Yoong H. Chang

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth‐regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell‐derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF‐deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth‐stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G‐proteins, the protein kinase C inhibitor H7 and the mitogen‐activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth‐regulatory signal during neurogenesis via pathways involving pertussis toxin‐sensitive G‐proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Canonical Transient Receptor Potential 1 Plays a Role in Basic Fibroblast Growth Factor (bFGF)/FGF Receptor-1-Induced Ca2+ Entry and Embryonic Rat Neural Stem Cell Proliferation

Basic fibroblast growth factor (bFGF) and its major receptor FGF receptor-1 (FGFR-1) play an important role in the development of the cortex. The mechanisms underlying the mitogenic role of bFGF/FGFR-1 signaling have not been elucidated. Intracellular Ca2+ concentrations ([Ca2+]i) in proliferating cortical neuroepithelial cells are markedly dependent on Ca2+ entry (Maric et al., 2000a). The absence of voltage-dependent Ca2+ entry channels, which emerge later, indicates that other membrane mechanisms regulate [Ca2+]i during proliferation. Canonical transient receptor potential (TRPC) family channels are candidates because they are voltage independent and are expressed during CNS development (Strübing et al., 2003). Here, we investigated the involvement of TRPC1 in bFGF-mediated Ca2+ entry and proliferation of embryonic rat neural stem cells (NSCs). Both TRPC1 and FGFR-1 are expressed in the embryonic rat telencephalon and coimmunoprecipitate. Quantitative fluorescence-activated cell sorting analyses of phenotyped telencephalic dissociates show that ∼80% of NSCs are TRPC1+, proliferating, and express FGFR-1. Like NSCs profiled ex vivo, NSC-derived progeny proliferating in vitro coexpress TRPC1 and FGFR1. Antisense knock-down of TRPC1 significantly decreases bFGF-mediated proliferation of NSC progeny, reduces the Ca2+ entry component of the Cai2+ response to bFGF without affecting Ca2+ release from intracellular stores or 1-oleoyl-2-acetyl-sn-glycerol-induced Ca2+ entry, and significantly blocks an inward cation current evoked by bFGF in proliferating NSCs. Both Ca2+ influx evoked by bFGF and NSC proliferation are attenuated by Gd3+ and SKF96365, two antagonists of agonist-stimulated Ca2+ entry. Together, these results show that TRPC1 contributes to bFGF/FGFR-1-induced Ca2+ influx, which is involved in self-renewal of embryonic rat NSCs.

Estrogen protects against β-amyloid-induced neurotoxicity in rat hippocampal neurons by activation of Akt

The cellular mechanisms underlying the neuroprotective effects of estrogen are only beginning to be elucidated. Here we examined the role of protein kinase B (Akt) activation in 17β-estradiol (E2) inhibition of β-amyloid peptide (31-35) (Aβ31−35)-induced neurotoxicity in cultured rat hippocampal neurons. Aβ31−35 (25-30 βM) significantly decreased the total number of microtubule associated protein-2 positive cells (MAP2+). This decrease was significantly reversed by pre-treatment with 100 nM E2. Further, 100 nM E2 alone significantly increased the total number of protein kinase B and microtubule associated protein-2 positive cells compared with controls. Such E2-induced increases were inhibited by LY294002 (20 μM), a specific PI3-K inhibitor, as well as by tamoxifen, an estrogen receptor antagonist/selective estrogen receptor modulator. These results indicate that the neuroprotective effects of E2 may be mediated at least in part via estrogen receptor-mediated protein kinase B activation.

