2. Phosphoylation of tau
• In AD tau protein get hyperphosphorylated
due to
Mutation
Activities of kinase
Regulation of protein phosphtase
Quinolinic Acid
3. 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
4. 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].
5. 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
7. MAP
• Hyper phosphorylation tau gain a toxic activity
to sequester normal tau and other MAPs, such
as MAP1 and MAP2, and cause microtubule
disassembly
8. Kinase involve in phospho..
• GSK-3β,
• cdk5,
• cAMP-dependent protein kinase (PKA),
• stress-activated protein kinases, and
calcium/calmodulin-dependent kinase II
(CaMK-II)
15. Retro viral infection
For single round of infection
• Add 4 μg/ml Polybrene to virus (from 100 x
stock), remove medium and cover cells with
virus + Polybrene
16. • 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
17. 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
18. 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.
19. 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.
20. 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.
21. 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.
22. 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.
