Activation of medial septum cholinergic neurons restores cognitive function in temporal lobe epilepsy

Cognitive impairment is the most common complication in patients with temporal lobe epilepsy with hippocampal scle rosis.There is no effective treatment for cognitive impairment.Medial septum cholinergic neurons have been reported to be a potential target for controlling epileptic seizures in tempo ral lobe epile psy.However,their role in the cognitive impairment of temporal lobe epilepsy remains unclear.In this study,we found that patients with temporal lobe epile psy with hippocampal sclerosis had a low memory quotient and severe impairment in verbal memory,but had no impairment in nonverbal memory.The cognitive impairment was slightly correlated with reduced medial septum volume and medial septum-hippocampus tra cts measured by diffusion tensor imaging.In a mouse model of chronic temporal lobe epilepsy induced by kainic acid,the number of medial septum choline rgic neurons was reduced and acetylcholine release was reduced in the hippocampus.Furthermore,selective apoptosis of medial septum cholinergic neurons mimicked the cognitive deficits in epileptic mice,and activation of medial septum cholinergic neurons enhanced hippocampal acetylcholine release and restored cognitive function in both kainic acid-and kindling-induced epile psy models.These res ults suggest that activation of medial septum cholinergic neurons reduces cognitive deficits in temporal lobe epilepsy by increasing acetylcholine release via projections to the hippocampus.

Impairment of the nerve growth factor pathway driving amyloid accumulation in cholinergic neurons the incipit of the Alzheimer's disease story?

The current idea behind brain pathology is that disease is initiated by mild disturbances of common physiological processes. Overtime, the disruption of the neuronal homeostasis will determine irreversible degeneration and neuronal apoptosis. This could be also true in the case of nerve growth factor （NGF） al- terations in sporadic Alzheimer＇s disease （AD）, an age-related pathology characterized by cholinergic loss, amyloid plaques and neurofibrillary tangles. In fact, the pathway activated by NGF, a key neurotrophin for the metabolism of basal forebrain cholinergic neurons （BFCN）, is one of the first homeostatic systems affected in prodromal AD. NGF signaling dysfunctions have been thought for decades to occur in AD late stages, as a mere consequence of amyloid-driven disruption of the retrograde axonal transport of neuro- trophins to BFCN. Nowadays, a wealth of knowledge is potentially opening a new scenario： NGF signaling impairment occurs at the onset of AD and correlates better than amyloid load with cognitive decline. The recent acceleration in the characterization of anatomical, functional and molecular profiles of early AD is aimed at maximizing the efficacy of existing treatments and setting novel therapies. Accordingly, the elucidation of the molecular events underlying APP metabolism regulation by the NGF pathway in the sep- to-hippocampal system is crucial for the identification of new target molecules to slow and eventually halt mild cognitive impairment （MCI） and its progression toward AD.

Age-dependent loss of cholinergic neurons in learning and memory-related brain regions and impaired learning in SAMP8 mice with trigeminal nerve damage

The tooth belongs to the trigeminal sensory pathway. Dental damage has been associated with impairments in the central nervous system that may be mediated by injury to the trigeminal nerve. In the present study, we investigated the effects of damage to the inferior alveolar nerve, an important peripheral nerve in the trigeminal sensory pathway, on learning and memory be-haviors and structural changes in related brain regions, in a mouse model of Alzheimer’s disease. Inferior alveolar nerve transection or sham surgery was performed in middle-aged （4-month-old） or elderly （7-month-old） senescence-accelerated mouse prone 8 （SAMP8） mice. When the middle-aged mice reached 8 months （middle-aged group 1） or 11 months （middle-aged group 2）, and the elderly group reached 11 months, step-down passive avoidance and Y-maze tests of learn-ing and memory were performed, and the cholinergic system was examined in the hippocampus （Nissl staining and acetylcholinesterase histochemistry） and basal forebrain （choline acetyltrans-ferase immunohistochemistry）. In the elderly group, animals that underwent nerve transection had fewer pyramidal neurons in the hippocampal CA1 and CA3 regions, fewer cholinergic ifbers in the CA1 and dentate gyrus, and fewer cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band, compared with sham-operated animals, as well as showing impairments in learning and memory. Conversely, no signiifcant differences in histology or be-havior were observed between middle-aged group 1 or group 2 transected mice and age-matched sham-operated mice. The present ifndings suggest that trigeminal nerve damage in old age, but not middle age, can induce degeneration of the septal-hippocampal cholinergic system and loss of hippocampal pyramidal neurons, and ultimately impair learning ability. Our results highlight the importance of active treatment of trigeminal nerve damage in elderly patients and those with Alzheimer’s disease, and indicate that tooth extraction should be avoided in these populations.

