Histone Deacetylase 2 Knockdown Ameliorates Morphological Abnormalities of Dendritic Branches and Spines to Improve Synaptic Plasticity in an APP/PS1 Transgenic Mouse Model.

Disease-modifying therapies, such as neuroprotective and neurorestorative interventions, are strongly desired for Alzheimer's disease (AD) treatment. Several studies have suggested that histone deacetylase 2 (HDAC2) inhibition can exhibit disease-modifying effects in AD patients. However, whether HDAC2 inhibition shows neuroprotective and neurorestorative effects under neuropathic conditions, such as amyloid ß (Aß)-elevated states, remains poorly understood. Here, we performed HDAC2-specific knockdown in CA1 pyramidal cells and showed that HDAC2 knockdown increased the length of dendrites and the number of mushroom-like spines of CA1 basal dendrites in APP/PS1 transgenic mouse model. Furthermore, HDAC2 knockdown also ameliorated the deficits in hippocampal CA1 long-term potentiation and memory impairment in contextual fear conditioning tests. Taken together, our results support the notion that specific inhibition of HDAC2 has the potential to slow the disease progression of AD through ameliorating Aß-induced neuronal impairments.

Discovery of Extremely Selective Fused Pyridine-Derived ß-Site Amyloid Precursor Protein-Cleaving Enzyme (BACE1) Inhibitors with High In Vivo Efficacy through 10s Loop Interactions.

ß-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is considered to be a promising target for treating Alzheimer's disease. However, all clinical BACE1 inhibitors have failed due to lack of efficacy, and some have even led to cognitive worsening. Recent evidence points to the importance of avoiding BACE2 inhibition along with careful dose titration. In this study, we focused on the fact that the 10s loop lining the S3 pocket in BACE1 can form both "open (up)" and "closed (down)" conformations, whereas in BACE2, it prefers to adopt a "closed" form; thus, more space is available in BACE1. By leveraging the difference, we designed fused pyridine analogues that could reach the 10s loop, leading to 6 with high selectivity and significant Aß reduction. The cocrystal structures confirmed that 6 significantly increased B-factors of the 10s loop in BACE2 relative to those in BACE1. Thus, the destabilization of BACE2 seems to offer structural insights into the reduced BACE2 potency of 6, explaining the significant improvement in BACE1 selectivity.

Balancing potency and basicity by incorporating fluoropyridine moieties: Discovery of a 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitor that affords robust and sustained central Aß reduction.

ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the treatment of Alzheimer's disease (AD). In this report, we describe the discovery of BACE1 inhibitors with a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine rings, which showed increased biochemical and cellular potency, along with reduced basicity on the amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained cerebrospinal fluid (CSF) Aß reduction in dog. The crystal structure of compound 11 bound to BACE1 confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.

Structure-Based Approaches to Improving Selectivity through Utilizing Explicit Water Molecules: Discovery of Selective ß-Secretase (BACE1) Inhibitors over BACE2.

BACE1 is an attractive target for disease-modifying treatment of Alzheimer's disease. BACE2, having high homology around the catalytic site, poses a critical challenge to identifying selective BACE1 inhibitors. Recent evidence indicated that BACE2 has various roles in peripheral tissues and the brain, and therefore, the chronic use of nonselective inhibitors may cause side effects derived from BACE2 inhibition. Crystallographic analysis of the nonselective inhibitor verubecestat identified explicit water molecules with different levels of free energy in the S2' pocket. Structure-based design targeting them enabled the identification of propynyl oxazine 3 with improved selectivity. Further optimization efforts led to the discovery of compound 6 with high selectivity. The cocrystal structures of 7, a close analogue of 6, bound to BACE1 and BACE2 confirmed that one of the explicit water molecules is displaced by the propynyl group, suggesting that the difference in the relative water displacement cost may contribute to the improved selectivity.

Discovery of Atabecestat (JNJ-54861911): A Thiazine-Based ß-Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor Advanced to the Phase 2b/3 EARLY Clinical Trial.

