Systemic LPS Administration Stimulates the Activation of Non-Neuronal Cells in an Experimental Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deficiency of the survival motor neuron (SMN) protein. Although SMA is a genetic disease, environmental factors contribute to disease progression. Common pathogen components such as lipopolysaccharides (LPS) are considered significant contributors to inflammation and have been associated with muscle atrophy, which is considered a hallmark of SMA. In this study, we used the SMNΔ7 experimental mouse model of SMA to scrutinize the effect of systemic LPS administration, a strong pro-inflammatory stimulus, on disease outcome. Systemic LPS administration promoted a reduction in SMN expression levels in CNS, peripheral lymphoid organs, and skeletal muscles. Moreover, peripheral tissues were more vulnerable to LPS-induced damage compared to CNS tissues. Furthermore, systemic LPS administration resulted in a profound increase in microglia and astrocytes with reactive phenotypes in the CNS of SMNΔ7 mice. In conclusion, we hereby show for the first time that systemic LPS administration, although it may not precipitate alterations in terms of deficits of motor functions in a mouse model of SMA, it may, however, lead to a reduction in the SMN protein expression levels in the skeletal muscles and the CNS, thus promoting synapse damage and glial cells' reactive phenotype.

The cytoplasmic localization of ADNP through 14-3-3 promotes sex-dependent neuronal morphogenesis, cortical connectivity, and calcium signaling.

Defective neuritogenesis is a contributing pathogenic mechanism underlying a variety of neurodevelopmental disorders. Single gene mutations in activity-dependent neuroprotective protein (ADNP) are the most frequent among autism spectrum disorders (ASDs) leading to the ADNP syndrome. Previous studies showed that during neuritogenesis, Adnp localizes to the cytoplasm/neurites, and Adnp knockdown inhibits neuritogenesis in culture. Here, we hypothesized that Adnp is localized in the cytoplasm during neurite formation and that this process is mediated by 14-3-3. Indeed, applying the 14-3-3 inhibitor, difopein, blocked Adnp cytoplasmic localization. Furthermore, co-immunoprecipitations showed that Adnp bound 14-3-3 proteins and proteomic analysis identified several potential phosphorylation-dependent Adnp/14-3-3 binding sites. We further discovered that knockdown of Adnp using in utero electroporation of mouse layer 2/3 pyramidal neurons in the somatosensory cortex led to previously unreported changes in neurite formation beginning at P0. Defects were sustained throughout development, the most notable included increased basal dendrite number and axon length. Paralleling the observed morphological aberrations, ex vivo calcium imaging revealed that Adnp deficient neurons had greater and more frequent spontaneous calcium influx in female mice. GRAPHIC, a novel synaptic tracing technology substantiated this finding, revealing increased interhemispheric connectivity between female Adnp deficient layer 2/3 pyramidal neurons. We conclude that Adnp is localized to the cytoplasm by 14-3-3 proteins, where it regulates neurite formation, maturation, and functional cortical connectivity significantly building on our current understanding of Adnp function and the etiology of ADNP syndrome.

RNA Editing Alterations Define Disease Manifestations in the Progression of Experimental Autoimmune Encephalomyelitis (EAE).

RNA editing is an epitranscriptomic modification, leading to targeted changes in RNA transcripts. It is mediated by the action of ADAR (adenosine deaminases acting on double-stranded (ds) RNA and APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) deaminases and appears to play a major role in the pathogenesis of many diseases. Here, we assessed its role in experimental autoimmune encephalomyelitis (EAE), a widely used non-clinical model of autoimmune inflammatory diseases of the central nervous system (CNS), which resembles many aspects of human multiple sclerosis (MS). We have analyzed in silico data from microglia isolated at different timepoints through disease progression to identify the global editing events and validated the selected targets in murine tissue samples. To further evaluate the functional role of RNA editing, we induced EAE in transgenic animals lacking expression of APOBEC-1. We found that RNA-editing events, mediated by the APOBEC and ADAR deaminases, are significantly reduced throughout the course of disease, possibly affecting the protein expression necessary for normal neurological function. Moreover, the severity of the EAE model was significantly higher in APOBEC-1 knock-out mice, compared to wild-type controls. Our results implicate regulatory epitranscriptomic mechanisms in EAE pathogenesis that could be extrapolated to MS and other neurodegenerative disorders (NDs) with common clinical and molecular features.

