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Proteolytic cleavage of amyloid β-protein precursor (AβPP) by β– and γ–secretases results in production of the amyloid β peptide (Aβ) that accumulates in the brains of sufferers of Alzheimer’s Disease (AD). We have developed a monoclonal antibody, 2B12, which binds in the vicinity of the β-secretase cleavage site on AβPP but does not bind within the Aβ region. We hypothesised that this antibody, directed against the substrate rather than the enzyme, could inhibit cleavage of AβPP by β-secretase via steric hindrance and thus reduce downstream production of Aβ. The antibody would enter cells by binding to AβPP when it is at the cell surface and then be internalised with the protein. We subsequently demonstrated that, after addition of 2B12 to standard growth media, this antibody was indeed capable of inhibiting Aβ40 production in neuroblastoma and astrocytoma cells expressing native AβPP, as measured by an ELISA. This inhibition was both concentration- and time-dependent and was specific to 2B12. We were only able to inhibit approximately 50% of Aβ40 production suggesting that not all APP is trafficked to the cell surface. We propose that this antibody could be used as a novel, putative therapy for the treatment of AD.

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The novel human differentiating factor peptide fragment HLDF6 (Thr-Gly-Glu-Asn-His-Arg) was synthesized and purified. HLDF6 (0.1mg/kg i.p. but not 1mg/kg i.p.) improved not only long-term (24h) memory in adult rats in the water maze behavioural paradigm but also performance in the delayed matching-to-position (DMTP) task (0.3 and 1.0 but not 0.1mg/kg i.p). Hence, HLDF6 not only enhanced allocentric spatial learning and reference memory (water maze) but also improved temporal, spatial and working memory processes in the DMTP behavioural paradigm. Immunoreactivity blotting analysis of HLDF (the protein precursor of HLDF6) was performed and the following rank order of visual intensities from brain structures was noted: hippocampus cerebral cortex cerebellum hypothalamus striatum. Subsequently, we found that the highest absolute levels of HLDF were expressed in the hippocampus and cerebral cortex as detected by ELISA. We also demonstrated that HLDF6 enhanced [(3)H]-thymidine and [(14)C]-leucine incorporation into whole brain and hippocampal homogenates (maxima occurring within the range 10 (-12)-10 (-6) M) suggesting that this hexapeptide promoted de novo DNA and protein biosynthesis. We discuss this data in terms of their implications for links with other integrative metabolic pathways involving immediate early gene activation which may underpin a potential application for HLDF6 in limiting memory impairments associated with neurodegenerative diseases.

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