MAP2 Immunoreactivity Research Articles

Human Milk Oligosaccharide 2'-Fucosyllactose Induces Neuroprotection from Intracerebral Hemorrhage Stroke.

Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFβ) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.

Naltrexone is neuroprotective against traumatic brain injury in mu opioid receptor knockout mice

AimsNaltrexone is a mu opioid receptor (MOR) antagonist used to treat drug dependence in patients. Previous reports indicated that MOR antagonists reduced neurodegeneration and inflammation after brain injury. The purpose of this study was to evaluate the neuroprotective effect of naltrexone in cell culture and a mouse model of traumatic brain injury (TBI).MethodsThe neuroprotective effect of naltrexone was examined in primary cortical neurons co‐cultured with BV2 microglia. Controlled cortical impact (CCI) was delivered to the left cerebral cortex of adult male MOR wild‐type (WT) and knockout (KO) mice. Naltrexone was given daily for 4 days, starting from day 2 after lesioning. Locomotor activity was evaluated on day 5 after the CCI. Brain tissues were collected for immunostaining, Western, and qPCR analysis.ResultsGlutamate reduced MAP2 immunoreactivity (‐ir), while increased IBA1‐ir in neuron/BV2 co‐culture; both responses were antagonized by naltrexone. TBI significantly reduced locomotor activity and increased the expression of IBA1, iNOS, and CD4 in the lesioned cortex. Naltrexone significantly and equally antagonized the motor deficits and expression of IBA1 and iNOS in WT and KO mice. TBI‐mediated CD4 protein production was attenuated by naltrexone in WT mice, but not in KO mice.ConclusionNaltrexone reduced TBI‐mediated neurodegeneration and inflammation in MOR WT and KO mice. The protective effect of naltrexone involves non‐MOR and MOR mechanisms.

Microtubule-Associated Protein 2 as a DHEA Binding Protein in Endometrial Cancer

Dehydroepiandrosterone (DHEA) is an androgen secreted by the adrenal glands, but its binding affinity for the androgen receptor is very low. DHEA is transformed into androstenedione by 3β-hydroxysteroid dehydrogenase (HSD) and then into testosterone by 17β-HSD type 5, or into estrone by aromatase. DHEA is also converted into androstenediol by 17β-HSD type 1. Therefore, DHEA is considered to play an important role as a precursor hormone for sex steroid hormones. We performed a search for a protein having an amino acid sequence homology to the DHEA binding site of 17β-HSD type 1, and found that microtubule-associated protein 2 (MAP2) binds to DHEA (Laurine E et al., J Biol Chem. 2003). MAP2 expression is necessary for neurite extension and cessation of cell division. MAP2 is known to suppress migration and invasion and affect the assembly, stabilization, and bundling of microtubules in melanoma cells, but the function of MAP2 in endometrial cancer has not been clarified. In this study, we investigated the expression of MAP2 and its association with DHEA in order to clarify the direct non-receptor action of DHEA in endometrial cancer. We employed frozen and formalin-fixed paraffin-embedded (FFPE) tissues of 35 endometrial cancer tissues (G1, n=12; G2, n=10; G3, n=9; Serous, n=4). Hormone concentrations were measured by liquid chromatograph-tandem mass spectrometer from the frozen sample, and immunohistochemistry of MAP2 was performed using FFPE tissues. We also examined MAP2 immunoreactivity using 59 normal endometrial tissues (proliferative phase, n=33; secretory phase, n=26) of FFPE tissue microarray slides. MAP2 immunoreactivity was found in the cytoplasm of endometrial cancer cells, and the MAP2-positive rate was significantly higher in type 1 (G1 and G2) than in type 2 (G2 and G3). The cell proliferation marker Ki-67 index was significantly lower in the MAP2-positive group. MAP2 was also detected in the glandular epithelial cells of the normal endometrium. The MAP2-positive rate was lower in the proliferative phase than in the secretory phase. Furthermore, the concentration of DHEA in the cancer tissue was significantly higher in the MAP2-positive group than in the MAP2-negative group. MAP2 is known to act on the stability of microtubules and is thought to be involved in the suppression of proliferation and infiltration in cancer cells. It was suggested that DHEA is involved in the stabilization of MAP2 and suppresses the progression of cancer in a hormone receptor-independent manner.