Exercise training alters autoimmune cell invasion into the brain in autoimmune encephalomyelitis.

BACKGROUND: The mechanisms by which exercise training (ET) elicits beneficial effects on the systemic immune system and the central nervous system (CNS) in autoimmune neuroinflammation are not fully understood. OBJECTIVES: To investigate (1) the systemic effects of high-intensity continuous training (HICT) on the migratory potential of autoimmune cells; (2) the direct effects of HICT on blood-brain-barrier (BBB) properties. METHODS: Healthy mice were subjected to high-intensity continuous training (HICT) by treadmill running. The proteolipid protein (PLP) transfer EAE model was utilized to examine the immunomodulatory effects of training, where PLP-reactive lymph-node cells (LNCs) from HICT and sedentary donor mice were analyzed in vitro and transferred to naïve recipients that developed EAE. To examine neuroprotection, encephalitogenic LNCs from donor mice were transferred into HICT or sedentary recipient mice and the BBB was analyzed. RESULTS: Transfer of PLP-reactive LNCs obtained from HICT donor mice attenuated EAE severity and inflammation in recipient mice. HICT markedly inhibited very late antigen (VLA)-4 and lymphocyte function-associated antigen (LFA)-1 expression in LNCs. Transfer of encephalitogenic LNCs into HICT recipients resulted in milder EAE and attenuated CNS inflammation. HICT reduced BBB permeability and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in CNS blood vessels. INTERPRETATION: HICT attenuates EAE development by both immunomodulatory and neuroprotective effects. The reduction in destructive CNS inflammation in EAE is attributed to systemic inhibition of autoreactive cell migratory potential, as well as reduction in BBB permeability, which are associated with reduced VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions.

Novel ADNP Syndrome Mice Reveal Dramatic Sex-Specific Peripheral Gene Expression With Brain Synaptic and Tau Pathologies.

BACKGROUND: ADNP is essential for embryonic development. As such, de novo ADNP mutations lead to an intractable autism/intellectual disability syndrome requiring investigation. METHODS: Mimicking humans, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 editing produced mice carrying heterozygous Adnp p.Tyr718∗ (Tyr), a paralog of the most common ADNP syndrome mutation. Phenotypic rescue was validated by treatment with the microtubule/autophagy-protective ADNP fragment NAPVSIPQ (NAP). RESULTS: RNA sequencing of spleens, representing a peripheral biomarker source, revealed Tyr-specific sex differences (e.g., cell cycle), accentuated in females (with significant effects on antigen processing and cellular senescence) and corrected by NAP. Differentially expressed, NAP-correctable transcripts, including the autophagy and microbiome resilience-linked FOXO3, were also deregulated in human patient-derived ADNP-mutated lymphoblastoid cells. There were also Tyr sex-specific microbiota signatures. Phenotypically, Tyr mice, similar to patients with ADNP syndrome, exhibited delayed development coupled with sex-dependent gait defects. Speech acquisition delays paralleled sex-specific mouse syntax abnormalities. Anatomically, dendritic spine densities/morphologies were decreased with NAP amelioration. These findings were replicated in the Adnp+/- mouse, including Foxo3 deregulation, required for dendritic spine formation. Grooming duration and nociception threshold (autistic traits) were significantly affected only in males. Early-onset tauopathy was accentuated in males (hippocampus and visual cortex), mimicking humans, and was paralleled by impaired visual evoked potentials and correction by acute NAP treatment. CONCLUSIONS: Tyr mice model ADNP syndrome pathology. The newly discovered ADNP/NAP target FOXO3 controls the autophagy initiator LC3 (microtubule-associated protein 1 light chain 3), with known ADNP binding to LC3 augmented by NAP, protecting against tauopathy. NAP amelioration attests to specificity, with potential for drug development targeting accessible biomarkers.

High-Intensity Exercise Training Protects the Brain Against Autoimmune Neuroinflammation: Regulation of Microglial Redox and Pro-inflammatory Functions.

