Differentiation Of Cholinergic Neurons Research Articles

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins βIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

Basic fibroblast growth factor-induced cholinergic neuronal differentiation of tonsil-derived mesenchymal stem cells

Background & Aim Mesenchymal stem cells (MSCs) have been considered as a potential tool in regenerative medicine for their great multipotency characteristics into various cell types. In past years, our group have been studying tonsil-derived MSCs (TMSCs) for their application and ease of obtaining large number and low immunogencity. Recently we attempted to expand potential regenerative implications of TMSCs not only for mesoderms but also for ectoderms, specifically neurons. Methods, Results & Conclusion Our initial study demonstrated that TMSCs incubated in the presence of 25 ng/mL of basic fibroblast growth factor and 10 μM of forskolin for 22 days resulted in large number of cells containing neurites which are morphologically similar with those observed with neurons. Western blot analyses determined that the TMSCs expressed the various neuronal markers, including neuron-specific enolase, growth-associated protein-43 (GAP43), postsynaptic density protein 95, synaptosomal nerve-associated protein 25, compared with the undifferentiated TMSC group. The expression of these markers dramatically increased at day 8 of the neuronal differentiation and further increased thereafter until day 22. In contrast, glial fibrillary acidic protein, a marker of neural stem cell/astrocyte, gradually declined as the neuronal differentiation progresses. In contrast, neither of oligodendrocyte marker O4 nor microglia marker CD11b was detected. Further studies revealed that the differentiated TMSCs showed enhanced immunoreactivities of mature neuronal markers such as neuronal nuclei, neuron-specific class III β tubulin, GAP43, and synaptophysin. Additionally, the differentiated TMSCs exhibited cholinergic neuronal phenotype as evidenced with the increased expression of choline acetyltransferase, a cholinergic neuronal marker but not vesicular glutamate transporter 1, a glutamatergic neuronal marker. Taken altogether, the findings suggest that TMSCs have a potential to differentiate into cholinergic neuron-like cells under the established differentiation condition. Although further optimization to increase neuronal cell population within heterogenous TMSC pools and functional validation are required, our study provides a glimpse of evidence that TMSC-derived cholinergic neurons could be used as a possible therapeutic tool in treating certain neurodegenerative disorders such as Alzheimer's diseases. The present study was supported by a research grant of the National Research Foundation of Korea (2019R1A2C108676711).

Comparative analysis of three different protocols for cholinergic neuron differentiation in vitro using mesenchymal stem cells from human dental pulp

ABSTRACTA decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer’s disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of β-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 µg/ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 µM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 µM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuron-like morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.

Stimulating brain recovery after stroke using theranostic albumin nanocarriers loaded with nerve growth factor in combination therapy

For many years, delivering drug molecules across the blood brain barrier has been a major challenge. The neuropeptide nerve growth factor is involved in the regulation of growth and differentiation of cholinergic neurons and holds great potential in the treatment of stroke. However, as with many other compounds, the biomolecule is not able to enter the central nervous system. In the present study, nerve growth factor and ultra-small particles of iron oxide were co-encapsulated into a chemically crosslinked albumin nanocarrier matrix which was modified on the surface with apolipoprotein E. These biodegradable nanoparticles with a size of 212 ± 1 nm exhibited monodisperse size distribution and low toxicity. They delivered NGF through an artificial blood brain barrier and were able to induce neurite outgrowth in PC12 cells in vitro. In an animal model of stroke, the infarct size was significantly reduced compared to the vehicle control. The combination therapy of NGF and the small-molecular MEK inhibitor U0126 showed a slight but not significant difference compared to U0126 alone. However, further in vivo evidence suggests that successful delivery of the neuropeptide is possible as well as the synergism between those two treatments.

Direct conversion of mouse astrocytes into neural progenitor cells and specific lineages of neurons

BackgroundCell replacement therapy has been envisioned as a promising treatment for neurodegenerative diseases. Due to the ethical concerns of ESCs-derived neural progenitor cells (NPCs) and tumorigenic potential of iPSCs, reprogramming of somatic cells directly into multipotent NPCs has emerged as a preferred approach for cell transplantation.MethodsMouse astrocytes were reprogrammed into NPCs by the overexpression of transcription factors (TFs) Foxg1, Sox2, and Brn2. The generation of subtypes of neurons was directed by the force expression of cell-type specific TFs Lhx8 or Foxa2/Lmx1a.ResultsAstrocyte-derived induced NPCs (AiNPCs) share high similarities, including the expression of NPC-specific genes, DNA methylation patterns, the ability to proliferate and differentiate, with the wild type NPCs. The AiNPCs are committed to the forebrain identity and predominantly differentiated into glutamatergic and GABAergic neuronal subtypes. Interestingly, additional overexpression of TFs Lhx8 and Foxa2/Lmx1a in AiNPCs promoted cholinergic and dopaminergic neuronal differentiation, respectively.ConclusionsOur studies suggest that astrocytes can be converted into AiNPCs and lineage-committed AiNPCs can acquire differentiation potential of other lineages through forced expression of specific TFs. Understanding the impact of the TF sets on the reprogramming and differentiation into specific lineages of neurons will provide valuable strategies for astrocyte-based cell therapy in neurodegenerative diseases.

