Comparison of Cortical and White Matter Traumatic Brain Injury Models Reveals Differential Effects in the Subventricular Zone and Divergent Sonic Hedgehog Signaling Pathways in Neuroblasts and Oligodendrocyte Progenitors

The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER^T2;R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.     

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals formed significantly fewer neurospheres. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon.     

Olig bHLH proteins interact with homeodomain proteins to regulate cell fate acquisition in progenitors of the ventral neural tube

BACKGROUND: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons. RESULTS: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region. CONCLUSIONS: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.     

Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.     

Stroke Increases Neural Stem Cells and Angiogenesis in the Neurogenic Niche of the Adult Mouse

The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction.     

Intranasal HB-EGF administration favors adult SVZ cell mobilization to demyelinated lesions in mouse corpus callosum

In the adult rodent brain, the subventricular zone (SVZ) represents a special niche for neural stem cells; these cells proliferate and generate neural progenitors. Most of these migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. SVZ-derived progenitors can also be recruited spontaneously to damaged brain areas to replace lost cells, including oligodendrocytes in demyelinated lesions. In this study, we searched for factors able to enhance this spontaneous recruitment of endogenous progenitors. Previous studies have suggested that epidermal growth factor (EGF) could stimulate proliferation, migration, and glial differentiation of SVZ progenitors. In the present study we examined EGF influence on endogenous SVZ cell participation to brain repair in the context of demyelinated lesions. We induced a focal demyelinated lesion in the corpus callosum by lysolecithin injection and showed that intranasal heparin-binding epidermal growth factor (HB-EGF) administration induces a significant increase in SVZ cell proliferation together with a stronger SVZ cell mobilization toward the lesions. Besides, HB-EGF causes a shift of SVZ-derived progenitor cell differentiation toward the astrocytic lineage. However, due to the threefold increase in cell recruitment by EGF treatment, the absolute number of SVZ-derived oligodendrocytes in the lesion of treated mice is higher than in controls. These results suggest that enhancing SVZ cell proliferation could be part of future strategies to promote SVZ progenitor cell mobilization toward brain lesions.     

Investigation of the proteolipid protein promoter activity during demyelination and repair

The transgenic plp-GFP mouse line expressing the green fluorescent protein (GFP) driven by the mouse myelin proteolipid protein (plp) gene promoter has been previously used to study the contribution of the plp lineage to oligodendrocyte development in the embryonic brain. Here, we show that the GFP fluorescence reflects the developmental expression of proteolipid protein during the postnatal development until adulthood in brain slices and in primary cultures of plp-GFP⁺ cells derived from postnatal animals. In the adult brain, plp-GFP-expressing cells are mature oligodendrocytes but not oligodendroglial progenitors. In the model of focal demyelination induced by lysolecithin (LPC) in the corpus callosum of adult plp-GFP animals, we observed an up-regulation of the morphogen Sonic Hedgehog (Shh) in the LPC-induced lesion but not in the control animals. Moreover, we show that the adenovirus-mediated transfer of Shh in the lesion results in the attenuation of the demyelination extent as evidenced by GFP fluorescence analysis in Shh-treated and control animals. Altogether these data show how plp-GFP fluorescence can be monitored to follow the oligodendrocyte lineage during demyelination and identify Shh morphogen as an important factor during repair.     

PI3K/Akt and CREB regulate adult neural hippocampal progenitor proliferation and differentiation

The phosphoinositide 3-OH kinase (PI3K)/Akt pathway has been implicated in regulating several important cellular processes, including apoptosis, survival, proliferation, and metabolism. Using both pharmacological and genetic means, we demonstrate here that PI3K/Akt plays a crucial role in the proliferation of adult hippocampal neural progenitor cells. PI3K/Akt transduces intracellular signals from multiple mitogens, including basic fibroblast growth factor (FGF-2), Sonic hedgehog (Shh), and insulin-like growth factor 1 (IGF-1). In addition, retroviral vector-mediated over-expression of wild type Akt increased cell proliferation, while a dominant negative Akt inhibited proliferation. Furthermore, wild type Akt over-expression reduced glial (GFAP) and neuronal (beta-tubulin III) marker expression during differentiation, indicating that it inhibits cell differentiation. We also show that activation of the cAMP response element binding protein (CREB), which occurs in cells stimulated by FGF-2, is limited when Akt signaling is inhibited, demonstrating a link between Akt and CREB. Over-expression of wild type CREB increases progenitor proliferation, whereas dominant negative CREB only slightly decreases proliferation. These results indicate that PI3K/Akt signaling integrates extracellular signaling information to promote cellular proliferation and inhibit differentiation in adult neural progenitors.     

