Study On The Protection And Mechanism Of Neurosteroids Against Aβ Induced Neurotoxicity In Vitro

Alzheimer's disease (AD) also called Alzheimer disease, Senile Dementia of the Alzheimer Type (SDAT) or simply Alzheimer's, is a progressive neurodegenerative disorder characterized clinically by memory and cognitive deficits. Alzheimer's disease is the most common form of dementia in the elderly. This incurable, degenerative, and terminal disease was first described by German psychiatrist and neuropathologist Alois Alzheimer in 1906 and was named after him. Generally, AD is diagnosed in people over 65 years of age. The prevalence of AD in the general population increased sharply in population beyond the age of 65. The confirmed diagnoses of AD in subject is about 4% under the age of 75 (aged from 65 to 74), but rise up to 50% of the population over 85 years of age suffers from this type of dementia. Today, the cause and progression of Alzheimer's disease are not well understood. There is no means presently available to treat the pathogenesis of AD. Currently used treatments offer only a small symptomatic benefit. As of 2008, more than 500 clinical trials have been conducted for identification of a possible treatment for AD, but it is unknown if any of the tested intervention strategies will show promising results. In developed countries, AD is one of the most costly diseases to society. Alzheimer's disease is one of known for placing a great burden on caregivers. To develop effective interventions and medications to cure AD has become a hot issure in AD research, and also an anxious economic social problem globally.The neuropathologic hallmarks of AD are extracellular senile plaques, intracellular neurofibrillary tangles, and loss of neurons and synapses in the cerebral cortex and certain subcortical regions. Being the main component of senile plaques in AD,β-amyloid (Aβ) is derived from amyloid precursor protein viaβ-secretase mediated pathway. Studies show that extracellular accumulation of fibrillar deposits of Aβin brain may be the core mechanism of AD pathogenesis, and it's also the potential target for AD therapy. The neurotoxicity of Aβhas been proven by many studies, which can be intensified by aggregation. There is a homeostasis of Aβproduction and in the physiological state. Genetic mutation and environment can interrupt this balance and lead to assembly and deposition of Aβ, which initiate a cascade of events leading to neuronal dysfunction and death, cause or accelerate the progress of AD, via oxidative stress, apoptosis, inflammation, etc.Increasing evidence suggests that selective neuronal loss in AD brain, expecialy the cerebral cortex and hippocampus, is closely linked with cognitive damagement in AD, which maybe chiefly indecued by Aβcytotoxcity. Apoptosis, the active process of cellular suicide under certain condition, has been the focus of intense research in the last several decades as a means of controlling cell populations in normal development and inflammation through programmed cell death (PCD) regulated by intracellular genetic mechanism or excellular signaling molecules. Excessive apoptosis is belived the main neurodegenerative cause of AD. Complicated molecular biological mechanisms are involved in apoptotic processes. Amomg those, activation of cysteine aspartyl proteases (caspases) play an essential role. Caspases is a family of cysteine proteases, which are first synthesized as inactive pro-caspases, and can be rapidly activated by stimuli. When caspase cascade reaction initiated, a series of caspase substrates cleaved within the cell, to trigger the apoptotic process. At present, at least forteen caspase proteins have been identified. Caspase 3, a member of the caspase family, plays a central role in the execution-phase of cell apoptosis, and is named"the terminator of apoptosis".The caspases can be activated through either the intrinsic (mitochondrial mediated) or extrinsic (death receptor mediated) apoptotic pathways. The former is considered the main apoptotic pathway in Aβcytotoxcity elucidated in vitro. The intrinsic apoptotic pathway is characterized by permeabilisation of the mitochondria and release of cytochrome c (Cyt-c) into the cytoplasm. Cyt-c then forms a multi protein complex known as