Investigation of the neuroprotective effects of Lycium barbarum water extract in apoptotic cells and Alzheimer's disease mice.

Alzheimer's disease (AD) affects people worldwide and is caused by chronic and progressive damage to the central nervous system. Lycium barbarum (LB), a renowned functional food and medicinal plant in Southeast Asia, may possess protective effects against nerve injury. The present study aimed to investigate the neuroprotective effects of LB water extract in a differentiated (D)PC12 cellular apoptosis model induced by L­glutamic acid (L­Glu), and a mouse model of AD, induced by the combination of AlCl3 and D­galactose. LB markedly increased DPC12 cell survival against L­Glu induced damage by increasing cell viability, reducing the apoptosis rate and G1 phase arrest, suppressing intracellular reactive oxygen species accumulation, blocking Ca2+ overload and preventing mitochondrial membrane potential depolarization. LB additionally normalized the expression levels of apoptosis regulator Bcl­2, apoptosis regulator BAX, and cleaved caspase­3, ­8 and ­9 in L­Glu exposed cells. In the AD mouse model, LB increased the amount of horizontal and vertical movement in the autonomic activity test, improved endurance time in the rotarod test and decreased escape latency time in the Morris water maze test. Additionally, the levels of acetylcholine and choline acetyltransferase were significantly increased in the serum and hypothalamus in the LB­treated AD mice. These data suggested that LB may exert neuroprotective effects and may aid in preventing neurodegenerative disease.

Anti-Oxidative Stress Activity Is Essential for Amanita caesarea Mediated Neuroprotection on Glutamate-Induced Apoptotic HT22 Cells and an Alzheimer's Disease Mouse Model.

Amanita caesarea, an edible mushroom found mainly in Asia and southern Europe, has been reported to show good antioxidative activities. In the present study, the neuroprotective effects of A. caesarea aqueous extract (AC) were determined in an l-glutamic acid (l-Glu) induced HT22 cell apoptosis model, and in a d-galactose (d-gal) and AlCl3-developed experimental Alzheimer's disease (AD) mouse model. In 25 mM of l-Glu-damaged HT22 cells, a 3-h pretreatment with AC strongly improved cell viability, reduced the proportion of apoptotic cells, restored mitochondrial function, inhibited the over-production of intracellular reactive oxygen species (ROS) and Ca2+, and suppressed the high expression levels of cleaved-caspase-3, calpain 1, apoptosis-inducing factor (AIF) and Bax. Compared with HT22 exposed only to l-Glu cells, AC enhanced the phosphorylation activities of protein kinase B (Akt) and the mammalian target of rapamycin (mTOR), and suppressed the phosphorylation activities of phosphatase and tensin homolog deleted on chromosome ten (PTEN). In the experimental AD mouse, 28-day AC administration at doses of 250, 500, and 1000 mg/kg/day strongly enhanced vertical movements and locomotor activities, increased the endurance time in the rotarod test, and decreased the escape latency time in the Morris water maze test. AC also alleviated the deposition of amyloid beta (Aß) in the brain and improved the central cholinergic system function, as indicated by an increase acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations and a reduction in acetylcholine esterase (AchE) levels. Moreover, AC reduced ROS levels and enhanced superoxide dismutase (SOD) levels in the brain of experimental AD mice. Taken together, our data provide experimental evidence that A. caesarea may serve as potential food for treating or preventing neurodegenerative diseases.

The Neuroprotective Properties of Hericium erinaceus in Glutamate-Damaged Differentiated PC12 Cells and an Alzheimer's Disease Mouse Model.

Hericium erinaceus, an edible and medicinal mushroom, displays various pharmacological activities in the prevention of dementia in conditions such as Parkinson's and Alzheimer's disease. The present study explored the neuroprotective effects of H. erinaceus mycelium polysaccharide-enriched aqueous extract (HE) on an l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cellular apoptosis model and an AlCl3 combined with d-galactose-induced Alzheimer's disease mouse model. The data revealed that HE successfully induced PC12 cell differentiation. A 3 h HE incubation at doses of 50 and 100 µg/mL before 25 mM of l-Glu effectively reversed the reduction of cell viability and the enhancement of the nuclear apoptosis rate in DPC12 cells. Compared with l-Glu-damaged cells, in PC12 cells, HE suppressed intracellular reactive oxygen species accumulation, blocked Ca2+ overload and prevented mitochondrial membrane potential (MMP) depolarization. In the Alzheimer's disease mouse model, HE administration enhanced the horizontal and vertical movements in the autonomic activity test, improved the endurance time in the rotarod test, and decreased the escape latency time in the water maze test. It also improved the central cholinergic system function in the Alzheimer's mice, demonstrated by the fact that it dose-dependently enhanced the acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations in both the serum and the hypothalamus. Our findings provide experimental evidence that HE may provide neuroprotective candidates for treating or preventing neurodegenerative diseases.

