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Multiple sclerosis (MS) and its preclinical models are characterized by marked changes in neuroplasticity, including excitatory/inhibitory imbalance and synaptic dysfunction that are believed to underlie the progressive cognitive impairment (CI), which represents a significant clinical hallmark of the disease. In this study, we investigated several parameters of neuroplasticity in the hippocampus of the experimental autoimmune encephalomyelitis (EAE) SJL/J mouse model, characterized by rostral inflammatory and demyelinating lesions similar to Relapsing-Remitting MS. By combining morphological and molecular analyses, we found that the hippocampus undergoes extensive inflammation in EAE-mice, more pronounced in the CA3 and dentate gyrus (DG) subfields than in the CA1, associated with changes in GABAergic circuitry, as indicated by the increased expression of the interneuron marker Parvalbumin selectively in CA3. By laser-microdissection, we investigated the impact of EAE on the alternative splicing of Arhgef9, a gene encoding a post-synaptic protein playing an essential role in GABAergic synapses and whose mutations have been related to CI and epilepsy. Our results indicate that EAE induces a specific increase in inclusion of the alternative exon 11a only in the CA3 and DG subfields, in line with the higher local levels of inflammation. Consistently, we found a region-specific downregulation of Sam68, a splicing-factor that represses this splicing event. Collectively, our findings confirm a regionalized distribution of inflammation in the hippocampus of EAE-mice. Moreover, since neuronal circuit rearrangement and dynamic remodeling of structural components of the synapse are key processes that contribute to neuroplasticity, our study suggests potential new molecular players involved in EAE-induced hippocampal dysfunction.

Autism spectrum disorder (ASD) includes a set of highly heritable neurodevelopmental syndromes characterized by social and communication impairment, repetitive behaviour, and intellectual disability. Although mutations in multiple genes have been associated to ASD, most patients lack detectable genetic alterations. For this reason, environmental factors are commonly thought to also contribute to ASD aetiology. Transcriptome analyses have revealed that autistic brains possess distinct gene expression signatures, whose elucidation can provide insights about the mechanisms underlying the effects of ASD-causing genetic and environmental factors. Herein, we have identified a coordinated and temporally regulated programme of gene expression in the post-natal development of cerebellum, a brain area whose defects are strongly associated with ASD. Notably, this cerebellar developmental programme is significantly enriched in ASD-linked genes. Clustering analyses highlighted six different patterns of gene expression modulated during cerebellar development, with most of them being enriched in functional processes that are frequently dysregulated in ASD. By using the valproic acid mouse model of ASD, we found that ASD-linked genes are dysregulated in the developing cerebellum of ASD-like mice, a defect that correlates with impaired social behaviour and altered cerebellar cortical morphology. Moreover, changes in transcript levels were reflected in aberrant protein expression, indicating the functional relevance of these alterations. Thus, our work uncovers a complex ASD-related transcriptional programme regulated during cerebellar development and highlight genes whose expression is dysregulated in this brain area of an ASD mouse model.

Microglia, besides being able to react rapidly to a wide range of environmental changes, are also involved in shaping neuronal wiring. Indeed, they actively participate in the modulation of neuronal function by regulating the elimination (or "pruning") of weaker synapses in both physiologic and pathologic processes. Mounting evidence supports their crucial role in early synaptic loss, which is emerging as a hallmark of several neurodegenerative diseases, including multiple sclerosis (MS) and its preclinical models. MS is an inflammatory, immune-mediated pathology of the white matter in which demyelinating lesions may cause secondary neuronal death. Nevertheless, primitive grey matter (GM) damage is emerging as an important contributor to patients' long-term disability, since it has been associated with early and progressive cognitive decline (CD), which seriously worsens the quality of life of MS patients. Widespread synapse loss even in the absence of demyelination, axon degeneration and neuronal death has been demonstrated in different GM structures, thus raising the possibility that synaptic dysfunction could be an early and possibly independent event in the neurodegenerative process associated with MS. This review provides an overview of microglial-dependent synapse elimination in the neuroinflammatory process that underlies MS and its experimental models.