Wnt3a expression during the differentiation of adipose-derived stem cells into cholinergic neurons

The present study analyzed changes in Wnt3a expression during differentiation of adipose-derived stern cells into cholinergic neurons. Immunocytochemistry and immunofluorescence revealed significantly increased nestin, neuron-specific enolase, microtubule-associated protein 2, and choline acetyltransferase expression in adipose-derived stem cells isolated from Sprague-Dawley rats and cultured in vitro in neural-induced medium. These expressions increased with prolonged induction time. Real-time reverse transcription-PCR and western blot assay results demonstrated significantly increased choline acetyltransferase and Wnt3a protein and mRNA expressions, respectively, in adipose-derived stem cells following induction. Choline acetyltransferase expression positively correlated with Wnt3a protein and mRNA expressions. These results demonstrated that neural-induced medium induced differentiation of adipose-derived stem cells into cholinergic neuronal-like cells, with subsequent increased Wnt3a expression.

The synthetic thyroid hormone, levothyroxine, protects cholinergic neurons in the hippocampus of naturally aged mice

The thyroid hormones, triiodothyronine and thyroxine, play important roles in cognitive func- tion during the mammalian lifespan. However, thyroid hormones have not yet been used as a therapeutic agent for normal age-related cognitive deficits. In this study, CD-1 mice （aged 24 months） were intraperitoneally injected with levothyroxine （L-T4; 1.6 gg/kg per day） for 3 consecutive months. Our findings revealed a significant improvement in hippocampal cyto- skeletal rearrangement of actin and an increase in serum hormone levels of L-T4-treated aged mice. Furthermore, the survival rate of these mice was dramatically increased from 60% to 93.3%. The Morris water maze task indicated that L-T4 restored impaired spatial memory in aged mice. Furthermore, level of choline acetyltransferase, acetylcholine, and superoxide dismutase were in- creased in these mice, thus suggesting that a possible mechanism by which L-T4 reversed cognitive impairment was caused by increased activity of these markers. Overall, supplement of low-dosage L-T4 may be a potential therapeutic strategy for normal age-related cognitive deficits.

Cell-based therapy in Alzheimer's disease: can human fetal cholinergic neurons “untangle the skein”?

Alzheimer＇s disease（AD）is a devastating neurodegenerative disorder and the most common form of old-age dementia.The disease is characterized by a progressive decline in cognitive functions,gradual loss of memory and ability to perform everyday activities,and leads to inevitable death within 3 to 9 years atter diagnosis.

Influence of interferon-gamma on the differentiation of cholinergic neurons in rat embryonic basal forebrain and septal nuclei