Carthamin yellow improves cerebral ischemia‑reperfusion injury by attenuating inflammation and ferroptosis in rats.

Carthamin yellow (CY), a flavonoid compound extracted from safflower, has been reported to attenuate cardiac ischemia and reperfusion injury. However, whether CY could ameliorate ischemic stroke is not completely understood. In the present study, the preventive effects of CY on experimental ischemic stroke were investigated using middle cerebral artery occlusion (MCAO) model rats. Neurological scores, brain edema, infarct area and microtubule-associated protein 2 (MAP-2) immunoreactivity were assessed to evaluate the effects of CY on ischemic brain injury. The involvement of inflammation and ferroptosis were examined to investigate the mechanism underlying the effects of CY. The results demonstrated that 2-week CY treatment attenuated the neurological deficit score, brain water content and infarct area, and increased MAP-2 immunoreactivity in the cortex in MCAO model rats. CY administration also deactivated the cortex NF-κB/NLR family pyrin domain containing 3 inflammasome signaling pathway, and decreased serum TNF-α, IL-1β and IL-6 concentrations. Moreover, CY treatment inhibited Fe2+ and reactive oxygen species accumulation, and reversed acyl-CoA synthetase long-chain family member 4, transferrin receptor 1, glutathione peroxidase 4 and ferritin heavy chain 1 protein expression levels in the brain. The levels of glutathione, superoxide dismutase and malondialdehyde in the serum were also reversed by CY treatment. Collectively, the results of the present study demonstrated that CY protected rats against ischemic stroke, which was associated with mitigation of inflammation and ferroptosis.

MAP2 is differentially phosphorylated in schizophrenia, altering its function

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz—that a large proportion of individuals have a “MAP2opathy”—in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

Astrocytic palmitoylethanolamide pre-exposure exerts neuroprotective effects in astrocyte-neuron co-cultures from a triple transgenic mouse model of Alzheimer's disease

Palmitoylethanolamide (PEA) is an endogenous lipid mediator that, also by blunting astrocyte activation, demonstrated beneficial properties in several in vitro and in vivo models of Alzheimer's disease (AD).In the present study, we used astrocyte-neuron co-cultures from 3xTg-AD mouse (i.e. an animal model of AD) cerebral cortex to further investigate on the role of astrocytes in PEA-induced neuroprotection. To this aim, we evaluated the number of viable cells, apoptotic nuclei, microtubule-associated protein-2 (MAP2) positive cells and morphological parameters in cortical neurons co-cultured with cortical astrocytes pre-exposed, or not, to Aβ42 (0.5 μM; 24 h) or PEA (0.1 μM; 24 h).Pre-exposure of astrocytes to Aβ42 failed to affect the viability, the number of neuronal apoptotic nuclei, MAP2 positive cell number, neuritic aggregations/100 μm, dendritic branches per neuron, the neuron body area, the length of the longest dendrite and number of neurites/neuron in 3xTg-AD mouse astrocyte-neuron co-cultures. Compared to neurons from wild-type (non-Tg) mouse co-cultures, 3xTg-AD mouse neurons co-cultured with astrocytes from this mutant mice displayed higher number of apoptotic nuclei, lower MAP2 immunoreactivity and several morphological changes. These signs of neuronal suffering were significantly counteracted when the 3xTg-AD mouse cortical neurons were co-cultured with 3xTg-AD mouse astrocytes pre-exposed to PEA.The present data suggest that in astrocyte-neuron co-cultures from 3xTg-AD mice, astrocytes contribute to neuronal damage and PEA, by possibly counteracting reactive astrogliosis, improved neuronal survival. These findings further support the role of PEA as a possible new therapeutic opportunity in AD treatment.