Background: Exercise training induces beneficial effects on neurodegenerative diseases, and specifically on multiple sclerosis (MS) and it's model experimental autoimmune encephalomyelitis (EAE). However, it is unclear whether exercise training exerts direct protective effects on the central nervous system (CNS), nor are the mechanisms of neuroprotection fully understood. In this study, we investigated the direct neuroprotective effects of high-intensity continuous training (HICT) against the development of autoimmune neuroinflammation and the role of resident microglia. Methods: We used the transfer EAE model to examine the direct effects of training on the CNS. Healthy mice performed HICT by treadmill running, followed by injection of encephalitogenic proteolipid (PLP)-reactive T-cells to induce EAE. EAE severity was assessed clinically and pathologically. Brain microglia from sedentary (SED) and HICT healthy mice, as well as 5-days post EAE induction (before the onset of disease), were analyzed ex vivo for reactive oxygen species (ROS) and nitric oxide (NO) formation, mRNA expression of M1/M2 markers and neurotrophic factors, and secretion of cytokines and chemokines. Results: Transfer of encephalitogenic T-cells into HICT mice resulted in milder EAE, compared to sedentary mice, as indicated by reduced clinical severity, attenuated T-cell, and neurotoxic macrophage/microglial infiltration, and reduced loss of myelin and axons. In healthy mice, HICT reduced the number of resident microglia without affecting their profile. Isolated microglia from HICT mice after transfer of encephalitogenic T-cells exhibited reduced ROS formation and released less IL-6 and monocyte chemoattractant protein (MCP) in response to PLP-stimulation. Conclusions: These findings point to the critical role of training intensity in neuroprotection. HICT protects the CNS against autoimmune neuroinflammation by reducing microglial-derived ROS formation, neurotoxicity, and pro-inflammatory responses involved in the propagation of autoimmune neuroinflammation.

Continuous and interval training attenuate encephalomyelitis by separate immunomodulatory mechanisms.

BACKGROUND: Studies have reported beneficial effects of exercise training on autoimmunity, and specifically on multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, it is unknown whether different training paradigms affect disease course via shared or separate mechanisms. OBJECTIVE: To compare the effects and mechanism of immune modulation of high intensity continuous training (HICT) versus high intensity interval training (HIIT) on systemic autoimmunity in EAE. METHODS: We used the proteolipid protein (PLP)-induced transfer EAE model to examine training effects on the systemic autoimmune response. Healthy mice performed HICT or HIIT by running on a treadmill. Lymph-node (LN)-T cells from PLP-immunized trained- versus sedentary donor mice were transferred to naïve recipients and EAE clinical and pathological severity were assessed. LN cells derived from donor trained and sedentary PLP-immunized mice were analyzed in vitro for T-cell activation and proliferation, immune cell profiling, and cytokine mRNA levels and cytokine secretion measurements. RESULTS: Both HICT and HIIT attenuated the encephalitogenicity of PLP-reactive T cells, as indicated by reduced EAE clinical severity and inflammation and tissue pathology in the central nervous system, following their transfer into recipient mice. HICT caused a marked inhibition of PLP-induced T-cell proliferation without affecting the T-cell profile. In contrast, HIIT did not alter T-cell proliferation, but rather inhibited polarization of T cells into T-helper 1 and T-helper 17 autoreactive populations. INTERPRETATION: HICT and HIIT attenuate systemic autoimmunity and T cell encephalitogenicity by distinct immunomodulatory mechanisms.

Application of antibody phage display to identify potential antigenic neural precursor cell proteins.

