Mechanisms Underlying Anti-amnesic Effects Of Neurosteroid Pregnenolone Sulfate In Mouse Models Of Alzheimer’s Disease

IntroductionWith the growth of aging population, the incidence of Alzheimer’s disease (AD) is increasing rapidly. In2050, the population of AD is expected more than90million worldwide. Due to lack of effective prevention, diagnosis and therapeutic strategies, AD has become the fourth leading cause of death in the world.AD is a progressive neurodegenerative disease that is characterized by cognitive impairment. The neuropathological hallmarks of AD include deposition of senile plaques created by β-amyloid peptide (Aβ), intracellular neurofibrillary tangles and neuronal death. The cholinergic system is involved in memory function, and cholinergic deficiency has been implicated in the early cognitive decline and behavioral changes of AD. Depletion of a7nicotinic acetylcholine receptor (a7nAChR) on the cholinergic projection neurons in AD is a universal feature of the disease. The a7nAChR has been shown to bind Aβ with high-affinity. This high-affinity binding not only specifically impairs the function of α7nAChRs, but also facilitates endocytosis and accumulation of Aβ in neurons. Intracerebroventricular injection of AP25.35causes dysfunction of a7nAChR and memory deficits. Moreover, Aβ has been reported to disrupt neuronal cells by calcium dyshomeostasis, excitotoxicity and apoptosis.It is widely accepted that the mammalian brain produces continuously newborn neurons in the hippocampal dentate gyrus (DG) throughout the adult life. The newborn neurons, similar to the established ones, are electrically active and connected to the hippocampal CA3field, indicating that they are functional. This is reinforced by evidence that hippocampus-dependent memory is strongly correlated with increased levels of newborn neurons, while the inhibition of neurogenesis has adverse effects on cognitive behaviors. Our previous studies have demonstrated that A(3impaired the neurite growth and survival of newborn neurons. A(3impairs neurite growth by down-regulating mammalian Target of Rapamycin (mTOR) signaling. Abnormal neurogenesis is one of the causes of cognitive impairment in AD.Neurosteroid pregnenolone sulfate (PREGS) is synthesized in the nervous system independently of peripheral glandular sources. The concentration of PREGS in the brain is negatively related with the incidence of AD and the deposition of senile plaques. The level of PREGS decreases in AD brain compared to the age-matched controls. Cognitive deficit in Aβ-rats can be improved by PREGS, however, the mechanism is still unclear. PREGS is a negative modulator of y-aminobutyric acid type A receptor (GABAAR) and a positive allosteric modulator of N-methyl-D-aspartate receptor (NMDA-R). PREGS has been demonstrated to enhance presynaptic glutamate release through activating a7nAChR. Furthermore, PREGS can also activate sigma-1receptor (σ1R). σ1R agonist PRE084can protect dendritic growth and survival of newborn neurons via up-regulating PI3K (phosphatidylinositol-3-kinase)-Akt signaling and reverse the Aβ-induced down-regulation of mTOR-p70S6k (70kDa ribosomal protein S6kinase). PREGS has been reported to stimulate the proliferation of progenitor cells in aged hippocampus via the negative modulation of GABAA receptors. The function of NMDA-R is correlated with the maturation, survival and neural circuit integration of newborn neurons. We have reported that α7nAChR agonist DMXB could improve synaptic transmission and cognitive function by dose-dependent preventing Aβ-induced dysfunction of a7nAChR. In order to systematically explain the mechanisms of PREGS against cognitive impairment in Aβ-mice, the present study explored the effects of PREGS on abnormal neurogenesis and cellular apoptosis induced by Aβ and its possible mechanisms.Objective(1) Effects of PREGS on A(3-impaired neurogenesis and its mechanisms.(2) Effects of PREGS on A(3-induced apoptosis and its mechanisms.—The First PartEffects of PREGS on Aβ-impaired neurogenesis and its mechanisms Materials and Methods1. Preparation of an animal model:genotypic identification of8-month-old male APP/PS1mice. ICR Male mice (25-30g) were used to explore partial mechanisms.2. PREGS administration and newborn neuron labelling:(1) APP/PS1mice:BrdU was intraperitoneally injected for12consecutive days to label proliferating cells. PREGS was subcutaneously injected at dose of20mg/kg once daily from day7before BrdU-injection and lasted34days. BrdU immunostaining was performed at day1(1-day-old) and day28(28-day-old) after the last BrdU-injection.(2) ICR Male mice:Three intraperitonealy injections of BrdU were given with the interval of6h. Date of drug administration was counted from the day of BrdU-injection. The injection of PREGS (3nmol) was given once daily for2consecutive days at days0-1, days5-6, days10-11, days15-16or days20-21after BrdU-injection respectively. BrdU immunostaining was performed at days22(22-day-old) after BrdU-injection. 