Time-dependent changes in gene expression induced by secreted amyloid precursor protein-alpha in the rat hippocampus.

BACKGROUND: Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer's disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15). RESULTS: Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer's disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα. CONCLUSIONS: Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.

Secreted amyloid precursor protein-alpha upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism.

Secreted amyloid precursor protein-alpha (sAPPalpha) is a neuroprotective and neurotrophic protein derived from the parent APP molecule. We have shown that sAPPalpha enhances long-term potentiation in vivo and can restore spatial memory in rats whose endogenous sAPPalpha production is impaired. These observations imply that the reduction of sAPPalpha levels seen in Alzheimer's disease, which occurs alongside increased levels of toxic amyloid-beta, may be aetiologically significant. The mechanism by which sAPPalpha brings about changes in plasticity at synapses remains unresolved. We hypothesised that sAPPalpha may stimulate changes in synaptodendritic protein synthesis, an important mechanism for normal plasticity. To test this hypothesis, we investigated the effect of sAPPalpha on protein synthesis in synaptoneurosomes prepared from the hippocampi of adult male Sprague-Dawley rats. sAPPalpha (10nM) significantly increased de novo protein synthesis as measured by the incorporation of [(35)S]-methionine into acid-insoluble proteins. This was dose-dependent and blocked completely by inhibitors of protein synthesis (cycloheximide) and of cGMP-dependent protein kinase (KT5823). Inhibitors of calcium/calmodulin-dependent protein kinases (KN62) and mitogen-activated protein kinase (PD98059) partially blocked the response. Further, the sAPPalpha-induced increase in protein synthesis was significantly attenuated when measured in synapses isolated from aged rats. These observations imply de novo protein synthesis at synapses may contribute to the long-lasting modulatory effects of sAPPalpha on synaptic plasticity.

Endogenous secreted amyloid precursor protein-alpha regulates hippocampal NMDA receptor function, long-term potentiation and spatial memory.

Secreted amyloid precursor protein-alpha (sAPP alpha) levels are reduced during the pathogenesis of Alzheimer's disease, but the significance of this for neural function is not well understood. Here, we show that intrahippocampal infusion of antibodies targeted to endogenous sAPP alpha reduced long-term potentiation (LTP) in the dentate gyrus of adult rats by approximately 50%. Conversely, infusion of recombinant sAPP alpha dose-dependently increased LTP and facilitated in vitro tetanically evoked NMDA receptor-mediated currents. Pharmacological inhibition of alpha-secretase and other a-disintegrin-and-metalloproteases by TAPI-1 reduced both LTP and tetanus-evoked NMDA receptor-mediated currents in dentate granule cells. Both effects were prevented by co-application of exogenous recombinant sAPP alpha. Similarly, spatial memory was inhibited by intrahippocampal TAPI-1, an effect that was prevented by co-application of recombinant sAPP alpha. Together these findings indicate that endogenous sAPP alpha is a key contributor to synaptic plasticity and spatial memory. Its reduced production in Alzheimer's disease may thus contribute to the clinical memory deficits.

Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.

The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.