Characterization of alginate/chitosan-based nanoparticles and mathematical modeling of their SpBMP-9 release inducing neuronal differentiation of human SH-SY5Y cells

The incidence of brain degenerative disease such as Alzheimer’s disease (AD) will increase as the world population is ageing. While current AD treatments have only a transient effect, there are many evidences indicating that some growth factors, such as BMP-9, may be used to treat AD. However, growth factors cannot readily access the brain because of their size and the presence of the blood brain barrier. We have therefore developed a small peptide derived from BMP-9, SpBMP-9, which can promote the differentiation of cholinergic neurons and inactivate GSK3beta, a Tau kinase. Here, we investigated the potential of a nanoparticle-based delivery system of SpBMP-9, made of alginate and chitosan (Alg/Chit NPs), as a new therapeutic strategy against AD. The Alg/Chit NPs size distribution revealed NPs with an average diameter of ∼240nm. The encapsulation efficiency of SpBMP-9 was ∼70% of the initial peptide mass loading. Release kinetics of SpBMP-9 were performed in physiological conditions and modelled with a mechanistic framework that took into account the size distribution of Alg/Chit NPs. The release of SpBMP-9 revealed to be mostly diffusive, but there were interactions between the peptide and the alginate chains. The Alg/Chit NPs could also increase the viability of SH-SY5Y cells in comparison to the control. Finally, the SpBMP-9 released from Alg/Chit NPs promoted the SH-SY5Y differentiation into mature neurons as demonstrated by a higher neurite outgrowth and an increased expression of the neuronal markers NSE and VAchT. In conclusion, the nano-scale SpBMP-9 delivery system made of Alg/Chit may be a promising therapeutic strategy against AD.

Cholinergic and dopaminergic neuronal differentiation of human adipose tissue derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as cartilage, bone, and fat cells. Recent studies have shown that induction of MSCs in vitro by growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF2) causes them to differentiate into neural like cells. These cultures also express ChAT, a cholinergic marker; and TH, a dopaminergic marker for neural cells. To establish a protocol with maximum differentiation potential, we examined MSCs under three experimental culture conditions using neural induction media containing FGF2, EGF, BMP-9, retinoic acid, and heparin. Adipose-derived MSCs were extracted and expanded in vitro for 3 passages after reaching >80% confluency, for a total duration of 9 days. Cells were then characterized by flow cytometry for CD markers as CD44 positive and CD45 negative. MSCs were then treated with neural induction media and were characterized by morphological changes and Q-PCR. Differentiated MSCs expressed markers for immature and mature neurons; β Tubulin III (TUBB3) and MAP2, respectively, showing the neural potential of these cells to differentiate into functional neurons. Improved protocols for MSCs induction will facilitate and ensure the reproducibility and standard production of MSCs for therapeutic applications in neurodegenerative diseases.

Intranasal BMP9 Ameliorates Alzheimer Disease-Like Pathology and Cognitive Deficits in APP/PS1 Transgenic Mice.

Alzheimer’s disease (AD) is the most common type of dementia and has no effective therapies. Previous studies showed that bone morphogenetic protein 9 (BMP9), an important factor in the differentiation and phenotype maintenance of cholinergic neurons, ameliorated the cholinergic defects resulting from amyloid deposition. These findings suggest that BMP9 has potential as a therapeutic agent for AD. However, the effects of BMP9 on cognitive function in AD and its underlying mechanisms remain elusive. In the present study, BMP9 was delivered intranasally to 7-month-old APP/PS1 mice for 4 weeks. Our data showed that intranasal BMP9 administration significantly improved the spatial and associative learning and memory of APP/PS1 mice. We also found that intranasal BMP9 administration significantly reduced the amyloid β (Aβ) plaques overall, inhibited tau hyperphosphorylation, and suppressed neuroinflammation in the transgenic mouse brain. Furthermore, intranasal BMP9 administration significantly promoted the expression of low-density lipoprotein receptor-related protein 1 (LRP1), an important membrane receptor involved in the clearance of amyloid β via the blood-brain barrier (BBB), and elevated the phosphorylation levels of glycogen synthase kinase-3β (Ser9), which is considered the main kinase involved in tau hyperphosphorylation. Our results suggest that BMP9 may be a promising candidate for treating AD by targeting multiple key pathways in the disease pathogenesis.

