Shalini Kumar

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.

Mice lacking NT-3, and its receptor TrkC, exhibit profound deficiencies in CNS glial cells.

Neurotrophin‐3 (NT‐3) and its receptor TrkC are known to be important for neuronal survival. More recently, NT‐3 has been implicated as playing a role in oligodendrocyte (OL) proliferation and survival in vitro. Examination of NT‐3 and TrkC knockout mice revealed a reduction in NT‐3‐dependent neurons. To date, no study has examined alterations in glial cell populations in these knockout mice. In this report, we demonstrate a decline in OL progenitor cell numbers within the CNS of NT‐3 and TrkC knockout mice. We also observed that immature and mature OL‐specific markers were attenuated in the NT‐3 and TrkC knockout animals. Deficiencies in other CNS glial cells, including astrocytes and ameboid microglia, were also observed. The subventricular zone (SVZ), a highly proliferative region for progenitor glial cells, was reduced in size. Furthermore, a nuclear‐specific stain revealed a decline in the numbers of pyknotic nuclei in and around the SVZ of the knockout mice. These data will support an in vivo NT‐3‐dependent mechanism for the normal development of CNS glial cells. GLIA 26:153–165, 1999.

Transferrin Is an Essential Factor for Myelination

It has been established that oligodendrocytes, the myelin forming cells, participate in iron homeostasis through the synthesis and secretion of transferrin. Here we investigated whether a correlation exists between myelination, the commonly studied function of oligodendrocytes, and that of transferrin synthesis and secretion. We used a proteolipid protein mutant, the myelin deficient rat, whose condition is characterized by severe hypomyelination. We compared the ontogenic profile for transferrin gene expression in mutants with that of unaffected rat pups through northern blot analysis and in situ hybridization. Surprisingly, transferrin synthesis was null in mutant oligodendrocytes. Next, we demonstrated that a single apo-transferrin intraparenchymal injection administered to P5 rat pups enabled mutant oligodendrocytes to synthesize myelin basic protein and to myelinate axons, indicating that transferrin effects mutant oligodendrocyte maturation regardless of its source. Thus, transferrin availability is essential for oligodendrocyte maturation and function, and oligodencrocytes are most vulnerable to transferrin deficiency during the premyelinating stage.

Estrogen receptor β ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis.

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) β ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and β) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERβ and activated the phosphatidylinositol 3-kinase (PI3K)/serine-threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERβ ligands immediate and favorable therapeutic candidates for demyelinating disease.

Regulation of mRNAs for Three Enzymes in the Glial Cell Model C6 Cell Line

Abstract: In the glial cell line C6, regulation of actinomycin D (Act‐D)‐sensitive translatable polysomal mRNAs of three key enzymes—glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) and glutamine synthetase (GS) by glucocorticoids and lactate dehydrogenase (LDH; EC 1.1.1.27) by catecholamines—is described. Though the first two enzymes are hydrocortisone (HC)‐inducible, the nature of their response to the hyperacetylating agent sodium butyrate is dramatically different. Furthermore the appearance of GPDH translatable poly (A)+ RNA in HC‐induced cells is inhibited by the presence of cycloheximide (CHX), whereas the induction of GS is unaffected by CHX. These observations necessitate further probing into an existing model system to explain the varied mechanisms of induction of these two enzymes by a single inducer. In combination with the third enzyme whose induction by catecholamines is glial specific, we believe that the C6 cell represents the most appropriate cell line for molecular neurobiologists to study the mechanisms of hormone action in glia.

Rat Brain Creatine Kinase Messenger RNA Levels Are High in Primary Cultures of Brain Astrocytes and Oligodendrocytes and Low in Neurons

