The Inhibit Effect Of ATRA On Aβ42-induced Apoptosis Of Astrocytes And Its Mechanism

BackgroundAlzheimer’s disease is a chronic progressive neurodegenerative disorder characterised by a deterioration of memory and other cognitive founctions. An estimated about36million people at present and with about115million people have the disease by2050. In addition, at present the available treatments have minimal or no effect on the course of the disease. Alzheimer’s disease has become one of the most important health care, social, and economic challenges of the21st century.Alzheimer’s disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in gross atrophy of the affected regions, including degeneration in the temporal lobe and parietal lobe, and parts of the frontal cortex and cingulate gyrus. At the microscopic level, amyloid plaques and neurofibrillary tangles are clearly visible in AD cases’ brains. But, the cause for most Alzheimer’s cases is still essentially unknown. Several competing hypotheses exist trying to explain the cause of the disease, including amyloid beta (AP) aggregation and deposition with plaque development, tau hyperphosphorylation with tangle formation, neurovascular dysfunction, and other mechanisms such as cell-cycle abnormalities, inflammatory processes, oxidative stress, and mitochondrial dysfunction. But the exactly etiology and pathogenesis is still not clear.An imbalance between production and clearance, and aggregation of peptides, causes AP to accumulate, and this excess may be the initiating factor in Alzheimer’s disease. This idea was called the "amyloid hypothesis".β-amyloid peptides originate from proteolysis of the amyloid precursor protein by the sequential enzymatic actions of beta-site amyloid precursor protein-cleaving enzyme1(BACE-1), a β-secretase, and y-secretase, a protein complex with presenilin1at its catalytic core. Soluble Aβ is thought to undergo a conformational change to high β-sheet content, rendering it prone to aggregate into soluble oligomers and larger insoluble fibrils in plaques. Aβ oligomers could directly inhibit hippocampal long-term potentiation and impair synaptic function, in addition to the inflammatory and oxidative stress caused by aggregated and deposited Aβ. Amyloid plaques are attached in the AD brain microvessel wall, causing cerebral vascular amyloid diseases, cerebrovascular disorders, reduced blood-brain symmetry, lack of regional energy use.Experimental evidence indicates that Aβ accumulation precedes and drives tau aggregation, disturbs axonal transpot. Activated microglia and reactive astrocytes localize to fibrillar plaques, and their biochemical markers are elevated in the brains of patients with Alzheimer’s disease. Chronically activated microglia release chemokines and a cascade of damaging cytokines. Aβ is a potent mitochondrial poison, especially affecting the synaptic pool. Consequently, electron transport, ATP production, oxygen consumption, and mitochondrial membrane potential all become impaired. Aβ oligomers can inhibit the base of synaptic transmission, and reduce the number of synapses in the tree, causing synaptic plasticity damage. Aβ impairs nicotinic acetylcholine (ACh) receptor (nAChr) signaling and ACh release from the presynaptic terminal.A first element to suggest the involvement of astrocytes in AD pathology is the observation of activated astrocytes at the site of Aβ deposits in both human AD postmortem brain tissue and in the brain tissue derived from animal models of AD. In human AD brains, large amounts of Aβ are observed in activated astrocytes, suggesting that they have an important role in Aβ clearance. Evidence obtained in astrocyte-neuron mixed cultures or co-culture experiments argues in favor of a deleterious effect of astrocyte-Aβ interactions on neuronal viability. Internalization of the aggregated form of AP by astrocytes has also been observed to affect their metabolic phenotype profoundly, both in terms of energy metabolism and oxidative stress status. Astrocytes play a very important role in the neurogenic immunity, has closely linked with a variety of neurodegenerative diseases. Functional changes of astrocytes involved in the pathogenesis AD. Astrocytes can synthesize and secrete various neurotrophic factors promote the growth of