BACKGROUND： Interferon-gamma （IFN-γ） can make neurons in basal forebrain and septal nuclei differentiate into cholinergic neurons by treating the cells in cerebral cortex of newborn rats, without the inhibition from IFN-γ antibody. The important effect of IFN-γ on the development and differentiation of neurons has been found by some scholars. OBJ EClIVE：To investigate whether IFN-γ has differentiational effect on cholinergic neurons in basal forebrain and septal nuclei, and make clear that the increased number of cholinergic neurons is resulted by cell differentiation or cell proliferation. DESIGN ： Controlled observation trial SETTING： Department of Cell Biology, Medical School, Beijing University MATERIALS： Sixty-eight female Wistar rats at embryonic 16 days, weighing 250 to 350 g, were enrolled in this study, and they were provided by the Experimental Animal Center, Medical School, Beijing University. IFN-γ was the product of Gibco Company. METHODS： This study was carried out in the Department of Cell Biology, Medical School, Beijing University and Daheng Image Company of Chinese Academy of Sciences during September 1995 to December 2002. The female Wistar rats at embryonic 16 days were sacrificed, and their fetuses were taken out. Primary culture of the isolated basal forebrain and septal nuclei was performed. The cultured nerve cells were assigned into 3 groups： control group （nothing added）, IFN-γ group（1×10^5 U/L interferon）, IFN-γ＋ IFN-γ antibody group （1 ×10^5 U/L IFN-γ ＋ IFN-γ antibody）. The specific marker enzyme （choline acetyl transferase） of cholinergic neuron was stained with immunohistochemical method. Choline acetyl transferase positive cells were counted, and ^14C-acetyl CoA was used as substrate to detect the activity of choline acetyl transferase, so as to reflect the differentiational effect of IFN-γ on cholinergic neuron in basal forebrain and septal nuclei. Flow cytometry was used to analyze cell circle and detect the proliferation of nerve cells. Nerve cells were marked with MAP2 and counted to evaluate the neuronal proliferation in basal forebrain and septal nuclei. MAIN OUTCOME MEASURES： Effect of interferon-γ on the number and activity of choline acetyl transferase-positive ceils in basal forebrain and septal nuclei as well as the effect on neuronal proliferation. RESULTS ： ① Nerve cells in the basal forebrain and septal nuclei of IFN-γ group grew well compared with control group.②The differentiation of cholinergic neurons： The number and activity of choline acetyl transferase positive cells in IFN-γ group were significantly higher than those in the control group [（49.30 ±4.92） /100 cells vs （7.50±1.58） /100 cells; （2 049.00±12.30） min^-1 vs （1 227.30±12.59） min^-1, p 〈 0.01], while there was no significant difference in the number and activity of choline acetyl transferase positive cells between IFN-γ ＋ IFN-γ antibody group and control group（P 〉 0.05）. ③The proliferation of cholinergic neurons： Cell percentage was 17.2% and 19.8% at S-stage, 6.2% and 6.1% at G2＋M stage in the control group and IFN-γ group respectively, without significant difference （P 〉 0.05）. CONCLUSION ： IFN-γ does not promote the neuronal proliferation in basal forebrain and septal nuclei, and the increased expression of cholinergic neurons is not resulted by the increase in the number of neurons, but its differentiation.

Disturbance of cholinergic Grb2-associated-binding protein 1 signaling participate in the pathological process of cognitive dysfunction

OBJECTIVE A causal relationshiphas been postulated between cholinergic dysfunction and the progression of cognitive decline in neurodegenerative disorders. However,the cause of the cognitive dysfunction remains unclear. METHODS Gab1^(loxP/loxP) were bred with ChAT-Cre mice to generate ChAT-Cre; Gab1^(f/f) mice. Excitability of cholinergic neurons wererecorded using whole-cel patch clump. A series of behavioral analyses were used to address the changes of cognitive function in ChAT-Cre; Gab1^(f/f) mice. Neurochemical changes on brain of conditional knockout mice were evaluated by using immunohistochemistry and Western blotting analysis. RESULTS Grb2-associated-binding protein 1(Gab1) is adocking/scaffolding molecule known to play an important role in cell growth and survival. Here,wereport that Gab1 is decreased in cholinergic neurons in a mousemodel of AD. We found that selective downregulation of Gab1 in the septum impaired learning and memory and hippocampal long-term potentiation,whereas overexpression of Gab1 in the same area rescued the cognitive deficitsseen in ChAT-Cre; Gab1^(f/f) and APP^(swe)/PS1 mice.^(18)F-FDGmicroP ET imaging data indicated that Gab1 treatment had no effect on metabolic activity of glucose in APPswe/PS1 mice. Moreover,we identify abnormal function of SKchannelscontributes to increased firing in cholinergic neuronsof ChAT-Cre; Gab1^(f/f) mice. CONCLUSION Gab1 signaling may serve as a potential treatment target for neurological disorders involving dysfunction of central cholinergic neurons.

Bone morphogenetic protein 4 induces cholinergic differentiation of brain-derived neural stem cells in vivo and in vitro

In vitro studies have demonstrated that many factors of bone morphogenetic proteins （BMPs） induce cholinergic differentiation of neural stem cells. However, BMP retains the potential to induce increased numbers of cholinergic neurons in central nervous system regions that are rich in cholinergic cells, which is an important determinant of BMP. Therefore, BMP-4 was added to neural stem cell culture medium or the adult rat hippocampal dentate gyrus. Results demonstrated that BMP-4 induced cholinergic differentiation of neural stem cells in vitro and increased the number of cholinergic neurons in the adult rat hippocampal dentate gyrus.

