Frederic Mercier

Anatomy of the brain neurogenic zones revisited : Fractones and the fibroblast/macrophage network

Cytogenesis in adult peripheral organs, and in all organs during development, occurs nearby basal laminae (BL) overlying connective tissue. Paradoxically, cytogenesis in the adult brain occurs primarily in the subependymal layer (SEL), a zone where no particular organization of BL and connective tissue has been described. We have reinvestigated the anatomy of the area considered the most neurogenic in the adult brain, the SEL of the lateral ventricle, in zones adjacent to the caudate putamen, corpus callosum, and lateral septal nucleus. Here, we report structural (confocal microscopy using laminin as a marker) and ultrastructural evidence for highly organized extravascular BL, unique to the SEL. The extravascular BL, termed fractones because of their fractal organization, were regularly arranged along the SEL and consisted of stems terminating in bulbs immediately underneath the ependyma. Fractones contacted local blood vessels by means of their stems. An individual fractone engulfed in its folds numerous processes of astrocytes, ependymocytes, microglial cells, and precursor cell types. The attachment site (base) of stems to blood vessels was extensively folded, overlying large perivascular macrophages that belong to a fibroblast/macrophage network coursing in the perivascular layer and through the meninges. In addition, collagen‐1, which is associated with BL and growth factors during developmental morphogenetic inductions, was immunodetected in the SEL and particularly regionalized within fractones. Because macrophages and fibroblasts produce cytokines and growth factors that may concentrate in and exert their effect from the BL, we suggest that the structure described is implicated in adult neurogenesis, gliogenesis, and angiogenesis. J. Comp. Neurol. 451:170–188, 2002.

Novel Extracellular Matrix Structures in the Neural Stem Cell Niche Capture the Neurogenic Factor Fibroblast Growth Factor 2 from the Extracellular Milieu

The novel extracellular matrix structures called fractones are found in the lateral ventricle walls, the principal adult brain stem cell niche. By electron microscopy, fractones were shown to contact neural stem and progenitor cells (NSPC), suggesting a role in neurogenesis. Here, we investigated spatial relationships between proliferating NSPC and fractones and identified basic components and the first function of fractones. Using bromodeoxyuridine (BrdU) for birth‐dating cells in the adult mouse lateral ventricle wall, we found most mitotic cells next to fractones, although some cells emerged next to capillaries. Like capillary basement membranes, fractones were immunoreactive for laminin β1 and γ1, collagen IV, nidogen, and perlecan, but not laminin‐α1, in the adult rat, mouse, and human. Intriguingly, N‐sulfate heparan sulfate proteoglycan (HSPG) immunoreactivity was restricted to fractone subpopulations and infrequent subependymal capillaries. Double immunolabel for BrdU and N‐sulfate HSPG revealed preferential mitosis next to N‐sulfate HSPG immunoreactive fractones. To determine whether N sulfate HSPG immunoreactivity within fractones reflects a potential for binding neurogenic growth factors, we identified biotinylated fibroblast growth factor 2 (FGF‐2) binding sites in situ on frozen sections, and in vivo after intracerebroventricular injection of biotinylated FGF‐2 in the adult rat or mouse. Both binding assays revealed biotinylated FGF‐2 on fractone subpopulations and on infrequent subependymal capillaries. The binding of biotinylated FGF‐2 was specific and dependent upon HSPG, as demonstrated in vitro and in vivo by inhibition with heparatinase and by the concomitant disappearance of N‐sulfate HSPG immunoreactivity. These results strongly suggest that fractones promote growth factor activity in the neural stem cell niche.

Connexin 26 and basic fibroblast growth factor are expressed primarily in the subpial and subependymal layers in adult brain parenchyma: roles in stem cell proliferation and morphological plasticity?

