Altered Expression of Autophagy Biomarkers in Hippocampal Neurons in a Multiple Sclerosis Animal Model.

Multiple Sclerosis (MS) is a chronic inflammatory disease that affects the brain and spinal cord. Inflammation, demyelination, synaptic alteration, and neuronal loss are hallmarks detectable in MS. Experimental autoimmune encephalomyelitis (EAE) is an animal model widely used to study pathogenic aspects of MS. Autophagy is a process that maintains cell homeostasis by removing abnormal organelles and damaged proteins and is involved both in protective and detrimental effects that have been seen in a variety of human diseases, such as cancer, neurodegenerative diseases, inflammation, and metabolic disorders. This study is aimed at investigating the autophagy signaling pathway through the analysis of the main autophagic proteins including Beclin-1, microtubule-associated protein light chain (LC3, autophagosome marker), and p62 also called sequestosome1 (SQSTM1, substrate of autophagy-mediated degradation) in the hippocampus of EAE-affected mice. The expression levels of Beclin-1, LC3, and p62 and the Akt/mTOR pathway were examined by Western blot experiments. In EAE mice, compared to control animals, significant reductions of expression levels were detectable for Beclin-1 and LC3 II (indicating the reduction of autophagosomes), and p62 (suggesting that autophagic flux increased). In parallel, molecular analysis detected the deregulation of the Akt/mTOR signaling. Immunofluorescence double-labeling images showed co-localization of NeuN (neuronal nuclear marker) and Beclin-1, LC3, and p62 throughout the CA1 and CA3 hippocampal subfields. Taken together, these data demonstrate that activation of autophagy occurs in the neurons of the hippocampus in this experimental model.

GLI1 and AXIN2 Are Distinctive Markers of Human Calvarial Mesenchymal Stromal Cells in Nonsyndromic Craniosynostosis.

All skeletal bones house osteogenic stem cell niches, in which mesenchymal stromal cells (MSC) provide progenitors for tissue growth and regeneration. They have been widely studied in long bones formed through endochondral ossification. Limited information is available on the composition of the osteogenic niche in flat bones (i.e., skull vault bones) that develop through direct membranous ossification. Craniosynostosis (CS) is a congenital craniofacial defect due to the excessive and premature ossification of skull vault sutures. This study aimed at analysing the expression of GLI1, AXIN2 and THY1 in the context of the human skull vault, using nonsyndromic forms of CS (NCS) as a model to test their functional implication in the aberrant osteogenic process. The expression of selected markers was studied in NCS patients' calvarial bone specimens, to assess the in vivo location of cells, and in MSC isolated thereof. The marker expression profile was analysed during in vitro osteogenic differentiation to validate the functional implication. Our results show that GLI1 and AXIN2 are expressed in periosteal and endosteal locations within the osteogenic niche of human calvarial bones. Their expression is higher in MSC isolated from calvarial bones than in those isolated from long bones and tends to decrease upon osteogenic commitment and differentiation. In particular, AXIN2 expression was lower in cells isolated from prematurely fused sutures than in those derived from patent sutures of NCS patients. This suggests that AXIN2 could reasonably represent a marker for the stem cell population that undergoes depletion during the premature ossification process occurring in CS.

Mesenchymal Stromal Cells from Fetal and Maternal Placenta Possess Key Similarities and Differences: Potential Implications for Their Applications in Regenerative Medicine.

Placenta-derived mesenchymal stromal cells (MSC) have attracted more attention for their immune modulatory properties and poor immunogenicity, which makes them suitable for allogeneic transplantation. Although MSC isolated from different areas of the placenta share several features, they also present significant biological differences, which might point to distinct clinical applications. Hence, we compared cells from full term placenta distinguishing them on the basis of their origin, either maternal or fetal. We used cells developed by Pluristem LTD: PLacenta expanded mesenchymal-like adherent stromal cells (PLX), maternal-derived cells (PLX-PAD), fetal-derived cells (PLX-R18), and amniotic membrane-derived MSC (hAMSC). We compared immune modulatory properties evaluating effects on T-lymphocyte proliferation, expression of cytotoxicity markers, T-helper and T-regulatory cell polarization, and monocyte differentiation toward antigen presenting cells (APC). Furthermore, we investigated cell immunogenicity. We show that MSCs and MSC-like cells from both fetal and maternal sources present immune modulatory properties versus lymphoid (T cells) and myeloid (APC) cells, whereby fetal-derived cells (PLX-R18 and hAMSC) have a stronger capacity to modulate immune cell proliferation and differentiation. Our results emphasize the importance of understanding the cell origin and characteristics in order to obtain a desired result, such as modulation of the inflammatory response that is critical in fostering regenerative processes.

Autophagy: a potential key contributor to the therapeutic action of mesenchymal stem cells.