S100B is a Ca 2+ -binding protein mainly concentrated in astrocytes. Its levels in biological fluids (cerebrospinal fluid, peripheral and cord blood, urine, saliva, amniotic fluid) are recognized as a reliable biomarker of active neural distress. Although the wide spectrum of diseases in which the protein is involved (acute brain injury, neurodegenerative diseases, congenital/perinatal disorders, psychiatric disorders) reduces its specificity, its levels remain an important aid in monitoring the trend of the disorder. Mounting evidence now points to S100B as a Damage-Associated Molecular Pattern molecule which, when released at high concentration, through its Receptor for Advanced Glycation Endproducts, triggers tissue reaction to damage in a series of different neural disorders. This review addresses this novel scenario, presenting data indicating that S100B levels and/or distribution in the nervous tissue of patients and/or experimental models of different neural disorders, for which the protein is used as a biomarker, are directly related to the progress of the disease: acute brain injury (ischemic/hemorrhagic stroke, traumatic injury), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis), congenital/perinatal disorders (Down syndrome, spinocerebellar ataxia-1), psychiatric disorders (schizophrenia, mood disorders), inflammatory bowel disease. In many cases, over-expression/administration of the protein induces worsening of the disease, whereas its deletion/inactivation produces amelioration. This review points out that the pivotal role of the protein resulting from these data, opens the perspective that S100B may be regarded as a therapeutic target for these different diseases, which appear to share some common features reasonably attributable to neuroinflammation, regardless their origin. (Figure presented.).

Nonsyndromic craniosynostosis (NCS) is the premature ossification of skull sutures, without associated clinical features. Mutations in several genes account for a small number of NCS patients; thus, the molecular etiopathogenesis of NCS remains largely unclear. Our study aimed at characterizing the molecular signaling implicated in the aberrant ossification of sutures in NCS patients. Comparative gene expression profiling of NCS patient sutures identified a fused suture-specific signature, including 17 genes involved in primary cilium signaling and assembly. Cells from fused sutures displayed a reduced potential to form primary cilia compared to cells from control patent sutures of the same patient. We identified specific upregulated splice variants of the Bardet Biedl syndrome-associated gene 9 (BBS9), which encodes a structural component of the ciliary BBSome complex. BBS9 expression increased during in vitro osteogenic differentiation of suture-derived mesenchymal cells of NCS patients. Also, Bbs9 expression increased during in vivo ossification of rat sutures. BBS9 functional knockdown affected the expression of primary cilia on patient suture cells and their osteogenic potential. Computational modeling of the upregulated protein isoforms (observed in patients) predicted that their binding affinity within the BBSome may be affected, providing a possible explanation for the aberrant suture ossification in NCS.

Despite the intense research on adult neural stem cell biology suggested possible translational outcomes in regenerative medicine for neurodegenerative diseases, neuroregeneration is unlikely to occur in adult brain, due to intrinsic features that characterize the neural stem cell niche. Mesenchymal stem cells (MSCs), osteogenic stem cells residing in the bone marrow stroma (also named bone marrow stromal cells), have been long considered highly plastic multipotent precursors, able to commit toward diversified lineages, including non-mesodermal ones. Their in vitro plasticity and ease of processing prompted their wide, sometimes untimely, exploitation in diversified regenerative medicine applications (Park et al., 2012; Bianco et al., 2013). They have been tested also for their putative, yet widely debated, neuroregenerative potential. This controversial issue stimulated this Research Topic, which aims to delve into relevant scientific milestones addressing the differences, possible interconnections, and overlaps between the osteogenic and the neurogenic niches' biology. The debated neuronal transdifferentiation potential of MSCs recently led to their inappropriate exploitation for the treatment of neurodegenerative disorders. The regulatory and ethical issues regarding this topic have been discussed in the Opinion paper by Solarino et al., delving into a recent Italian case of medical malpractice, which triggered significant international dispute (Abbott, 2013; Blasimme and Rial-Sebbag, 2013). Indeed, a better clarification of the specific features displayed by the osteogenic and the neurogenic stem cell niches is needed, as discussed by Lattanzi et al. This mini-review provides a pairwise comparison of the two niches within their in vivo environments, highlighting functionally relevant similarities and differences that should be considered to achieve a more rational clinical translation. The contribution by Salgado et al. provides an exhaustive description of oste