Genetic and microstructural differences in the cortical plate of gyri and sulci during gyrification in fetal sheep.

Gyrification of the cerebral cortex is a developmentally important process, but the mechanisms that drive cortical folding are not fully known. Theories propose that changes within the cortical plate (CP) cause gyrification, yet differences between the CP below gyri and sulci have not been investigated. Here we report genetic and microstructural differences in the CP below gyri and sulci assessed before (at 70days of gestational age [GA] 70), during (GA 90), and after (GA 110) gyrification in fetal sheep. The areal density of BDNF, CDK5, and NeuroD6 immunopositive cells were increased, and HDAC5 and MeCP2 mRNA levels were decreased in the CP below gyri compared with sulci during gyrification, but not before. Only the areal density of BDNF-immunopositive cells remained increased after gyrification. MAP2 immunoreactivity and neurite outgrowth were also increased in the CP below gyri compared with sulci at GA 90, and this was associated with microstructural changes assessed via diffusion tensor imaging and neurite orientation dispersion and density imaging at GA 98. Differential neurite outgrowth may therefore explain the localized changes in CP architecture that result in gyrification.

HIV influences microtubule associated protein-2: potential marker of HIV-associated neurocognitive disorders.

Postmortem brains of patients diagnosed with HIV-1-associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood. Dendrite length and remodeling occurs via microtubules, the dynamics of which are regulated by microtubule-binding proteins, including microtubule-associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal microtubules. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120. First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss. PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2-positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to microtubules, conjugated to a nanolipoprotein particle delivery platform. Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.

Altered expression of parvalbumin immunoreactivity in rat main olfactory bulb following pilocarpine-induced status epilepticus

Epilepsy is a chronic neurological disease characterized by spontaneous recurrent seizures and caused by various factors and mechanisms. Malfunction of the olfactory bulb is frequently observed in patients with epilepsy. However, the morphological changes in the olfactory bulb during epilepsy-induced neuropathology have not been elucidated. Therefore, in the present study, we investigated the expression of parvalbumin (PV), one of the calcium-binding proteins, and morphological changes in the rat main olfactory bulb (MOB) following pilocarpine-induced status epilepticus (SE). Pilocarpine-induced SE resulted in neuronal degeneration in the external plexiform layer (EPL) and glomerular layer (GL) of the MOB. PV immunoreactivity was observed in the neuronal somas and processes in the EPL and GL of the control group. However, six hours after pilocarpine administration, PV expression was remarkably decreased in the neuronal processes compared to the somas and the average number of PV-positive interneurons was significantly decreased. Three months after pilocarpine treatment, the number of PV-positive interneurons was also significantly decreased compared to the 6 hour group in both layers. In addition, the number of NeuN-positive neurons was also significantly decreased in the EPL and GL following pilocarpine treatment. In double immunofluorescence staining for PV and MAP2, the immunoreactivity for MAP2 around the PV-positive neurons was significantly decreased three months after pilocarpine treatment. Therefore, the present findings suggest that decreases in PV-positive GABAergic interneurons and dendritic density in the MOB induced impaired calcium buffering and reciprocal synaptic transmission. Thus, these alterations may be considered key factors aggravating olfactory function in patients with epilepsy.

Low-pressure fluid percussion minimally adds to the sham craniectomy-induced neurobehavioral changes: Implication for experimental traumatic brain injury model