Self-Renewing and Differentiating Properties of Cortical Neural Stem Cells Are Selectively Regulated by Basic Fibroblast Growth Factor (FGF) Signaling via Specific FGF Receptors

Developmental processes mediating the initiation of lineage commitment from self-renewing neural stem cells (NSCs) remain mostly unclear because of the persisting ambiguity in identifying true NSCs from proliferative lineage-restricted progenitors (LRPs), which are directly or indirectly derived from NSCs. Our multilineage immunohistochemical analyses of early embryonic rat telencephalon at the onset of neurogenesis revealed clear dorsoventral gradients in the emergence of two types of neuronal progenitors (NPs) from multilineage-negative NSCs. Enumeration of NSCs using comprehensive flow cytometric analysis demonstrated that their precipitous decline in vivo involved both active differentiation into NPs and an increased propensity toward apoptosis. Both processes paralleled the dorsoventral changes in fibroblast growth factor receptor (FGFR) expressions. NSCs residing in the dorsal telencephalon coexpressed FGFR1 and FGFR3, whereas those residing in the ventral telencephalon also expressed FGFR2. NSCs exposed to basic fibroblast growth factor (bFGF) in vitro generated four stereotypical clonal expansion states: efficiently self-renewing, inefficiently self-renewing limited by apoptosis, exclusively neurogenic, and multipotential, generating up to five types of LRPs. The plasticity among these expansion states depended on ambient [bFGF], telencephalic developmental stage, and differential activation/inactivation of specific FGFRs. Coactivation of FGFR1 and FGFR3 promoted symmetrical divisions of NSCs (self-renewal), whereas inactivation of either triggered asymmetrical divisions and neurogenesis from these cells. Developmental upregulation of FGFR2 expression correlated with a shift of NSCs into a multipotential state or apoptosis. These results provide new insights regarding the roles of FGFRs in diversification of NSC properties and initiation of neural lineage-restricted differentiation.

α-lipoic acid protects rat cortical neurons against cell death induced by amyloid and hydrogen peroxide through the Akt signalling pathway

Substantial evidence suggests that the accumulation of beta-amyloid (Abeta)-derived peptides contributes to the aetiology of Alzheimers disease (AD) by stimulating formation of free radicals. Thus, the antioxidant alpha-lipoate, which is able to cross the blood-brain barrier, would seem an ideal substance in the treatment of AD. We have investigated the potential effectiveness of alpha-lipoic acid (LA) against cytotoxicity induced by Abeta peptide (31-35) (30 microM) and hydrogen peroxide (H(2)O(2)) (100 microM) with the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction and fluorescence dye propidium iodide assays in primary neurons of rat cerebral cortex. We found that treatment with LA protected cortical neurons against cytotoxicity induced by Abeta or H(2)O(2). In addition, LA-induced increase in the level of Akt in the neurons was observed by Western blot. The LA-induced neuroprotection and Akt increase were attenuated by pre-treatment with the phosphatidylinositol 3-kinase inhibitor, LY294002 (50 microM). Our data suggest that the neuroprotective effects of the antioxidant LA are partly mediated through activation of the PKB/Akt signaling pathway.

Dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) regulate apoptosis during neurogenesis by triggering the Akt signaling pathway in opposing ways

Dehydroepiandrosterone (DHEA) can function to protect neural precursors and their progeny targeted with toxic insults; however, the molecular mechanisms underlying the neuroprotective effects of DHEA are not understood. We cultured neural precursors from the embryonic forebrain of rats and examined the effects of DHEA and its sulfated derivative (DHEAS) on the activation of the serine-threonine protein kinase Akt, which is widely implicated in cell survival signaling. We found that DHEA activated Akt in neural precursor culture, in association with a decrease in apoptosis. In contrast, DHEAS decreased activated Akt levels and increased apoptosis. The effects of DHEA on neural cell survival and activation of Akt were not blocked by the steroid hormone antagonists flutamide and tamoxifen, but both were blocked by a PI3-K inhibitor, LY294002. These findings suggest that during neurogenesis in the developing cortex, DHEA and DHEAS regulate the survival of neural precursors and progeny through the Akt signaling pathway.

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells.

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum‐free medium in response to basic fibroblast growth factor. RT‐PCR revealed the presence of mRNAs encoding AMPAA, AMPAC, KA1, KA2, NMDA1, and NMDA2D subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca2+ imaging and whole‐cell patch‐clamp recording techniques in cells pulse‐labeled with bromodeoxyuridine (BrdU) for 1–4 hr. The recorded cells were then double‐immunostained for BrdU incorporation and neuron‐specific β‐tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca2+ and inward currents only in differentiating neurons. In contrast, proliferating (BrdU+TuJ1‐) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca2+ imaging revealed that a subpopulation of BrdU+TuJ1+ cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA‐responsive cells pulse‐labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU+TuJ1+ cells under physiological conditions and may play a role in terminal cell division. J. Neurosci. Res. 61:652–662, 2000.