neurons, maintaining neuronal survival, maintaining brain homeostasis.RA is the key regulator of cell growth, proliferation, differentiation and programmed apoptosis, regulates the formation of neurosteroids in the brain, and participates in the development of neurodegenerative disease. A large number of experimental evidence support that RA signaling pathways are involved at every step of AD development in the brain. RA can protect against antioxidative stress and has anti-inflammatory effects, promotes a-secretase cleavage of APP, induces expression of APOE, and inhibit tau protein phosphorylation. Though unclear, RA signaling pathway has become a hotspot in the studies on molecular mechanism of AD. RARa, PPARγ, APOE, ADAM10play a key role in the RA signaling pathway, how to regulate the AD pathological changes will be the focus of RA signaling pathway study. So we would like to confirm whether ATRA can promote astrocyte synthesis and secretion of APOE, and the ability to suppress Aβ-induced apoptosis of astrocytes.Methods1.Astrocytes in primary culture and identificationIn this study,we use the primary cultural cortex astrocytes,and isolate and purifiy astrocytes by differential velocity adherent technique Immunofluorescence stains the specific astrocyte marker GFAP.At the same time, we use the DAPI to mark the cell nuclear.2.The effect of ATRA on apoptosis of astrocytesAdd2μg/ml of Aβ42in the medium, inducing apoptosis of astrocytes. Hoechst33258staining, observed the morphologic changes of apoptosis of astrocytes. The astrocytes are incubated with different concentrations of ATRA24hours.MTT assay for detection of clusters of astrocytes survival rates.3. The effect of ATRA on APOE, BACE1mRNA expression in astrocytes. Add2μg/ml of Aβ342in the medium, inducing apoptosis of astrocytes. Then,the astrocytes are incubated with different concentrations of ATRA24hours. Using TRIzol extraction of clusters of astrocytes total RNA. And using a reverse transcription kit and qPCR kit (Roche) to detect the APOE, BACE1mRNA expression in astrocytes.4. The effect of ATRA on APOE, BACE1expression in astrocytesAdd2μg/ml of Aβ342in the medium, inducing apoptosis of astrocytes. Then,the astrocytes are incubated with different concentrations of ATRA24hours. Extraction of total cellular protein in each group.Using the BCA method,measure the total protein concentration in astrocytes.Using Western Blot Assay for detection of clusters of APOE,BACE1in astrocytes.5. The effect of ATRA on astrocytes AβclearanceAdd2μg/ml of Aβ42in the medium, inducing apoptosis of astrocytes.Then,the astrocytes are incubated with different concentrations of ATRA24hours.Collection the medium and astrocytes. Using the ELISA kit to detect the AP levels in the cell culture medium and astrocytes.Results1.Astrocytes in primary culture and identificationImmunofluorescence stains the specific astrocyte marker GFAP.At the same time, we use the DAPI to mark the cell nuclear. Astrocytes purity>95%.2.The effect of ATRA on apoptosis of astrocytesBy hoechst33258staining of the astrocytes,we find that AP can accelerated the astrocytes apoptosis.The effectof different concentrations of ATRA on the apoptosis of astrocytes induced by Aβ is not same.The0.1μM and1μM ATRA can inhibit the apoptosis of astrocytes induced by Aβ42(P<0.05).3.The effect of ATRA on APOE, BACE1mRNA expression in astrocytes.Compared with the control group,The0.1μM and1μM ATRA can increase the expression of APOE mRNA(P<0.05).BACE1mRNA expression was no significant change.4.The effect of ATRA on APOE, BACE1expression in astrocytes Compared with the control group, APOE protein expression was significantly increased in the normal group.Comparison of the experimental and control groups, ATRA can increased the APOE protein expression(P<0.01).ATRA has no effect on the BACE1expression of astrocytes.5.The effect of ATRA on astrocytes ApclearanceCompared with the control group, ATRA can reduce the Aβ levels in the culture medium(P<0.05),and astrocytes(P<0.01).ConclusionsATRA can up-regulate the expression of APOE, prohibit the astrocytes apoptosis induced by Aβ42. Low concentrations of ATRA on astrocytes have a protective effect.There is no effect for ATRA regulating the expression of BACEl in astrocytes. The high concentration of ATRA has a cell toxicity.