The gap junction protein connexin 26 (Cx26) has been detected previously in the parenchyma of the developing brain and in the developing and adult meninges, but there is no clear evidence for the presence of this connexin in adult brain parenchyma. Confocal mapping of Cx26 through serial sections of the meningeal‐intact rat brain with four antibodies revealed an intense Cx26 immunoreactivity in both parenchyma and extraparenchyma. In the extraparenchyma, a continuum of Cx26‐immunoreactive puncta was observed throughout the three meningeal layers, the perineurium of cranial nerves, and meningeal projections into the brain, including sheaths of blood vessels and stroma of the choroid plexus. In the parenchyma, Cx26‐immunoreactive puncta were located primarily in subependymal, subpial, and perivascular zones and were associated primarily with glial fibrillary acidic protein‐positive (GFAP+) astrocytes, the nuclei of which are strongly immunoreactive for basic fibroblast growth factor (bFGF). Although it was found to a lesser extent than in astrocytes, bFGF immunoreactivity also was intense in the nuclei of meningeal fibroblasts. In addition, we have found a close correlation between the distribution of Cx26 and vimentin immunoreactivities in the meninges and their projections into the brain. We previously showed vimentin and S100β immunoreactivities through a network of meningeal fibroblasts in the three layers of meninges, perivascular cells, and ependymocytes and in a population of astrocytes. The related topography of this network with GFAP+ astrocytes has also been demonstrated. Considering that connexin immunoreactivity may reflect the presence of functional gap junctions, the present results are consistent with our hypothesis that all of these various cell types may communicate in a cooperative network. J. Comp. Neurol. 431:88–104, 2001.

Immunocytochemical basis for a meningeo-glial network

Evidence is presented here for a cellular network that courses through all layers of meninges, the vasculature of both the brain and meninges, and extends into the brain parenchyma. Confocal mapping of calcium‐binding protein S100β immunoreactivity (S100β‐ir) and of the intermediate filament vimentin‐ir through serial sections of the meningeal‐intact adult rat brain revealed this network. In all tissues examined, S100β‐ir and vimentin‐ir were primarily colocalized, and were found in cells with elongated processes through which these cells contacted one another to form a network. The location of labeling and the morphology of the cells labeled were consistent with the possibility that this network consists of fibroblasts in the meninges and the walls of large blood vessels, of pericytes at the level of capillaries, and of ependymocytes and a population of astrocytes in the brain parenchyma. At many sites along the borders of the brain parenchyma itself and of the brain blood vessels, it was possible to detect S100β‐ir and vimentin‐ir cell processes that cross the basal laminae. This suggested the probable means by which the S100β‐ir cells of the extraparenchymal tissues anatomically contact the cells that express the same markers in the brain. Privileged anatomical relationships of the S100β/vimentin network with the glial fibrillary acidic protein (GFAP) astrocytes further suggested that, together, they form the structural basis for a general meningeo‐glial network. This organization challenges the current model of brain architecture, calls for a reconsideration of the role of meninges and vascular tissues, and appears to reflect the existence of hitherto unsuspected systems of communication. J. Comp. Neurol. 420:445–465, 2000.

Fractones and other basal laminae in the hypothalamus

The physiological role of basal laminae (BL) and connective tissue (meninges and their projections) in the adult brain is unknown. We recently described novel forms of BL, termed fractones, in the most neurogenic zone of the adult brain, the subependymal layer (SEL) of the lateral ventricle. Here, we investigated the organization of BL throughout the hypothalamus, using confocal and electron microscopy. New types of BL were identified. First, fractones, similar to those found in the lateral ventricle wall, were regularly arranged along the walls of the third ventricle. Fractones consisted of labyrinthine BL projecting from SEL blood vessels to terminate immediately beneath the ependyma. Numerous processes of astrocytes and of microglial cells directly contacted fractones. Second, another form of BL projection, termed anastomotic BL, was found between capillaries in dense capillary beds. The anastomotic BL enclosed extraparenchymal cells that networked with the perivascular cells coursing in the sheaths of adjacent blood vessels. Vimentin immunoreactivity was often detected in the anastomotic BL. In addition, the anastomotic BL overlying macrophages contained numerous fibrils of collagen. We also found that the BL located at the pial surface formed labyrinthine tube‐like structures enclosing numerous fibroblast and astrocyte endfeet, with pouches of collagen fibrils at the interface between the two cell types. We suggest that cytokines and growth factors produced by connective tissue cells might concentrate in BL, where their interactions with extracellular matrix proteins might contribute to their effects on the overlying neural tissue, promoting cytogenesis and morphological changes and participating in neuroendocrine regulation. J. Comp. Neurol. 455:324–340, 2003.

Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex.

Significance The stimulation of certain surface receptors on immune cells triggers the release of calcium (Ca2+) stored in the endoplasmic reticulum (ER). This Ca2+ flux is required for efficient activation and function of immune cells, and involves the ER membrane Ca2+ channel, the inositol 1,4,5-triphosphate receptor (IP3R). We found that stable expression of IP3R requires the addition of a fatty acid through a process called palmitoylation catalyzed by an enzyme complex composed of DHHC6 (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) and selenoprotein K (Selk) proteins. These findings provide new mechanistic insight into the selenium-sensitive fine-tuning of immune cell activation through posttranslational modification of the IP3R Ca2+ channel. This study also reveals a novel DHHC6/Selk enzyme complex responsible for regulating stable expression of the IP3R. Calcium (Ca2+) is a secondary messenger in cells and Ca2+ flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca2+ flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca2+ flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca2+ flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.