Modeling experimental traumatic brain injury (TBI) in rodents is necessarily required to understand the pathophysiological and neurobehavioral consequences of neurotrauma. Numerous models have been developed to study experimental TBI. Fluid percussion injury (FPI) is the most extensively used model to represent clinical phenotypes. Nevertheless, the surgical ‘sham’ procedure (craniectomy), a prerequisite of FPI, is the impeding factor in experimental TBI. We hypothesized that if craniectomy causes substantial structural and functional changes in the brain, it might mimic the mild FPI-induced neurobehavioral dysfunctions. To understand the hypothesis, C57BL/6 mice were exposed to lateral FPI at 1.2 atm pressure and changes in the neuronal architecture, hippocampal neurogenesis, neuroinflammation, and behavioral functions were compared to the sham (craniectomy) and control mice at day 7 post-FPI. We observed that both the craniectomy and FPI significantly augmented the ipsilateral hippocampal neurogenesis as evaluated by DCX and Beta-III tubulin immunoreactivity. Similarly, a significant increase in GFAP and TMEM immunoreactivity in CA1 and CA3 regions showed that craniectomy mimics FPI-induced neuroinflammation. The additive damaging effect of craniectomy with FPI was also reported in the term of axonal and dendritic fragmentation, swelling and neuronal death using silver staining, Fluoro-jade, and MAP-2 immunoreactivity. Sham-exposed mice showed a significant functional decrease in grip strength. Our results indicate that sham craniectomy itself is enough to cause TBI like characteristics, and thus fluid percussion at mild pressure is minimally additive with craniectomy. Considering the method as a mixed (focal & diffused) injury model, the ‘net neurotrauma severity’ should be compared with naïve control instead of the sham as it is an outcome of cumulative damage due to fluid pressure and craniectomy. Nevertheless, to understand the long term consequences of neurotrauma, the extent of recovery in surgical sham may separately be quantified.

Human Milk Oligosaccharide 2'-Fucosyllactose Reduces Neurodegeneration in Stroke Brain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca++ (Ca++i) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Ca++i; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Ca++i, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.

<P>Abnormal DNA Methylation in Pluripotent Stem Cells from a Patient with Prader-Willi Syndrome Results in Neuronal Differentiation Defects</P>

DNA methylation is a common method of gene expression regulation, and this form of regulation occurs in the neurodevelopmental disorder Prader Willi syndrome (PWS). Gene expression regulation via methylation is important for humans, although the role of methylation in neuronal differentiation is unknown. We characterized the cellular differentiation potential of iPS cells derived from a patient with PWS with abnormal methylation (M-iPWS cells). A CGH array revealed that, unlike iPWS cells, the abnormally methylated M-iPWS cells had no deletion in the15q11.2-q13 chromosome region. In addition, methylation-specific PCR showed that M-iPWS cells had strong methylation of the SNRPN CpG island. To assess the effect of abnormal methylation on cell differentiation, the M-iPWS cells and iPWS cells were induced to differentiate into embryoid bodies, which contain endoderm, mesoderm, and ectoderm. The results suggest that iPS cells from patients with PWS are defective at differentiation into ectoderm. When directionally differentiated into neuronal cells, the expression of neural stem cells (NSCs) markers in M-iPWS cells was relatively low compared to those from control cells. Nestin and Musashi-1 immunoreactivity was also low in NSCs from M-iPWS cells. MAP2 and βIII-tubulin immunoreactivity was also sparse, and marker expression was low in neuronal cells differentiated from M-iPWS cells. We conclude that abnormal methylation in the PWS region is involved in neuronal differentiation.

Royal jelly increased map-2 expression in hippocampal neurons of hypothyroid rats: an immunohistochemical study

ABSTRACTWe investigated microtubule-associated protein 2 (MAP-2) immunoreactivity of hippocampal neurons and the potential role of royal jelly (RJ) in regulating MAP-2 during experimental hypothyroidism (HT). Thirty adult female Wistar albino rats were randomized into five groups: the control group was untreated, the sham control group was treated with 10 mg/kg 0.9% sterile saline injected intraperitoneally (i.p.), The RJ group was treated with 100 mg/kg RJ by oral gavage, the HT group was treated with 10 mg/kg propylthiouracil (PTU) i.p. to induce experimental hypothyroidism, and the HT + RJ group was treated with 10 mg/kg PTU i.p. + 100 mg/kg RJ by oral gavage. Oral and i.p. administrations were performed once daily for 20 days. Thyroid stimulating hormone (TSH) and free thyroxine (fT4) levels in the serum were measured biochemically, MAP-2 was measured immunohistochemically in the hippocampus and an immunohistochemical H score was calculated. MAP-2 immunoreactivity appeared in the cytoplasm of neuron cell bodies and dendrites in the hippocampal CA3 and CA1 regions in the control, sham control and RJ groups. MAP-2 immunoreactivity decreased significiantly in the HT group compared to control, sham control and RJ groups. Also, vascular dilation and swollen cells were observed following PTU administration. The degeneration that was observed in the HT group decreased by RJ administration. By contrast, MAP-2 immunoreactivity increased following administration of RJ. Experimental hypothyroidism reduced significiantly MAP-2 immunoreactivity in both the CA3 and CA1 regions and caused degeneration, including edema and vascular dilation, in the hippocampus. RJ increased MAP-2 expression and exhibited a therapeutic effect on the degenerative changes.