Fluid compartmentation and electrolytes of cat cerebral cotex in vitro. 3. Ontogenetic and comparative aspects

Studies on swelling and fluid compartmentation have been carried out in vitro on incubated slices of cerebral cortex from kittens 1.5-120 days post-natal age and on incubated sections of corpus callosum and slices of liver and kidney cortex from adult cats. The findings have been compared with analogous data for incubated slices of adult cat cerebral cortex, studied under identical conditions (Bourke and Tower , 1966a, b), in order to identify the probable morphological correlates of fluid and electrolyte distribution. Incubated cortical slices from neonatal (1.5-4-day-old) kittens exhibit none of the relevant characteristics of slices from adult cerebral cortex. By 1 month post-natal age, K⁺-dependent swelling of slices becomes demonstrable, and the K⁺ and Na⁺ contents of slices approximate adult levels. Both these developments coincide with the morphological and physiological maturation of cortical neurons. At 3 months post-natal age, slice swelling accessible to C1^? but not to sucrose becomes observable and the dependence of sucrose space size on time, during incubation, of solute addition becomes demonstrable. Both these developments follow completion of axonal myelination in the cortex but coincide with the period of cortical glial cell proliferation. Incubated sections of corpus callosum from adult cats exhibit none of the relevant characteristics observed for cortical slices under identical conditions. Tissue swelling is minimal and uninfluenced by K⁺ concentrations of incubation media. Tissue fluid spaces accessible to sucrose are approximately twice the size of spaces accessible to inulin. In general, qualitatively similar results have been obtained for incubated slices of cat liver or kidney cortex or for incubated sections of rat diaphragm under the same conditions. A behaviour for glial cells (? astrocytes) in cerebral cortex under such in vitro conditions distinctly different from behaviour of subcortical glial cells is suggested.     

成年哺乳动物皮质区神经发生的研究进展

神经发生是神经干细胞在适当的条件下分化成功能性整合神经元的过程,主要包括细胞的增殖、迁移、分化和存活。成年神经发生区以前脑室管膜下区(Subventricular zones,SVZ)和海马齿状回颗粒层下区(Subgranular zones,SGZ)为主,但皮质作为神经元和神经胶质细胞数量最多、分布最广,同时也是哺乳动物高度发展的脑区,是否有成年神经元新生,已成为近年来神经科学领域的研究热点[1,2]。现本文就未成熟神经元在皮质区的研究方法、分布、来源与转归、病理生理功能影响等方面探讨成年哺乳动物皮质神经发生现象。     

Netrin1 is required for neural and glial precursor migrations into the olfactory bulb

Netrin1 (NTN1) deficiency in mouse brain causes defects in axon guidance and cell migration during embryonic development. Here we show that NTN1 is required for olfactory bulb (OB) development at late embryogenesis and at early postnatal stages to facilitate the accumulation of proper numbers of granular and glomerular neuron subtypes and oligodendrocytes into the OB. In addition to the analysis of Ntn1−/− mice we made tissue and neurosphere cultures to clarify the role of NTN1 in the anterior forebrain. We propose that a subset of neural progenitors/precursors requires NTN1 to efficiently enter the rostral migratory stream to migrate into the OB. The analysis of postnatal Ntn1−/− OBs revealed a reduction of specific types of interneurons which have been shown to originate from particular subregions of the lateral ventricle walls. Based on Ntn1 expression in ventral parts of the ventricle walls, we observed a decrease in the mainly ventrally derived type II interneurons that express calcium-binding proteins calretinin and calbindin. Instead, no change in the numbers of dorsally derived tyrosine hydroxylase expressing interneurons was detected. In addition to the specific reduction of type II interneurons, our results indicate that NTN1 is required for oligodendroglial migration into the OB. Furthermore, we characterised the Ntn1 expressing subpopulation of neurosphere-forming cells from embryonic and adult brain as multipotent and self-renewing. However, NTN1 is dispensable for the proliferation of neurosphere forming progenitor cells and for their differentiation.     

Morphological studies on neuroglia

Immunohistochemical studies with the use of the peroxidase-antiperoxidase (PAP) method revealed that "amoeboid microglial cells", in the brains of neonatal rats and "brain macrophages" in lesioned brains of adult rats react positively to an antiserum raised against macrophages. In brains of neonatal rats, "amoeboid microglial cells" stained by means of the PAP-method were observed in the corpus callosum, internal capsule, dorso-lateral region of the thalamus, subventricular zone of the lateral ventricle, and the subependymal layer of the ventricular system. These cellular elements were not detected in brains of rats aged 21 days or older. Resting microglial cells displaying a typical ramified structure were not specifically stained. Cells reacting positively to the macrophage antiserum appeared (i) in the cerebral cortex of adult rats following placement of a stab wound, or (ii) in the hippocampal formation after kainic acid-induced lesions; in the damaged areas immunoreactive cells exhibited the typical features of "brain macrophages". "Brain macrophages" and "amoeboid microglial cells" are considered to belong to the class of exudate macrophages derived from blood monocytes. Thus, elements of hematogenous origin do exist in the intact brain parenchyma of neonatal rats and in lesioned brains of adult rats. The relationship between brain macrophages and resting microglial cells is discussed.     

Long-term survival of human neural stem cells in the ischemic rat brain upon transient immunosuppression

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders.     

Hedgehog-Gli signalling and the growth of the brain

The development of the vertebrate brain involves the creation of many cell types in precise locations and at precise times, followed by the formation of functional connections. To generate its cells in the correct numbers, the brain has to produce many precursors during a limited period. How this is achieved remains unclear, although several cytokines have been implicated in the proliferation of neural precursors. Understanding this process will provide profound insights, not only into the formation of the mammalian brain during ontogeny, but also into brain evolution. Here we review the role of the Sonic hedgehog-Gli pathway in brain development. Specifically, we discuss the role of this pathway in the cerebellar and cerebral cortices, and address the implications of these findings for morphological plasticity. We also highlight future directions of research that could help to clarify the mechanisms and consequences of Sonic hedgehog signalling in the brain.