BACKGROUND: The discovery of neural precursor cells (NPCs) and the concomitant intensive research in the field offer regenerative medicine novel approaches, enabling it to tackle conditions, such as neurodegenerative diseases. Transplantation of NPCs is nowadays considered a cutting-edge treatment for these conditions and many related clinical trials have been already completed or are still ongoing. However, little is known about the antigenicity of NPCs, with most studies addressing the question whether their antigenicity could lead to rejection of the transplanted cells. RESULTS: In this study we investigated the antigenic potential of syngeneic NPCs emulsion, upon subcutaneous (s.c.) administration to wild type C57BL/6 mice, following a standard immunization protocol. The whole IgG repertoire expressed upon immunization was cloned into a Fab phage display vector. From the created phage display library, Fab expressing clones interacting with NPCs lysate proteins were selected with the biopanning technique. The IgG Fab fragment from clone 65 proved to be reactive against antigens originating from NPCs lysates and/or whole brain lysate in diverse immunological assays. CONCLUSIONS: Using a standard immunization protocol to administer NPCs antigens, and applying the Fab fragment phage display technique, we were able to isolate at least a monoclonal IgG Fab fragment, which interacts with different mouse brain proteins. It is not clear whether such antibodies are produced in the host organisms, following NPCs transplantation.

Tauopathy in the young autistic brain: novel biomarker and therapeutic target.

Given our recent discovery of somatic mutations in autism spectrum disorder (ASD)/intellectual disability (ID) genes in postmortem aged Alzheimer's disease brains correlating with increasing tauopathy, it is important to decipher if tauopathy is underlying brain imaging results of atrophy in ASD/ID children. We concentrated on activity-dependent neuroprotective protein (ADNP), a prevalent autism gene. The unique availability of multiple postmortem brain sections of a 7-year-old male, heterozygous for ADNP de novo mutation c.2244Adup/p.His559Glnfs*3 allowed exploration of tauopathy, reflecting on a general unexplored mechanism. The tested subject exhibited autism, fine motor delays, severe intellectual disability and seizures. The patient died after multiple organ failure following liver transplantation. To compare to other ADNP syndrome mutations, immortalized lymphoblastoid cell lines from three different patients (including ADNP p.Arg216*, p.Lys408Valfs*31, and p.Tyr719* heterozygous dominant mutations) and a control were subjected to RNA-seq. Immunohistochemistry, high-throughput gene expression profiles in numerous postmortem tissues followed. Comparisons to a control brain and to extensive datasets were used. Live cell imaging investigated Tau-microtubule interaction, protecting against tauopathy. Extensive child brain tauopathy paralleled by multiple gene expression changes was discovered. Tauopathy was explained by direct mutation effects on Tau-microtubule interaction and correction by the ADNP active snippet NAP. Significant pathway changes (empirical P value < 0.05) included over 100 genes encompassing neuroactive ligand-receptor and cytokine-cytokine receptor interaction, MAPK and calcium signaling, axon guidance and Wnt signaling pathways. Changes were also seen in steroid biosynthesis genes, suggesting sex differences. Selecting the most affected genes by the ADNP mutations for gene expression analysis, in multiple postmortem tissues, identified Tau (MAPT)-gene-related expression changes compared with extensive normal gene expression (RNA-seq) databases. ADNP showed relatively reduced expression in the ADNP syndrome cerebellum, which was also observed for 25 additional genes (representing >50% of the tested genes), including NLGN1, NLGN2, PAX6, SMARCA4, and SNAP25, converging on nervous system development and tauopathy. NAP provided protection against mutated ADNP disrupted Tau-microtubule association. In conclusion, tauopathy may explain brain-imaging findings in ADNP syndrome children and may provide a new direction for the development of tauopathy protecting drug candidates like NAP in ASD/ID.

Spatio-temporal expression profile of NGF and the two-receptor system, TrkA and p75NTR, in experimental autoimmune encephalomyelitis.