Scopolamine causes delirium-like brain network dysfunction and reversible cognitive impairment without neuronal loss

Delirium is a severe acute neuropsychiatric syndrome that commonly occurs in the elderly and is considered an independent risk factor for later dementia.However,given its inherent complexity,few animal models of delirium have been established and the mechanism underlying the onset of delirium remains elusive.Here,we conducted a comparison of three mouse models of delirium induced by clinically relevant risk factors,including anesthesia with surgery(AS),systemic inflammation,and neurotransmission modulation.We found that both bacterial lipopolysaccharide(LPS)and cholinergic receptor antagonist scopolamine(Scop)induction reduced neuronal activities in the delirium-related brain network,with the latter presenting a similar pattern of reduction as found in delirium patients.Consistently,Scop injection resulted in reversible cognitive impairment with hyperactive behavior.No loss of cholinergic neurons was found with treatment,but hippocampal synaptic functions were affected.These findings provide further clues regarding the mechanism underlying delirium onset and demonstrate the successful application of the Scop injection model in mimicking delirium-like phenotypes in mice.

Effects of L-3-n-butylphthalide on caspase-3 and nuclear factor kappa-B expression in primary basal forebrain and hippocampal cultures after beta-amyloid peptide 1-42 treatment

BACKGROUND： L-3-n-butylphthalide （L-NBP） can inhibit phosphorylation of tau protein and reduce the neurotoxicity of beta-amyloid peptide 1-42 （Aβ1-42）. OBJECTIVE： To observe the neuroprotective effects of L-NBP on caspase-3 and nuclear factor kappa-B （NF- K B） expression in a rat model of Alzheimer＇s disease. DESIGN, TIME AND SETTING： A cell experiment was performed at the Central Laboratory of Provincial Hospital affiliated to Shandong University between January 2008 and August 2008. MATERIALS： L-NBP （purity 〉 98%） was provided by Shijiazhuang Pharma Group NBP Pharmaceutical Company Limited. Aβ1-42, 3-[4,5-dimethylthiazolo-2]-2,5 iphenyltetrazolium bromide （MTT）, and rabbit anti-Caspase-3 polyclonal antibody were provided by Cell Signaling, USA; goat anti-choactase and rabbit anti-NF- kB antibodies were provided by Santa Cruz, USA. METHODS： Primary cultures were generated from rat basal forebrain and hippocampal neurons at 17 or 19 days of gestation. The cells were assigned into five groups： the control group, the Aβ1-42 group （2 μmol/L）, the Aβ1-42 ＋ 0.1 μmol/L L-NBP group, the Aβ1-42 ＋ 1 μ mol/L L-NBP group, and the Aβ1-42 ＋ 10μmol/L L-NBP group. The neurons were treated with Aβ1-42 （2 μmol/L） alone or in combination with L-NBP （0.1, 1, 10 μmol/L） for 48 hours. Cells in the control group were incubated in PBS. MAIN OUTCOME MEASURES： Morphologic changes were evaluated using inverted microscopy, viability using the M-I-I- method, and the changes in caspase-3 and NF- k B expression using Western blot. RESULTS： Induction with Aβ1-42 for 48 hours caused cell death and soma atrophy, and increased caspase-3 and NF- K B expression （P 〈 0.05）. L-NBP blocked these changes in cell morphology, decreased caspase-3 and NF- k B expression （P 〈 0.05）, and improved cell viability, especially at the high dose （P 〈 0.05）. CONCLUSION： AI3^-42 is toxic to basal forebrain and hippocampal primary neurons; L-NBP protects against this toxicity and inhibits the induction of caspase-3 and NF- K B expression.

Familial Alzheimer's disease modelling using induced pluripotent stem cell technology

Alzheimer’s disease(AD)is a progressive neurodegenerative disease in which patients exhibit gradual loss of memory that impairs their ability to learn or carry out daily tasks.Diagnosis of AD is difficult,particularly in early stages of the disease,and largely consists of cognitive assessments,with only one in four patients being correctly diagnosed.Development of novel therapeutics for the treatment of AD has proved to be a lengthy,costly and relatively unproductive process with attrition rates of】90%.As a result,there are no cures for AD and few treatment options available for patients.Therefore,there is a pressing need for drug discovery platforms that can accurately and reproducibly mimic the AD phenotype and be amenable to high content screening applications.Here,we discuss the use of induced pluripotent stem cells(iPSCs),which can be derived from adult cells,as a method of recapitulation of AD phenotype in vitro.We assess their potential use in high content screening assays and the barriers that exist to realising their full potential in predictive efficacy,toxicology and disease modelling.At present,a number of limitations need to be addressed before the use of iPSC technology can be fully realised in AD therapeutic applications.However,whilst the use of AD-derived iPSCs in drug discovery remains a fledgling field,it is one with immense potential that is likely to reach fruition within the next few years.