Accumulation of amyloid ß peptides (Aß) is thought to be one of the causal factors of Alzheimer's disease (AD). The aspartyl protease ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting protease for Aß production, and therefore, BACE1 inhibition is a promising therapeutic approach for the treatment of AD. Starting with a dihydro-1,3-thiazine-based lead, Compound J, we discovered atabecestat 1 (JNJ-54861911) as a centrally efficacious BACE1 inhibitor that was advanced into the EARLY Phase 2b/3 clinical trial for the treatment of preclinical AD patients. Compound 1 demonstrated robust and dose-dependent Aß reduction and showed sufficient safety margins in preclinical models. The potential of reactive metabolite formation was evaluated in a covalent binding study to assess its irreversible binding to human hepatocytes. Unfortunately, the EARLY trial was discontinued due to significant elevation of liver enzymes, and subsequent analysis of the clinical outcomes showed dose-related cognitive worsening.

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

Trifluoromethyl Dihydrothiazine-Based ß-Secretase (BACE1) Inhibitors with Robust Central ß-Amyloid Reduction and Minimal Covalent Binding Burden.

The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as ß-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aß reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

Discovery of an Extremely Potent Thiazine-Based ß-Secretase Inhibitor with Reduced Cardiovascular and Liver Toxicity at a Low Projected Human Dose.

Genetic evidence points to deposition of amyloid-ß (Aß) as a causal factor for Alzheimer's disease. Aß generation is initiated when ß-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aß reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aß reduction of 80% at trough level.

Structure-Based Design of Selective ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitors: Targeting the Flap to Gain Selectivity over BACE2.

BACE1 inhibitors hold potential as agents in disease-modifying treatment for Alzheimer's disease. BACE2 cleaves the melanocyte protein PMEL in pigment cells of the skin and eye, generating melanin pigments. This role of BACE2 implies that nonselective and chronic inhibition of BACE1 may cause side effects derived from BACE2. Herein, we describe the discovery of potent and selective BACE1 inhibitors using structure-based drug design. We targeted the flap region, where the shape and flexibility differ between these enzymes. Analysis of the cocrystal structures of an initial lead 8 prompted us to incorporate spirocycles followed by its fine-tuning, culminating in highly selective compounds 21 and 22. The structures of 22 bound to BACE1 and BACE2 revealed that a relatively high energetic penalty in the flap of the 22-bound BACE2 structure may cause a loss in BACE2 potency, thereby leading to its high selectivity. These findings and insights should contribute to responding to the challenges in exploring selective BACE1 inhibitors.

Hypoxia-induced upregulation of angiogenic factors in immortalized human periodontal ligament fibroblasts.

Hypoxia induces complex cellular responses that are mediated by a key transcription factor, hypoxia-inducible factor-1 (HIF-1). HIF-1 promotes production of cytokines and angiogenic factors and contributes to recovery of injured tissues. In the present study, expressions of angiogenin (ANG) and vascular endothelial growth factor (VEGF), which are potent angiogenic factors in mammalian tissues, were examined in immortalized fibroblasts exposed to hypoxia. After 24 h of exposure to hypoxia, ANG and VEGF mRNAs expressions were significantly elevated in periodontal ligament (PDL) fibroblasts but not in embryonic fibroblasts. Hypoxia also increased productions of ANG and VEGF proteins in PDL fibroblasts. HIF-1α mRNA expression was not affected by hypoxia in either fibroblast, although HIF-1α protein expression was enhanced after exposure to hypoxia. Treatment of PDL fibroblasts with dimethyloxaloylglycine, a prolyl hydroxylase inhibitor that stabilizes the HIF-1α protein, significantly increased expressions of ANG and VEGF mRNAs under normoxia. This suggests that stabilization of HIF-1α is crucial for upregulation of ANG and VEGF in PDL fibroblasts. These results indicate that, under hypoxic conditions, HIF-1α upregulates synthesis of ANG and VEGF in PDL fibroblasts and promotes angiogenesis.

Effect of hypoxia on the expression of CCAAT/enhancer-binding protein ß and receptor activator of nuclear factor kappa-B ligand in periodontal ligament cells.