BACKGROUND: Nerve growth factor (NGF) and its receptors, tropomyosin receptor kinase A (TrkA) and pan-neurotrophin receptor p75 (p75NTR), are known to play bidirectional roles between the immune and nervous system. There are only few studies with inconclusive results concerning the expression pattern and role of NGF, TrkA, and p75NTR (NGF system) under the neuroinflammatory conditions in multiple sclerosis (MS) and its mouse model, the experimental autoimmune encephalomyelitis (EAE). The aim of this study is to investigate the temporal expression in different cell types of NGF system in the central nervous system (CNS) during the EAE course. METHODS: EAE was induced in C57BL/6 mice 6-8 weeks old. CNS tissue samples were collected on specific time points: day 10 (D10), days 20-22 (acute phase), and day 50 (chronic phase), compared to controls. Real-time PCR, Western Blot, histochemistry, and immunofluorescence were performed throughout the disease course for the detection of the spatio-temporal expression of the NGF system. RESULTS: Our findings suggest that both NGF and its receptors, TrkA and p75NTR, are upregulated during acute and chronic phase of the EAE model in the inflammatory lesions in the spinal cord. NGF and its receptors were co-localized with NeuN+ cells, GAP-43+ axons, GFAP+ cells, Arginase1+ cells, and Mac3+ cells. Furthermore, TrkA and p75NTR were sparsely detected on CNPase+ cells within the inflammatory lesion. Of high importance is our observation that despite EAE being a T-mediated disease, only NGF and p75NTR were shown to be expressed by B lymphocytes (B220+ cells) and no expression on T lymphocytes was noticed. CONCLUSION: Our results indicate that the components of the NGF system are subjected to differential regulation during the EAE disease course. The expression pattern of NGF, TrkA, and p75NTR is described in detail, suggesting possible functional roles in neuroprotection, neuroregeneration, and remyelination by direct and indirect effects on the components of the immune system.

Exercise intensity-dependent immunomodulatory effects on encephalomyelitis.

BACKGROUND: Exercise training (ET) has beneficial effects on multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the intensity-dependent effects of ET on the systemic immune system in EAE remain undefined. OBJECTIVE: (1) To compare the systemic immune modulatory effects of moderate versus high-intensity ET protocols in protecting against development of EAE; (2) To investigate whether ET affects autoimmunity selectively, or causes general immunosuppression. METHODS: Healthy mice performed moderate or high-intensity treadmill running programs. Proteolipid protein (PLP)-induced transfer EAE was utilized to examine ET effects specifically on the systemic immune system. Lymph node (LN)-T cells from trained versus sedentary donor mice were transferred to naïve recipients and EAE severity was assessed, by clinical assessment and histopathological analysis. LN-T cells derived from donor trained versus sedentary PLP-immunized mice were analyzed in vitro for proliferation assays by flow cytometry analysis and cytokine and chemokine receptor gene expression using real-time PCR. T cell-dependent immune responses of trained versus sedentary mice to the nonautoantigen ovalbumin and susceptibility to Escherichia coli-induced acute peritonitis were examined. RESULTS: High-intensity training in healthy donor mice induced significantly greater inhibition than moderate-intensity training on proliferation and generation of encephalitogenic T cells in response to PLP-immunization, and on EAE severity upon their transfer into recipient mice. High-intensity training also inhibited LN-T cell proliferation in response to ovalbumin immunization. E. coli bacterial counts and dissemination were not affected by training. INTERPRETATION: High-intensity training induces superior effects in preventing autoimmunity in EAE, but does not alter immune responses to E. coli infection.

The autism/neuroprotection-linked ADNP/NAP regulate the excitatory glutamatergic synapse.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, was discovered as a leading de novo mutated gene causing the autism-like ADNP syndrome. This syndrome is phenotypically characterized by global developmental delays, intellectual disabilities, speech impediments, and motor dysfunctions. The Adnp haploinsufficient mouse mimics the human ADNP syndrome in terms of synapse density and gene expression patterns, as well as in developmental, motor, and cognitive abilities. Peripheral ADNP was also discovered as a biomarker for Alzheimer's disease and schizophrenia, with nasal administration of the ADNP snippet peptide NAP (enhancing endogenous ADNP activity) leading to partial cognitive and functional protection at the cellular, animal and clinical settings. Here, a novel formulation for effective delivery of NAP is provided with superior brain penetration capabilities. Also provided are methods for treating pertinent clinical implications such as autism, cognitive impairments, olfactory deficits, and muscle strength using the formulation in the Adnp haploinsufficient mouse. Results showed a dramatically specific increase in brain/body bioavailability with the new formulation, without breaching the blood brain barrier. Additional findings included improvements using daily intranasal treatments with NAP, at the behavioral and brain structural levels, diffusion tensor imaging (DTI), translatable to clinical practice. Significant effects on hippocampal and cerebral cortical expression of the presynaptic Slc17a7 gene encoding vesicular excitatory glutamate transporter 1 (VGLUT1) were observed at the RNA and immunohistochemical levels, explaining the DTI results. These findings tie for the first time a reduction in presynaptic glutamatergic synapses with the autism/Alzheimer's/schizophrenia-linked ADNP deficiency coupled with amelioration by NAP (CP201).