Neurotrophin-3 promotes proliferation and cholinergic neuronal differentiation of bone marrow- derived neural stem cells via notch signaling pathway

AimsRecently, the potential for neural stem cells (NSCs) to be used in the treatment of Alzheimer's disease (AD) has been reported; however, the therapeutic effects are modest by virtue of the low neural differentiation rate. In our study, we transfected bone marrow-derived NSCs (BM-NSCs) with Neurotrophin-3 (NT-3), a superactive neurotrophic factor that promotes neuronal survival, differentiation, and migration of neuronal cells, to investigate the effects of NT-3 gene overexpression on the proliferation and differentiation into cholinergic neuron of BM-NSCs in vitro and its possible molecular mechanism. Main methodsBM-NSCs were generated from BM mesenchymal cells of adult C57BL/6 mice and cultured in vitro. After transfected with NT-3 gene, immunofluorescence and RT-PCR method were used to determine the ability of BM-NSCs on proliferation and differentiation into cholinergic neuron; Acetylcholine Assay Kit was used for acetylcholine (Ach). RT-PCR and WB analysis were used to characterize mRNA and protein level related to the Notch signaling pathway. Key findingsWe found that NT-3 can promote the proliferation and differentiation of BM-NSCs into cholinergic neurons and elevate the levels of acetylcholine (ACh) in the supernatant. Furthermore, NT-3 gene overexpression increase the expression of Hes1, decreased the expression of Mash1 and Ngn1 during proliferation of BM-NSCs. Whereas, the expression of Hes1 was down-regulated, and Mash1 and Ngn1 expression were up-regulated during differentiation of BM-NSCs. SignificanceOur findings support the prospect of using NT-3-transduced BM-NSCs in developing therapies for AD due to their equivalent therapeutic potential as subventricular zone-derived NSCs (SVZ-NSCs), greater accessibility, and autogenous attributes.

N-BENZYLCINNAMIDE INDUCES CHOLINERGIC NEURONAL DIFFERENTIATION FROM HUMAN AMNIOTIC FLUID MESENCHYMAL STEM CELLS

N-BENZYLCINNAMIDE INDUCES CHOLINERGIC NEURONAL DIFFERENTIATION FROM HUMAN AMNIOTIC FLUID MESENCHYMAL STEM CELLS

The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens, including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However, these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis.Here, we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary, high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes, resulting in subsequent cholinergic neuron differentiation. Thus, our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells.

Xenon-mediated neuroprotection in response to sustained, low-level excitotoxic stress.

Noble gases such as xenon and argon have been reported to provide neuroprotection against acute brain ischemic/anoxic injuries. Herein, we wished to evaluate the protective potential of these two gases under conditions relevant to the pathogenesis of chronic neurodegenerative disorders. For that, we established cultures of neurons typically affected in Alzheimer's disease (AD) pathology, that is, cortical neurons and basal forebrain cholinergic neurons and exposed them to L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to generate sustained, low-level excitotoxic stress. Over a period of 4 days, PDC caused a progressive loss of cortical neurons which was prevented substantially when xenon replaced nitrogen in the cell culture atmosphere. Unlike xenon, argon remained inactive. Xenon acted downstream of the inhibitory and stimulatory effects elicited by PDC on glutamate uptake and efflux, respectively. Neuroprotection by xenon was mimicked by two noncompetitive antagonists of NMDA glutamate receptors, memantine and ketamine. Each of them potentiated xenon-mediated neuroprotection when used at concentrations providing suboptimal rescue to cortical neurons but most surprisingly, no rescue at all. The survival-promoting effects of xenon persisted when NMDA was used instead of PDC to trigger neuronal death, indicating that NMDA receptor antagonism was probably accountable for xenon’s effects. An excess of glycine failed to reverse xenon neuroprotection, thus excluding a competitive interaction of xenon with the glycine-binding site of NMDA receptors. Noticeably, antioxidants such as Trolox and N-acetylcysteine reduced PDC-induced neuronal death but xenon itself lacked free radical-scavenging activity. Cholinergic neurons were also rescued efficaciously by xenon in basal forebrain cultures. Unexpectedly, however, xenon stimulated cholinergic traits and promoted the morphological differentiation of cholinergic neurons in these cultures. Memantine reproduced some of these neurotrophic effects, albeit with less efficacy than xenon. In conclusion, we demonstrate for the first time that xenon may have a therapeutic potential in AD.

