Figure 4.

Increased tau phosphorylation leads to a cholinergic imbalance with decreased acetylcholine levels in retinoic acid (RA)‐differentiated SH‐SY5Y cells. RA‐differentiated SH‐SY5Y cells were transfected with pCI empty vector (control) or plasmid cDNAs that encode GSK3β and tau (GSK3β + tau). (a) Ultracentrifugation on sucrose gradient served to verify the expression of tetramers of acetylcholinesterase (AChE) in non‐transfected RA‐differentiated cells. Molecular forms of AChE (tetramers: G₄; and light monomers: G₁) were identified by comparison with the position of molecular weight markers catalase (11.4S) and alkaline phosphatase (6.1S). Representative profile is shown. (b) Immunoblots probed to total tau (T‐tau), GSK3β and phosphorylated tau (P‐tau) were then performed to verify transfection and the effect on tau phosphorylation. Equal amounts of protein were loaded in all lanes. Representative blots are shown. (c) AChE enzymatic activity was assayed in cellular extracts and expressed as percentage (%) of the activity respect to pCI control‐transfected cells. (d) AChE protein levels were assayed by western blotting of 30 μg of protein from cellular extracts using the anti‐AChE antibody N‐19. Immunoreactivity of the AChE bands was quantified and normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Immunoreactive levels were expressed as % of control levels. Representative blot and densitometric quantification are presented. (e) The levels of choline acetyltransferase (ChAT) were assayed by western blot in cellular extracts, with GAPDH as loading control. (f) Cellular levels of the neurotransmitter acetylcholine (ACh) were also measured using a commercial fluorometric method and expressed as % respect to control. Results were confirmed in at least of n = 18 cell wells from three independent cell cultures experiments. Mean value ± SEM are represented. *Significantly different (p < .05) from the control group, as assessed by the Student's t test