Atypical Auditory Brainstem Response and Protein Expression Aberrations Related to ASD and Hearing Loss in the Adnp Haploinsufficient Mouse Brain.

Autism is a wide spread neurodevelopmental disorder with growing morbidity rates, affecting more boys than girls worldwide. Activity-dependent neuroprotective protein (ADNP) was recently recognized as a leading gene accounted for 0.17% of autism spectrum disorder (ASD) cases globally. Respectively, mutations in the human ADNP gene (ADNP syndrome), cause multi-system body dysfunctions with apparent ASD-related traits, commencing as early as childhood. The Adnp haploinsufficient (Adnp+/-) mouse model was researched before in relations to Alzheimer's disease and autism. Adnp+/- mice suffer from deficient social memory, vocal and motor impediments, irregular tooth eruption and short stature, all of which corresponds with reported phenotypes in patients with the ADNP syndrome. Recently, a more elaborated description of the ADNP syndrome was published, presenting impediments such as hearing disabilities in > 10% of the studied children. Irregular auditory brainstem response (ABR) has been connected to ASD-related cases and has been suggested as a potential hallmark for autism, allowing diagnosis of ASD risk and early intervention. Herein, we present detriment hearing in the Adnp+/- mice with atypical ABR and significant protein expression irregularities that coincides with ASD and hearing loss studies in the brain.

Exposure to 3-Nitropropionic Acid Mitochondrial Toxin Induces Tau Pathology in Tangle-Mouse Model and in Wild Type-Mice.

Oxidative stress, particularly of mitochondrial origin, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease (AD) and other tauopathies. Controversies regarding the responses of tau phosphorylation state to various stimuli causing oxidative stress have been reported. Here we investigated the effect of 3-nitropropionic acid (3NP), a mitochondrial toxin which induces oxidative stress, on the tangle-pathology in our previously generated double mutant (E257T/P301S, DM) -Tau-tg mice and in WT-mice. We detected an increase in tangle pathology in the hippocampus and cortex of the DM-Tau-tg mice following exposure of the mice to the toxin, as well as generation of tangles in WT-mice. This increase was accompanied with alterations in the level of the glycogen synthase kinase 3ß (GSK3ß), the kinase which phosphorylates the tau protein, and in the phosphorylation state of this kinase. A response of microglial cells was noticed. These results point to the involvement of mitochondrial dysfunction in the development of the tangle-pathology and may suggest that interfering with mitochondrial dysfunction may have an anti-tangle therapeutic potential.

Beneficial effects of environmental enrichment on behavior, stress reactivity and synaptophysin/BDNF expression in hippocampus following early life stress.

Exposure to environmental enrichment can beneficially influence the behavior and enhance synaptic plasticity. The aim of the present study was to investigate the mediated effects of environmental enrichment on postnatal stress-associated impact with regard to behavior, stress reactivity as well as synaptic plasticity changes in the dorsal hippocampus. Wistar rat pups were submitted to a 3 h maternal separation (MS) protocol during postnatal days 1-21, while another group was left undisturbed. On postnatal day 23, a subgroup from each rearing condition (maternal separation, no-maternal separation) was housed in enriched environmental conditions until postnatal day 65 (6 weeks duration). At approximately three months of age, adult rats underwent behavioral testing to evaluate anxiety (Elevated Plus Maze), locomotion (Open Field Test), spatial learning and memory (Morris Water Maze) as well as non-spatial recognition memory (Novel Object Recognition Test). After completion of behavioral testing, blood samples were taken for evaluation of stress-induced plasma corticosterone using an enzyme-linked immunosorbent assay (ELISA), while immunofluorescence was applied to evaluate hippocampal BDNF and synaptophysin expression in dorsal hippocampus. We found that environmental enrichment protected against the effects of maternal separation as indicated by the lower anxiety levels and the reversal of spatial memory deficits compared to animals housed in standard conditions. These changes were associated with increased BDNF and synaptophysin expression in the hippocampus. Regarding the neuroendocrine response to stress, while exposure to an acute stressor potentiated corticosterone increases in maternally-separated rats, environmental enrichment of these rats prevented this effect. The current study aimed at investigating the compensatory role of enriched environment against the negative outcomes of adverse experiences early in life concurrently on emotional and cognitive behaviors, HPA function and neuroplasticity markers.