Intranasal Administration of Conditioned Medium from Cultured Mesenchymal Stem Cells Improves Cognitive Impairment in Olfactory Bulbectomized Mice

Alzheimer’s disease (AD) is the common cause of dementia which shows the neuro-pathologies like an accumulation of amyloid-β (Aβ) and degeneration of cholinergic neuron. Olfactory bulbectomized (OBX) mice show some of AD features, so they have been used to research as AD model. Mesenchymal stem cells (MSCs) can differentiate into many kinds of cells, including neuronal cells. In this study, we intranasally administrated the conditioned medium derived from cultured umbilical cord (UC) MSCs. The intranasal administration of the MSCs medium restored the cognitive impairment observed in OBX mice. In addition, the decreased number of choline acetyltransferase-positive cells in the medial septum was restored by the conditioned medium administration. In conclusion, MSCs-derived conditioned medium may have protective effects of cholinergic neurons in the medial septum, thereby rescuing the cognitive impairment of OBX.

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

Background:Cell 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.Methods:Mouse 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.Results:Astrocyte-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.Conclusions:Our 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.

Effects of acupuncture stimulation of the sacral region on EEG and urinary bladder activity in rats

Objective To observe the effect of acupuncture stimulation of the sacral segment on the excitability of the cerebral cortex and the activity of the urinary bladder and the involvement of the cholinergic neurons in the laterodorsal tegmental （LDT） nucleus of the brainstem in acupuncture-induced electroencephalogram （EEG） changes. Methods A total of 109 SD rats were used in the present study. Under anesthesia （urethane）, a pair of stainless steel electrodes was separately implanted into the frontal and parietal bony sutures to record EEG. Glass microelectrodes were used to record extracellular discharges of single neuron of the LDT nucleus in the brainstem. Urinary bladder pressure was recorded through a catheter inserted in the bladder and the contraction was induced by infusion of normal saline. A filiform acupuncture needle was inserted into the sacral segment Ecorresponding to Zhongliao （中髎BL 33）] and rotated manually for 1 min. Results In 27 rats whose bladder was full of normal saline, acupuncture stimulation of the sacral region suppressed the contraction activity of the bladder, the fast EEG with lower amplitude and higher frequency tuned into slow EEG with higher amplitude and lower frequency in 6 cases （22.2%）. The inhibitory effect occurred from 45 s to 12 min after acupuncture manipulation. In 82 rats whose bladder was empty, acupuncture stimulation caused the fast EEG to turn into slow EEG in 71 cases （86.6%）. Simultaneously, LDT cholinergic neurons reduced their firing rates from （2.9±1.5） Hz to （1.2±0.6） Hz （n = 12, P〈0.05）, and the reduction of LDT neuronal discharge was earlier in time than the change of EEG. Conclusion Acupuncture stimulation of the sacral region can lower the excitability of the cerebral cortex and suppress bladder activity, which is closely associated with its resultant inhibitory effect on the electrical activity of LDT cholinergic neurons.

The promise of stem cells in the therapy of Alzheimer’s disease

Alzheimer’s disease(AD),a common neurodegenerative disorder associated with gradually to dramatic neuronal death,synaptic loss and dementia,is considered to be one of the most obscure and intractable brain disorders in medicine.Currently,there is no therapy clinically available to induce marked symptomatic relief in AD patients.In recent years,the proof-of-concept studies using stem cell-based approaches in transgenic AD animal models provide new hope to develop stem cell-based therapies for the effective treatment of AD.The degeneration of basal forebrain cholinergic neurons(BFCNs)and the resultant cholinergic abnormalities in the brain contribute substantially to the cognitive decline of AD patients.The approches using stem cell-derived BFCNs as donor cells need to be developed,and to provide proof of principle that this subtype-specific neurons can induce functional recovery of AD animal models.With the continuous scientific advances in both academic and industrial fields,the potentials of stem cells in cellular neuroprotection and cell replacement in vivo have been elucidated,and stem cell-based therapy for repairing degenerative brains of AD is promising.