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Reelin is an extracellular matrix glycoprotein involved in the modulation of synaptic plasticity and essential for the proper radial migration of cortical neurons during development and for the integration and positioning of dentate granular cell progenitors; its expression is down-regulated as brain maturation is completed. Trimethyltin (TMT) is a potent neurotoxicant which causes selective neuronal death mainly localised in the CA1-CA3/hilus hippocampal regions. In the present study we analysed the expression of reelin and the modulation of endogenous neurogenesis in the postnatal rat hippocampus during TMT-induced neurodegeneration (TMT 6 mg/kg). Our results show that TMT administration induces changes in the physiological postnatal decrease of reelin expression in the hippocampus of developing rats. In particular, quantitative analysis of reelin-positive cells evidenced, in TMT-treated animals, a persistent reelin expression in the stratum lacunosum moleculare of Cornu Ammonis and in the molecular layer of Dentate Gyrus. In addition, a significant decrease in the number of bromodeoxyuridine (BrdU)-labeled newly-generated cells was also detectable in the subgranular zone of P21 TMT-treated rats compared with P21 control animals; no differences between P28 TMT-treated rats and age-matched control group were observed. In addition the neuronal commitment of BrdU-positive cells appeared reduced in P21 TMT-treated rats compared with P28 TMT-treated animals. Thus TMT treatment, administrated during development, induces an early reduction of endogenous neurogenesis and influences the hippocampal pattern of reelin expression in a temporally and regionally specific manner, altering the physiological decrease of this protein.

Neuropeptides are emerging as key regulators of stem cell niche activities in health and disease, both inside and outside the central nervous system (CNS). Among them, neuropeptide Y (NPY), one of the most abundant neuropeptides both in the nervous system and in non-neural districts, has become the focus of much attention for its involvement in a wide range of physiological and pathological conditions, including the modulation of different stem cell activities. In particular, a pro-neurogenic role of NPY has been evidenced in the neurogenic niche, where a direct effect on neural progenitors has been demonstrated, while different cellular types, including astrocytes, microglia and endothelial cells, also appear to be responsive to the peptide. The marked modulation of the NPY system during several pathological conditions that affect neurogenesis, including stress, seizures and neurodegeneration, further highlights the relevance of this peptide in the regulation of adult neurogenesis. In view of the considerable interest in understanding the mechanisms controlling neural cell fate, this review aims to summarize and discuss current data on NPY signaling in the different cellular components of the neurogenic niche in order to elucidate the complexity of the mechanisms underlying the modulatory properties of this peptide.

Modulation of endogenous neurogenesis is regarded as a promising challenge in neuroprotection. In the rat model of hippocampal neurodegeneration obtained by Trimethyltin (TMT) administration (8 mg/kg), characterised by selective pyramidal cell loss, enhanced neurogenesis, seizures and cognitive impairment, we previously demonstrated a proliferative role of exogenous neuropeptide Y (NPY), on dentate progenitors in the early phases of neurodegeneration. To investigate the functional integration of newly-born neurons, here we studied in adult rats the long-term effects of intracerebroventricular administration of NPY (2 µg/2 µl, 4 days after TMT-treatment), which plays an adjuvant role in neurodegeneration and epilepsy. Our results indicate that 30 days after NPY administration the number of new neurons was still higher in TMT+NPY-treated rats than in control+saline group. As a functional correlate of the integration of new neurons into the hippocampal network, long-term potentiation recorded in Dentate Gyrus (DG) in the absence of GABAA receptor blockade was higher in the TMT+NPY-treated group than in all other groups. Furthermore, qPCR analysis of Kruppel-like factor 9, a transcription factor essential for late-phase maturation of neurons in the DG, and of the cyclin-dependent kinase 5, critically involved in the maturation and dendrite extension of newly-born neurons, revealed a significant up-regulation of both genes in TMT+NPY-treated rats compared with all other groups. To explore the early molecular events activated by NPY administration, the Sonic Hedgehog (Shh) signalling pathway, which participates in the maintenance of the neurogenic hippocampal niche, was evaluated by qPCR 1, 3 and 5 days after NPY-treatment. An early significant up-regulation of Shh expression was detected in TMT+NPY-treated rats compared with all other groups, associated with a modulation of downstream genes. Our data indicate that the neurogenic effect of NPY administration during TMT-in

Trimethyltin (TMT) is an organotin compound exhibiting neurotoxicant effects selectively localized in the limbic system and especially marked in the hippocampus, in both experimental animal models and accidentally exposed humans. TMT administration causes selective neuronal death involving either the granular neurons of the dentate gyrus or the pyramidal cells of the Cornu Ammonis, with a different pattern of localization depending on the different species studied or the dosage schedule. TMT is broadly used to realize experimental models of hippocampal neurodegeneration associated with cognitive impairment and temporal lobe epilepsy, though the molecular mechanisms underlying the associated selective neuronal death are still not conclusively clarified. Experimental evidence indicates that TMT-induced neurodegeneration is a complex event involving different pathogenetic mechanisms, probably acting differently in animal and cell models, which include neuroinflammation, intracellular calcium overload, and oxidative stress. Microarray-based, genome-wide expression analysis has been used to investigate the molecular scenario occurring in the TMT-injured brain in different in vivo and in vitro models, producing an overwhelming amount of data. The aim of this review is to discuss and rationalize the state-of-the-art on TMT-associated genome wide expression profiles in order to identify comparable and reproducible data that may allow focusing on significantly involved pathways.