MAP2 immunoreactivity deficit is conserved across the cerebral cortex within individuals with schizophrenia

Several postmortem studies have reported lower levels of immunoreactivity (IR) for microtubule-associated protein 2 (MAP2) in several cortical regions of individuals with schizophrenia (SZ). However, whether this effect is conserved across multiple brain areas within an individual with SZ or if it is regionally-specific remains unclear. We characterized patterns of MAP2-IR across three cortical regions at different levels of the rostral-caudal axis within individual subjects with and without SZ. MAP2-IR levels were measured in deep layer 3 of dorsolateral prefrontal cortex (DLPFC), lateral intraparietal cortex (LIP), and primary visual cortex (V1). Postmortem tissue containing each cortical region was derived from 20 pairs of SZ subjects and nonpsychiatric comparison (NPC) subjects matched perfectly for sex, and as closely as possible for age and postmortem interval. MAP2-IR was assessed by quantitative fluorescence microscopy. We observed significantly lower levels of MAP2-IR in SZ subjects relative to NPC subjects, without a significant region by diagnosis interaction. Logs of the within-pair ratios (SZ:NPC) of MAP2-IR were significantly correlated across the three regions. These findings demonstrate that MAP2-IR deficits in SZ are consistent across three neocortical regions within individual subjects. This pattern of MAP2-IR deficit has implications for therapeutic development and future investigations of MAP2 pathology in SZ.

Herpes Simplex Virus Type-1 Infection Impairs Adult Hippocampal Neurogenesis via Amyloid-β Protein Accumulation

We previously reported that Herpes simplex virus type-1 (HSV-1) infection of cultured neurons triggered intracellular accumulation of amyloid-β protein (Aβ) markedly impinging on neuronal functions. Here, we demonstrated that HSV-1 affects in vitro and in vivo adult hippocampal neurogenesis by reducing neural stem/progenitor cell (NSC) proliferation and their neuronal differentiation via intracellular Aβ accumulation. Specifically, cultured NSCs were more permissive for HSV-1 replication than mature neurons and, once infected, they exhibited reduced proliferation (assessed by 5'-bromo-deoxyuridine incorporation, Ki67 immunoreactivity, and Sox2 mRNA expression) and impaired neuronal differentiation in favor of glial phenotype (evaluated by immunoreactivity for the neuronal marker MAP2, the glial marker glial fibrillary astrocyte protein, and the expression of the proneuronal genes Mash1 and NeuroD1). Similarly, impaired adult neurogenesis was observed in the subgranular zone of hippocampal dentate gyrus of an in vivo model of recurrent HSV-1 infections, that we recently set up and characterized, with respect to mock-infected mice. The effects of HSV-1 on neurogenesis did not depend on cell death and were due to Aβ accumulation in infected NSCs. Indeed, they were: (a) reverted, in vitro, by the presence of either β/γ-secretase inhibitors preventing Aβ production or the specific 4G8 antibody counteracting the action of intracellular Aβ; (b) not detectable, in vivo, in HSV-1-infected amyloid precursor protein knockout mice, unable to produce and accumulate Aβ. Given the critical role played by adult neurogenesis in hippocampal-dependent memory and learning, our results suggest that multiple virus reactivations in the brain may contribute to Alzheimer's disease phenotype by also targeting NSCs. Stem Cells 2019;37:1467-1480.