Generation of Human Embryonic Stem Cell Line Expressing zsGreen in Cholinergic Neurons Using CRISPR/Cas9 System.

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation, physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation, cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons.

Directed differentiation of basal forebrain cholinergic neurons from human pluripotent stem cells

BackgroundBasal forebrain cholinergic neurons (BFCNs) play critical roles in learning, memory and cognition. Dysfunction or degeneration of BFCNs may connect to neuropathology, such as Alzheimer’s disease, Down’s syndrome and dementia. Generation of functional BFCNs may contribute to the studies of cell-based therapy and pathogenesis that is related to learning and memory deficits. New methodHere we describe a detail method for robust generation of BFCNs from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In this method, BFCN progenitors are patterned from hESC or hiPSC-derived primitive neuroepithelial cells, with the treatment of sonic hedgehog (SHH) or combination with its agonist Purmorphamine, and by co-culturing with human astrocytes. ResultsAt day 20, ∼90% hPSC-derived progenitors expressed NKX2.1, which is a transcriptional marker for MGE. Moreover, around 40% of NKX2.1+ cells co-expressed OLIG2 and ∼15% of NKX2.1+ cells co-expressed ISLET1, which are ventral markers. At day 35, ∼40% neurons robustly express ChAT, most of which are co-labeled with NKX2.1, ISLET1 and FOXG1, indicating the basal forebrain-like identity. At day 45, these neurons express mature neuronal markers MAP2, Synapsin, and VAChT. Comparison with existing method(s)In this method, undefined conditions including genetic modification or cell-sorting are avoided. As a choice, feeder free conditions are used to avoid ingredients of animal origin. Moreover, Purmorphamine can be substituted for SHH to induce ventral progenitors effectively and economically. ConclusionWe provide an efficient method to generate BFCNs from multiple hPSC lines, which offers the potential application for disease modeling and pharmacological studies.

Differentiation of Cholinergic Neurons in Rat Spinal Cord Under Conditions of Allotransplantation into a Peripheral Nerve and In Situ Development.

The method of ectopic transplantation of embryonic anlages of CNS allows studying histoblastic potencies of progenitor cells developing under conditions of changed microenvironment. Some progenitor cells in the transplants of rat embryonic spinal cord retained their ability to express choline acetyltransferase after transplantation into the sciatic nerve of adult animals. Comparative analysis of cholinergic neurons in the neurotransplants and neurons formed in rat spinal cord during normal ontogeny showed that choline acetyltransferase-positive cells after transplantation into the nerve reached morphological differentiation of motor neurons at later terms than cells developing in situ. They were scattered one by one and did not form nuclear nerve centers. We did not fi nd structures similar to presynaptic cholinergic buds typical of intact spinal cord near these cells throughout the observation period. Solitary cholinergic neurons survived in the transplants for 19 months.

Overexpression of NTRK1 Promotes Differentiation of Neural Stem Cells into Cholinergic Neurons.

Neurotrophic tyrosine kinase type 1 (NTRK1) plays critical roles in proliferation, differentiation, and survival of cholinergic neurons; however, it remains unknown whether enhanced expression of NTRK1 in neural stem cells (NSCs) can promote their differentiation into mature neurons. In this study, a plasmid encoding the rat NTRK1 gene was constructed and transfected into C17.2 mouse neural stem cells (NSCs). NTRK1 overexpression in C17.2 cells was confirmed by western blot. The NSCs overexpressing NTRK1 and the C17.2 NSCs transfected by an empty plasmid vector were treated with or without 100 ng/mL nerve growth factor (NGF) for 7 days. Expression of the cholinergic cell marker, choline acetyltransferase (ChAT), was detected by florescent immunocytochemistry (ICC). In the presence of NGF induction, the NSCs overexpressing NTRK1 differentiated into ChAT-immunopositive cells at 3-fold higher than the NSCs transfected by the plasmid vector (26% versus 9%, P < 0.05). The data suggest that elevated NTRK1 expression increases differentiation of NSCs into cholinergic neurons under stimulation of NGF. The approach also represents an efficient strategy for generation of cholinergic neurons.

Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines

Cholinergic neurons have been identified with the acetylcholine synthetic enzyme choline acetyltransferase (ChAT). However, ChAT is difficult to localize in newly differentiated peripheral neurons making the study of cholinergic neuronal development problematic. Consequently, researchers have used mouse reporter lines to indicate the presence of ChAT. Our objective was to determine which ChAT reporter line was the most sensitive indicator of ChAT expression. We utilized two different fluorescent ChAT reporter lines (ChAT-GFP and ChAT-Cre;R26R:floxSTOP:tdTomato) together with immunolocalization of ChAT protein (ChAT-IR) to characterize the spatial and temporal expression of ChAT in myenteric neurons throughout enteric nervous system (ENS) development. ChAT-IR cells were first seen in the intestine at E10.5, even within the migration wavefront of neural precursors. Myenteric neurons within the distal small intestine (dSI) and proximal colon were first labeled by ChAT-IR, then ChAT-GFP, and finally ChAT-Cre tdTomato. The percentage of ChAT-IR neurons is equivalent to adult levels in the dSI by E13.5 and proximal colon by P0. After these stages, the percentages remained relatively constant throughout development despite dramatic changes in neuronal density. These observations indicate that neurotransmitter expression occurs early and there is only a brief gap between neurogenesis and neurotransmitter expression. Our finding that the proportion of ChAT myenteric neurons reached adult levels during embryonic development suggests that the fate of cholinergic neurons is tightly regulated and that their differentiation might influence further neuronal development. ChAT-GFP is a more accurate indicator of early ENS cholinergic neuronal differentiation than the ChAT-Cre;R26R:floxSTOP:tdTomato reporter mouse.

Ginsenoside Re and Rd Enhance the Expression of Cholinergic Markers and Neuronal Differentiation in Neuro-2a Cells

In Alzheimer's disease (AD), extensive neuronal loss and a deficiency of the neurotransmitter acetylcholine (ACh) are the major characteristics during pathogenesis in the brain. In the present study, we aimed to investigate whether representative ginsenosides from ginseng can regulate choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are required for cholinergic neurotransmission. Our results revealed that Re and Rd induced effectively the expression of ChAT/VAChT genes in Neuro-2a cells as well as ACh elevation. Microtubule-associated protein-2 (MAP-2), nerve growth factor receptor (p75), p21, and TrkA genes and proteins were also significantly expressed. Moreover, both activated extracelullar signal-regulated protein kinase (ERK) and Akt were inhibited by K252a, a selective Trk receptor inhibitor. These findings strongly indicate that Re and Rd play an important role in neuronal differentiation and the nerve growth factor (NGF)-TrkA signaling pathway. High performance liquid chromatography analysis showed that Re and Rd administered orally were transported successfully into brain tissue and increased the level of ChAT and VAChT mRNA. The present study demonstrates that Re and Rd are selective candidates for upregulation of the expression of cholinergic markers, which may counter the symptoms and progress of AD.

Upregulation of Lhx8 increase VAChT expression and ACh release in neuronal cell line SHSY5Y

Lhx8 is a transcription factor for cholinergic differentiation. Our previous experiments found upregulation of Lhx8 promoted cholinergic neuronal differentiation of hippocampal neural stem/progenitor cells or hippocampal newborn neurons in vitro. However, the role of Lhx8 in VAChT expression and ACh release is still less understood. In this report, we transfected Lhx8 cDNA into neuronal cell line SHSY5Y by lentiviral vectors to acquire the cells which stably expressed high level of Lhx8. Using this cell model, we provided experimental evidence that increasing Lhx8 upregulated the expression of ChAT and VAChT, and also increased the ACh release in culture medium. We suggested that Lhx8 overexpression is a useful strategy to increase the release of ACh and maybe of therapeutic value to neurodegenerative diseases.

Stimulation of cholinogenesis in human fetal nerve cell cultures

The aim of the study was to obtain a neural cell population enriched in culture with cholinergic neurons and their determined precursors. It has been established that the joint application of retinoic acid and acetylcholine appears to be the most efficient combination of neuroinductors, stimulating the differentiation of cholinergic neurons from neural stem cells. During the entire period of culturing, the amount of ChAT+-positive cells increased reliably to 21.1 ± 6.2% from 5.3 ± 2.9, whereas the ir concentration in the control samples was 9.1 ± 4.8% of the total cell count. The cell population enriched with cholinergic neurons and their determined precursors correlated well with the increased acetylcholinesterase activity. Thus, the addition of retinoic acid and acetylcholine stimulate both neurogenesis and cholinogenesis in human fetal neural stem cell cultures.