Investigating Pathology In The Eye Of Alzheimer、Disease5xFAD Transgenic Mouse Model

Objectives1. To investigate APP expression in the eye of5xFAD mice. To compare the level ofAPP, phosphor-APP, BACE1, PS1and rhodopsin in the eye of5xFAD mice and B6SJLmice in different age groups.2. To explore the subcellular location of APP, phosphor-APP, BACE1, PS1andrhodopsin in the retina of5xFAD mice.3. To investigate the expression of total tau and phospho-tau protein in the eye of5xFAD mice. To detect differernt phospho-sites of tau in the retina of B6SJL mice and5xFAD mice. To compare the level of tau and phospho-tau protein in the eye of5xFADmice and B6SJL mice in different age groups.4. To detect the level of GSK3β and P-GSK3β in the retina of5xFAD mice and B6SJLmice, to explore whether GSK3β pathway is involve in the eye pathological process of5xFAD mice.Methods1. Detected the expression of APP in the cornea, lens, retina, optic nerve and brain of2months old5xFAD mice by Western Blot. Compared the level of APP, phosphor-APP,BACE1, PS1and rhodopsin in the retina, optic nerve and brain of5xFAD mice(2months,9months old) and B6SJL mice (1week,1month,2months,9months old) by Western Blot.2. Tissue subcellular fractionation was used to detect the subcellular location of APP,phosphor-APP, BACE1, PS1and rhodopsin in the retina of5xFAD mice.3. Detect the expression of total tau and phospho-tau in the cornea, lens, retina, opticnerve and brain of2months old5xFAD mice by Western Blot. Observe the expressionof differernt phospho-sites of tau protein in the retina of B6SJL mice and5xFAD mice.Compare the level of tau and phospho-tau in the retina, optic nerve and brain of5xFADmice(2months,9months old) and B6SJL mice (1week,1month,2months,9monthsold) by Western Blot.4. Detected the level of GSK3β, P-GSK3β in the retina and brain of5xFAD mice andB6SJL mice at2months and9months old by Western Blot.Results1. APP expressed in all parts of eyeball, including cornea, lens, retina and optic nerve,but it has differential expression patterns in different parts of eye, the expression patternin retina and optic nerve are mostly like in brain. With the developing(from1w to1m),APP levels are increased in the retina of B6SJL mice, and the expression pattern of APPturns more mature. With the aging(from2m to9m), APP levels are significantly higherin the retina of B6SJL mice and5xFAD mice(p=0.02, p<0.01). While APP levels aredramaticly increased in the retina and optic nerve of9months old5xFAD micecompared to age-matched B6SJL mice(p<0.01). APP levels in retina are closelycorrespond to the levels in brain.2. With the developing(from1w to1m), phospho-APP levels are low in the retina ofB6SJL mice. With the aging(from2m to9m), phospho-APP levels are significantlyhigher in the retina of B6SJL mice and5xFAD mice(p=0.03, p<0.01), While it is alsoincreased in the retina of5xFAD mice compared to age-matched B6SJL mice at2months old and9months old (p<0.01). Phospho-APP levels are also sharply increasedin the optic nerve of9months old5xFAD mice compared to age-matched B6SJL mice.Phosphor-APP levels in retina are closely correspond to the levels in brain.3. With the developing(from1w to1m), rhodopsin levels are sharply increased to the max amount. With the aging(from2m to9m), rhodopsin levels are decreased in theretina of5xFAD mice(p=0.01) but not B6SJL mice. While rhodopsin levels are alsodecreased in the retina of5xFAD mice compared to age-matched B6SJL mice(p=0.01).4. With the developing(from1w to1m), levels of BACE1and PS1are notabledecreased in the retina of B6SJL mice. With the aging(from2m to9m), levels ofBACE1and PS1are increased in the retina of B6SJL mice and5xFAD mice(p<0.01,p=0.01). While the levels of BACE1and PS1are increased in the retina of5xFAD micecompare to B6SJL mice at2months old and9months old(p<0.01). BACE1and PS1levels in retina are closely correspond to the levels in brain.5. APP majorly located in endoplasmic reticulum, golgi and early endosome.Phosphor-APP located in the golgi, BACE1located both in endoplasmic reticulum andearly endosome, PS1located in endoplasmic reticulum and glogi, while rhodopsinlocated in glogi.6. Total tau and phosphor-tau only expression in the retina, optic nerve and brain of5xFAD mice. The mainly phospho-sites of tau protein expressed in the retina of B6SJLmice and5xFAD mice is Ser396. Ser202only expressed in the retina of9months old5xFAD mice, while Ser199/Thr205got nothing.7. With the developing(from1w to1m), levels of total tau are notable increased in theretina of B6SJL mice. With the aging(from2m to9m), levels of total tau are increasedin the retina of B6SJL mice and5xFAD mice(p<0.01, p<0.01). While the levels of totaltau are increased in the retina of5xFAD mice compare to B6SJL mice at2months oldand9months old(p<0.01, p<0.01). Total tau levels in retina are closely correspond tothe levels in brain.8. With the developing(from1w to1m), levels of PHF-1and the ratio of PHF-1to totaltau are notable decreased in the retina of B6SJL mice. With the aging(from2m to9m),levels of PHF-1and the ratio of PHF-1to total tau are increased in the retina of B6SJLmice and5xFAD mice(p<0.01, p<0.01). While the levels of total tau are increased in theretina of5xFAD mice compare to B6SJL mice at9months old(p<0.01, p<0.01). PHF-1levels in retina are closely correspond to the levels in brain. 9. With the aging(from2m to9m), levels of GSK3β are increased in the retina of9months old5xFAD mice while levels of P-GSK3β are decreased(p=0.02, p=0.01).Levels of GSK3β and P-GSK3β are closely correspond to the levels in brain.Conclusions1. With the developing and aging, levels of APP, phosphor-APP, BACE1, PS1, total tauand PHF-1are closely correspond to the levels in brain.2. Levels of APP, phosphor-APP, BACE1, PS1are increased in the retina of9monthsold5xFAD mice compared to age-matched B6SJL mice and2months old5xFAD mice,while rhodopsin levels are decreased.3. Subcellular location of APP, phosphor-APP, BACE1, PS1, rhodopsin in the retinawere in the endoplasmic reticulum, golgi and early endosome which similar with thebrain.4. Levels of total tau, PHF-1and the ratio of PHF-1to total tau are increased with agingin the retina of9months old5xFAD mice compared to age-matched B6SJL mice and2months old5xFAD mice.5. The mainly phospho-sites of tau protein expressed in the retina of B6SJL mice and5xFAD mice is Ser396.6. GSK3β signal pathway may participated in the mechanism of overexpressed PHF-1in the retina of aged5xFAD mice.