Nerve growth factor is expressed and stored in central neurons of adult zebrafish.

Nerve growth factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro-NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates that pro-NGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF is highly similar, and zebrafish NGF protein has been reported in mature and two-precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF-positive cells. NGF mRNA was visualized by insitu hybridization on whole-mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize pro-NGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF-positive cells, anti-NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double-immunolabeling against NGF/proliferative cell nuclear antigen (PCNA; proliferation marker) and NGF/microtube-associated protein2 (MAP2; neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish, and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF-positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store pro-NGF.

Density of small dendritic spines and microtubule-associated-protein-2 immunoreactivity in the primary auditory cortex of subjects with schizophrenia.

Previously, we demonstrated that dendritic spine density (DSD) in deep layer 3 of the primary auditory cortex (A1) islower, due to having fewer small spines, in subjects with schizophrenia (SZ) than non-psychiatric control (NPC) subjects. We also previously demonstrated that microtubule-associated-protein-2 immunoreactivity (MAP2-IR) in A1 deep layer 3 is lower, and positively correlated with DSD, in SZ subjects. Here, we first sought to confirm these findings in an independent cohort of 25 SZ-NPC subject pairs (cohort 1). We used immunohistochemistry and confocal microscopy to measure DSD and MAP2-IR in A1 deep layer 3. Consistent with previous studies, both DSD and MAP2-IR were lower in SZ subjects. We then tested the hypothesis that MAP2-IR mediates the effect of SZ on DSD in a cohort of 45 SZ-NPC subject pairs (combined cohort)that included all subjects from cohort 1 and two previously studied cohorts. Based on the distribution of MAP2-IR values in NPC subjects, we categorized each SZ subject as having either low MAP2-IR (SZ MAP2-IR(low)) or normal MAP2-IR (SZ MAP2-IR(normal)). Among SZ MAP-IR(low) subjects, mean DSD was significantly lower than in NPC subjects. However, mean DSD did not differ between SZ MAP2-IR(normal) and NPC subjects. Moreover, MAP2-IR statistically mediated small spine differences, with lower MAP2-IR values associated with fewer small spines. Our findings confirm that low density of small spines and low MAP2-IR are robust SZ phenotypes and suggest that MAP2-IR mediates the effect of SZ on DSD.

Formononetin inhibits neuroinflammation and increases estrogen receptor beta (ERβ) protein expression in BV2 microglia

Formononetin is a bioactive non-steroidal polyphenol found in a variety of plants. In this study we evaluated the effects of formononetin on neuroinflammation in LPS-stimulated BV2 microglia. Results showed that formononetin significantly reduced the production of TNF-α, IL-6 and IL-1β, nitrite and PGE2, as well as protein levels of iNOS and COX-2. Reporter gene assays showed that formononetin produced inhibition of NF-κB luciferase activity in HEK293 cells stimulated with TNF-α. Immunoblotting experiments revealed an inhibition of IKKα phosphorylation, with the resultant attenuation of phosphorylation and degradation of IκBα following LPS stimulation. Formononetin also produced an inhibition of nuclear translocation and DNA binding by NF-κB following LPS stimulation. RNAi experiments showed that transfection of BV2 microglia with ERβ siRNA resulted in the loss of anti-inflammatory action of formononetin. MTT assay and MAP2 immunoreactivity experiments showed that formononetin produced significant neuroprotective activity by preventing BV2 microglia conditioned media-induced toxicity to HT22 neurons. Investigations on the effect of formononetin on MCF7 breast cancer cells revealed that, while the compound significantly increased ER-luciferase activity, its effects on proliferation were modest. This study has established that formononetin inhibits neuroinflammation by targeting NF-κB signalling pathway in BV2 microglia, possibly through mechanisms involving ERβ. Formononetin appears to modulate ERβ in MCF7 breast cancer cells with limited proliferative effect. Formononetin could therefore serve as a chemical scaffold for the development of novel compounds which have selective neuroprotective actions in the brain.

