Disease Modifying Effect Of Prenatal To Postnatal Maternal Dietary Treatment With A Neurotrophic Peptidergic Compound In 3×Tg-AD Mouse

Part 1:Disease modifying effect of prenatal to postnatal maternal dietary treatment with a neurotrophic peptidergic compound in 3×Tg-AD mouseObjective Currently,the four FDA(Food and Drug Administration)approved drugs(donepezil,galantamine,rivastigmine,and memantine)available for AD treatment only provide symptomatic benefit with little effect on the underlying pathology.Obviously,there is impending urgency to find an effective disease-modifying therapy.Previously,Dr.Iqbal’s group found that pharmacological stimulation of neural stem cells by chronic treatment with a neurotrophic peptidergic compound not only rectified defects in neurogenesis,neuronal and synaptic plasticity,and cognition but also reduced the underlying disease pathology in 3×Tg-AD mice.In the present study,we tried to find the disease modifying effect of prenatal to postnatal maternal dietary treatment with this compound in a triple transgenic mouse model of Alzheimer’s disease.Methods 1.The female 3×Tg-AD mice(2 months old)(n=20)and age and gender matched WT controls(n=15)were treated orally with P021 or vehicle diet from embryonic day 8 to postnatal day 21.Treatment was administered as 200 nmol peptide/g formulated diet.The vehicle-treated control animals received the same diet but without the peptide.On average,each mouse consumed ~2.7 g diet/day.2.The animals were behaviorally tested when they were 2.5 months old(elevated plus maze,open field,novel object recognition,morris water maze,rotarod,fear conditioning).3.After completion of the behavioral tasks,4 mice of each group were sacrificed and brain tissue was collected for immunohistochemical and biochemical analysis.Results 1.The result of elevated plus maze showed that WT mice spent less time on the open arms than 3×Tg-AD mice.Treatment of P021 didn’t affect anxity of the mice.2.The result of open field showed that 3×Tg-AD mice spent less time in the center than WT mice.Treatment of P021 didn’t have effect on anxity of the mice.3.The result of novel object recognition showed 3×Tg-AD mice treated with P021 spent more time exploring new objects than the untreated mice.4.The result of Morris water maze showed the spatial reference memory of 3×Tg-AD mice were impaired.Treatment of P021 didn’t show beneficial effect on the spatial reference memory.5.The result of rotarod showed 3×Tg-AD mice spent more time on the rotarting rod.Treatment of P021 didn’t show any effect.6.The result of fear conditioning showed the percentage of freezing time of 3×Tg-AD mice were much higher than WT mice,treatment of P021 didn’t show any effect.7.The result of western blots showed P021 promoted maturation of BDNF in WT mice.The phosphorylation of tau at Ser396,Ser404 and Thr212 sites decreased in the 3×Tg-AD mice treated with P021,but there was no effect on the phosphorylation of tau at Ser199 and Ser 202 sites.Glu R1 and PSD95 increased in the 3×Tg-AD mice treated with P021,but not synaptophysin.Conclusions 1.P021 treatment improved short term memory of 3×Tg-AD mice;it had no effect on anxiety,spatial reference memory,motor function and conditional memory.2.P021 treatment not only rectified defects in neuronal and synaptic plasticity,and cognition,but also reduced the underlying disease pathology in 3×Tg-AD mice.Part 2:Rapid alteration of phosphorylation of proteins during postmortem: implication in the study of protein phosphorylationObjective Protein phosphorylation is the most common and important regulatory physiological mechanism,which regulate the activity and function of proteins.Alteration of phosphorylation of proteins during postmortem is not clear yet.This study is designed to determine the actual level of protein phosphorylation after mouse death,which is very crucial to the study in the future.Methods 1.To know if there’s any change of the protein phophorylation after mice dead,we sacrificed the mice by dislocation and left the dead bodies in room temperature for 2,5,and 10 min.The forebrains were collected and stored at-80℃.Phosphorylation of tau in the brains was analyzed by Western blots developed with phosphorylation dependent-and site-specific tau antibodies.2.To know dynamic of protein dephophorylation during postmortem,we recorded brain collecting time of each mouse after death and analyzed phosphorylation levels of proteins with Western blots.The phosphorylation levels were plotted against time after death and the non-linear regression was performed.3.To know what if there’s same phenomenon in other organs,we sacrificed the mice by dislocation and left the dead bodies in room temperature for 2,5,and 10 min.The hearts,kidneys and livers were collected and stored at-80 oC.Phosphorylation of GSK-3β,CREB,AMPK and PKAc were analyzed by Western blots.4.We perfused brain through heart after dislocation with cold PBS or room-temperature PBS for 2 min,and then the brains were used for the analyses of protein phosphorylation by Western blots.5.We immediately transferred the brains into ice-cold PBS(0oC)for different time,and then measured phosphorylation of above proteins.Results 1.Tau protein in mouse brains was rapidly dephosphorylated site-specifically after death.Among them,Thr212 and Ser262 were the most sensitive sites toward postmortem interval.Phosphorylations of tau at Thr205,Ser214,and Ser396 were reduced significantly in 2 to 10 min after death.However,the dephosphorylation at Ser199,Ser404 and Ser422 progressed very slowly.AKT(Ser473),GSK-3β(Ser9),CREB(Ser133),and Erk(Thr202/Tyr204)in mouse brains all were dephosphorylated very rapidly and postmortem delay dependently.Interestingly,phosphorylation levels of AMPK(Thr172)and PKA catalytic subunit(Thr197)were increased time-dependently in the mouse brains with 10 min PMI.2.Phosphorylation of tau at Ser 199 did not alter significantly by 200 sec after death.However,the phosphorylationsof tau at Thr212 and GSK-3β at Ser 9 were decreased rapidly and reduced to 50 % in the mouse brain with ~40 sec PMI.The phosphorylation of tau at Thr 205,Ser 396 and CREB at Ser 133 were also reduced,but with a little less aggression.3.Phosphorylation of GSK-3β and CREB in hearts,livers and kidneys were significantly decreased after death.However,the phosphorylation of AMPK and PKAc did not change significantly in those tissues after death.4.Perfusion with either RT-PBS or cold PBS all caused dephosphorylation of brain proteins including tau at Thr 205,Thr 212,Ser 214,Ser 262,Ser 396 and AKT at Ser473,GSK-3β at Ser9,CREB at Ser133,and Erk at Thr 202/Tyr 204,but the phosphorylation levels of AMPK at Ser 172 and PKAc were increased.However,dephosphorylation of these proteins caused by cold-PBS perfusion was slightly less than RT-PBS perfusion.5.Keeping the brains in ice-cold PBS significantly prevented the alteration,including decreased and increased of the phosphorylation of proteins.Keeping the brains in ice-cold PBS longer time did not make dramatic effects on the phosphorylation of these proteins,suggesting 2-5 min in ice-cold PBS is enough for the protective roles on the protein phosphorylation during PMI.Conclusions Changes of protein phosphorylation appear protein-or site-or tissue-specific manner.Keeping brain in ice-cold PBS effectively prevented the alteration of protein phosphorylation.For the study of protein phosphorylation,the perfusion should not be suggested.