Summary/Abstract Alzheimer’s disease (AD), one of the most prevalent degenerative diseases, is characterized by the prevalence of senile plaques that consist of fibrils of amyloid b (Ab). In the amyloidogenic pathway, sequential proteolysis of the b-amyloid precursor protein (APP) by b and g secretases generate Ab (40- 42 amino acid) peptides. Ab multimerizes into neurotoxic oligomers that target the synapse, leading to disruption of long-term potentiation, cell death, and memory loss. Alternatively, in the non-amyloidogenic pathway, APP is cleaved sequentially by a and g secretase to generate sAPPa that exhibits neuroprotective properties and precludes the formation of Ab. Studies have identified ADAM10, a member of the adamlysin family of zinc metalloproteinases, as the constitutive a-secretase in neurons. Activation of ADAM10 is currently being evaluated as a means of therapeutic intervention. However, this strategy has severe disadvantages: ADAM10 is known to cleave multiple substrates and is upregulated in a wide range of malignancies. A non-specific (non-selective) up-regulation/enhancement of ADAM10 as a means of therapeutic intervention in AD would likely have deleterious side effects. The importance of the non-catalytic (disintegrin and cysteine rich) domains of ADAM10 in substrate recognition was shown previously. A conformation specific mAb, 8C7, which recognizes an active conformation of ADAM10 found on tumor cells was developed. Importantly, while 8C7 selectively enhances APP cleavage in cell-based assay, it inhibits Notch-Delta and Eph-ephrin oncogenic signaling in preclinical models, without any discernible toxicity effects in mice, thereby ruling out deleterious side effects that might result from its potential use as AD therapeutic. The proposed studies will characterize the ADAM10-APP interactions in vitro to better understand how the ADAM10 a-secretase interacts with and cleaves APP. The molecular mechanism of action of the 8C7 mAb in promoting this cleavage will also be investigated. A human version of the anti-ADAM10 mAb 8C7, named 1H5, that augments cleavage of peptide substrates and binds to the substrate binding domain of ADAM10, was generated recently. Similar to 8C7, this human mAb (1H5), as well as a single-chain variable fragment (scFv) derived from it, will now be evaluated in cell-based assays in order to determine their efficacies in promoting APP cleavage. The shedding of APP will further be examined in rat neuronal and human glioblastoma cell lines in the presence of the mAbs (8C7 and 1H5) and the scFv. This proposal, to enhance the non- amyloidogenic pathway with anti-ADAM10 mAbs towards the development of novel AD treatments, serves as a proof of principle of therapeutic potential.