Study Of Oxidative Damage And Variation Of Monoamine Neurotransmitter In Conditional Presenilin-1/Presenilin-2 Double Knockout Mice

Alzheimer's disease(AD),which is characterized by progressive loss of neurons, accumulations of senile plaques and neurofibrillary tangles,is the most common cause of dementia.It has been reported that mutations in presenilin genes are associated with the early onset of Familial Alzheimer's disease(FAD). Presenilin1(PS1) and presenilin2(PS2),two homologous genes that shared～60% amino acids,are considered to be the active sites ofγ-secretase complex which is responsible for the proteolytic processing of amyloid precursor protein(APP). Forebrain-specific conditional PS1 and PS2 double knockout mice(dKO mice) indeed appeared decreased Aβcontent,but also exhibited several of the Alzheimer-like neurodegenerative symptoms,including impairments in synaptic plasticity and hippocampus-dependent memory,severe shrinkage of the cortex,neuronal atrophy, tau phosphorylation and enlargement of the lateral and third ventricles.Oxidative stress refers to the imbalance between the antioxidative defense system and the oxidative system.Among various tissues,brain is very prone to be attacked as a result of its high oxygen consumption rate,plentiful polyunsaturated fatty acids and abundant transition metals but relative paucity of antioxidants.In recent years,multiple lines of evidence have suggested that oxidative stress is a causal, or at least an ancillary factor in the neuropathology of AD.Furthermore,an increasing amount of experiments indicated that aggregated Aβis capable of generating free radicals,and thus induce the oxidative damage observed in AD-affected brains;vice versa,Aβis also subjected to being attacked by free radicals,which then leads to the alteration of Aβproperties and forms Aβfibrils more easily. Monoamine neurotransmitter contained catecholamines,serotonin and histamine, While catecholamines are further constitute of dopamine,epinephrine and noradrenaline.Monoamine neurotransmitters are involved in many physiology progresses,such as thermoregulation,activity of spirit and emotion and sleeping.Lots of studies showed that disruption of the equilibrium state of monoamine neurotransmitters can lead to neurodegenerative disease such as AD.1.Oxidative damage in dKO miceThis report aims to describe the oxidative damage profile in brain of presenilin1 and presenilin2 conditional double knockout mice(dKO) both at early and at late age stages,and discuss the correlation between oxidative stress and the Alzheimer-like phenotypes of dKO mice.The protein level of Aβ₄₂ in dKO cortex and free 8-OHdG in urine were measured by ELISA.Thiobarbituric acid method and spectrophotometric DNPH assay were used to determine the lipid peroxidation and protein oxidation in cortex,respectively.SOD activity and GSH-PX activity were assessed by SOD Assay Kit-WST and GSH-PX assay kit,separately.When compared to control mice,significant decrease of Aβ₄₂ was verified in dKO cortex at 6 months age.Although lipid peroxidation(assessed by MDA) was increased only in dKO cortex at 3 months and protein oxidation(assessed by Carbonyl groups) was basically unchanged in dKO cortex,ELISA analysis indicated that free 8-OHdG for measuring DNA lesion was significantly decreased in urine of dKO mice from 3 months to 12 months.Activity of SOD and GSH-PX showed no statistical difference except a significant increase of GSH-PX in dKO mice at 9 months.Oxidative damage, especially DNA lesion,was correlated with the neurodegenerative symptoms appeared in dKO mice without the deposition of Aβ₄₂.2.Change of monoamine neurotransmitter in dKO miceBy using capillary electrophoresis assay,monoamine neurotransmitters in cortex, hippocampus and other forebrain region of dKO mice aged at 6,9 and 12 months were measured to illustrate the relationship among presenilins function deficiency, neurodegenerative phenotypes and monoamine neurotransmitters.Data showed that levels of monoamine neurotransmitters in cortex of dKO mice were significantly decreased at 6 months when compared to controls,while as mice getting older,levels of monoamine neurotransmitters increased to that of controls,or even higher.In hippocampus,5-hydroxytryptamin and epinephrine in dKO mice had a significant increase at 6 months,followed with a significant increase of each monoamine neurotransmitter at 12 months age.In other forebrain region,5-hydroxytryptamin and dopamine had a similar level between control and dKO mice at 6 and 9 months but a significant decrease at 12 months;however,level of norepinephrine and epinephrine were significantly decreased at 6 and 12 months except epinephrine of 6 months. These results demonstrated that knockout of presenilins genes could lead to the variation of monoamine neurotransmitters,which had a parallelism with neurodegenerative phenotypes of dKO mice.However,whether presenilins deficiency caused the variation of monoamine neurotransmitter directly or not,and how about the effects of variation of monoamine neurotransmitters on AD-like pathology need to be further analyzed.