the'apoptosome'and initates activation of the caspase cascade through caspase 9.Intensive research carried out in recent years has shown that neurosteroids may be of particular importance in the treatment of diseases where neurodegeneration is predominant including AD. Endogenous neurosteroids are steroid substances, including pregnenolone (PREG), dehydroepi- androsterone (DHEA), progesterone (PROG) and their derivatives, that are synthesized in the brain tissure (neurons and glia) in the presence of the steroidogenic enzymes, either de novo from cholesterol or by in situ metabolism of precursors imported from peripheral sources. Neurosteroids have a wide variety of diverse functions, and were classified as 4th generation neuromessengers in the brain. Studies show that neurosteroids are important in nerverous system development. They are a relatively new class of neuroproctive compounds brought to prominence in the past 2 decades.A general trend toward decreased levels of all neurosteroids were found in post-mortem AD patients'brain region with increase of key pathologic proteins, Aβand tau. The results indicated an involvement of neurosteroid in AD, suggesting their possible neuroprotective role. Other studies in vitro demonstrated the neuroprotective effects of PREG and DHEA to against damages induced by Aβvia reduction of free-radical peroxidation and prevention of glucocorticoid receptor (GR) localization to the nucleus. There was a hypothesis suggesting, high concentration of Aβmay induce toxic effects on never cells by inhaibition of the endogenous production of neuroprotective nerosteroids. Although some neurosteroids, such as PREG and DHEA, have demonstrated their protective effects against Aβneurotoxicity, little is known on the role of endogenous neurosteroids in the regulaton of cellular mechanism involved in human neverous system pathophysiology, such as AD, expecially the changes of neurosteroidgenesis, neurosteroids synthesis in brain, induced by Aβneurotoxicity. In nervous system, PREG and DHEA lie in the upstream of neurosteroidgenesis. As precursors of other downstream neurosteroids, the neuroprotective effects they demonstrated maybe generated by themselves and/or their dowmsteam metaboloics. Furthermore, much more mechanisms of neurosteroids protection maybe involved, including cell-membrane receptors, cytoplasmic organelles, e.g., mitochondria, or intracellular targets.Aim: In the presented study, an in virto AD cell model was established based the primary rat cortical neurons culture technique developed previously in our lab. The model used Aβ25-35, the wildly used experimental active fragment of Aβas neurotoxicity proxy. Based on this model, a valid solid-phase extraction combind HPLC-MS assay was applied to investigate four chief neurosteroids, PREG, DHEA, PROG, and AP levels in cultured neurons induced by Aβcytotoxicity. Afterwards, PROG and AP, whose level found obviously changed by Aβdamagement, were choosen as intervening substances to check out their protective effects against Aβcytotoxicity. Then the reversre effects of PROG and mechanisms involved were studied. The purpose of this paper is to add something valuble for AD prevention and medication.Methods:1. Primary culture of rat cortical neuronsBriefly, cortical neurons were obtained from new born SD rats by mild digestion and mechanical dissection. The cells were seeded on the culture plates pre-coated with poly-L-lysine in the DMEM containing 10% fetal bovine serum at the density of 1.0×109 L-1. The neurobasal medium were changed 24 hours after culture and followed by changing half medium every 2 days. Cultures were maintained at 37oC, saturation humidity in an atmosphere containing 5% CO2 for 8 to 10 days prior to experimentation. Inverted microscope was used to observe morphological changes of cultured neurons.2. Sampling and quantitationTreated neurons were collected, after cell lysis, supernatant protein concentrations were quantitatived by ultraviolet (UV) spectrometry.3. Cell viability assayThe MTT assay was used to evaluate the viability of the neurons cultured in 96-well plates. Briefly, 20μL MTT was added to the culture media with a final concentration of 3.6 mmol·L-1. After 4 hours of culture, the culture media was removed and 200μL DMSO was added