Jean de Vellis

Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin

A monoclonal antibody to vimentin (RBA1) and a polyclonal antiserum to glial fibrillary acidic protein (GFAP) were used in double labeling experiments to examine astrocyte intermediate filaments in development and wounding. RBA1 bound to radial glia in newborn rat parietal cortex that are predominantly anti-GFAP-negative. The RBA1-positive radial fibers disappeared by postnatal day 20 with the greatest rate of disappearance occurring between day 8 and day 15. Between birth and day 20, the anti-GFAP staining increased to the adult pattern in mature shaped astrocytes. Some overlay was observed between the binding patterns of the two antibodies. Stab wounds to cortical areas were made at a developmental time when there were normally no RBA1-positive astrocytes. RBA1-positivity was present in some astrocytes but only at the edges of the wounds. The distribution patterns of RBA1-positive cells led to hypotheses concerning the possible function of vimentin in astrocytes and its regulation during development and wounding.

Stem cell-based cell therapy in neurological diseases: A review

Human neurological disorders such as Parkinsons disease, Huntingtons disease, amyotrophic lateral sclerosis (ALS), Alzheimers disease, multiple sclerosis (MS), stroke, and spinal cord injury are caused by a loss of neurons and glial cells in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells, mesenchymal stem cells, and neural stem cells, and extensive efforts by investigators to develop stem cell‐based brain transplantation therapies have been carried out. We review here notable experimental and preclinical studies previously published involving stem cell‐based cell and gene therapies for Parkinsons disease, Huntingtons disease, ALS, Alzheimers disease, MS, stroke, spinal cord injury, brain tumor, and lysosomal storage diseases and discuss the future prospects for stem cell therapy of neurological disorders in the clinical setting. There are still many obstacles to be overcome before clinical application of cell therapy in neurological disease patients is adopted: 1) it is still uncertain what kind of stem cells would be an ideal source for cellular grafts, and 2) the mechanism by which transplantation of stem cells leads to an enhanced functional recovery and structural reorganization must to be better understood. Steady and solid progress in stem cell research in both basic and preclinical settings should support the hope for development of stem cell‐based cell therapies for neurological diseases.

A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis

During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokine-induced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.

CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP+ cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133+. Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133+ ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133+ ependymal cells represent an additional—perhaps more quiescent—stem cell population in the mammalian forebrain.

Brain neurons develop in a serum and glial free environment: effects of transferrin, insulin- insulin-like growth factor-I and thyroid hormone on neuronal survival, growth and differentiation

We have developed a pure cortical neuronal culture free of glial cells, grown in a serum-free environment. The cultured cells immunostained positively with neurofilament antibody while they displayed virtually no glial cell characteristics, such as glial fibrillary acidic protein, glycerol phosphate dehydrogenase or glutamine synthetase. Insulin and transferrin were necessary and sufficient for neuronal survival, neurite extension and glutamic acid decarboxylase (GAD) expression. Insulin-like growth factor-I was able to replace insulin and was active close to its physiological concentration, suggesting it might be the in vivo factor influencing neuronal growth in the brain. The dynamics of the developmental process were striking. The neurons moved on the poly-D-lysine covered plastic dish, and rearrangements in contacts between cells were observed. At first the neurons underwent a general cellular growth manifested by a large increase in the culture total protein content and by the initiation of neurites. A more specific differentiation, as indicated by the sharp increase in GAD levels which was concurrent with an increase in interneuronal contacts, lagged behind the initial growth. Thyroid hormone (TH) affected the differentiation process, causing a future increase in GAD levels during the same time of increase in neurite growth, in interneuronal contacts, in thyroid hormone receptors and thyroid gland maturation. Removal of each of the hormones after a few days of cell growth revealed that transferrin was still required for neuronal survival while insulin became essential for general cellular growth but not specific neuronal differentiation, since it caused an increase in both the total protein and GAD levels but not in GAD specific activity. TH, on the other hand, affected the differentiation process as evident by its ability to increase GAD specific activity. This action of TH, however, required the presence of insulin, without which no increase in GAD level by TH was observed. This neuronal culture, glial and serum-free, provides a new system for investigating neuronal development and function in the complex mammalian central nervous system.

