Alzheimer’s-specific effects of soluble b-amyloid on protein kinase C-a and -g degradation in human fibroblasts

Alzheimer’s disease (AD) is a multifactorial disease in which b-amyloid peptide (bAP) plays a critical role. We report here that the soluble fraction 1–40 of bAP differentially degrades protein kinase C-a and -g (PKCa and PKCg) isoenzymes in normal (age-matched controls, AC) and AD fibroblasts most likely through proteolytic cascades. Treatment with nanomolar concentrations of bAP(1–40) induced a 75% decrease in PKCa, but not PKCg, immunoreactivity in AC fibroblasts. In the AD fibroblasts, a 70% reduction of the PKCg, but not PKCa, immunoreactivity was observed after bAP treatment. Preincubation of AC or AD fibroblasts with 50 mM lactacystine, a selective proteasome inhibitor, prevented b-AP(1–40)-mediated degradation of PKCa in the AC cells, and PKCg in the AD fibroblasts. The effects of bAP(1–40) on PKCa in AC fibroblasts were prevented by inhibition of protein synthesis and reversed by PKC activation. A 3-hr treatment with 100 nM phorbol 12myristate 13-acetate restored the PKCa signal in treated AC cells but it did not reverse the effects of bAP(1–40) on PKCg in the AD fibroblasts. Pretreatment with the protein synthesis inhibitor, cycloheximide (CHX, 100 mM), inhibited the effects of bAP(1–40) on PKCa and blocked the rescue effect of phorbol 12-myristate 13-acetate in AC fibroblasts but did not modify PKCg immunoreactivity in AD cells. These results suggest that bAP(1–40) differentially affects PKC regulation in AC and AD cells via proteolytic degradation and that PKC activation exerts a protective role via de novo protein synthesis in normal but not AD cells. The b-amyloid protein (bAP) is the major constituent of the neuritic plaques that are, together with the neurofibrillar tangles, physiologic hallmarks of Alzheimer’s disease (AD) (1, 2). Excessive release of bAP in different cerebral areas, promoted by a mutant form of amyloid precursor protein (APP), contributes to its accumulation within the neuritic plaques (3, 4). In many cell types from AD tissues, including fibroblasts, changes have been demonstrated in signal transduction systems (5) that involve calcium homeostasis (6–9), ion channel permeability (10–12), cyclic AMP (13, 14), and phosphoinositide metabolites (15). Altered production of bAP also has been shown (16–18). Furthermore, bAP itself can affect the same transduction systems. Low concentrations (10 nM) of bAP(1–40) can affect K1 channel opening (19) and reduce intracellular levels of GTPbinding proteins (20) in human fibroblasts. Although a biphasic effect of bAP treatment on rat neuronal cell cultures also has been reported (21, 22), the mechanisms underlying the acute effects of bAP, including increased sensitivity to oxidative stress (23, 24), are still not well understood. Previous demonstrations of PKC deficits in the frontal cortex (25) and alterations in PKC-dependent phosphorylation in the brains of Alzheimer’s patients suggested an early role for PKC dysfunction in the pathogenesis of AD (26–31). Furthermore, reduced PKC phosphorylating activity and a lower affinity for phorbol ester binding as well as a decreased PKC immunoreactivity have also been reported for AD fibroblasts (32, 33). In this study, we investigate the acute effects of bAP(1–40) on PKC regulation in Alzheimer’s fibroblasts. MATERIALS AND METHODS Cell Culture. Human skin fibroblasts were purchased from Coriel Cell Repositories, seeded, and maintained as described (20). Cells from 6 familial AD [AG07872, AG06840B, AG8170B, AG08527A*, AG06848B*, AG08563A; four males and two females, 57.7 6 2.1 years of age (mean 6 SD); *, autopsy confirmation], 4 nonfamilial AD (AG05770D*, AG07377, AG06263, AG06838; two males and two females, 53.8 6 1.1 years of age; *, autopsy confirmation), 10 agematched AC (AG07665A, AG06842B, AG07867, AG07603A, AG04560B, AG06241B, AG3652C, AG07141, AG08044A, AG07310; six males and four females, 55.2 6 2.9 years of age) were used. Passage numbers were almost exactly the same for AC (7.3 6 SD 1.4, n 5 11) and AD (6.7 6 SD 0.6, n 5 11) cell lines. There were no differences in growth rates or time to senescence between AD and control fibroblasts (34). Cells were grown to confluence in DMEM (GIBCO) supplemented with 10% fetal bovine serum (GIBCO). Rat cerebellar granule cells were prepared as described previously (35). Briefly, cerebella from 8-day-old rat were dissociated after trypsinization (0.025% trypsin solution) and trituration in the presence of DNase (0.01%) and trypsin inhibitor (0.05%). Cells were then dispersed and cultured into basal medium Eagle’s (BME) supplemented with 25 mM KCly2 mM glutaminey10% fetal bovine serum (GIBCO). The growth of nonneuronal cells was inhibited by the addition of 20 mM cytosine b-D-arabinofuranoside. Cortical neuron cultures were performed as described (36) with slight modifications. Cortical tissue was obtained from fetuses extracted by a C-section from a 17-day pregnant rat. Brains were dissected, dissociated in a solution containing 26 unitsymg papain and 1 mM cysteine, and dispersed in DMEM supplemented with 10% fetal bovine serum and 100 unitsyml penicillin and 100 mgyml streptomycin. Cells then were seeded for 72 h before the addition of 1 mM cytosine b-D-arabinofuranoside. b-Amyloid Treatment. bAP(1–40) (Bachem) was dissolved initially in dimethyl sulfoxide (DMSO, 100 mM) (21, 22) and further diluted in saline solution to desired final concentrations (1 nM–5 mM). bAP(1–40) was added 24 h after seeding The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 0027-8424y98y955562-6$0.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: AD, Alzheimer’s disease; AC, age-matched controls; bAP, beta-amyloid protein; Lacta, lactacystine; CHX, cycloheximide; PMA, phorbol 12-myristate 13-acetate; APP, amyloid precursor protein; DIV, days in vitro. †To whom reprint requests should be addressed at: National Institutes of Health, Building 36, Room 4A-21, Bethesda, MD 20892. e-mail: [email protected].