Hypoxia after traumatic injuries to a tooth is one of the causes of subsequent root resorption. Inflammatory cytokines produced under hypoxic conditions are associated with root resorption, but the mechanism has not been fully understood. In this study, the role of hypoxia-inducible factor-1 (HIF-1) signaling in the regulation of CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)/enhancer-binding protein-ß (C/EBPß) and the receptor activator of nuclear factor kappa-B ligand (RANKL) expressions in immortalized human periodontal ligament (PDL) cells was investigated. PDL cells cultured under a hypoxic condition showed an increase in the expression of C/EBPß and RANKL messenger RNAs (mRNAs), whereas the expression of osteoprotegerin and HIF-1α mRNAs was unaffected. Hypoxia had no effects on the secretion of interleukin (IL)-1ß, IL-6, IL-8, IL-17A, tumor necrosis factor-alpha, macrophage migration inhibitory factor, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor in the culture media. Treatment of the cells with dimethyloxaloylglycine, a competitive HIF prolyl hydroxylase inhibitor, significantly increased the expression of C/EBPß and RANKL mRNAs. This suggested that the hypoxia-induced elevation of C/EBPß and RANKL mRNAs was dependent on the HIF-1 activity. PDL cells transfected with a specific small interfering RNA designed to target the C/EBPß gene showed a significant suppression of the RANKL mRNA. These findings indicated that C/EBPß may play an important role in tooth root resorption via RANKL activation in hypoxia-exposed PDL cells.

Rational Design of Novel 1,3-Oxazine Based ß-Secretase (BACE1) Inhibitors: Incorporation of a Double Bond To Reduce P-gp Efflux Leading to Robust Aß Reduction in the Brain.

Accumulation of Aß peptides is a hallmark of Alzheimer's disease (AD) and is considered a causal factor in the pathogenesis of AD. ß-Secretase (BACE1) is a key enzyme responsible for producing Aß peptides, and thus agents that inhibit BACE1 should be beneficial for disease-modifying treatment of AD. Here we describe the discovery and optimization of novel oxazine-based BACE1 inhibitors by lowering amidine basicity with the incorporation of a double bond to improve brain penetration. Starting from a 1,3-dihydrooxazine lead 6 identified by a hit-to-lead SAR following HTS, we adopted a p Ka lowering strategy to reduce the P-gp efflux and the high hERG potential leading to the discovery of 15 that produced significant Aß reduction with long duration in pharmacodynamic models and exhibited wide safety margins in cardiovascular safety models. This compound improved the brain-to-plasma ratio relative to 6 by reducing P-gp recognition, which was demonstrated by a P-gp knockout mouse model.

Discovery of Potent and Centrally Active 6-Substituted 5-Fluoro-1,3-dihydro-oxazine ß-Secretase (BACE1) Inhibitors via Active Conformation Stabilization.

ß-Secretase (BACE1) has an essential role in the production of amyloid ß peptides that accumulate in patients with Alzheimer's disease (AD). Thus, inhibition of BACE1 is considered to be a disease-modifying approach for the treatment of AD. Our hit-to-lead efforts led to a cellular potent 1,3-dihydro-oxazine 6, which however inhibited hERG and showed high P-gp efflux. The close analogue of 5-fluoro-oxazine 8 reduced P-gp efflux; further introduction of electron withdrawing groups at the 6-position improved potency and also mitigated P-gp efflux and hERG inhibition. Changing to a pyrazine followed by optimization of substituents on both the oxazine and the pyrazine culminated in 24 with robust Aß reduction in vivo at low doses as well as reduced CYP2D6 inhibition. On the basis of the X-ray analysis and the QM calculation of given dihydro-oxazines, we reasoned that the substituents at the 6-position as well as the 5-fluorine on the oxazine would stabilize a bioactive conformation to increase potency.

Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7.

BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-ß (TGF-ß) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells. METHODS: The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay. RESULTS: All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

Distinct relations among plasma concentrations required for different pharmacological effects in oxycodone, morphine, and fentanyl.