to dissolve the crystals. The absorbance of the solution was read at 570nm on a spectrophotometer and the cell viability was calculated by compare to controls.4. Measurement of lactate dehydrogenase (LDH) activity in culture mediaThe culture media were collected and LDH activity was determined using a LDH activity kit. The absorbance of the solution was determined by UV spectrometry. The LDH activity was calculated according to the operation instruction and compared to controls.5. Determination of neurosteroids'concentrations in culture media using HPLC-MSOne milliliter of cultured media samples were mixed with methyltestosterone (MT), the internal standard, and methanol, then purified by solide phase extraction (SPE) using Waters Oasis HLB SPE columns (30 mg,1 cc). Thereafter, samples were derivatized with 2-nitro-4-trifluoromethyl- phenylhydrazine (2NFPH) and determinated using high performance liquid chromatography-mass spectrometry (HPLC-MS).The HPLC-MS method used a Thermo BDS Hypersil C18 column (2.1×150 mm, 5μm) as stationary phase, with an Agilent Zorbax SB C18 guard column (4.6×12.5 mm, 5μm). The mobile phase consists of acetonitrile and water under a gradient programe and pumped in 1 mL·min-1. Twenty milliliters of sample was analyzed onto the system by an Agilent autosampler. APCI ionization was used in mass detection in selected ion monitoring mode. The negative quasi-molecular ions monitored for neurosteroid derivants were m/z 518 (PREG-2NFPH,[M-H]-), m/z 490 (DHEA-2NFPH,[M-H]-), m/z 719 (PROG-2(2NFPH),[M-H]-), m/z 520 (AP-2NFPH,[M-H]-), and m/z 504 (MT-2NFPH,[M-H]-), respectively.6. Apoptosis of neurons indentified by Hoechest 33342 dyingCulture media were removed after treatments. Neurons were carefully washed by cold saline solutions, and fixed for 20 minutes in 4% parafoemaldehyde solution. After washing, the fixed neurons were dyed for 5 minutes using Hoechest 33342 (5 mg·L-1). Apoptosis were identified by nuclear morphometry under fluorescence microscope. The apoptosis rate was calculated by 6 randomly selected eye filds.7 Western-blot for plasma Cyt-c, caspase-9, and caspase-3 levelsCell plasma protein lysates were mixed with denaturing lysis buffer. The mixture was boiled for 10 min, and cooled to room temperature. Western-blot was used to observe the changes of Cyt-c, caspase-9, and caspase-3 levels in neurons after treatment respectively. Ten milliliters of each protein sample was separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking in skimmed milk, the membranes were incubated with antibody over night. The membranes were then developed with peroxidase-conjugated secondary antibody. The immunoreactive proteins were visulized by enhanced chemiluminescence system. The immunoblots were quantitated using analystic software.8. Data and statisticsStatistic was performed with SPSS 11.5 for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA with the Dunnett t test and binary variable correlation analysis. Significance was considered when P<0.05.Results:1. Neurotoxicity induced by Aβ25-35 in primary rat cortical neurons1.1 Morphological alterations of neuronsCortical neurons cultured for 8 days, were generally bipolar or multipolar cells under inverted microscopy, most of them with triangular soma, and usually aggregated with their branching dendrites interconnected as netwok.Compared with control group, 24 h after 1 mmol·L-1 Glu treatment, numbers of neurons decreased obviously, with swelling soma, cavitation of cytoplasm, granuliform pieces and reduced refraction. A decreased trend was found with increased concentrations of Aβ25-35. Neuons number was roughly equivalent in 1μmol·L-1 Aβ25-35 treatment group and GLU group, while that is obviously low in 10μmol·L-1 Aβ25-35 treatment group.1.2 Influence on neurons viabilityCompared with controls, neurons damaged obviously in Glu group, whose cell viability massured by MTT assay was (53.1±5.5) % (P<0.01). Meanwhile Aβ25-35 decreased MTT absorbance value and cell survival rate of neurons in a dose-dependent manner. The Pearson correlation coefficient was -0.897 between the concentration of Aβ25-35 and neuron viability (P<0.01). The massured cell viability is (52.7±3.0) % in 1μmol·L-1 Aβ25-35 group, equivalent to Glu group (P>0.05).1.3 Influence on LDH releaseLactate