Alzheimer’s diseaseBy

Mohammed Razeeth s

Phosphoylation of tau• In AD tau protein get hyperphosphorylated

due toMutationActivities of kinaseRegulation of protein phosphtaseQuinolinic Acid

Serine and threonine residues

• To date, at least 37 serine and threonine residues have been found to be phosphorylated in PHF-tau

• Thr39, Ser46, Thr69,Thr123, Ser137, Thr153, Thr175, Thr181, Ser198, Ser199,Ser202, Thr205, Ser208, Ser210, Thr212, Ser214, Thr217,Thr231, Ser235, Ser237, Ser238, Ser241, Ser262, Ser285,Ser305, Ser324, Ser352, Ser356, Ser396, Ser400, Thr403,Ser404, Ser409, Ser412, Ser413, Ser416, and Ser422

Serine and threonine residues

• Some of the phosphorylation sites seen in PHF tau are not phosphorylated at all in normal brains.

• These sites include Thr212/Ser214, Thr231/Ser235 [90], and Ser422[91, 92].

Phosphoryltion at Tyr• The phosphorylation site of tau was mapped

as Tyr18.• Williamson et al [94] demonstrated that in

primary human and rat brain cortical cultures tau is phosphorylated at Tyr 29 upon treatment with Aβ

Regulation

MAP• Hyper phosphorylation tau gain a toxic activity

to sequester normal tau and other MAPs, such as MAP1 and MAP2, and cause microtubule disassembly

Kinase involve in phospho..• GSK-3β, • cdk5, • cAMP-dependent protein kinase (PKA), • stress-activated protein kinases, and

calcium/calmodulin-dependent kinase II (CaMK-II)

Pathway

Evidance

My concern• To up regulate protein phosphates by

activating PP2A

Evidance

Methodology• Cell lineHT22 hippocampal neuronal cell line, SH-

SY5Y• Retroviral infection• Immuno chemical method-immuno histo

compatability• SDS page

Retro viral infectionFor single round of infection• Add 4 μg/ml Polybrene to virus (from 100 x

stock), remove medium and cover cells with virus + Polybrene

• 12-well plate: 300-500 μl/well• 6-well plate: 750-1000 μl/well• T25/5 cm dish: 1.5 ml• 10 cm dish: 4.5 ml

Day 1, 6-8 hrs later• Add medium (the type in which the target

cells grow) to the virus incubations to dilute the Polybrene which is toxic at concentrations higher than 2 μg/ml.

• Eg; • 500 μl of virus, add 750 μl of medium• 1 ml of virus, add 1.5 ml medium

For 2 rounds of infection• 1. Start with the first infection on the morning

of Day 1 as described above• In the morning of Day 2, remove the virus

containing medium• Leave the cells for 24 hrs, until Day 3.

For 3 rounds of infection• Start with the first infection in the late

afternoon/evening of Day 1 by removing medium from target cells and adding virus + Polybrene, leave overnight, do not dilute polybrene.

For 3 rounds of infection• The next morning remove virus and replace by

new virus + Polybrene.• In evening do not remove virus of second

infection, but dilute it by adding an equal amount of new virus without polybrene.

For 4 rounds of infection• In the morning of Day 2, remove the virus containing medium

of the first infection and repeat the infection as described above.

• In the evening of Day 2, remove the virus containing medium of the first infection and repeat the infection.

• In the morning of Day 3 repeat the infection and discard the Phoenix cells.

For 4 rounds of infection• In the evening of Day 3, change the medium of

the infected cells.• 24 hours later, start antibiotic selection. For

puromyocin, select for 3 days. For hygromyocin, select for 7 days.

Thank you