Abstract: Rat brain creatine kinase (CKB) gene expression is highest in the brain but is also detectable at lower levels in some other tissues. In the brain, the CKB enzyme is thought to be involved in the regeneration of ATP necessary for transport of ions and neurotransmitters. To understand the molecular events that lead to high CKB expression in the brain, we have determined the steady‐state levels of CKB mRNA in homogeneous cultures of primary rat brain astrocytes, oligodendrocytes, and neurons. Northern blot analysis showed that whereas the 1.4‐kb CKB mRNA was detectable in neurons, the level was about 17‐fold higher in oligodendrocytes and 15‐fold higher in astrocytes. The blots were hybridized with a CKB‐specific 32P‐antisense RNA probe, complementary to the 3’untranslated sequence of CKB, which hybridizes to CKB mRNA but not CKM mRNA. Also, the 5’and 3’ends of CKB mRNA from the glial cells were mapped, using exon‐specific antisense probes in the RNase‐protection assay, and were found to be the same in astrocytes and oligodendrocytes. This indicated that (a) the site of in vivo transcription initiation in astrocytes and oligodendrocytes was directed exclusively by the downstream, nonconcensus TTAA sequence at ‐25 bp in the CKB promoter that is also utilized by all other cell types that express CKB and (b) the 3’end of mature CKB mRNA was the same in astrocytes and oligodendrocytes. In addition, there was no detectable alternate splicing in exon 1, 2, or 8 of CKB mRNA in rat astrocytes and oligodendrocytes. Also, our studies showed that 1.4‐kb CKB mRNA is expressed in established C6 glioma cells at an intermediate level about threefold higher than that in primary neurons.

Multiple functional therapeutic effects of the estrogen receptor β agonist indazole-Cl in a mouse model of multiple sclerosis

Significance In the search for effective multiple sclerosis treatment, much effort has been invested in estrogens and estrogen receptor (ER) agonists because of their neuroprotective benefits. However, because estrogens can produce ERα-based feminizing effects and cancer, ERβ agonists represent more desirable therapeutic candidates. The structurally unique ERβ ligand indazole chloride (Ind-Cl), a halogen-substituted phenyl-2H-indazole core, is a preclinical development candidate with a strong dossier. Our results indicate that Ind-Cl is effective in functionally ameliorating disease even when treatment is initiated at peak experimental autoimmune encephalomyelitis clinical disease. Ind-Cl’s immunomodulatory and direct remyelinating effects result in motor dysfunction amelioration. These findings support Ind-Cls potential to provide unique therapeutic benefits to patients with multiple sclerosis, as well as patients affected by other demyelinating disorders. Currently available immunomodulatory therapies do not stop the pathogenesis underlying multiple sclerosis (MS) and are only partially effective in preventing the onset of permanent disability in patients with MS. Identifying a drug that stimulates endogenous remyelination and/or minimizes axonal degeneration would reduce the rate and degree of disease progression. Here, the effects of the highly selective estrogen receptor (ER) β agonist indazole chloride (Ind-Cl) on functional remyelination in chronic experimental autoimmune encephalomyelitis (EAE) mice were investigated by assessing pathologic, functional, and behavioral consequences of both prophylactic and therapeutic (peak EAE) treatment with Ind-Cl. Peripheral cytokines from autoantigen-stimulated splenocytes were measured, and central nervous system infiltration by immune cells, axon health, and myelination were assessed by immunohistochemistry and electron microscopy. Therapeutic Ind-Cl improved clinical disease and rotorod performance and also decreased peripheral Th1 cytokines and reactive astrocytes, activated microglia, and T cells in brains of EAE mice. Increased callosal myelination and mature oligodendrocytes correlated with improved callosal conduction and refractoriness. Therapeutic Ind-Cl-induced remyelination was independent of its effects on the immune system, as Ind-Cl increased remyelination within the cuprizone diet-induced demyelinating model. We conclude that Ind-Cl is a refined pharmacologic agent capable of stimulating functionally relevant endogenous myelination, with important implications for progressive MS treatment.

Transferrin Regulates Transcription of the MBP Gene and Its Action Synergizes with IGF-1 to Enhance Myelinogenesis in the md Rat

Myelin-deficient (md) rats and their unaffected littermates were injected at postnatal day 4 either with a single dose of transferrin (Tf) or insulin-like growth factor one (IGF-1) singly or combined. Two weeks later, their brains were perfused and coronal sections were analyzed for MBP by in situ hybridization and for transferrin and myelin basic protein (Tf and MBP) by double immunofluorescence. Each of the factors separately had an effect on mutant animals as seen by both increased OL maturation, and MBP mRNA and protein synthesis. The combination of factors resulted in a profound enhancement of the myelinogenic properties of oligodendrocytes (OL) with a consequent increase in the number of MBP-labeled fibers. The brains of unaffected littermates also responded to growth factor(s) injection either by increasing myelination in some brain areas or by regulating the synthesis of MBP in OL. Using rat OL cultures we studied the site of transferrin action for the expression of MBP gene. We found by run off transcription that the MBP mRNA was significantly increased at the nuclear level but the PLP message was unaffected. Thus, transferrin selectively regulates MBP at the transcriptional level and together with IGF-1 synergizes to increase both the maturation and myelinogenic properties of md and normal OL.