dehydrogenase activity measured in control group was (23.6±2.3) U·g-1 Prot. Compared with controls, LDH release increased obviously in Glu group, whose deteced LDH activity was (39.9±2.9) U·g-1 Prot (P<0.01). That is likely in 10μmol·L-1 Aβ25-35 group vs Glu group (P>0.05), which was (38.8±2.0) U·g-1 Prot. There were no differences among other Aβ25-35 treatment (0.1μmol·L-1 and 1μmol·L-1) with controls (P>0.05).2. Effect of Aβ25-35 on neurosteroidogenesis in primary rat cortical neurons2.1 Influence on neurons viabilityCompared with controls, cell survival rate of neurons was (49.3±4.3) % in 1μmol·L-1 Aβ25-35 group (P<0.01), while cholesterol treatment made no difference with controls (P>0.05). But, in Aβ25-35 plus cholesterol group, the cell viability was (83.6±5.1) %, obviously higer than Aβ25-35 treatment alone (P<0.01).2.2 Influence on neurosteroidgenesisAbsence of the necessary substance, cholesterol, no neurosteroids found in control and Aβ25-35 alone groups. In cholesterol and Aβ25-35 plus cholesterol groups, DHEA levels were found below LLOQ. In cholesterol group, PREG, PROG, and AP levels were (1.3±0.2)μg, (26.2±2.3)μg, and (10.1±0.8)μg per grams protein, respectively; those were (1.1±0.1)μg, (18.4±1.2)μg, and (15.3±1.0)μg per grams protein in Aβ25-35 plus cholesterol group respectively. Compared with cholesterol treatment alone, No change in PREG level was observed after treated with Aβ25-35 (P>0.05), while PROG and AP levels greatly varied (P<0.01), the former decrease by 29.8%; the later increased by 51.5%.3. Effects of PROG and AP treatments on neuronal damagement in Aβ25-35 induced AD model3.1 Influence of PROG treatment on neuronal damagement in Aβ25-35 induced AD modelObserved under inverted microscope, numbers of neurons decreased obviously in model group (1μmol·L-1 Aβ25-35 treatment) compared with controls. An ascending trend was found with increasing PROG concentrations.Compared with controls, cell survival rate of neurons was (56.7±4.1) % in model group (P<0.01). Compared with model group, PROG treatment increased neurons viabilities in a dose-relervant way in range of 0.001μmol·L-1~ 0.1μmol·L-1. The Pearson correlation coefficient was 0.757 between the concentrations of PROG and neuron viability (P<0.01). The cell viability was (100.7±7.9) % in 0.1μmol·L-1 PROG group, having no difference with controls (P>0.05).3.2 Influence of AP treatment on neuronal damagement in Aβ25-35 induced AD modelNo ascending trend was found with increasing AP concentrations.Cell survival rate of neurons was (50.9±3.9) % in model group Compared with controls (P<0.01). Compared with model group, AP treatment decreased neurons viabilities in a dose-relervant way in range of 0.001μmol·L-1~ 0.1μmol·L-1. The Pearson correlation coefficient was -0.841 between AP concentrations and neuron viability (P<0.01).4. Effects of PROG treatment on neuronal apoptosis in Aβ25-35 induced AD model4.1 Protection of PROG treatment on neurons in Aβ25-35 induced AD modelNeuron numbers decreased obviously in model group compared with controls under inverted microscope. Cell survival rate of neurons was (47.9±2.9) % in model group (P<0.01). Compared with model group, an ascending trend was found with increasing PROG concentrations. In the range of 0.001μmol·L-1~ 0.1μmol·L-1, PROG treatment increased neurons viabilities in a dose-relervant way. The Pearson correlation coefficient was 0.779 between PROG concentration and neuron viability (P<0.01). The cell viability in 0.1μmol·L-1 PROG group had no difference with controls (P>0.05).4.2 Influence on neurons apoptosisIdentified by staining of nuclei under fluorescence microscope, neuronal apoptosis increased in model group. A decreasing trend was found when co-treated with PROG in neurons.Apoptosis rate in controls was (11.7±0.8) %, that was (52.0±5.1) % in model group (P<0.01). Compared with model group, PROG reduced the neuronal apoptosis rate in a dose-relervant manner in range of 0.001μmol·L-1 ~ 0.1μmol·L-1. The Pearson correlation coefficient between PROG concentration and neuron apoptosis rate was -0.887 (P<0.01). The neuronal apoptosis rate is (11.8±1.2) % in 0.1μmol·L-1 PROG group, had no difference with controls (P>0.05).4.3 Influence on cytoplasmic caspase-3 levelMeasured by western-blot, cytoplasmic