Heparan sulfate niche for cell proliferation in the adult brain

In adulthood, new neurons and glial cells are generated from stem cells in restricted zones of the brain, namely the olfactory bulb (OB), rostral migratory stream (RMS), subventricular zone (SVZ) of the lateral ventricle, sub-callosum zone (SCZ) and sub-granular layer (SGL) of the dentate gyrus. What makes these zones germinal? We previously reported that N-sulfated heparan sulfates (N-sulfated HS) present in specialized extracellular matrix structures (fractones) and vascular basement membranes bind the neurogenic factor FGF-2 (fibroblast growth factor-2) next to stem cells in the anterior SVZ of the lateral ventricle, the most neurogenic zone in adulthood. To determine to which extent cell proliferation is associated with N-sulfated HS, we mapped N-sulfated HS and proliferating cells by immunohistochemistry throughout the adult mouse brain. We found that cell proliferation is associated with N-sulfated HS in the OB, RMS, the whole germinal SVZ, and the SCZ. Cell proliferation was weakly associated with N-sulfated HS in the SGL, but the SGL was directly connected to a sub-cortical N-sulfated HS+ extension of the meninges. The NS-sulfated HS+ structures were blood vessels in the OB, RMS and SCZ, and primarily fractones in the SVZ. N-sulfated HS+ fractones, blood vessels and meninges formed a continuum that coursed along the OB, SVZ, RMS, SCZ and SGL, challenging the view that these structures are independent germinal entities. These results support the possibility that a single anatomical system might be globally responsible for mitogenesis and ultimately the production of new neurons and glial cells in the adult brain.

Perlecan is required for FGF-2 signaling in the neural stem cell niche.

In the adult subventricular zone (neurogenic niche), neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

Hippocampus/amygdala alterations, loss of heparan sulfates, fractones and ventricle wall reduction in adult BTBR T+ tf/J mice, animal model for autism

Multiple studies converge to implicate alterations of the hippocampus and amygdala in the pathology of autism. We have previously reported anatomical alterations of the meninges, vasculature and fractones, the specialized extracellular matrix (ECM) of the subventricular zone, in the forebrain of adult BTBR T+ tf/J mice, animal model for autism. Here, we used bisbenzidine cell nucleus staining and dual immunofluorescence histochemistry for laminin and N-sulfated heparan sulfate proteoglycans (NS-HSPG) to examine a series of brain sections containing the amygdala and hippocampus in the adult BTBR T+ tf/j mouse. We observed an excessive separation of the two hippocampi, a modified trajectory of the meninges leading to a shrunken choroid plexus in the lateral ventricle, a shorter granular layer of the dentate gyrus, and a reduced size of the amygdala nuclei. The lateral ventricle near the amygdala, and the third ventricle were shrunken. The number and size of fractones, and their immunoreactivity for NS-HSPG, were reduced throughout the third and lateral ventricles walls. Enlarged blood vessels were found at the endopiriform cortex/amygdala interface. These results show anatomical alterations of the hippocampal/amygdala that are associated with defects of the choroid plexus/ventricular system and the ECM in the BTBR T+ TF/J mouse. Similar alterations of the hippocampus/amygdala axis in humans with autism to these observed in BTBR T+ tf/J mice make this animal model highly valuable for the study of autism. Moreover, the meningo/vascular and ECM alterations in BTBR T+ Tf/J mice suggest a possible role of the brain connective tissue in autism.

Fractone‐heparan sulphates mediate FGF‐2 stimulation of cell proliferation in the adult subventricular zone

Objectives: Fractones are extracellular matrix structures that form a niche for neural stem cells and their immediate progeny in the subventricular zone of the lateral ventricle (SVZa), the primary neurogenic zone in the adult brain. We have previously shown that heparan sulphates (HS) associated with fractones bind fibroblast growth factor‐2 (FGF‐2), a powerful mitotic growth factor in the SVZa. Here, our objective was to determine whether the binding of FGF‐2 to fractone‐HS is implicated in the mechanism leading to cell proliferation in the SVZa.