Emerging roles of CXCL12-bearing microvesicles in glio-vascular communication during human brain development

The CXC chemokine axis formed by CXCL12 and its receptors CXCR4/CXCR7 is involved in CNS development enhancing migration and differentiation of neuronal precursors [1, 2]. According to these data, our recent studies have demonstrated that during human cerebral cortex development, radial glia (RG) cells express high levels of CXCL12, which finds its receptor on migrating postmitotic neuroblasts [3]. Moreover, during this first survey we have also revealed the ligand concentrated in RG processes and in astrocyte endfeet in contact with the microvessel wall. Features and distribution of these glio-vascular contacts have been further studied to ascertain the possible involvement of the glial CXC ligand/receptor system in vessel growth and differentiation. The study was carried out on human telencephalon by laser confocal and transmission electron microscopy to detect the expression of chemokine CXCL12 together with specific glio-vascular markers and reveal subcellular details of the identified cell structures. Immunolabelling for CXCL12 showed the highest level of RG CXCL12-enriched vascular contacts in the subcortical layers, where the chemokine concentrated in small swellings that appeared at intervals along the RG fibres. These RG varicosities formed en passant vascular contacts or asymmetrical enlargements that were seen to specifically bent and come in contact with the vessel wall. At the highest confocal resolution, both symmetrical and asymmetrical RG varicosities appeared filled with CXCL12 labelled dot-like structures that have been regarded as cell microvesicles (MVs). Their vesicular nature was confirmed by ultrastructural observations that allowed to recognize small membrane-bound MVs gathered in RG perivascular varicosities. Aspects of CXCL12 MV shedding also appeared a common feature of these specialized glio-vascular contacts confirming the hypothesis that RG-derived CXCL12 can be conveyed to the recipient vascular cells participating in glio-vascular communication and coordinating neuro-vascular interactions during cerebral cortex development and vascularization

BBB-endothelial tight junction response to NG2 in murine model of chronic EAE

The blood brain barrier (BBB) phenotype of brain endothelial cells (ECs) is the result of the influence and interaction from all the cell components of neurovascular unit (pericytes, astrocytes, oligodendrocytes, microglia, neurons) and basal lamina molecules. Pericyte-associated NG2, a transmembrane chondroitin sulphate proteoglycan, modulates EC proliferation and migration through its interaction with the involved cell growth factors and receptors (Fukushi et al., 2004). Our previous studies carried out on a model of cerebral cortex EAE (experimental autoimmune encephalomyelitis), induced by MOG in C57BL/6 mice, demonstrated the impairment of BBB-microvessels with dismantled tight junction (TJ) strands and scarce perivascular infiltrations (Errede et al., 2012). Interestingly, the datum of a minimal inflammatory infiltrate has been also reported in a model of EAE induced in knout-out mice for the proteoglycan NG2 (Kucharova et al., 2011). On the basis of these data, the present study was carried out on the same model of EAE to clarify the role of NG2 on ECs of brain microvessels, utilizing two groups of mice, wild type (WT) and homozygous NG2 KO (NG2-/-). The expression of two integral proteins of the endothelial TJs, claudin-5 and occludin, the relevance of IBA1 reactive microglia cells and the level of BBB leakage by an exogenous permeability tracer, FITC-Dextran have been analyzed by immunohistochemistry and high resolution confocal microscopy. The results on the junctional staining pattern showed that unlike WT EAE, NG2 KO EAE microvessels were characterized by TJs continuous junctional strands with an unusual distribution of junctional proteins organized in honeycomb-like meshes. These findings suggest that NG2 proteoglycan can be directly implicated in pericyte/EC relations, including the mutual organization of TJ proteins in BBB- microvessels during neurological disease

Cell-to-cell communication within the neurovascular unit (NVU) in a model of cerebral cortex demyelination

The concept of neurovascular unit (NVU) emphasizes the critical role of cell-to-cell interaction and communication between glial, neuronal, and vascular cell components during blood-brain barrier (BBB) development, and in adult normal and pathological conditions. In this study we have analysed the involvement of the nerve glial antigen 2, NG2, a chondroitin sulphate proteoglycan, highly expressed in developing and adult CNS, in cell cross-talk within the NVU. During CNS development NG2 is expressed by activated pericyte and appears downregulated as these cells undergo terminal differentiation. NG2 has also been identified on the surface of oligodendrocyte precursor cells, OPCs, evenly distributed throughout the CNS already by the end of the first postnatal week in mice and throughout adulthood. In a previous study on cerebral cortex experimental autoimmune encephalomyelitis (EAE) in mice, we firstly observed and described the glia-limitans-like position of NG2-bearing OPCs that during neuroinflammation extend processes to the pial surface and acquire a perivascular arrangement, coming in contact with the wall of EAE cortex microvessels. With the aim of understanding if a subset of OPCs specifically contributes to the cell composition of the NVU during EAE, we have explored, by morphometric analyses applied to laser confocal microscopy, OPCs distribution and vascular relationships in the cerebral cortex of WT controls and naïve NG2KO and in EAE WT and EAE NG2KO mice, at both early (20 dpi) and late (40 dpi) disease stages. In EAE WT mice, juxtavascular (JV) and perivascular (PV) OPCs were identified in a higher number compared to healthy mice. On the contrary, absence of NG2 in EAE NG2 KO mice seemed to affect the proliferative response of OPCs, specifically inhibiting the emergence of the JV and PV OPC subsets. The results indicate that in WT mice during EAE, the NVU microenvironment, classically formed by perivascular astrocytes, receives the insertion of OPCs as a specific vascular subset and suggest NG2 as the molecule involved in the observed NVU damage