The selective vulnerability of specific neuronal subpopulations to trimethyltin (TMT), an organotin compound with neurotoxicant effects selectively involving the limbic system and especially marked in the hippocampus, makes it useful to obtain in vivo models of neurodegeneration associated with behavioural alterations, such as hyperactivity and aggression, cognitive impairment as well as temporal lobe epilepsy. TMT has been widely used to study neuronal and glial factors involved in selective neuronal death, as well as the molecular mechanisms leading to hippocampal neurodegeneration (including neuroinflammation, excitotoxicity, intracellular calcium overload, mitochondrial dysfunction and oxidative stress). It also offers a valuable instrument to study the cell-cell interactions and signalling pathways that modulate injury-induced neurogenesis, including the involvement of newly generated neurons in the possible repair processes. Since TMT appears to be a useful tool to damage the brain and study the various responses to damage, this review summarises current data from in vivo and in vitro studies on neuroprotective strategies to counteract TMT-induced neuronal death, that may be useful to elucidate the role of putative candidates for translational medical research on neurodegenerative diseases.

S100B is a calcium-binding protein concentrated in glial cells, although it has also been detected in definite extra-neural cell types. Its biological role is still debated. When secreted, S100B is believed to have paracrine/autocrine trophic effects at physiological concentrations, but toxic effects at higher concentrations. Elevated S100B levels in biological fluids (CSF, blood, urine, saliva, amniotic fluid) are thus regarded as a biomarker of pathological conditions, including perinatal brain distress, acute brain injury, brain tumors, neuroinflammatory/neurodegenerative disorders, psychiatric disorders. In the majority of these conditions, high S100B levels offer an indicator of cell damage when standard diagnostic procedures are still silent. The key question remains as to whether S100B is merely leaked from injured cells or is released in concomitance with both physiological and pathological conditions, participating at high concentrations in the events leading to cell injury. In this respect, S100B levels in biological fluids have been shown to increase in physiological conditions characterized by stressful physical and mental activity, suggesting that it may be physiologically regulated and raised during conditions of stress, with a putatively active role. This possibility makes this protein a candidate not only for a biomarker but also for a potential therapeutic target.

Trimethyltin (TMT), an organotin compound considered a useful tool to obtain an experimental model of neurodegeneration, exhibits neurotoxicant effects selectively localised in the limbic system and especially in the hippocampus, which are different in the rat and in mice. In the rat hippocampus, we investigated the expression of aldehyde 4-hydroxynonenal, a major bioactive marker of membrane lipid peroxidation, heat shock protein (HSP) 110/105 family members, markers of oxidative stress, and the neuroinflammatory marker cyclooxygenase-2 after TMT-intoxication at various time points after treatment. Our data show that TMT-induced neurodegeneration in the rat hippocampus is associated specifically with oxidative stress and lipid peroxidation, but not with HSP expression, indicating species-specific differences in the neurotoxicity of TMT between rats and mice.

Due to its abundance, easy retrieval, and plasticity characteristics, adipose-tissue-derived stromal cells (ATSCs) present unquestionable advantages over other adult-tissue-derived stem cells. Based on the in silico analysis of our previous data reporting the ATSC-specific expression profiles, the present study attempted to clarify and validate at the functional level the expression of the neurospecific genes expressed by ATSC both in vitro and in vivo. This allowed evidencing that ATSCs express neuro-specific trophins, metabolic genes, and neuroprotective molecules. They were in fact able to induce neurite outgrowth in vitro, along with tissue-specific commitment along the neural lineage and the expression of the TRKA neurotrophin receptor in vivo. Our observation adds useful information to recent evidence proposing these cells as a suitable tool for cell-based applications in neuroregenerative medicine.