Immunocytochemical localization of glycerol-3-phosphate dehydrogenase in rat oligodendrocytes

In this study, two indirect immunoperoxidase staining procedures were used to investigate the cellular localization of rat brain glycerol-3-phospate dehydrogenase (EC 1.1.1.8;GPDH). At the light and electron microscopic level, we found that the use of monospecific rabbit antibodies to GPDH consistently resulted in the specific staining of only one glial cell population. GPDH-positive cells in perineuronal, interfascicular and perivascular positions were identified as oligodendrocytes by classical morphological criteria. The specificity of GPDH antigen-antibody reaction was determined by qualitative and quantitative immunochemical methods and by imunocytochemical controls for immunologic and methodologic sources of nonspecific reaction product. The illustrative data from this study serve to qualitatively define GPDH as a biochemical marker for oligodendrocytes in rat central nervous tissue. In view of the fact that the synthesis of rat brain GPDH is specifically regulated by glucocorticoids, the positive results obtained in this study further warrant the interpretation that rat oligodendrocytes are target cells for glucocorticoids.

HORMONAL CONTROL OF GLYCEROLPHOSPHATE DEHYDROGENASE IN THE RAT BRAIN

—Following hypophysectomy or adrenalectomy, glycerolphosphate dehydrogenase (GPDH) (EC 1.1.1.8) activity decreased exponentially in the cerebral hemispheres and brain stem of adult male rats. The latter region was more affected than the former. Malate dehydrogenase (EC 1.1.1.40), isocitrate dehydrogenase (EC 1.1.1.42), lactate dehydrogenase (EC 1.1.1.27) and mitochondrial glycerolphosphate dehydrogenase (EC 1.1.95.5) activities remained unchanged. Injection of adrenocorticotrophic hormone or cortisol in hypophysectomized rats or cortisol in adrenalectomized rats restored GPDH activity. Thyroidectomy and gonadectomy had no effect on GPDH activity. Liver GPDH was not decreased by hypophysectomy or adrenalectomy. Muscle GPDH was diminished slightly by adrenalectomy and as much as brain GPDH by hypophysectomy. In young rats GPDH developmental increase in activity was inhibited by hypophysectomy. These results clearly show that brain GPDH activity is specifically regulated by cortisol (and probably closely related corticosteroids).

Notch Signaling in Astrocytes and Neuroblasts of the Adult Subventricular Zone in Health and after Cortical Injury

The postnatal subventricular zone (SVZ) is a niche for continuous neurogenesis in the adult brain and likely plays a fundamental role in self-repair responses in neurodegenerative conditions. Maintenance of the pool of neural stem cells within this area depends on cell-cell communication such as that provided by the Notch signaling pathway. Notch1 receptor mRNA has been found distributed in different areas of the postnatal brain including the SVZ. Although the identity of Notch1-expressing cells has been established in the majority of these areas, it is still unclear what cell types within the SVZ are expressing components of this pathway. Here we demonstrate that most of expression of Notch1 in the adult SVZ occurs in polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neural precursors and in glial fibrillary acidic protein-positive SVZ astrocytes. Notch1 was also found in PSA-NCAM-positive neuroblasts located within the rostral migratory stream (RMS) but much less in those that have reached the olfactory bulb. We show that two of the naturally occurring Notch1 activators, Jagged1 and Delta1, are also expressed in the SVZ and within the RMS in the adult mouse brain. Finally, using a model of cortical stab wound, we show that the astrogliogenic response of the SVZ to injury is accompanied by activation of the Notch pathway.