BBB-endothelial cell response to cerebral cortex demyelination in a mouse model of chronic EAE

Changes in blood-brain barrier (BBB) function have been implicated in demyelinating diseases. This study aimed to investigate the response of cerebral cortex microvessels to nerve fibre demyelination in a chronic model of murine experimental autoimmune encephalomyelitis (EAE) characterized by areas of extensive subpial demyelination along with well-demarcated lesions extended to deeper cortex layers. These cortices showed activation of microglia and astrogliosis with absence of typical perivascular inflammatory infiltrates. On the basis of these data, we have analyzed the expression of two integral proteins of endothelial tight junctions, claudin-5 and occludin, a structural protein of caveolae, caveolin-1, as well as the BBB-specific endothelial transporter, Glut1 in the cerebral cortex of EAE-affected mice by immunofluorescence confocal microscopy. Microvascular endothelial cells showed an increased expression of caveolin-1 and a coincident decrease of both claudin-5 and occludin junctional staining pattern. At a very early disease stage, claudin-5 molecules formed aggregates and vacuoles that also stained for Glut 1, whereas occludin pattern became diffusely cytoplasmic at advanced stages of the disease. Internalization/dismantling and loss of tight junction proteins and impairment of BBB function were confirmed by coexpression of claudin-5 whit the autophagosomal marker MAP1LC3A and by FITC-dextran experiments that showed leakage of the tracer into the perivascular neuropil. Overall, these observations indicate that in the cerebral cortex of EAE mice, during demyelination and independently from the inflammatory involvement of the cortex, a ‘microvascular disease’ characterized by a differential involvement of claudin-5 and occludin occurs, thereby possibly contributing to demyelinating disease progression

Characterization of oligodendrocyte lineage precursor cells in the mouse cerebral cortex: a confocal microscopy approach to demyelinating diseases

The identification of stem cells resident in the adult central nervous system has redirected the focus of research into demyelinating diseases, such as multiple sclerosis, mainly affecting the brain white matter. This immunocytochemical and morphometrical study was carried out by confocal microscopy in the adult mouse cerebral cortex, with the aim of analysing, in the brain grey matter, the characteristics of the oligodendrocyte lineage cells, whose capability to remyelinate is still controversial. The observations demonstrated the presence in all the cortex layers of glial restricted progenitors, reactive to A2B5 marker, oligodendrocyte precursor cells, expressing the NG2 proteoglycan, and pre­oligodendrocytes and pre­myelinating oligodendrocytes, reactive to the specific marker O4. NG2 expressing cells constitute the major immature population of the cortex, since not only oligodendrocyte precursor cells and pre-oligodendrocytes but also a part of the glial restrict progenitors express the NG2 proteoglycan. Together with the popula­ tion of these immature cells, a larger population of mature oligodendrocytes was revealed by the classical oligodendrocyte and myelin markers, 2’,3’-cyclic nucleotide 3’­phosphodiesterase, myelin basic protein and myelin oligodendrocyte glycoprotein. The results indicate that oligodendrocyte precursors committed to differentiate into myelin forming oligodendrocytes are present through all layers of the adult cortex and that their phenotypic features exactly recall those of the oligodendroglial lineage cells during development

Activation of autophagy and suspended apoptosis in skeletal muscle of inclusion body myositis