Exercise training attenuates experimental autoimmune encephalomyelitis by peripheral immunomodulation rather than direct neuroprotection.

BACKGROUND: Conflicting results exist on the effects of exercise training (ET) on Experimental Autoimmune Encephalomyelitis (EAE), nor is it known how exercise impacts on disease progression. OBJECTIVE: We examined whether ET ameliorates the development of EAE by modulating the systemic immune system or exerting direct neuroprotective effects on the CNS. METHODS: Healthy mice were subjected to 6weeks of motorized treadmill running. The Proteolipid protein (PLP)-induced transfer EAE model in mice was utilized. To assess effects of ET on systemic autoimmunity, lymph-node (LN)-T cells from trained- vs. sedentary donor mice were transferred to naïve recipients. To assess direct neuroprotective effects of ET, PLP-reactive LN-T cells were transferred into recipient mice that were trained prior to EAE transfer or to sedentary mice. EAE severity was assessed in vivo and the characteristics of encephalitogenic LN-T cells derived from PLP-immunized mice were evaluated in vitro. RESULTS: LN-T cells obtained from trained mice induced an attenuated clinical and pathological EAE in recipient mice vs. cells derived from sedentary animals. Training inhibited the activation, proliferation and cytokine gene expression of PLP-reactive T cells in response to CNS-derived autoantigen, but strongly enhanced their proliferation in response to Concanavalin A, a non-specific stimulus. However, there was no difference in EAE severity when autoreactive encephalitogenic T cells were transferred to trained vs. sedentary recipient mice. CONCLUSION: ET inhibits immune system responses to an auto-antigen to attenuate EAE, rather than generally suppressing the immune system, but does not induce a direct neuro-protective effect against EAE.

Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents.

BACKGROUND: Neural precursor cells (NPCs) located in the subventricular zone (SVZ), a well-defined NPC niche, play a crucial role in central nervous system (CNS) homeostasis. Moreover, NPCs are involved in the endogenous reparative process both in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the possibility that NPCs may be vulnerable to immune-related components may not be ruled out. Therefore, we investigated the potential affinity of myelin oligodendrocyte glycoprotein (MOG)-induced humoral response(s) to NPCs. METHODS: MOG35-55-EAE was induced in C57BL/6 mice; blood-sampling was performed on days 17-21 (acute phase) along with a naive group and corresponding antisera (AS) were collected (EAE-AS, NAIVE-AS). The presence of anti-CNS autoantibodies was examined with western blotting. Furthermore, using the collected antisera and anti-MOG antibody (as positive control), immunohistochemistry and double immunofluorescence were implemented on normal neonatal, postnatal, and adult mouse brain sections. Targeted NPCs were identified with confocal microscopy. In vitro immunoreactivity assessment on NPCs challenged with autoantibodies was evaluated for apoptotic/autophagic activity. RESULTS: Western blotting verified the existence of autoantibodies in EAE mice and demonstrated bands corresponding to yet unidentified NPC surface epitopes. A dominant selective binding of EAE-AS in the subventricular zone in all age groups compared to NAIVE-AS (p < 0.001) was observed. Additionally, anti-BrdU+/EAE-AS+ colocalization was significantly higher than anti-BrdU+/anti-MOG+, a finding suggesting that the EAE humoral response colocalized with NPCs(BrdU+), cells that do not express MOG. Well-established NPC markers (Nestin, m-Musashi-1, Sox2, DCX, GFAP, NG2) were used to identify the distinct cell types which exhibited selective binding with EAE-AS. The findings verified that EAE-AS exerts cross-reactivity with NPCs which varies throughout the neonatal to adult stage, with a preference to cells of early developmental stages. Finally, increased expressions of Caspase 3 and Beclin 1 on NPCs were detected. CONCLUSION: We provide evidence for the first time that MOG35-55 EAE induces production of antibodies with affinity to SVZ of naive mice in three different age groups. These autoantibodies target lineage-specific NPCs as brain develops and have the potential to trigger apoptotic pathways. Thus, our findings provide indication that cross-talk between immunity and NPCs may lead to functional alteration of NPCs regarding their viability and potentially oligodendrogenesis and effective remyelination.