Estrogen receptor (ER) β expression in oligodendrocytes is required for attenuation of clinical disease by an ERβ ligand

Significance Multiple sclerosis (MS) is a debilitating neurodegenerative disease characterized by inflammation and demyelination. Immunomodulatory agents slow, but do not prevent, disease progression and offer only indirect neuroprotection. The estrogen receptor β (ERβ) ligand diarylpropionitrile (DPN) has direct neuroprotective effects in a mouse model of MS by stimulating endogenous myelination. Here we elucidate the cell type(s) mediating DPN’s effects. Conditional knockout of ERβ in oligodendrocyte (OL) lineage cells prevented DPN-induced improvement in clinical disease and myelination, partially prevented callosal conduction improvement, and prevented activation of a pathway implicated in OL survival/axon myelination. Our results indicate that DPN-conferred neuroprotection in a mouse model of MS is mediated by ERβ in OLs and inform highly targeted pharmacotherapeutic approaches to MS treatment. Treatment of experimental autoimmune encephalomyelitis (EAE) mice with the estrogen receptor (ER) β ligand diarylpropionitrile (DPN) has been shown to have neuroprotective effects via stimulation of endogenous myelination. The direct cellular mechanisms underlying the effects of this ERβ ligand on the central nervous system are uncertain because different cell types in both the peripheral immune system and central nervous system express ERs. ERβ is the target molecule of DPN because DPN treatment fails to decrease EAE clinical symptoms in global ERβ-null mice. Here we investigated the potential role of ERβ expression in cells of oligodendrocyte (OL) lineage in ERβ ligand-mediated neuroprotection. To this end, we selectively deleted ERβ in OLs using the well-characterized Cre-loxP system for conditional gene knockout (CKO) in mice. The effects of this ERβ CKO on ERβ ligand-mediated neuroprotective effects in chronic EAE mice were investigated. ERβ CKO in OLs prevented DPN-induced decrease in EAE clinical disease. DPN treatment during EAE did not attenuate demyelination, only partially improved axon conduction, and did not activate the phosphatidylinositol 3-kinase/serine-threonine-specific protein kinase/mammalian target of rapamycin signaling pathway in ERβ CKO mice. However, DPN treatment significantly increased brain-derived neurotrophic factor levels in ERβ CKO mice. These findings demonstrate that signaling through ERβ in OLs is essential for the beneficial myelination effects of the ERβ ligand DPN in chronic EAE mice. Further, these findings have important implications for neuroprotective therapies that directly target OL survival and myelination.

Neurotrophin activates signal transduction in oligodendroglial cells: Expression of functional TrkC receptor isoforms

The role of the NT‐3 has been implicated in the survival of progenitor oligodendrocytes in culture. The object of this study was to investigate the expression of the TrkC receptor and its responsiveness in glial cells. We report the expression of two TrkC receptor isoforms in rat primary oligodendrocyte cultures, a glial progenitor cell line, CG‐4, and in C6 glioma cells. The reverse transcription‐polymerase chain reaction‐aided amplification of glial trkC with specific primers from the kinase domain, followed by its cloning and sequencing, shows the presence of two trkC transcripts. The sequence of one of the transcripts is homologous to a previously identified trkC isoform which encodes a functional receptor. The other transcript contains a 42‐bp insert in the kinase domain. A Western blot of CG‐4 and C6 probed with antibody to a TrkC revealed the presence of gp145‐kDa protein band. The investigations revealed a rapid autophosphorylation of gp145TrkC in CG‐4 and C6 cells in the presence of its specific ligand, NT‐3. Furthermore, K252a, a neurotrophin‐specific inhibitor, abolishes the NT‐3‐mediated receptor autophosphorylation. We also examined other NT‐3‐dependent phosphorylation of cellular substrates in oligodendroglial cells. Interestingly, we observed phosphorylation of phospholipase Cγ‐1 in CG‐4 and C6 cells, and phosphorylation of phosphatidylinositol 3‐kinase in C6 cells in the presence of NT‐3. Both the NT‐mediated phosphorylation of phospholipase Cγ‐1 and phosphorylation of phosphatidylinositol 3‐kinase are blocked in the presence of K252a. The detection of the NT‐3‐mediated early signal transduction events demonstrates that TrkC receptor exhibits NT‐3‐mediated intracellular response in oligodendroglial cells.