Regulated Extracellular Choline Acetyltransferase Activity- The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway.

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer's disease and multiple sclerosis in particular.

A high cholesterol diet ameliorates hippocampus-related cognitive and pathological deficits in ovariectomized mice.

Both sex hormone deficiency and hypercholesterolemia are related to cognitive decline or Alzheimer's disease. However, their interactive influence on the neurodegenerative progress is not clear. This study was designed to assess the effects of ovarian hormone depletion and high cholesterol diet alone or in combination on hippocampus-related cognitive and pathological deficits in adult female ICR mice. Depletion of ovarian hormones by ovariectomy for 9 weeks resulted in significant spatial learning and memory deficits as revealed by the water maze testing. Such cognitive alteration was accompanied with increases in neuron death and decreases in choline acetyltransferase activity and synaptopysin expression in the hippocampus. On the other hand, the high cholesterol diet (3% cholesterol plus normal chow) did not exacerbate, but slightly alleviated cognitive decline and significantly attenuated hippocampal pathological changes in ovariectomized mice. Moreover, ovariectomized mice fed high cholesterol had increased serum estrogen levels compared with those fed a normal chow. These results indicate that high cholesterol intake increases the sex hormone synthesis and in turn partially attenuates hippocampus-related cognitive and pathological deficits caused by ovariectomy.

In utero methanesulfonyl fluoride differentially affects learning and maze performance in the absence of long-lasting cholinergic changes in the adult rat.

There is increasing evidence that acetylcholinesterase (AChE) may have various specific developmental roles in brain development. Nevertheless, specific effects of AChE inhibition during early brain development have not been adequately described. Therefore, methanesulfonyl fluoride (MSF), an irreversible AChE inhibitor that shows high selectivity for the CNS was used to produce AChE inhibition in utero to study subsequent adult behaviors, sleep, and cholinergic markers. Rats exposed to MSF in utero showed a deficit in spatial learning tasks using appetitive motivation but, surprisingly, they performed equally well or better than controls when aversive motivation was used. One hypothesis was that MSF treatment in utero affected the response to stress. Tests of anxiety however showed no differences in basal levels of anxiety. Studies of sleep behavior, however, indicated a higher level of REM sleep which is only seen during the light phase of male rats exposed to MSF in utero as compared to controls. No differences in cholinergic markers in the brains of adults were found except that females exposed to MSF in utero had a higher level of ChAT activity in the synaptosomal fraction of the hippocampus. Even so, whether cholinergic alterations accompany the in utero MSF exposure remains to be determined. The failure to find widespread changes in cholinergic markers in the adult brains suggests changes in behaviors should be further investigated by testing the participation of postsynaptic mechanisms, measuring of cholinergic markers during earlier development periods and the possible participation of other neurotransmitter systems to clearly reveal the role of the cholinergic system following in utero MSF exposure.

Peripheral choline acetyltransferase is expressed by monocytes and upregulated during renal allograft rejection in rats.

Acetylcholine (ACh) has been shown to modulate the function of mononuclear leukocytes, both by muscarinic and nicotinic ACh receptors. Acute stimulation of lymphocytes with ACh or muscarinic agonists enhances proinflammatory functions, whereas chronic application of the ACh agonist nicotine has an anti-inflammatory effect (Geng et al., 1996; Kawashima and Fujii, 2003). In macrophages, acute treatment with nicotine down-modulates effector functions (Wang et al., 2003, 2004). ACh regulating leukocytes might originate from the nervous system. However, once released, ACh is quickly degraded. Relevant concentrations occur only in the direct vicinity of nerve endings. Non-neuronal ACh acting in a paracrine or autocrine fashion is more likely to influence immune functions. Lymphocytes express all enzymes needed for ACh synthesis, including choline acetyltransferase (ChAT). In the rat, alternative splicing generates common ChAT and peripheral ChAT (pChAT). Up to now, ChAT expression by monocytes has not been demonstrated. We investigate pChAT in monocytes in an experimental model of acute renal allograft rejection. Inside the blood vessels of the transplant, huge numbers of activated, cytotoxic monocytes accumulate and probably contribute to graft destruction (Grau et al., 2001).

Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes.

Cholinergic markers and the expression of M(2)-M(5) muscarinic cholinergic receptor subtypes were investigated in human peripheral blood lymphocytes by Western blot analysis and immunocytochemistry. The totality of peripheral blood lymphocytes express acetylcholine (ACh) immunoreactivity, choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular ACh transporter (VAChT) and M(2)-M(5) muscarinic cholinergic receptor protein immunoreactivity. Western blot analysis performed independently on T and B lymphocytes using anti-ChAT and anti-AChE antibodies revealed labelling of single bands of approximately 68-70 and 70 kDa, respectively, whereas VAChT was bound to two bands of approximately 80 and 45 kDa. The pattern of immunoblotting was similar in membranes of lymphocytes and striatum, used as a reference brain tissue. Western blot analysis using anti M(2)-M(5) receptor antibodies revealed labelling of single bands of approximately 55, 85-90, 50 and 81 kDa, respectively. Confocal laser immunofluorescence showed the localization of ACh and VAChT immunoreactivity in punctiform areas likely corresponding to cytoplasmic vesicles. ChAT and AChE were diffused to the cytoplasm and plasma membrane. Muscarinic receptor immunoreactivity was located in lymphocyte plasma membrane. Although the role of lymphocyte cholinergic system is still unclear, the demonstration of cholinergic markers in T and B human blood lymphocytes supports the view that a cholinergic systems may contribute to the regulation of immune function. The characterization of these cholinergic markers may also contribute to define if their evaluation can be used for assessing the status of brain cholinergic system.