In the nervous system, protease-activated receptors (PARs), which are activated by thrombin and other extracellular proteases, are expressed widely at both neuronal and glial levels and have been shown to be involved in several brain pathologies. As far as the glial receptors are concerned, previous experiments performed in rat hippocampus showed that expression of PAR-1, the prototypic member of the PAR family, increased in astrocytes both in vivo and in vitro following treatment with trimethyltin (TMT). TMT is an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. In the present experiments, the authors extended their investigation to microglial cells. In particular, by 7 days following TMT intoxication in vivo, confocal immunofluorescence revealed an evident PAR-1-related specific immunoreactivity in OX-42-positive microglial cells of the CA3 and hilus hippocampal regions. In line with the in vivo results, when primary rat microglial cells were treated in vitro with TMT, a strong upregulation of PAR-1 was observed by immunocytochemistry and Western blot analysis. These data provide further evidence that PAR-1 may be involved in microglial response to brain damage.

Trimethyltin (TMT), an organotin compound with neurotoxicant effects selectively localised in the limbic system and especially marked in the hippocampus, is considered a useful tool to obtain an experimental model of neurodegeneration. Animals exposed to TMT develop behavioural alterations (hyperactivity, aggression), cognitive impairment (memory loss and learning impairment) and spontaneous seizures. TMT induces selective neuronal death involving the granular neurons of the Fascia Dentata and the pyramidal cells of the Cornu Ammonis, with a different pattern of severity and extension according to the various species studied and the dosage schedule. TMT-induced neurodegenerative events are associated with the activation of astrocytes and microglial cells and with the upregulation of proinflammatory cytokines. While the mechanisms by which TMT induces neurodegeneration are still not understood, many hypotheses seem to suggest that neuronal damage could be largely dependent on calcium overload. This review summarizes current data from in vivo and in vitro studies of the neurotoxic effects of TMT, focusing on the hypotheses regarding the mechanisms leading to neuronal death induced by the toxin.

Nitric oxide (NO) and purinergic ionotropic receptors (P2X) mediate cellular events in the central nervous system (CNS) under physiological conditions as well as during pathological events, and they have been recently proposed to interact in mediating CNS response to injury (Viscomi et al. [2004] Neuroscience 123:393-404; Florenzano et al. [2008] Pflugers Arch. 452:622-644). Trimethyltin (TMT) is an organotin compound that generates neurotoxic effects, and it has been used in a model of neurodegenerative disease and memory dysfunction. TMT causes neuronal death and reactive gliosis primarily in the hippocampus and other limbic regions. In the present study, we examined the degenerative events and the expression of nitric oxide synthase (NOS) and P2X receptor subtypes (P2X(1,2,4,7)Rs) that were induced by TMT administration at different time points (3, 7, 14, and 21 days) by conventional and confocal microscopy and Western blotting. Massive glial activation and neuronal death in the CA1 and CA3 regions were observed after TMT treatment. In these areas, astrocytic P2X(2)R and neuronal NOS were temporarily enhanced in association with the progression of neuronal death. In the hippocampus, the physiological expression of P2X(1)R, P2X(4)R, and P2X(7)R was not modified by TMT. The present data demonstrate that, as in other neurodegenerative models, TMT-induced hippocampal degeneration is associated with nitrergic and purinergic activations. Nevertheless, at odds with previous data, in this model the two systems are active in segregated cell populations, namely, P2XR in astrocytes and NOS in neurons. Finally, the temporal relations between P2XR and NOS expression and neuronal degeneration suggest interactions between P2XR/NO signaling and cell survival

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139–151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMSp139). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMSp139 was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMSp139 modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4+ T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMSp139 because lymph node APCs infected with rMSp139 selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMSp139 expanded p139-specific CD8+ cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross–reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

We infected SJL mice with a recombinant M. smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein (PLP) 139-151 (p139) fused to MPT64, a secreted protein of M. tb (rMS p139). Infected mice developed a relapsing EAE showing a prevailing demyelination of the CNS and disease severity was significantly lower in comparison to the one that follows immunization with p139. rMS p139 was not detected in LN or spleen in the course of clinical disease development, or in the CNS during relapse. Infection with rMS p139 modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4+ T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease, were not activated by infection with rMSp139 since lymph node APC infected with rMS p139 selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMSp139 expanded p139-specific CD8+ cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross reactive non-pathogenic mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

