Processing of Alzheimer's amyloid precursor protein in cultured cells.

The deposition in the brain of the 4 kDa beta-amyloid peptide (betaA4), from amyloid precursor protein (APP), is a key pathology in Alzheimer's disease (AD). The single APP gene is spliced to give 3 major isoforms. In the majority of body tissues, the most common APP isoforms are APP[751] and APP[770], which both contain a Kunitz protease inhibitor (KPI) domain, APP[695] is predominant in the brain. APP is processed through several pathways, not all of which lead to betaA4 production. Central nervous system (CNS) neurones in vivo secrete betaA4, which can be detected in the cerebrospinal fluid, though it is unknown why betaA4 is deposited in the brain in AD. NTera2 (NT2) cells derived from a human teratocarcinoma were used as a model of APP processing. Retinoic acid induces these cells to differentiate into a neuronal phenotype (NT2N cells), which has been shown to closely resemble immature human CNS neurones. Both cell types produce high levels of endogenous APP.Intracellular and secreted APP was studied in both cell types by means of western blotting and immunoprecipitation with a panel of antibodies. It was found that NT2 cells predominantly make and secrete KPI containing APP. NT2N cells make and secrete predominantly APP[695] though some KPI containing APP is also present.There is evidence that neurones in the AD brain are in a state of stress, which could increase levels of APP due to a heat shock promotor region in its gene. To investigate this, NT2 cells were subjected to a heat shock, which resulted in increased levels of heat shock protein (HSP) and APP. KPI containing APP predominated, but there was no corresponding increase in secreted APP. Both cell types were also serum deprived, which resulted in little effect on protein production in NT2 stem cells. However, the neuronal cells showed a small increase in intracellular, KPI-containing APP and in HSP. A reduction in overall APP secretion, and cessation of KPI secretion accompanied this.To further investigate the effects of shock on APP production, mRNA levels in control and serum deprived NT2 and NT2N cells were studied using in situ hybridisation. Control NT2 cells contain low levels of APP751, APP695 and HSP mRNA, with higher levels of APP[770] mRNA. After serum deprivation HSP, APP[751] and APP[770] mRNA levels all rose significantly, while APP[695] mRNA levels were unchanged. Control NT2N cells contained high levels of APP695 mRNA, lower levels of APP[751] mRNA, and very low levels of APP[770] and HSP mRNA. Serum deprivation resulted in unchanged levels of APP[695] and APP[770] mRNA, while APP[751] and HSP levels were increased.These findings indicate that cellular stress can result in increased levels of APP, specifically APP[751], in both neuronal and non-neuronal cells. Increased levels of this isoform have also been reported in AD. Hence cellular stress leads to an increase in an APP isoform implicated in AD, and could also provide an explanation for the increased levels of betaA4 in the disease.