Neurotrophic factors in memory disorders.

Neurotrophic factors are target-derived proteins capable of affecting survival, target innervation, and/or function of neuronal cell populations. These factors are structurally and functionally related to the classical neurotrophic molecule nerve growth factor (NGF) and resemble a genetic family called neurotrophins. Besides NGF and brain-derived neurotrophic factor (BDNF), there is little knowledge whether these neurotrophins play a pathophysiological role in dementing brain disease(s). BDNF-mRNA levels are reported to be decreased in the hippocampus of individuals with Alzheimer's disease (AD). Decreased NGF production does not seem to play a causal role both in age-related cognitive impairment and AD which is usually associated with neurodegenerative processes in the cholinergic basal forebrain system. However, there are several experimental indications that NGF might be of importance for the stimulation of compensatory changes and repair mechanisms; given in pharmacological dose, NGF might be of therapeutical benefit, as reported in a preliminary clinical case study. Thus, the availability of sufficient quantities of recombinant human neurotrophins should allow comprehensive research programs in future.

A reevaluation of choline acetyltransferase activity in human cerebrospinal fluid and serum.

Choline acetyltransferase (EC 2.3.1.6) was studied in human cerebrospinal fluid and blood. Sensitivity, precision and reproducibility of the radioactive assay were evaluated with a homogenate from pig brain and with partially purified enzymes from bovine brain and human placenta. Contrary to other reports, with the assay used in the present study, choline acetyltransferase activity was not detectable in the cerebrospinal fluid or blood of 50 patients with various neurological disorders. Only a very slight but significant non-enzymatic formation of a radioactive product by concentrated cerebrospinal fluid was observed. Nonenzymatic but enzyme-like formation of acetylcholine by thiol reagents and other compounds is discussed.

Inhibition of human sperm motility by inhibitors of choline acetyltransferase.

Human spermatozoa contain choline acetyltransferase (ChA), acetylcholinesterase and acetylcholine (ACh). There is no storage pool for ACh in spermatozoa. Therefore, ChA inhibitors should exhibit dramatic effects in the alteration of levels and turnover of ACh and sperm motility. The effects of two groups of ChA inhibitors, 2-benzoylethyltrimethylammonium (BETA) and related compounds and halogenoacetylcholines (cholinesters of iodo-, bromo- and chloroacetic acids; XACh, where X = l, Br or Cl), on the motility index of human ejaculated sperm were studied. These investigations gave the following results: 1) BETA was a potent inhibitor of ChA from monkey brain (I50, 4.8 x 10(-6) M), homogenates of rat spermatozoa (I50, 6.5 x 10(-5) M) and homogenates of human spermatozoa (I50, 5.6 x 10(-5) M). It decreased the motility index of human spermatozoa (I50, 8.5 x 10(-8) M) at concentrations higher than 10(-8) M after a contact time of 5 to 60 min. It decreased the motility index of human spermatozoa by about 80% after 5 min and by 95% after 1 hr at a concentration of 10(-6) M. 2) There was a positive relationship between the inhibition of ChA and the depression of the motility index of human spermatozoa among these inhibitors. Both the number of motile cells and the graded motility were decreased. 3) All ChA inhibitors studied are quaternary ammonium compounds that do not significantly cross membrane barriers. 4) Both human sperm cells and human sperm cell homogenates had the same ChA activity. 5) Seventy-five percent of ChA activity could be washed away from human spermatozoa. 6) The same amount of ChA inhibition was observed when BETA was added to the homogenate of sperm cells or whole sperm cells. All of these observations indicate that sperm cell ChA is accessible to BETA and related quaternary ammonium compounds. These studies indicate that ACh is possibly synthesized by the tail and is a local hormone in the coordination of contraction and relaxation cycles of spermatozoan flagella.

Choline acetyltransferase activity in human blood and its diurnal oscillation--an artifact?

Diurnal oscillation of choline acetyltransferase (ChAT)-like activity in human blood has recently been reported in the literature. From studies on the pH-optimum of acetylcholine synthesizing activity in human blood we suggest, however, that this activity is only partly related to ChAT, and that the diurnal oscillation observed might be an artifact resulting from the analytical procedure.