Type II collagen is a DR4/DR1 restricted target of self-reactive T cells that sustain rheumatoid arthritis. The aim of the present study was to analyze the T-cell receptor repertoire at the onset of and at different phases in rheumatoid arthritis. We used the CDR3 BV-BJ spectratyping to study the response to human collagen peptide 261-273 in 12 patients with DR4+ rheumatoid arthritis (six at the onset of disease and six during the course of disease) and in five healthy DR4+ relatives. The collagen-specific T-cell repertoire is quite restricted at the onset of disease, involving approximately 10 rearrangements. Within the studied collagen-specific rearrangements, nearly 75% is shared among patients. Although the size of the repertoire used by control individuals is comparable to that of patients, it is characterized by different T-cell receptors. Part of the antigen-specific T-cell repertoire is spontaneously enriched in synovial fluid. The specific T-cell repertoire in the periphery was modulated by therapy and decreased with the remission of the disease. Failure of immunoscopy to detect this repertoire was not due to suppression of collagen-driven proliferation in vitro by CD4+ CD25+ T cells. Clinical relapse of the disease was associated with the appearance of the original collagen-specific T cells. The collagen-specific T-cell receptor repertoire in peripheral blood and synovial fluid is restricted to a limited number of rearrangements in rheumatoid arthritis. The majority of the repertoire is shared between patients with early rheumatoid arthritis and it is modulated by therapy.

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/ NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair.

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair.

Like humans, canines develop with aging beta-amyloid (Abeta) plaques and a progressive cognitive deficit on tasks similar to those used in diagnosis and follow-up of Alzheimer's disease. Owing to that, dogs are quite unique to investigate the early events taking place in the diffuse Abeta plaque maturation and its relationship with cognitive deficit. The aim of the present investigation was to study the link between the diffuse Abeta plaque maturation and the astro- and microglial reactivity. The involvement of insulin and beta-subunit of S100 protein (S100beta) overexpression in the process was also investigated. Abeta plaques were measured and counted in prefrontal cortex of 16 pet dogs of different breeds, weight and sex, classified as control and with a light or severe cognitive deficit. A correlation between canine graded cognitive deficit, diffuse plaque maturation, and S100beta (-) astrocytosis, but not with cerebrospinal fluid insulin level, was found that may reflect the very early events of Abeta deposition in Alzheimer's disease.

The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated

Thrombin, its main inhibitor (protease nexin-1) and its related receptors (protease-activated receptors, PAR-1,-2, -3, -4) were studied in rat hippocampus following administration of trimethyltin (TMT), a neurotoxin inducing neuronal degeneration and reactive gliosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry revealed that while expression of prothrombin and protease nexin-1 did not change significantly in TMT-treated hippocampi, PARs (in particular PAR-1 and to a lesser extent PAR-2 and PAR-3) were upregulated in reactive astrocytes, suggesting their involvement in neurodegeneration and in the consequent response of the nervous tissue.

In this study we used an immunocytochemical approach to study nestin expression in the rat hippocampus during trimethyltin-induced neurodegeneration at different time points (5, 10, 15, 21 and 50 days) after intoxication. Nestin is transiently expressed by a subpopulation of astroglial cells strictly associated with pyramidal neurons in those hippocampal areas severely affected by degeneration. This observation shows that cerebral tissue re-expresses this developmental protein during neurodegenerative diseases in early stages of astroglial activation.

The aged dog is considered a promising model for examining molecular and cellular processes involved in a variety of human neurological disorders. By using the canine counterpart of senile dementia of the Alzheimer's type (ccSDAT), we investigated the specific vulnerability of the gamma-aminobutyric acid (GABA) cortical subset of interneurons, characterized by their calcium-binding protein content, to neuronal death. Dogs representing a large variety of breeds were classified into three groups: young control, aged control, and ccSDAT. In all dogs, the general distribution and cell typology of parvalbumin-, calretinin-, and calbindin-positive neurons were found to be similar to those in the human. As in Alzheimer's disease patients, neurons displaying parvalbumin or calretinin immunoreactivity were resistant and the calbindin-positive ones depleted. Together with aging, amyloid deposition in its early phase (stage II) participates in this specific neuronal death, but with a lower potency. In conclusion, our data provide evidence that preservation of GABAergic cortical interneurons has to be focused on the early stage of beta-amyloid deposition. We also demonstrate the usefulness of dogs of all breeds for investigating the early phases of human brain aging and Alzheimer's disease.

