autonomous multipolar-to-bipolar transition, independent of cell-cycle exit. Finally, we found that RP58 represses Ngn2 transcription to regulate the Ngn2-Rnd2 pathway; Ngn2 knockdown rescued migration defects of the RP58(-/-) neurons. Our findings highlight the critical role of RP58 in multipolar-to-bipolar transition via suppression of the Ngn2-Rnd2 pathway in the developing cerebral cortex.

Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons

Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture. Here, we leveraged surface nano/micro-modification techniques to fabricate a template substrate for constraining neurite lengths of cultured neurons. Using the template, we asked (i) Do neurons polarize even if all neurites cannot grow sufficiently long? (ii) Would the neurite be fated to become an axon if only one was allowed to grow long? A pattern with symmetrical short paths (20 μm) was used to address the former question, and an asymmetrical pattern with one path extended to 100 μm for the latter. Axon formation was evaluated by tau-1/MAP2 immunostaining and live-cell imaging of constitutively-active kinesin-1. We found that (1) neurons cannot polarize when extension of all neurites is restricted and that (2) when only a single neurite is permitted to grow long, neurons polarize and the longest neurite becomes the axon. These results provide clear evidence that axon outgrowth is required for its specification.

Efficient neuronal differentiation and maturation of human pluripotent stem cells encapsulated in 3D microfibrous scaffolds

Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here, we report the use of chitin-alginate 3D microfibrous scaffolds, previously developed for hPSC propagation, to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid, the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons, as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore, flow cytometry confirmed that >90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines, a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons, without tumorigenesis, suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications.

Synaptic Silencing and Plasma Membrane Dyshomeostasis Induced by Amyloid-β Peptide are Prevented by Aristotelia chilensis Enriched Extract

Alzheimer's disease (AD) is characterized by the presence of different types of extracellular and neurotoxic aggregates of amyloid-β (Aβ). Recently, bioactive compounds extracted from natural sources showing neuroprotective properties have become of interest in brain neurodegeneration. We have purified, characterized, and evaluated the protective potential of one extract enriched in polyphenols obtained from Aristotelia chilensis (MQ), a Chilean berry fruit, in neuronal models of AD induced by soluble oligomers of Aβ1-40. For example, using primary hippocampal cultures from rats (E18), we observed neuroprotection when the neurons were co-incubated with Aβ (0.5 μM) plus MQ for 24 h (Aβ = 23 ± 2%; Aβ + MQ = 3 ± 1%; n = 3). In parallel, co-incubation of Aβ with MQ recovered the frequency of Ca2+ transient oscillations when compared to neurons treated with Aβ alone (Aβ = 72 ± 3%; Aβ + MQ = 86 ± 2%; n = 5), correlating with the changes observed in spontaneous synaptic activity. Additionally, MAP-2 immunostaining showed a preservation of the dendritic tree, suggesting that the toxic effect of Aβ is prevented in the presence of MQ. A new complex mechanism is proposed by which MQ induces neuroprotective effects including antioxidant properties, modulation of cell survival pathways, and/or direct interaction with the Aβ aggregates. Our results suggest that MQ induces changes in the aggregation kinetics of Aβ producing variations in the nucleation phase (Aβ: k1 = 2.7 ± 0.4 × 10-3 s-1 MQ: k1 = 8.3 ± 0.6 × 10-3 s-1) and altering Thioflavin T insertion in β-sheets. In conclusion, MQ induces a potent neuroprotection by direct interaction with the Aβ aggregates, generating far less toxic species and in this way protecting the neuronal network.

Abstract 114: Neuroprotective Role of Brain Pericytes through PDGFRβ-Akt Signaling in Ischemic Stroke

Brain pericytes are a constituent of the neurovascular unit and play various important roles in brain functions, such as regulation of capillary blood flow, maintenance of blood-brain barrier and angiogenesis. Previous reports have elucidated that PDGF-B prevents neuronal cell death during ischemic insults in adult rodent models; however, the detailed mechanisms by which PDGF-B signaling protects neurons from ischemic damage are not fully understood. In the present study, we investigated whether brain pericytes play neuroprotective roles in brain ischemia, using a permanent middle cerebral artery occlusion stroke model (MCAO) and cultured human brain pericytes. Immunohistochemistry revealed that the expression of PDGF receptorβ(PDGFRβ) was induced predominantly in pericytes in peri-infarct areas. PDGF-B induced marked phosphorylation of Akt in cultured pericytes. Consistently, Akt was markedly phosphorylated in the PDGFRβ-expressing pericytes in peri-infarct areas. PDGF-B upregulated the expression of neurotrophins, such as neuronal growth factor (NGF) and neurotrophin-3 (NT-3), through Akt activation in the cultured pericytes. We subjected PDGFRβheterozygous knockout (PDGFRβ+/-) mice to MCAO. Infarct volume, as assessed by MAP2 immunostaining, was significantly greater in PDGFRβ+/- than wild-type mice ( 48% increase at day 7, p &lt; 0.01 , n=5). The number of TUNEL positive apoptotic cells was significantly greater in PDGFRβ+/- mice (54 % increase at day 4, p &lt; 0.001 , n=6). Production of NGF and NT-3 at mRNA and protein levels in infarct areas was significantly decreased in PDGFRβ+/- mice (NGF: 28% decrease, p&lt;0.05, NT-3: 22% decrease, p&lt;0.05). Since it has been established that neurotrophin receptors are induced in peri-infarct areas, the decreases in neurotrophin production may increase apoptotic neuronal cell death in the PDGFRβ+/- mice. In conclusion, brain pericytes may have a direct neuroprotective role through secreting neurotrophins via PDGFRβ-Akt signaling, thereby decreasing infarct volume in ischemic stroke.