Several clinical reports showed that adverse effect profiles are not the same in morphine, oxycodone, and fentanyl. The authors investigated whether the relationship between plasma concentrations for antinociceptive effect and for various pharmacological effects differed among oxycodone, morphine, and fentanyl under controlled experimental setting using animal models. Oxycodone induced constipation and an antinociceptive effect in a similar concentration-dependent manner, whereas morphine required approximately 9-fold higher plasma concentration for antinociceptive effect compared with that for constipation when 50% effective plasma concentration (EC(50)) levels were compared. The EC(50) values for inhibition of behavioral activity were 2.1-, 2.7-, and 1.3-fold higher than those for antinociceptive effect in oxycodone, morphine, and fentanyl, respectively. Respiratory inhibition was observed even at higher plasma concentrations in all three opioids, and the differences in the EC(50) values compared with those for antinociceptive effects were 234.5-fold (oxycodone), 233.1-fold (morphine), or 104.2-fold (fentanyl). These results showed that oxycodone, morphine, and fentanyl exhibited unique patterns of plasma concentrations required for different pharmacological effects. The different adverse effect profiles observed in a clinical setting appear to be resulted from, at least in part, distinct intrinsic pharmacological profiles among these µ-opioid receptor agonists.

Morphine, oxycodone, and fentanyl exhibit different analgesic profiles in mouse pain models.

Morphine, oxycodone, and fentanyl are clinically prescribed drugs for the management of severe pain. We investigated whether these opioids possess different efficacy profiles on several types of pain in mouse pain models. When the three opioids were tested in the femur bone cancer model, all of them significantly reversed guarding behavior, whereas the effects on limb-use abnormality and allodynia-like behavior differed among the opioids. Particularly, although oxycodone (5 - 20 mg/kg) and fentanyl (0.2 mg/kg) significantly reversed limb-use abnormality, not even a high dose of morphine (50 mg/kg) could reverse it. When the effects of these opioids were examined in a sciatic nerve ligation (SNL) model of neuropathic pain, oxycodone was the most effective, producing an antinociceptive effect without affecting the withdrawal threshold of sham-treated animals. When the effects of these opioids were examined with the tail-flick test using naive animals, oxycodone, morphine, and fentanyl exhibited antinociceptive effects on thermal nociception. These results show that the three opioids exhibit different efficacy outcomes in multiple pain models and that the efficacy profile of oxycodone does not overlap those of morphine and fentanyl.

Oxycodone-induced analgesic effects in a bone cancer pain model in mice.

The femur bone cancer pain model was developed by implanting mouse osteolytic tumor cells (NCTC 2472) into the intramedulla of the femur in C3H/HeN mice. In vivo imaging analysis revealed that the implanted tumor cells grew progressively over 14 days. Associated with the tumor growth, guarding behavior, which was an indication of ongoing pain, time-dependently increased. Limb use abnormality and allodynia, which were indications of ambulatory and neuropathic pain, respectively, also appeared. The analgesic effects of oxycodone and other opioids, such as morphine and fentanyl, were evaluated at 14 days when all pain-related behaviors clearly appeared. Oxycodone (2-20 mg/kg, s.c.), morphine (10-50 mg/kg, s.c.) and fentanyl (0.05-0.2 mg/kg, s.c.) significantly reduced guarding behavior. Oxycodone (5-20 mg/kg, s.c.) and fentanyl (0.1 and 0.2 mg/kg, s.c.) significantly reversed limb use abnormality, but morphine (5-50 mg/kg, s.c.) did not. Moreover, oxycodone (5-20 mg/kg, s.c.) dose-dependently reversed allodynia without affecting the sham-treated mice. Morphine (50 mg/kg, s.c.) and fentanyl (0.075-0.2 mg/kg, s.c.) also reversed allodynia, but morphine (50 mg/kg, s.c.) tended to affect and fentanyl (0.1 and 0.2 mg/kg, s.c.) affected the withdrawal threshold in sham-treated mice. These results suggested that oxycodone relieved not only ongoing pain, but also ambulatory and neuropathic pain, and that the analgesic profile of oxycodone could be different from that of either morphine or fentanyl.