Thrombin, its main inhibitor (protease nexin-1) and its related receptors (protease-activated receptors, PAR-1,-2, -3, -4) were studied in rat hippocampus following administration of trimethyltin (TMT), a neurotoxin inducing neuronal degeneration and reactive gliosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry revealed that while expression of prothrombin and protease nexin-1 did not change significantly in TMT-treated hippocampi, PARs (in particular PAR-1 and to a lesser extent PAR-2 and PAR-3) were upregulated in reactive astrocytes, suggesting their involvement in neurodegeneration and in the consequent response of the nervous tissue.

The association of motor neuron disease (MND) with rapidly progressive aphasic dementia has been recognized as a distinct clinical syndrome within the group of frontotemporal dementias (FTDs). Although the clinical and neuropsychological features of this syndrome have been defined, a small number of post-mortem studies have been published with heterogeneous neuropathological findings. We performed cognitive, neuro-imaging and neuropathological studies on a 71-year-old male with rapidly progressive aphasic dementia and MND. We initially found a selective non-fluent aphasia associated with hypoperfusion of the left frontotemporal cortex. Proton magnetic resonance spectroscopy revealed an asymmetric change of brain metabolites, with greater changes in the left temporal lobe. The bulbar manifestations of MND occurred over the following 6 months, and the patient died of bronchopneumonia. The neuropathological examination revealed loss of neurons in the hypoglossal nucleus and anterior horns of the cervical spinal cord with microvacuolation and dot-like ubiquitin-positive deposits in the frontoparietotemporal cortex, but no changes suggestive of Alzheimer's, Pick's or Lewy body disease. These findings support the conclusion that MND with rapidly progressive aphasic dementia is a distinctive clinical entity within the group of FTD-MND.

S100B is a calcium-binding protein that, in the nervous system, is mainly concentrated in glial cells. Although its biological role is still unclear, the protein is hypothesized, at high concentrations, to act in the pathogenesis of neurodegenerative processes, possibly through oxidative stress mechanisms. To investigate this hypothesis we studied the spinal cord of wobbler mice, an animal model of motor neuron degeneration. Using immunocytochemistry, we detected an overexpression of S100B in astrocytes of the cervical spinal cord of these animals. We also confirmed this finding by reverse transcriptase polymerase chain reaction. In the same spinal cord regions, scattered neurons appeared to be immunostained for 4-hydroxynonenal-modified proteins, an indicator of lipid peroxidation. This finding constitutes a sign of oxidative stress-induced neurodegeneration.

Endothelial monocyte-activating polypeptide-II (EMAP-II), a novel cytokine with proinflammatory and antiangiogenic properties, has previously been shown to be expressed by activated monocytes/microglial cells in the rat brain and was therefore considered a useful marker to stage microglial activation in inflammatory lesions. The aim of the present immunohistochemical study was to investigate expression of EMAP-II in the rat hippocampus after intoxication with the organotin compound trimethyltin (TMT). Administration of this neurotoxicant is known to produce brain damage mainly affecting the hippocampal formation, with severe neuronal cell loss being observed predominantly in regions CA-1 and CA-3. The maximum severity of TMT-induced brain damage is observed 21 days after a single ip administration. In this well-characterized model of neurodegeneration, activated microglial cells have been described to occur mainly in the early stages of TMT-induced neurotoxicity. Following TMT intoxication, we observed a significant increase in EMAP-II(+) monocytes/microglial cells in the CA-1 and the CA-3 regions. The CA-2 region, however, was largely spared. While appearance of single EMAP-II(+) microglial cells was observed already after 5 days, EMAP-II immunoreactivity reached its maximum after 21 days and persisted in some of the rats up to 35 days. These findings show a close correlation to the temporal and spatial pattern of neuronal damage described in the rat hippocampus after TMT administration previously. Thus, upregulation of EMAP-II by activated monocytes/microglial cells may serve as a sensitive marker of neurotoxic lesions in the rat brain.

The neurotoxicant trimethyltin (TMT) induces massive neuronal loss in vivo in the hippocampus of rodents, accompanied by behavioral alterations. The present study investigates the pattern of cell death after in vivo administration of TMT to adult mice. In the granular cell layer of the Dentate Gyrus, TUNEL staining detected DNA fragmentation, and apoptotic bodies were also evident. In addition, a ladder pattern of internucleosomal DNA fragmentation was shown in agarose gel electrophoresis. We show that activated caspase-3, which is known to play a pivotal role in apoptotic processes, is clearly expressed by degenerating neurons. Inducible cyclooxygenase is also expressed at cytoplasmic level by degenerating granular neurons, suggesting that this enzyme may participate in TMT-induced neurodegeneration.