caspase-3 level elevated in model group compared with controls, from (3.47±0.26) to (5.84±0.51) (P<0.01). PROG inhibited this elevation of caspase-3 level in a dose-relervant way in range of 0.001μmol·L-1~ 0.1μmol·L-1. The Pearson correlation coefficient between PROG concentration and cytoplasmic caspase-3 level was -0.904 (P<0.01). The caspase-3 level was (3.58±0.21) in 0.1μmol·L-1 PROG group, having no difference with controls (P>0.05).5. Effects of PROG treatment on mitochondral apoptosis relative proteins in Aβ25-35 induced AD model4.1 Protection of PROG treatment on neurons in Aβ25-35 induced AD modelNeuron numbers decreased obviously in model group compared with controls under inverted microscope. Cell survival rate of neurons was (51.9±4.5) % in model group (P<0.01). Compared with model group, an ascending trend was found with increasing PROG concentrations. PROG treatment increased neurons viabilities in a dose-relervant way in range of 0.001μmol·L-1~ 0.1μmol·L-1. The Pearson correlation coefficient was 0.860 between PROG concentration and neuron viability (P<0.01). The cell viability in 0.1μmol·L-1 PROG group had no difference with controls (P>0.05).5.2 Influence on cytoplasmic Cyt-c levelCompared with controls, cytoplasmic Cyt-c level increased in model group, from (2.86±0.23) to (4.58±0.39) (P<0.01). PROG inhibited this up-regulation of Cyt-c level in a dose-relervant way in range of 0.001μmol·L-1~ 0.1μmol·L-1. The Pearson correlation coefficient was -0.850 between PROG concentration and cytoplasmic Cyt-c level (P<0.01). The Cyt-c level is (2.98±0.19) in 0.1μmol·L-1 PROG group, having no difference with controls (P>0.05).5.3 Influence on cytoplasmic caspase-9 levelCytoplasmic caspase-9 level in model group deceased compared with controls, from (6.00±0.46) to (4.99±0.42) (P<0.01). PROG inhibited this down-regulation of caspase-9 level in a dose-relervant way in range of 0.001μmol·L-1~ 0.1μmol·L-1. The Pearson correlation coefficient between PROG concentration and cytoplasmic caspase-9 level was -0.836 (P<0.01). The caspase-9 level of 0.1μmol·L-1 PROG group is (5.99±0.35), having no difference with controls (P>0.05).Conclusions:1. An in virto cell model for AD research was established based the study of damagement of beta-amyloid active fragment 25-35 (Aβ25-35) on primary rat cortical neurons. Treated with 1μmol·L-1 Aβ25-35 for 24 h lead to obviously morphological changes in neurons, decreasing cell viability by about 50% measured by MTT assay.2. A sensitive, facilitated, and reliable solid-phase extraction combind HPLC-MS assay was developed to mesure the four chief neurosteroids, PREG, DHEA, PROG, and AP simultaniuosly in culture media. The method was suitable to study metabolim of neurosteroids in cultured cortical neurons. 3. The PREG-PROG-AP metabolic pathway changed obviously during Aβcytotoxicity, with PROG level greatly decreased, and AP level sharply increased, indicating a selective effection on neuronal neurosteroidgenesis. The results also suggested that optionally applied of neurosteroids, or regulation related steroidal synthetase activity, to regain the balance of neurosteroidgenesis broken by Aβcytotoxicity, may be of benefit to inhibition of Aβneurotoxcoty.4. PROG treatment inhibited the declination of cell viability induced by Aβ25-35 cytotoxcoty in a dose-dependent way. On the contrary, AP, the metabolite of PROG, aggravated the Aβ25-35 induced declination of cell viability in a dose-dependent way.5. Neuronal apoptosis elevated while cell viability declined in Aβ25-35 induced AD cell model, with cytoplasmic caspase-3 level up-regulated, indicated the involvement of activation of appoptosis. These effects can be inhibited by PROG treatment in a dose-dependent way, suggesting the anti-appotosic effects are involved in PROG protection against Aβinduced neuronal damagement.6. The relate proteins of metocondrial apoptosis pathway changed obviously in Aβ25-35 induced AD cell model, with cytoplasmic Cyt-c level level up-regulated and caspase-9 level down-regualted, indicated the involvement of activation of metocondrial apoptosis pathway. These effects can be inhibited by PROG treatment in a dose-dependent way, suggesting action on metocondrial apoptosis pathway maybe the possible mechanism of PROG neuroprotenction.