Inclusion Body Myositis (IBM) is characterized by rimmed vacuole formation and misfolded protein accumulation, both depending on lysosome dysfunction. In skeletal muscle, selective protein degradation is allowed by macroautophagy. A proper balance in degradation and accumulation of proteins and organelles is critical for cell survival. Extracellular signal-regulated protein kinase (ERK1/2) is essential in cell survival, but recent evidence suggests that it is also necessary for autophagy. Alteration in subcellular localization of ERK promotes cell death either via autophagic death or via apoptosis upstream caspase-3. Moreover, in IBM myocytes there is no convincing evidence for apoptosis. Here, we correlated the expression level of autophagic and apoptotic molecules with that of ERK2 by analysing, with immunohistochemistry (IHC) and western blot (WB) methods, immunolocalization and expression of a panel of molecules directly involved and/or associated with the disease histopathogenesis: coated vesicles protein clathrin, mannose-6-phosphate receptor (M6PR), autophagy related proteins Beclin1 and ATG5, microtubule associated protein light chain LC3a and LC3b, Apoptotic Protease Activating Factor 1 (APAF1), Caspase-3, ERK2, and the specific IBM marker SMI31. Muscle biopsy specimens were obtained from 10 patients with sporadic IBM, 1 familial IBM patient, 1 amyotrophic lateral sclerosis patient, 1 patient with polymyositis with prominent mitochondrial pathology and 9 non myophatic patients as control specimens. IHC studies of expression and colocalization revealed an increase of clathrin, Beclin1, ATG5, and LC3 immunoreactivity, mainly observed in the sarcoplasm of small, atrophic fibres in all diseased specimens compared to controls. By WB analysis, expression level of both APAF1 and Caspase-3 did not significantly change between patients and controls, whereas the level of expression of ERK2 and autophagy markers seemed to inversely correlate. The results demonstrated that transport of newly synthesized lysosome enzymes and formation of autophagic vacuoles are both activated in IBM muscle. ERK2 phosphorylating activity is probably involved in rescue attempt to overcome the cell injury rather than directly stimulating the cell death. During IBM, the apoptotic cascade seems to be suspended, however,under the effect of cytotoxic stimuli, protective autophagy may switch to autophagic programmed cell death

BBB-endothelial tight junction response to mesenchymal stem cells in a model of MOG EAE

Experimental autoimmune encephalomyelitis (EAE), an induced autoimmune disease of the central nervous system, simulates the main histopathological and clinical aspects of multiple sclerosis including the impairment of the blood-brain barrier (BBB). In several experimental models of human neurodegenerative diseases, the intravenous (iv) injection of bone marrow-derived mesenchymal stem cells (MSCs) ameliorates clinical symptoms and histopathological features [1,2]. On the basis of these data, we have analyzed the status of BBB tight junctions (TJs) of cerebral cortex microvessels in a model of MOG-EAE with iv injection of MSCs (EAE-MSC). The observations were carried out on EAE-MSC mice sacrificed at 6-24 hrs and 10 days after MSCs iv injection. The expression of endothelial TJ proteins, claudin-5 and occludin, was analyzed in healthy, EAE, and EAE-MSC mice by immunofluorescence confocal microscopy, together with the evaluation of barrier function by FITC-Dextran, as an exogenous permeability tracer. The results demonstrate that unlike EAE animals, characterized by an interrupted junctional staining and a barrier leakage, EAE-MSC mice show together with attenuate disease symptoms, a continuous, control- like claudin-5 and occludin junctional pattern and a functionally recovered barrier efficiency. Overall, these findings suggest that during EAE, the neuroprotective effect of the injected MSCs includes a reparative BBB response that in turn may contribute to the reduction of the inflammatory infiltrates and to the significant amelioration of the disease

Microvascular pericytes involvement in experimental autoimmune encephalomyelitis

In the CNS, pericytes are microvessel wall-encircling cells that, together with endothelial cells, perivascular glial endfeet and basement membrane, form the blood-brain barrier (BBB). Dysfunction of the BBB and migration of autoreactive T lymphocytes into the CNS are histopathological hallmarks of both Multiple Sclerosis (MS), a chronic demyelinating disease, and experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. The proteoglycan NG2, which has been described to accumulate within MS plaques and at spinal cord (SC) injury sites, is a primary component of pericytes, engaged in pericyte/endothelial cell interaction, proliferation and migration. To explore the role of NG2-expressing pericytes during neuroinflammation and BBB dysfunction, pericyte coverage (pericyte number/vessel length) and density (pericyte number/tissue volume) ratios were studied in brain microvessels by immunohistochemistry and laser confocal microscopy using specific pericyte markers, NG2, RGS5, and CD13. The observations were made in mice affected by MOG-induced chronic EAE with two different genetic C57BL/6 backgrounds: wild type (WT) and homozygous NG2 null (NG2-/-). In literature, NG2-/- mice did not exhibit gross phenotypic or vascular alterations, whereas our results demonstrated an unaltered pericyte density associated with slightly decreased pericyte coverage index and pericyte/endothelial cell ratio. These observations were confirmed in NG2-/- EAE-affected mice, that showed an attenuated disease severity and demyelination, and a milder BBB leakage and leukocyte infiltration, as compared with EAE WT. Taken together these results lend support to the idea of a direct involvement of NG2 proteoglycan in pericyte-endothelial cell interactions essential for the preservation of a proper BBB function