In the model of trimethyltin (TMT)-induced neurodegeneration in developing rat hippocampus, calretinin (CR)-immunoreactive neurons are selectively spared and even more numerous than in controls. We investigated the possibility of an additional synthesis of CR using RT-PCR. The amount of CR mRNA increased significantly after TMT treatment. CR mRNA production after TMT treatment could hypothetically be regarded as a compensatory phenomenon in developing rats.

The effect of acetyl-l-carnitine (ALC) on behavioral deficits following neonatal anoxia (N2 100% for 25 min at 30 h after birth) was studied in the rat. Transient hyperactivity at P20–P45 postnatal days and permanent spatial memory deficits were shown by anoxic rats. A chronic ALC treatment (50 mg/kg per die injected intraperitoneally from P2, after anoxia, to P60) significantly reduced the transient increase in sniffing, rearing and locomotory activity of anoxic rats, but, mostly, ameliorated the spatial memory performances in a maze at P30–P40 and in a water maze at P50–P60. No behavioral changes were seen in ALC-treated animals that received sham-exposure at birth. On the basis of these results, the use of ALC for the treatment of perinatal asphyctic insults in children is suggested

Behavioral and neurochemical changes following enriched housing were studied in Wistar rats neonatally exposed to anoxia (100% N2 for 25 min at approximately 30 h after birth) or to sham treatment. Neonatal anoxia provoked transient hyperactivity during the P25–P40 period, and spatial memory disturbances persisting into adult life. Enriched housing, from P21, at weaning, to P60, improved behavior in open field and spatial memory abilities in a water maze, reducing the deficits that followed neonatal anoxia. Changes in the expression of the calcium binding protein parvalbumin were present in the CA1, CA3, and DG regions of the hippocampus in both sham-treated and anoxic rats exposed to enrichment. The present findings give further support to the evidence of a positive effect of enriched housing on behavior and learning of normal and lesioned animals, which is sustained by modifications in neuronal activity, and suggest that modifications in the environment can be useful to counteract the development of some neurological disturbances that follow neonatal insults, e.g., perinatal asphyxia

The consequences of neonatal anoxia (N2 100% for 25 min at 30 h after birth) on the rat hippocampus were studied 7-60 days postnatally with immunocytochemistry for gamma-aminobutyric acid (GABA), parvalbumin (PV) and calbindin-D28k (CB). In both sham-treated and anoxic rats, GABA imlmunoreactivity presented a mature expression since early stages, while PV and CB immunoreactivity showed a major postnatal development. In anoxic animals, a significant reduction in the number of hippocampal GABAimmunoreactive neurons was observed at all tilme-points analysed, a transitory effect on PV immunoreactivity was seen at P7 and P21, while no modifications in the number of CEI-immunoreactive neurons could be found. Thus, selective vulnerability of GABA-containing neurons and relative resistance of neurons in which PV or CB immunoreactivity is present or is expressed later, occur in the hippocampus after neonatal anoxia. The role of calcium binding proteins (CBP) in nerve cell protection is discussed.

Early and delayed neuronal and glial changes in the hippocampus were studied in Wistar rats following neonatal anoxia induced by 100% Nz exposure for 25 min at approximately 30 h postnatally. Sham-treatment induced a transient increase in the number of fos immunoreactive neurons in the CAl, CA2, and CA3 regions, with a peak at 120 min following handling. In contrast, a significant decrease in the number of fosstained cells was seen in the CA1 and CA2 regions at 120 min after the exposure to anoxia, compared to sham-treatment. At 150 and 240 min increased fos immunoreactivity was detected in the CA2 region of anoxic rats. Enhanced glial fibrillary acidic protein staining was seen at Postnatal Day 7 (P7) in the hippocampus of the rats exposed to neonatal anoxia, while no differences between anoxic and sham-treated animals were observed at later time-points. No alteration in nerve cell density was found at P7, while at P15 and later stages a significant reduction in neuronal density was seen in the CA1 region of anoxic rats. Thus, the rapid induction in hippocampal neuronal activity that followed sham-treatment was blocked by the neonatal anoxia, as revealed by changes in immediate early gene expression. A transient reactive astrocytosis developed in the days after the anoxic insult, followed by a loss of neurons in the CA1 region. The findings indicate that a sequence of specific neuronal and glial alterations takes place in the hippocampus after neonatal anoxia, which finally leads to a detectable, regionally restricted, neuronal loss. Moreover, inhibition in fos protein expression may be an early marker for the anoxic damage in CA1 neurons