Macroautophagy/autophagy occurs at basal levels in all eukaryotic cells and plays an important role in maintaining bio-energetic homeostasis through the control of molecule degradation and organelle turnover. It can be induced by environmental conditions such as starvation, and is deregulated in many diseases including autoimmune diseases, neurodegenerative disorders, and cancer. Interestingly, the modulation of autophagy in mesenchymal stem cells (MSCs) represents a possible mechanism which, affecting MSC properties, may have an impact on their regenerative, therapeutic potential. Furthermore, the ability of MSCs to modulate autophagy of cells in injured tissues/organs has been recently proposed to be involved in the regeneration of damaged tissues and organs. In particular, MSCs can affect autophagy in immune cells involved in injury-induced inflammation reducing their survival, proliferation, and function and favoring the resolution of inflammation. In addition, MSCs can affect autophagy in endogenous adult or progenitor cells, promoting their survival, proliferation and differentiation supporting the restoration of functional tissue. This review provides, for the first time, an overview of the studies which highlight a possible link between the therapeutic properties of MSCs and their ability to modulate autophagy, and it summarizes examples of disorders where these therapeutic properties have been correlated with such modulation. A better elucidation of the mechanism(s) through which MSCs can modulate the autophagy of target cells and how autophagy can affect MSCs therapeutic properties, can provide a wider perspective for the clinical application of MSCs in the treatment of many diseases.Abbreviations: 3-MA: 3-methyladenine; AD: Alzheimer disease; ATG: autophagy-related; BECN1: beclin 1; BM: bone marrow; CD: cluster of differentiation; EAE: experimental autoimmune encephalomyelitis; IL: interleukin; INF: interferon; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; PD: Parkinson disease; PtdIns3K: class III phosphatidylinositol 3-kinase; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; SQSTM1: sequestosome 1; TBI: traumatic brain injury; TGF: transforming growth factor; TNF: tumor necrosis factor.

Autophagy is Activated In Vivo during Trimethyltin-Induced Apoptotic Neurodegeneration: A Study in the Rat Hippocampus.

Trimethyltin (TMT) is an organotin compound known to produce significant and selective neuronal degeneration and reactive astrogliosis in the rodent central nervous system. Autophagy is the main cellular mechanism for degrading and recycling protein aggregates and damaged organelles, which in different stress conditions, such as starvation, generally improves cell survival. Autophagy is documented in several pathologic conditions, including neurodegenerative diseases. This study aimed to investigate the autophagy and apoptosis signaling pathways in hippocampal neurons of TMT-treated (Wistar) rats to explore molecular mechanisms involved in toxicant-induced neuronal injury. The microtubule-associated protein light chain (LC3, autophagosome marker) and sequestosome1 (SQSTM1/p62) (substrate of autophagy-mediated degradation) expressions were examined by Western blotting at different time points after intoxication. The results demonstrate that the LC3 II/I ratio significantly increased at 3 and 5 days, and that p62 levels significantly decreased at 7 and 14 days. Immunofluorescence images of LC3/neuronal nuclear antigen (NeuN) showed numerous strongly positive LC3 neurons throughout the hippocampus at 3 and 5 days. The terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay indicated an increase in apoptotic cells starting from 5 days after treatment. In order to clarify apoptotic pathway, immunofluorescence images of apoptosis-inducing factor (AIF)/NeuN did not show nuclear translocation of AIF in neurons. Increased expression of cleaved Caspase-3 was revealed at 5-14 days in all hippocampal regions by Western blotting and immunohistochemistry analyses. These data clearly demonstrate that TMT intoxication induces a marked increase in both autophagy and caspase-dependent apoptosis, and that autophagy occurring just before apoptosis could have a potential role in neuronal loss in this experimental model of neurodegeneration.

Antigen-dependent T lymphocytes (TcRß+) are primarily differentiated in the thymus rather than in other lymphoid tissues in sea bass (Dicentrarchus labrax, L.).

All jawed vertebrates share lymphocyte receptors that allow the recognition of pathogens and the discrimination between self and non-self antigens. The T cell transmembrane receptor (TcR) has a central role in the maturation and function of T lymphocytes in vertebrates via an important role in positive selection of the variable region of TcR αß/γδ chains. In this study, the TcRß transcript expression and TcRß(+) cell distribution during the ontogeny of the immune system of sea bass (Dicentrarchus labrax, L.) were analysed. RT-PCR analysis of larvae during early development demonstrated that the ß chain transcript is expressed by 19 days post-fertilisation (p.f.). RNA probes specific for the ß chain were synthesised and used for in situ hybridisation experiments on 30 day p.f. to 180 day old juvenile larvae. A parallel immunohistochemical study was performed using the anti-T cell monoclonal antibody DLT15 developed in our laboratory [Scapigliati et al., Fish Shellfish Immunol 1996; 6:383-401]. The first thymus anlage was detectable at 32-33 days p.f. (Corresponding to about 27 days post-hatch). DLT15(+) cells were detected at day 35 p.f. in the thymus whereas TcRß(+) cells were recognisable at day 38 p.f. in the thymus and at day 41 p.f. in the gut. TcRß(+) cells were observed in capillaries from 41 to 80 days p.f. At day 46 p.f., TcRß(+) cells were identified in the head kidney and were detected in the spleen 4 days later. The present results demonstrate that TcRß(+) cells can be differentiated first in the thymus and then in other organs/tissues, suggesting potential TcRß(+) cell colonisation from the thymus to the middle gut. Once the epithelial architecture of the thymus is completed with the formation of the cortical-medullary border (around 70-75 days p.f.), DLT15(+) cells or TcRß(+) cells are confined mainly to the cortex and cortical-medullary border. In particular, a large influx of TcRß(+) cells was observed at the cortical-medullary border from 72 to 90 days p.f., suggesting a role in positive selection for this thymic region during the ontogeny of the fish immune system. This study provides novel information about the primary differentiation and distribution of TcRß(+) cells in sea bass larvae and juveniles.

