Therapeutic effects of PKC activators in Alzheimer's disease transgenic mice

Abstract

Alzheimer's disease (AD) characteristically presents with early memory loss. Regulation of K(+) channels, calcium homeostasis, and protein kinase C (PKC) activation are molecular events that have been implicated during associative memory which are also altered or defective in AD. PKC is also involved in the processing of the amyloid precursor protein (APP), a central element in AD pathophysiology. In previous studies, we demonstrated that benzolactam (BL), a novel PKC activator, reversed K(+) channels defects and enhanced secretion of APP alpha in AD cells. In this study we present data showing that another PKC activator, bryostatin 1, at subnanomolar concentrations dramatically enhances the secretion of the alpha-secretase product sAPP alpha in fibroblasts from AD patients. We also show that BL significantly increased the amount of sAPP alpha and reduced A beta 40 in the brains of APP[V717I] transgenic mice. In a more recently developed AD double-transgenic mouse, bryostatin was effective in reducing both brain A beta 40 and A beta 42. In addition, bryostatin ameliorated the rate of premature death and improved behavioral outcomes. Collectively, these data corroborate PKC and its activation as a potentially important means of ameliorating AD pathophysiology and perhaps cognitive impairment, thus offering a promising target for drug development. Because bryostatin 1 is devoid of tumor-promoting activity and is undergoing numerous clinical studies for cancer treatment in humans, it might be readily tested in patients as a potential therapeutic agent for Alzheimer's disease.

Fig. 1.

Secretion of APP-α in human fibroblasts. Bryostatin (Bry, 0.1 nM, solid bar) dramatically enhanced the amount of sAPP-α in the medium after 3 h of incubation in a well characterized autopsy confirmed AD cell line (P < 0.0001, ANOVA). The graph units are relative to the vehicle, DMSO, alone (1). Bryostatin was significantly (P < 0.001, Tukey's posttest) more potent than another PKC activator, BL, at the same concentration (0.1 nM). Pretreatment (rightmost bar) with staurosporin (Sta, 100 nM) completely abolished the effect of bryostatin (0.1 nM). Bryostatin was also effective in enhancing secretion in two control cell lines, although to a lesser extend than in the AD cell line (hatched bar). A time course (for the AD cell line) is depicted in Inset. Secretion is clearly near enhanced by 15 min of incubation [bryostatin (Bryo), 0.1 nM] and near maximal at 160 min of incubation, remaining elevated up to 3 h. Bryostatin at a lower concentration, 0.01 nM, was much slower but had about the same effect on secretion after 120 min of incubation.

Fig. 2.

PKC translocation in human fibroblasts. Bar graphs show the ratios between the immunoreactivity (normalized by total protein content) of the membrane-bound PKC (P, particulate) and the immunoreactivity detected in the cytosolic fraction (S, soluble). (A) PKC-α translocation was marked after 30 min of incubation with 0.1 nM bryostatin (Bry) (solid bar). Translocation was still present (P > S) at 180 min of incubation (rightmost bar). (B) Translocation of other isoenzymes was comparable to that observed for PKC-α.

Fig. 3.

Transgenic mice APP[V717I]. Mice (four treated and four controls) began treatment form just after weaning (3 weeks) with BL (1 mg/kg i.p., daily) for 17 weeks. (A) sAPP-α increased significantly (P = 0.04) in the brains of the treated group compared with vehicle alone. (B) The same animals had a proportionally significant (P = 0.02, Student's t test) reduction of Aβ40.

Fig. 4.

Transgenic mice APP[V717I]/PS1[A246E]: Mortality. (A) Life table analysis of all mice. Both treated (n = 23) and untreated (n = 29) mice showed premature death; however, the rate was slowed by bryostatin. (B) Life table analysis of male mice. The difference was particularly salient in males (n = 22). Nontreated animals exhibited elevated mortality that was significantly reduced by bryostatin (P = 0.006, Fisher's exact test, two-tailed). (C) Life table analysis of female mice. Females (n = 30) did not show the same level of premature death, and treatment did not change the rate significantly.

Fig. 5.

Transgenic mice APP[V717I]/PS1[A246E]: Amyloid peptides. Mice completing the treatment with bryostatin (≈5.5 months) as a group (n = 15) showed reductions in the amount of both Aβ40 (A) and Aβ42 (B; P = 0.04, Student's t test) peptides compared with controls (n = 13) receiving vehicle alone. Contl., control; Bryo, bryostatin.