A cellular model of Alzheimer's disease therapeutic efficacy: PKC activation reverses Abeta-induced biomarker abnormality on cultured fibroblasts

Abstract

PKC signaling is critical for the non-toxic degradation of amyloid precursor protein (APP) and inhibition of GSK3beta, which controls phosphorylation of tau protein in Alzheimer's disease (AD). Thus the misregulation of PKC signaling could contribute to the origins of AD. Bryostatin, a potent PKC modulator, has the potential to ameliorate both the neurodegeneration and the recent memory loss associated with AD. As reported herein bryostatin and a potent synthetic analog (picolog) are found to cause stimulation of non-amyloidogenic pathways by increasing alpha-secretase activity and thus lowering the amount of toxic Abeta produced. Both bryostatin and picolog increased the secretion of the alpha-secretase product (s-APP-alpha) of APP at sub-nanomolar to nanomolar concentrations. A peripheral AD-Biomarker has previously been autopsy-validated. This Biomarker, based on bradykinin-induced differential phosphorylation of Erk1 and Erk2, has been used here to test the therapeutic efficacy both for bryostatin and picolog. Both of these PKC activators are then shown to convert the AD Erk1/2 phenotype of fibroblasts into the phenotype of "normal" control skin fibroblasts. This conversion occurred for both the abnormal Erk1/2 phenotype induced by application of Abeta(1-42) to the fibroblasts or the phenotype observed for fibroblasts of AD patients. The Abeta(1-42)-induction, and PKC modulator reversal of the AD Erk1/2 biomarker phenotype demonstrate the AD-Biomarker's potential to monitor both disease progression and treatment response. Additionally, this first demonstration of the therapeutic potential in AD of a synthetically accessible bryostatin analog warrants further preclinical advancement.

Fig. 1

Chemical formula of bryostatin and picolog.

Fig. 2

PKC activation by bryostatin and synthetic analog, picolog.

Fig. 3

Increase of α-secretase activity by picolog Alzheimer’s disease fibroblasts: (A) (i) Western blot analysis of secretion of s-APP-α by picolog treatment, (ii) Illustration of recognition of s-APP-α by 6E10 antibody. (B) Concentration dependence of α-secretase product formation (s-APP-α) by picolog. (C) Comparison of α-secretase activity induced by sub-nanomolar concentrations of bryostatin and picolog. (N=Number of independent measurements, *P<0.028).

Fig. 4

Bryostatin and picolog treatment reverses AD-Index of human Alzheimer’s disease fibroblasts: (A) Skin fibroblasts cells from AD patients were treated with 0.2 nM bryostatin for 16 h. AD-Index was measured before and after the treatment and found to be decreased after each treatment. (N=Number of skin fibroblast from individual patient cell lines, *P<0.02, Error bars=SEM of all AD-Index values). (B) Scatterplot of individual data points from each cell lines of panel A. (C) Skin fibroblasts cells from AD patients were treated with 5 nM picolog for 16 h in culture medium. AD-Index was measured before and after the treatment and found to be decreased after each treatment. (N=Number of skin fibroblast from individual patient cell lines, *P<0.008, Error bars=SEM of all AD-Index values). (D) Scatterplot of individual data points from each cell lines of panel C

Fig. 5

Bryostatin/picolog treatment reverses AD-Index of Aβ_1–42-treated control fibroblasts: (A) Skin fibroblasts from control patients were treated with 1.0 μM Aβ_1–42 in culture medium for 16 h to produce the AD phenotype. After Aβ_1–42-treatment, the AD phenotype skin fibroblasts were exposed with 0.2 nM bryostatin in culture medium for 16 h. The abnormal AD-Index was reversed to normal (negative) values. (N=Number of skin fibroblast from individual patient cell lines, P< 0.005). (B) Scatterplot of individual data points from each cell lines of panel A. (C) The skin fibroblasts from control patients were treated with 1.0 μM Aβ_1–42 for 16 h in culture medium to produce the AD phenotype. After Aβ_1–42-treatment, the AD phenotype skin fibroblasts were exposed to 5 nM picolog in culture medium for 16 h. The abnormal AD-Index was reversed to normal (negative) values. (N=Number of skin fibroblast from individual patient cell lines, *P<0.01, **P<0.01). (D) Scatterplot of individual data points from each cell lines of panel C.

Fig. 6

Illustration of effects of Aβ and PKC activators (bryostatin and picolog) on the AD-Index.