Dysregulation of intracellular calcium homeostasis is responsible for neuronal death in an experimental model of selective hippocampal degeneration induced by trimethyltin.

Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca(2+)-binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca(2+) overload. The present study was aimed at determining whether intracellular Ca(2+) homeostasis dysregulation is involved in the TMT-induced neurodegeneration and if intracellular Ca(2+)-buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time- and concentration-dependent [Ca(2+)](i) increases that were primarily due to Ca(2+) release from intracellular stores although Ca(2+) entry through Ca(v)1 channels also contributed to [Ca(2+)](i) increases in the early phase of TMT action. Cell pre-treatment with the Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (2 muM) significantly reduced the TMT-induced neuronal death. Moreover, CR(+) neurons responded to TMT with smaller [Ca(2+)](i) increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca(2+) homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR(+) neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca(2+) overload.

Canine cognitive dysfunction and the cerebellum: acetylcholinesterase reduction, neuronal and glial changes.

The specific functional and pathological alterations observed in Alzheimer's disease are less severe in the cerebellum than in other brain areas, particularly the entorhinal cortex and hippocampus. Since dense core amyloid-beta plaque formation has been associated with an acetylcholinesterase heterogeneous nucleator action, we examined if an acetylcholinesterase imbalance was involved in cerebellum plaque deposition. By using the canine counterpart of senile dementia of the Alzheimer's type, a promising model of human brain aging and early phases of Alzheimer's disease, we investigated how cerebellar pathology and acetylcholinesterase density could be related with cognitive dysfunction. As in Alzheimer's disease, the late affectation of the cerebellum was evidenced by its lack of amyloid-beta plaque and the presence of diffuse deposition throughout all cortical grey matter layers. The highest acetylcholinesterase optic density corresponded to cerebellar islands of the granular layer and was predominantly associated with synaptic glomeruli and the somata of Golgi cells. Its reduction correlated with aging and loss of granule cells, whereas cognitive deficit only correlated with loss of Purkinje cells. The observed Bergmann glia alterations may correspond to a reactive response to the loss and damage of the Purkinje cells, their specific neuronal partner. Regarding the role of acetylcholinesterase mediation in amyloid-beta deposition, our data argue against an interaction between these two proteins because acetylcholinesterase reduction correlates with aging but not with cognitive deficit. Finally, our data support the use of companion dogs of all breeds to study aging and early phases of Alzheimer's disease.

Early nuclear factor-kappaB activation and inducible nitric oxide synthase expression in injured spinal cord neurons correlating with a diffuse reduction of constitutive nitric oxide synthase activity.

OBJECT: Because of toxicity at high concentrations, nitric oxide (NO) contributes to spinal cord injury (SCI) secondary lesions. At low concentrations NO modulates nuclear factor-kappaB (NF-kappaB) activation. The authors investigated the activity of neuronal and endothelial NO synthase (nNOS and eNOS) to determine correlations with NF-kappaB activation and inducible NOS (iNOS) expression soon after SCI. METHODS: In 48 adult male Wistar rats clip-based (50 g/mm2/10 seconds) SCI was induced, and spinal cords were removed at different intervals for the following evaluations: 1) assaying specific activity of nNOS and eNOS; 2) electrophoresis mobility shift assay for activated NF-kappaB; 3) Northern blotting for iNOS; 4) immunohistochemistry for iNOS and NF-kappaB; and 5) immunofluorescence for iNOS and NF-kappaB. At 15 minutes postinjury, eNOS activity decreased significantly (p < 0.001), as did nNOS activity at 1 hour compared with these levels in control animals and rats killed at 15 and 30 minutes after SCI (p < 0.001). Basal NF-kappaB levels were variable in controls and at 15 and 30 minutes after injury. One hour postinjury, NF-kappaB activation was diffuse. Inducible NOS messenger RNA localized diffusely, peaking 6 hours after injury and remaining stable until 24 hours postinjury. Immunohistochemical analysis showed diffuse iNOS and NF-kappaB staining, especially in neurons inside and around the lesion. Immunofluorescence demonstrated that injured neurons were a source of NF-kappaB and iNOS soon after injury. CONCLUSIONS: Both nNOS and eNOS exhibited different regulation and roles soon after injury: nNOS correlated with NF-kappaB activation, whereas eNOS may have participated in vascular changes of the injured spinal cord. Neurons seemed to play a pivotal role in modulating and amplifying the inflammatory response in the injured spinal cord.