3. Proliferating cell nuclear antigen (Ki67) immunostaining was used to evaluate the proliferation of progenitor.4. Doublecortin (DCX) immunostaining was used to quantify the dendritic growth of newborn neurons.5. BrdU and NeuN (neuron specific nuclear protein) or GFAP (glial fibrillary acidic protein) double label immuno-histofluorescence staining were used to evaluate the differentiation of progenitor and the maturation of the newborn neurons.6. Aβ immunostaining was used to observe the deposition of amyloid plaques.7. Brain derived neurotrophic factor (BDNF) levels were determined by enzyme-linked immunosorbent assay (ELISA).8. Field potential recordings:At60min after the lasting injection of PREGS in ICR mice, the brains were rapidly removed and coronal brain slice were prepared to record the excitatory postsynaptic potentiation (EPSP) and paired-pulse facilitation (PPF) in the hippocampal DG.9. Morris water maze task was used to test the cognitive function.Results1.8-month-old APP/PS1mice showed a mass of amyloid plaques in the hippocampal region. In Morris water maze test, APP/PS1mice spent a longer escape-latency to reach the hidden platform than that in control mice.2. Compared to control mice, the number of1-day-old BrdU immuno-positive (BrdU+) cells increased approximately30%in the hippocampal DG of8-month-old APP/PS1mice. The number of Ki67immuno-positive (Ki67+) cells was also significantly upregulated in the same brain region of8-month-old APP/PS1mice, indicating an over-proliferation of progenitor cells in the DG. The treatment with PREGS did not affect the over-proliferation of progenitor cells in APP/PS1mice. 3. The dendritic length of DCX immuno-positive (DCX+) cells in APP/PSl mice was significantly shorter than that of control mice. The treatment with PREGS enhanced the neurite growth of newborn neurons.4. The number of28-day-old BrdU+cells in APP/PS1mice decreased by approximately50%relative to control mice. There was a less population of BrdU+/NeuN+cells in APP/PS1mice than that in control mice, while the number of BrdU+/GFAP+cells in APP/PS1mice was nearly the same as control mice. These results demonstrate that A(3impairs the maturation and survival of newborn neurons. The administration of PREGS could significantly increase the number of newborn neurons in APP/PS1mice.5. PREGS could partially attenuate the depositon of Aβ in the hippocampal region.6. In comparison with control mice, the level of BDNF was reduced in APP/PS1mice, which could be improved by the treatment with PREGS.7. The treatment of PREGS at days10-16after BrdU-injection could increase the number of22-old-day BrdU+cells in ICR mice. PREGS could induce a sustained increasing in EPSP slope and decreasing in PPR, indicating an increase in presynaptic glutamate release. The a7nAChR, σ1R and NMDA-R antagonists could prevent the PREGS-induced increase in presynaptic glutamate release and survival of newborn neurons. These results indicate that PREGS reduces the inactive newborn neural death through potentiating excitatory input activity of newborn neurons.7. The treatment with PREGS could attenuate the prolongation of escape-latency in APP/PS1mice.Conclusion1. The treatment with PREGS can rescue the Aβ-impaired neurite growth of newborn neurons and protect the survival of newly generated neurons through reducing Aβ deposition and increasing the expression of BDNF.2. PREGS protects the survival and maturation of newborn neurons in APP/PS1mice through enhancing the input activity of newborn neurons.3. PREGS improves cognitive deficits by protecting impairment of neurogenesis induced by Aβ. —The Second Part Effects of PREGS on Aβ-induced apoptosis and its mechanisms.Materials and Methods1. Preparation of AD model:ICR male mice were intracerebroventriculy injected with the "aggregated" AP25-35(9nmol).2. Drug administration:PREGS (20mg/kg) was intraperitoneally injected once daily on1-7days after Aβ25-35-injection.3. Morris water maze task was used to test the cognitive performance.4. The number of pyramidal cells in hippocampal CA1region was assessed by histological examination.5. TUNEL staining was used to examine the number of apoptotic cells.6. Western blot analyses the phosphorylation level of ERK1/2, Akt and caspase-3.Results1. AP25-35-mice spent longer escape-latency to reach the hidden platform compared to control mice. The number of pyramidal cells was reduced approximately50% and the number of apoptotic cells (TUNEL positive cells) was significantly increased in Aβ25-35-mice.2. The treatment with PREGS could attenuate the prolongation of escape-latency and the increase of apoptotic cells in Aβ25-35-mice.3. The σ1R and a7nAChR antagonists could antagonize the anti-apoptotic effects of PREGS.4. In comparison with control mice, the phosphorylation level of Akt (p-Akt) or ERK2(p-ERK2) in hippocampus was decreased and the caspase-3was elevated in Aβ25-35-mice. The administration of PREGS in Aβ25-35-mice prevented the decrease in p-Akt and p-ERK2and the increase in caspase-3, which was sensitive to the σ1R antagonist NE100and the a7nAChR antagonist.5. The PI3K inhibitor LY294002and the ERK kinase (MEK) inhibitor U0126could attenuate the anti-apoptotic effects of PREGS.6. The PI3K inhibitor LY294002could block the anti-amnesic effect of PREGS in Aβ25-35-mice.Conclusion1. Aβ inhibits the phosphorylation of ERK2and Akt to enhance the activity caspase-3, which induces apoptosis of hippocampal neurons.2. PREGS cascades σ1R and a7nAChR-mediated PI3K-Akt and ERK signaling pathway to protect hippocampal neurons from Aβ-toxicity, which exerts a potential anti-amnesic effects.