Pretreatment with Human Chorionic Gonadotropin Protects the Neonatal Brain against the Effects of Hypoxic-Ischemic Injury.

Though the human fetus is exposed to placentally derived human chorionic gonadotropin (hCG) throughout gestation, the role of hCG on the fetal brain is unknown. Review of the available literature appears to indicate that groups of women with higher mean levels of hCG during pregnancy tend to have offspring with lower cerebral palsy (CP) risk. Given that newborn cerebral injury often precedes the development of CP, we aimed to determine whether hCG may protect against the neurodegenerative effects of neonatal brain injury. We utilized the Rice-Vannucci model of neonatal cerebral hypoxia-ischemia (HI) in postnatal day 7 mice to examine whether intraperitoneal administration of hCG 15-18 h prior, 1 h after or immediately following HI decrease brain tissue loss 7 days after injury. We next studied whether hCG has pro-survival and trophic properties in neurons by exposing immature cortical and hippocampal neurons to hCG in vitro and examining neurite sprouting and neuronal survival prior and after glutamate receptor-mediated excitotoxic injury. We found that intraperitoneal injection of hCG 15 h prior to HI, but not at or 1 h after HI induction, resulted in a significant decrease in hippocampal and striatal tissue loss 7 days following brain injury. Furthermore, hCG reduced N-methyl-d-aspartate (NMDA)-mediated neuronal excitotoxicity in vitro when neurons were continuously exposed to this hormone for 10 days or when given at the time and following neuronal injury. In addition, continuous in vitro administration of hCG for 6-9 days increased neurite sprouting and basal neuronal survival as assessed by at least a 1-fold increase in MAP2 immunoreactivity and a 2.5-fold increase in NeuN + immunoreactivity. Our findings suggest that hCG can decrease HI-associated immature neural degeneration. The mechanism of action for this neuroprotective effect may partly involve inhibition of NMDA-dependent excitotoxic injury. This study supports the hypothesis that hCG during pregnancy has the potential for protecting the developing brain against HI, an important CP risk factor.

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OBJECTIVES/SPECIFIC AIMS: The objectives of this study are to determine whether high-frequency ipsi-lesion or low-frequency contra-lesion ECS improves forelimb function following experimental stroke in aged animals with focal and large strokes. We also want to investigate whether ECS-induced improvements in motor function are related to an enhancement of neural structural plasticity (dendrites and synapses) and changes in growth promoting (BDNF) and growth inhibiting (NOGO-A) expression in the infarcted motor cortex in young and aged animals. METHODS/STUDY POPULATION: We will investigate whether excitatory ECS of the infarcted cortex or inhibition of the noninfarcted cortex combined with daily impaired-forelimb rehabilitative training (RT) results in greater motor functional recovery compared to RT alone. Immunohistochemical (IHC) analyses and unbiased stereological techniques will be performed to investigate changes in proteins associated with dendritic restructuring (MAP2), synaptic plasticity (PSD95 and synaptophysin), and alteration in the expression of BDNF and NOGO-A. RESULTS/ANTICIPATED RESULTS: We expect that inhibitory ECS of the noninfarcted motor cortex will improve behavioral outcomes in moderate to severe stroke animals compared with excitatory ECS or no stimulation (RT alone) animals. We predict that the ECS condition that improves motor performance most significantly compared with RT alone will have a corresponding greater increase in remaining ipsi-infarct motor cortical dendritic and synaptic plasticity (demonstrated by a greater density of MAP2, synaptophysin, and PSD-95 immunoreactivity), and greater expression of BDNF. It is unknown, but also expected that better behavioral recovery will coincide with a greater reduction in NOGO-A in the injured motor cortex. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will aid in creating a model that will allow for a better understanding of the relationship between brain stimulation, severity of injury and, in future studies, aging. These studies will also help clarify previous conflicting brain stimulation results.