Sex-related differences in MAPKs activation in rat astrocytes: effects of estrogen on cell death.

Gender-related differences in the unstimulated and estrogen-induced activation of the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2, cell proliferation, and cell death were examined using rat cortical astrocytes in culture. Females have higher unstimulated levels of phosphorylated ERK1 and ERK2 than males. 17beta-Estradiol (E(2)) decreases activation of ERK1 and ERK2, with females showing a greater response than males. Further, E(2) results in more inhibition of DNA synthesis and greater increase in cell death in females than in males. The inhibitory effects of E(2) on DNA synthesis are mimicked and enhanced by a specific MAPK kinase (MEK) inhibitor, PD98059. Finally, the inhibitory effects of E(2) are blocked by the estrogen receptor antagonist tamoxifen in astrocytes from females but not males, with ER-alpha (estrogen receptor alpha) present in the former but not the latter. Taken together, these results suggest that the sex differences in unstimulated and estrogen-modulated activation of MAPKs may result in differential regulation of cell proliferation and death in astrocytes and possibly contribute to sexual dimorphisms in brain development.

Basic FGF‐responsive telencephalic precursor cells express functional GABAA receptor/Cl− channels in vitro

We have previously described the expression of specific gamma-aminobutyric acid (GABA)A receptor subunits and their transcripts in the cortical neuroepithelium (Ma and Barker, 1995, 1998). However, it is not clear whether neural precursor cells exposed to basic fibroblast growth factor (bFGF) in vitro reproduce the biological properties of neuroepithelial cells in vivo within the embryonic ventricular zone. In the present study, neural precursor cells were isolated from the telencephalic neuroepithelium of embryonic day 13-13.5 rats and exposed to bFGF in serum-free medium. Basic FGF-responsive cells were capable of dividing and differentiating into neurons and astrocytes. The rapidly dividing cells formed multicellular spheres and then a rosette-like formation in which a majority of cells expressed GABA(A) receptor alpha4, beta1, or gamma1 subunit proteins. We found in perforated patch-clamp recordings that GABA depolarized bromodeoxyundine (BrdU)+ precursor cells, and under voltage-clamp induced a bicuculline-sensitive current that reversed at the Cl- equilibrium potential. GABA also increased cytoplasmic Ca2+ in a significant number of BrdU+ cells that was blocked by bicuculline. The bicuculline sensitivity of these pharmacological effects implicates GABA(A) receptor/Cl- channels, since bicuculline is a competitive GABA(A) antagonist at these channels in well-differentiated cells. It is possible that the three GABA(A) receptor subunits (alpha4, beta1, and gamma1) found in precursor cells form the Cl- channels detected electrophysiologically. The functional GABA(A) receptor/Cl- channels and associated regulation of their cytoplasmic Ca2+ levels via bicuculline-sensitive mechanisms may play significant roles in the regulation of neural cell proliferation in this model neuroepithelium.

TNF-α induced over-expression of GFAP is associated with MAPKs

Increased levels of tumor necrosis factor-alpha (TNF-α), a pluripotent cytokine that is reportedly mitogenic to astrocytes, are associated with the expression of glial fibrillary acidic protein (GFAP), the most specific marker for astrocytes, in many neuropathological conditions, including brain injury, CNS infection, Creutzfeldt-Jakob disease and Alzheimers disease. Here, we show that treatment of cultured astrocytes with TNF-α resulted in dramatic over-expression of GFAP, associated with a substantial activation of the mitogen activated protein kinase (MAPK) Erk2 (extracellular signal-regulated protein kinase). We also demonstrate that TNF-α-induced over-expression of GFAP was significantly attenuated by the MAPK inhibitor PD98059. We conclude that TNF-α may up-regulate GFAP through the MAPK signaling pathway. Because increased GFAP is a hallmark of reactive gliosis, understanding the mechanisms that regulate GFAP expression may facilitate development of strategies to minimize the gliosis associated with many brain diseases.