PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Does senile impairment of cholinergic system in rats concern only disturbances in cholinergic phenotype or the progressive degeneration of neuronal cell bodies?

The trophic effect of continuous intraventricular infusion of nerve growth factor (NGF) on morphology of the basal forebrain (BF) cholinergic neurons was tested in 4- and 28-month-old male Wistar rats. All studies were conducted using behaviorally uncharacterized animals from the same breeding colony. Immunohistochemical procedure for choline acetyltransferase (ChAT) and p75NTR receptor has been applied to identify cholinergic cells in the structures of basal forebrain (BF). Using a quantitative image analyzer, morphometric and densitometric parameters of ChAT- and p75NTR-positive cells were measured immediately after cessation of NGF infusion. In 28-month-old non-treated rats the number of intensively ChAT-positive cells in all forebrain structures was reduced by 50-70% as compared with young animals. The remaining ChAT-positive cells appeared shrunken and the neuropil staining was NTR markedly reduced. In contrast, the same neurons when stained for p75 were numerous and distinctly visible with perfect morphology. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neuronal cell bodies. NGF restored the number of intensely stained ChAT-positive cells to about 90% of that for young controls and caused a significant increase in size of those cells in 28-month-old rats as compared with the control, age-matched group. NGF did not influence the morphology of p75NTR-positive neurons, which were well labeled, irrespective of treatment and age of the rats. In 4-month-old rats, NGF infusion decreased the intensity of both ChAT and p75NTR immunostaining. These data provide some evidence for preservation of BF cholinergic neurons from atrophy during aging and indicate that senile impairment of the cholinergic system in rats concerns decrease in ChAT-protein expression rather than an acute degeneration of neuronal cell bodies. Treatment with NGF resulted in restoration of cholinergic phenotype in the BF neurons of aged rats. However, the present study also rises issue of possible detrimental effects of NGF in young normal animals.     

Transforming growth factor-α expands progenitor cells of the basal forebrain,but does not promote cholinergic differentiation

Transforming growth factor-α (TGF-α), a member of the epidermal growth factor (EGF) family, binds to the EGF-receptor (EGF-R). The early expression and widespread distribution of TGF-α and EGF-R in the developing central nervous system (CNS) suggest that TGF-α may play a role in the developing CNS. To study possible effects of TGF-α on cholinergic differentiation in the basal forebrain, we cultured septal nuclei with adjacent basal forebrain from embryonic rat brain in the presence and absence of TGF-α. At the highest dose of TGF-α used (100 ng/mL), activity of choline acetyltransferase (ChAT; EC 2.3.1.6) and the number of cholinergic neurons doubled. However, because protein levels tripled, specific ChAT activity actually declined. To determine the mechanism accounting for the increase in ChAT, we labeled dividing precursors present in the cultures with a replication-deficient retrovirus expressing β-galactosidase in the presence and absence of TGF-α. By staining the cultures for both LacZ and ChAT, we determined that the precursor population expanded in size (individually labeled clones contained more cells), but the percentage of cholinergic neurons present in the clones was unchanged. Therefore, while TGF-α expands the precursor pool, it does not promote cholinergic differentiation. Interleukin-9, included to prompt neuronal differentiation, did not by itself increase ChAT activity, nor did it enhance the action of TGF-α. This was true even when basic fibroblast growth factor was included. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 405–412, 1998     

Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons

A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells.     

Hormonal modulation of neuronal cells behaviour in vitro

In this study we have investigated the effect of insulin and/or of nerve growth factor (NGF) on enzyme activities of cholinergic neurotransmission, in cultured embryonic rat mesencephali. Our data show that choline-O-acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity display a prominent change in the embryonic brain tissues as a function of time in vitro. The change depends on the age of embryos from which the brain cell cultures have been set up. Namely, ChAT activity increases in the cultures taken from 13-17-day-old embryos as a function of time in vitro. AChE activity shows a striking decrease if the cultures have been set up from the older embryos (17-day-old), while AChE activity increases in the cultures prepared from 13-day-old embryos continuously. Insulin (amount ranging 10-27 micrograms/ml) causes a significant inhibition in the ChAT activity in comparison with the increased enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng). AChE activity of 13-day-old embryos was not influenced by insulin (20-27 micrograms/ml) but the same amount of insulin prevents the decrease of AChE activity in cultured brain cells originating from 17-day-old-embryos. Biochemical studies of NGF treated cultures (30 ng/ml) revealed that nerve growth factor resulted in 5-12-fold increase in specific activity of the cholinergic enzyme, choline acetyltransferase (ChAT). NGF did not influence the AChE activity in cultured brain cells (13-17-day-old).     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.     