[Priming of neuronal differentiation of cultured neural stem cells by neutrophins].

Neurogenesis is promoted by basic helix-loop-helix (bHLH) transcription factors Mash1, Math1 and/or NeuroD but suppressed by another set, Hes1 and Hes5. It is still unknown what kinds of extracellular signals are involved in their regulation. Therefore, the effects of neurotrophins on the expression of bHLH factors and neuronal differentiation were investigated by the use of cultured mouse neural stem cells. Each neurotrophin increased Mash1 and Math1 mRNAs of the stem cells growing in the presence of fibroblast growth factor-2 (FGF-2), but did not alter Hes1, Hes5 or NeuroD mRNA levels. Simultaneously, most of the cells expressed nestin, but not microtubule-associated protein 2 (MAP2), and still remained undifferentiated. FGF-2 removal from the medium reduced the levels of Hes1 and Hes5 mRNAs and increased those of Mash1, Math1, and NeuroD mRNAs, resulting in substantial neuronal differentiation. However, when the cells were pretreated with a neurotrophin, brain-derived neurotrophic factor, FGF-2 removal enhanced earlier NeuroD expression and generated many more MAP2-positive cells. The high level of Mash1 and Math1 that had been elevated at FGF-2 withdrawal accelerated NeuroD expression in cooperation with the reduced Hes1 and Hes5 expression. Our present results suggest that neurotrophins stimulate neuronal differentiation via altering the balance of expression of various bHLH transcription factors.

Growth arrest of PC12 cells by nerve growth factor is dependent on the phosphatidylinositol 3-kinase/Akt pathway via p75 neurotrophin receptor.

We recently isolated mutant PC12 cell clones (PC84 cells) by transfection of PC12 cells with nerve growth factor (NGF) cDNA. These cells secreted active NGF and extended short processes, but proliferated faster than the parental PC12 cells. Because the expression level of p75, a low-affinity receptor for NGF, was significantly low, we suspected that NGF signaling via p75 was necessary for the growth arrest of the PC12 cells, and this was shown to be the case by repressing p75 function in PC12 cells. In this study, we examined the downstream signaling of p75, which would ultimately evoke the growth arrest. NGF is known to induce rapid phosphorylation of MAP kinase and Akt in PC12 cells, whereas in PC84 cells, MAP kinase was phosphorylated but the phosphorylation level of Akt was very low under the serum-free condition. This finding suggested that the low expression level of p75 in PC84 cells was the reason for the low Akt activation. Because Akt is known to be activated via phosphatidylinositol (PI) 3-kinase, we treated PC12 cells with a PI3-kinase inhibitor, Wortmannin, and found these cells did not cease proliferation in the presence of NGF. Furthermore, anti-p75 neutralizing antibody reduced NGF-induced phosphorylation of Akt in PC12 cells under the serum-free condition. Because we had already shown that PC12 cells treated with anti-p75 neutralizing antibody did not cease proliferation in the presence of NGF, these results suggest that NGF activates Akt via p75, which is necessary for the NGF-induced growth arrest of PC12 cells.

Neurotrophins facilitate synthesis of choline acetyltransferase and tyrosine hydroxylase in cultured mouse neural stem cells independently of their neuronal differentiation.

Effects of three neurotrophins, i.e., nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3, on the expression of four neurotransmitter-synthesizing enzymes, i.e. choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH), and glutamate decarboxylase 65 were investigated in cultured mouse neural stem cells. All three neurotrophins enhanced the mRNA expression of ChAT, TH, or DBH of the cells caused to differentiate by the removal of fibroblast growth factor (FGF)-2 from the culture medium, and increased the protein and mRNA levels of ChAT and TH of even the undifferentiated proliferating neural stem cells due to the presence of FGF-2. These results demonstrate that neurotrophins stimulate the synthesis of ChAT and TH of the neural stem cells prior to neuronal differentiation, and suggest that neurotrophins may play roles in the commitment to neuronal cells and choice of specific neurotransmitter phenotypes in early stages of neurogenesis.