Cyclic MOG35-55 ameliorates clinical and neuropathological features of experimental autoimmune encephalomyelitis.

EAE is induced to susceptible mice using linear peptides of myelin proteins of the central nervous system. Specific peptide motifs within the peptide-binding groove of the MHC peptide-complex determines the affinity of the peptide in each animal and the consequent T-cell receptor recognition and activation of the cell. Altered peptide ligand (APL) vaccination is a novel approach based on an effort to induce T-cell tolerance or alter cytokine profile from pro-inflammatory to anti-inflammatory. In the present study we synthesized the MOG35-55 peptide and altered its 3-dimensional conformation to make it a cyclic one (c-MOG35-55). EAE was induced in C57BL/6 mice and pathology was studied on acute and chronic phase of the disease. Our data indicates that c-MOG35-55 peptide alone induces a mild transient acute phase without chronic axonopathy. Administration of the c-MOG35-55 peptide at a 1:1 ratio during disease induction significantly ameliorates clinical disease and underlying pathology, such as demyelination and axonopathy in the acute and chronic phases. Binding and structural studies revealed milder interactions between the c-MOG35-55 and mouse or human MHC class II alleles (H2-IAb and HLA-DR2). Collectively, we provide data supporting for the first time the concept that the cyclic modification of an established encephalitogenic peptide ameliorates the clinical outcomes and underlying pathological processes of EAE. Such a cyclic modification of linear peptides could provide a novel treatment approach for future, patient-selective, immunomodulative treatments of multiple sclerosis.

Long-term effects of enriched environment following neonatal hypoxia-ischemia on behavior, BDNF and synaptophysin levels in rat hippocampus: Effect of combined treatment with G-CSF.

Increasing evidence shows that exposure to an enriched environment (EE) is neuroprotective in adult and neonatal animal models of brain ischemia. However, the mechanisms underlying this effect remain unclear. The aim of the current study was to investigate whether post-weaning EE would be effective in preventing functional deficits and brain damage by affecting markers of synaptic plasticity in a neonatal rat model of hypoxia-ischemia (HI). We also examined the possibility that granulocyte-colony stimulating factor (G-CSF), a growth factor with known neuroprotective effects in a variety of experimental brain injury models, combined with EE stimulation could enhance the potential beneficial effect of EE. Seven-day-old Wistar rats of either sex were subjected to permanent ligation of the left common carotid artery followed by 60min of hypoxia (8% O2) and immediately after weaning (postnatal day 21) were housed in enriched conditions for 4weeks. A group of enriched-housed rats had been treated with G-CSF immediately after HI for 5 consecutive days (50µg/kg/day). Behavioral examination took place approximately at three months of age and included assessments of learning and memory (Morris water maze) as well as motor coordination (Rota-Rod). Infarct size and hippocampal area were estimated following behavioral assessment. Synaptic plasticity was evaluated based on BDNF and synaptophysin expression in the dorsal hippocampus. EE resulted in recovery of post-HI motor deficits and partial improvement of memory impairments which was not accompanied by reduced brain damage. Increased synaptophysin expression was observed in the contralateral to carotid ligation hemisphere. Hypoxia-ischemia alone or followed by enriched conditions did not affect BDNF expression which was increased only in enriched-housed normal rats. The combined therapy of G-CSF and EE further enhanced cognitive function compared to EE provided as monotherapy and prevented HI-induced brain damage by altering synaptic plasticity as reflected by increased synaptophysin expression. The above findings demonstrate that combination of neuroprotective treatments may result in increased protection and it might be a more effective strategy for the treatment of neonatal hypoxic-ischemic brain injury.