K-252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures

Abstract: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 n M K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neurodegenerative diseases.     

Fibrillar β‐amyloid 1‐42 alters cytokine secretion,cholinergic signalling and neuronal differentiation

Adult neurogenesis is impaired by inflammatory processes, which are linked to altered cholinergic signalling and cognitive decline in Alzheimer's disease. In this study, we investigated how amyloid beta (Aβ)‐evoked inflammatory responses affect the generation of new neurons from human embryonic stem (hES) cells and the role of cholinergic signalling in regulating this process. The hES were cultured as neurospheres and exposed to fibrillar and oligomeric Aβ_1‐42 (Aβf, AβO) or to conditioned medium from human primary microglia activated with either Aβ_1‐42 or lipopolysaccharide. The neurospheres were differentiated for 29 days in vitro and the resulting neuronal or glial phenotypes were thereafter assessed. Secretion of cytokines and the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and choline acetyltransferase (ChAT) involved in cholinergic signalling was measured in medium throughout the differentiation. We report that differentiating neurospheres released various cytokines, and exposure to Aβf, but not AβO, increased the secretion of IL‐6, IL‐1β and IL‐2. Aβf also influenced the levels of AChE, BuChE and ChAT in favour of a low level of acetylcholine. These changes were linked to an altered secretion pattern of cytokines. A different pattern was observed in microglia activated by Aβf, demonstrating decreased secretion of TNF‐α, IL‐1β and IL‐2 relative to untreated cells. Subsequent exposure of differentiating neurospheres to Aβf or to microglia‐conditioned medium decreased neuronal differentiation and increased glial differentiation. We suggest that a basal physiological secretion of cytokines is involved in shaping the differentiation of neurospheres and that Aβf decreases neurogenesis by promoting a microenvironment favouring hypo‐cholinergic signalling and gliogenesis.     

Human Skeletal Muscle Cells Synthesise a Neuronotrophic Factor Reactive with Spinal Neurons

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.     

Progranulin regulates neurogenesis in the developing vertebrate retina

We evaluated the expression and function of the microglia‐specific growth factor, Progranulin‐a (Pgrn‐a) during developmental neurogenesis in the embryonic retina of zebrafish. At 24 hpf pgrn‐a is expressed throughout the forebrain, but by 48 hpf pgrn‐a is exclusively expressed by microglia and/or microglial precursors within the brain and retina. Knockdown of Pgrn‐a does not alter the onset of neurogenic programs or increase cell death, however, in its absence, neurogenesis is significantly delayed—retinal progenitors fail to exit the cell cycle at the appropriate developmental time and postmitotic cells do not acquire markers of terminal differentiation, and microglial precursors do not colonize the retina. Given the link between Progranulin and cell cycle regulation in peripheral tissues and transformed cells, we analyzed cell cycle kinetics among retinal progenitors following Pgrn‐a knockdown. Depleting Pgrn‐a results in a significant lengthening of the cell cycle. These data suggest that Pgrn‐a plays a dual role during nervous system development by governing the rate at which progenitors progress through the cell cycle and attracting microglial progenitors into the embryonic brain and retina. Collectively, these data show that Pgrn‐a governs neurogenesis by regulating cell cycle kinetics and the transition from proliferation to cell cycle exit and differentiation. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 77: 1114–1129, 2017     

Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor

Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.     

Clinical trials of bestatin for leukemia and solid tumors

Dissociated cells from 13- and 17-day-old embryonic rat mesencephali have grown in primary cultures in order to compare the early and late influences of different agents - insulin, dexamethasone and nerve growth factor (NGF) - on the expression of cholinergic maturation process. We have studied cholin acetyltransferase (ChAT) activity, which is regarded as a specific marker for cholinergic function of the brain, and a widely used differentiation marker, the acetyl-cholinesterase (AchE) enzyme. Biochemical maturation of increasing specific activity of ChAT in both younger and older cells was taken into consideration. During cultivation the AchE activity was slightly increased in younger cells, but a dramatic decrease could be noted in older ones. Insulin in concentration from 10 to 27 µg mL^–1 causes a significant inhibition in ChAT activity in comparison with the enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng), independently of embryos age. This polypeptide hormone is able to enhance AchE activity in the cultured cells, especially in older ones. With continuous treatment of the culture with dexamethasone, a synthetic glucocorticoid, the ChAT activity in younger cells reaches a maximum curve by day 9 (nine). At this time the AchE activity shows a slighter, no significant increase than at any other time during cultivation. In cell cultures taken from 17-day-old embryos however dexamethasone treatment evoked a significant decrease in ChAT activity with a concomitant increase of AchE activity which was compared to insulin treatment. In spite of the fact that the NGF is able to enhance the ChAT activity, no significant alteration in AchE activity can be measured in younger cell cultures. These results suggest an uneven expression of the enzymes in embryonic rat mesencephali in the presence of above agents depending on the age of cells.     

Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol-3'-kinase pathway

Nerve growth factor (NGF) exerts anti-apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen-activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up-regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF-induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter-luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)-Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)-treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A-treated rats, NGF increased cholinergic enzyme activity in a dose-dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A-treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A-treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.     

Human endometrial stem cell neurogenesis in response to NGF and bFGF

The potential of cell therapy is promising in nerve regeneration, but is limited by ethical considerations about the proper and technically safe source of stem cells. We report the successful differentiation of human EnSCs (endometrial stem cells) as a rich source of renewable and safe progenitors into high-efficiency cholinergic neurons. The extracellular signals of NGF (nerve growth factor) and bFGF (basic fibroblast growth factor) could induce cholinergic neuron differentiation. ChAT (choline acetyltransferase), MAP2 (microtubule associated protein 2) and NF-l (neurofilament L) increased after administration of bFGF and NGF to the EnSC cultures. trkC and FGFR2 (fibroblast growth factor receptor 2), which belong to the NGF and bFGF receptors respectively, were determined in populations of EnSCs. NGF, bFGF and their combination differentially influenced human EnSCs high efficiency differentiation. By inducing cholinergic neurons from EnSCs in a chemically defined medium, we could produce human neural cells without resorting to primary culture of neurons. This in vitro method provides an unlimited source of human neural cells and facilitates clinical applications of EnSCs for neurological diseases.     

Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats

Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NGF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NGF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NGF. The anti-NGF antibody administration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons.     

Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors

Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via alpha 7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express alpha 7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunohistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-alpha release in murine-derived microglial cells, an effect attenuated by alpha 7 selective nicotinic antagonist, alpha-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha 7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.     

Intracellular Storage and Evoked Release of Acetylcholine from Postnatal Rat Basal Forebrain Cholinergic Neurons in Culture with Nerve Growth Factor

Cholinergic neurons from the septum area, the vertical limb of the diagonal band of Broca, and the nucleus basalis of Meynert of postnatal 13-day-old rats were cultured with or without nerve growth factor (NGF) conditions. Total choline acetyltransferase (ChAT) activities, acetylcholine (ACh) contents, and survival numbers of cholinergic neurons in culture from each of three distinct regions were increased by NGF treatment, but little difference was found in cellular ChAT activities and ACh contents obtained in cultures with or without NGF. The result shows that NGF promotes the survival of cholinergic neurons from 13-day-old rats. Furthermore, the release of ACh from cultured neurons was investigated. The cells cultured with NGF showed a larger increase of the high K+-evoked ACh release than those cultured without NGF. However, NGF had no effect on spontaneous release. This suggests that NGF could regenerate and sustain the stimulation-evoked release mechanisms of ACh in cultured cholinergic neurons from postnatal rats.     

Involvement of dopaminergic neuronal cystatin C in neuronal injury-induced microglial activation and neurotoxicity

Factors released from injured dopaminergic (DA) neurons may trigger microglial activation and set in motion a vicious cycle of neuronal injury and inflammation that fuels progressive DA neurodegeneration in Parkinson's disease. In this study, using proteomic and immunoblotting analysis, we detected elevated levels of cystatin C in conditioned media (CM) from 1-methyl-4-phenylpyridinium and dieldrin-injured rat DA neuronal cells. Immunodepletion of cystatin C significantly reduced the ability of DA neuronal CM to induce activation of rat microglial cells as determined by up-regulation of inducible nitric oxide synthase, production of free radicals and release of proinflammatory cytokines as well as activated microglia-mediated DA neurotoxicity. Treatment of the cystatin C-containing CM with enzymes that remove O- and sialic acid-, but not N-linked carbohydrate moieties markedly reduced the ability of the DA neuronal CM to activate microglia. Taken together, these results suggest that DA neuronal cystatin C plays a role in the neuronal injury-induced microglial activation and neurotoxicity. These findings from the rat DA neuron-microglia in vitro model may help guide continued investigation to define the precise role of cystatin C in the complex interplay among neurons and glia in the pathogenesis of Parkinson's disease.