NT-3-MEDIATED TRKC RECEPTOR ACTIVATION PROMOTES PROLIFERATION AND CELL SURVIVAL OF RODENT PROGENITOR OLIGODENDROCYTE CELLS IN VITRO AND IN VIVO

We have previously described the expression of a functional full‐length trkC transcript for neurotrophin‐3 (NT‐3) receptor in oligodendroglia (OL) cells (Kumar and de Vellis, 1996). To date, the role of NT‐3 and its signal transduction cascade in OL remains poorly defined. We report that the NT‐3 responsive population of cells in the OL lineage are the progenitor cells and that the addition of NT‐3 results in the autophosphorylation of p145TrkC. Furthermore, NT‐3‐mediated activation of p21ras and mitogen‐activated protein kinase (MAPK), extracellular signal‐regulated protein kinase2 (ERK2), were also observed in the progenitor OL cells. These protein tyrosine kinase (PTK)‐induced responses were sensitive to the presence of K252a, an inhibitor for tyrosine kinase. We have determined that NT‐3 promotes progenitor OL cell commitment to enter into S‐phase of cell cycle to initiate DNA synthesis, in a manner similar to platelet‐derived growth factor‐AA (PDGF‐AA). NT‐3 thus plays a role in cell proliferation when present alone, while augmenting the proliferation capacity of PDGF‐AA as indicated by the nuclear binding activity of the transcription factor, E2F‐1. Both the initiation and progression of mitotic events were confirmed by the expression of c‐myc and cdc2 in the presence of NT‐3, PDGF‐AA or NT‐3 plus PDGF‐AA. A cell survival assay examining interleukin 1‐β‐converting enzyme (ICE)‐like protease‐mediated cleavage of poly (ADP‐ribose) polymerase (PARP) revealed an increase in OL progenitor cell death in the absence of NT‐3 or PDGF‐AA. In corroboration with our in vitro studies, in vivo results show an increased expression of the progenitor OL cell marker, glycerol phosphate dehydrogenase (GPDH) within 48 hr following an intracranial injection of NT‐3, PDGF‐AA, or NT‐3 plus PDGF‐AA in PN4–5 rats. These novel findings suggest that PDGF‐AA potentiates the OL progenitor cells ability to enter into the S‐phase of the cell cycle and that NT‐3 can augment this activity. Furthermore, PDGF‐AA and NT‐3 can block ICE‐like protease‐mediated PARP fragmentation in progenitor OL cells. These results provide important information which further delineates the signal transduction cascades and the role of NT‐3 and PDGF‐AA on OL progenitor cells. J. Neurosci. Res. 54:754–765, 1998.

Synergistic action of thyroid hormone, insulin and hydrocortisone on astrocyte differentiation

We report here on the synergistic regulation of astrocyte development by 3 hormones: thyroid hormone (TH), insulin, and hydrocortisone (HC). Their effect, in a defined serum-free media, on astrocyte morphology, on glia fibrillary acidic protein (GFAP) immunostaining pattern, and on glutamine synthetase (GS) was investigated. TH transformed the flat, polygonal astrocytes into process-bearing cells. This effect was accentuated by insulin, which by itself had no effect on astrocyte morphology. The morphological transformations were accompanied by changes in the pattern of GFAP immunostaining which indicated a more organized and directed cytoskeleton arrangement in the TH-insulin treated cultures. Over 95% of the cells in the culture expressed GFAP. All 3 hormones regulated GS levels. TH increased GS levels by 50% and insulin raised its levels by 3-fold. While having no effect on astrocyte morphology, HC increased GS levels by 3.7-fold in both the hormone-free and insulin-supplemented medium. HC acted synergistically with insulin in its action on GS bringing about a 12-fold increase in the enzyme activity. In contrast, TH did not interact with insulin and was additive with HC in its action on GS. The continuous presence of insulin and TH was required to maintain their morphological and GS effect, suggesting that these hormones might not only be important for astrocyte differentiation, but later on for astrocyte function as well. Since astrocytes interact with and affect neurons and oligodendrocytes, the findings reported here might have bearing on the development and function of these other brain cells as well.