Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the leading cause of dementia in elderly. Accumulating experimental and clinical evidence points to the decisive role of amyloid-beta peptide (Aβ) accumulation in AD pathogenesis. Therefore, much effort was invested in inhibiting the activity of two essential enzymes involved in processing of amyloid precursor protein (APP) and Aβ generation: the β-secretase amyloid precursor protein cleavage site enzyme-1 (BACE-1) (Vassar et al. Science 1999) and the γ-secretase complex. Recent failures of clinical trials with γ-secretase inhibitors have shifted the focus to BACE1 as a key AD drug target (Yan and Vassar Lancet Neurol 2014). However, the complex phenotypes of BACE1-null mice (summarized in review Yan and Vassar Lancet Neurol 2014) and the numerous recently identified BACE1 substrates (Kuhn et al. EMBO J 2012) suggest that the inhibition of BACE1 may cause unacceptable side-effects. This emphasizes the need to investigate more thoroughly the mechanisms of action of this enzyme and the functions of its substrates. The goal of this project is to characterize the processing and distribution of the two top BACE1 substrates, seizure protein 6 (Sez6) and seizure 6-like protein 1 (Sez6L1) in a transgenic (tg) mouse model of Alzheimer’s disease. These BACE1 substrates were selected because of their predominant expression in neurons and their potential role in synaptic function, learning and memory and neurodegeneration (Gunnersen et al. Neuron 2007; Osaki et al. Brain Res 2011).  In line with the recently identified accumulation of BACE1 in human and tg-mouse AD brains (Yang et al. Nat Med 2003; Kandalepas et al. Acta Neuropathol 2013) we propose that accumulation of BACE1 within presynaptic terminals and enhanced cleavage of its substrates at or near the synapse, in addition to APP, may add to synaptic (dys)function, learning and memory deficits, neurodegeneration and the pathogenesis of Alzheimer’s disease. Elucidation of BACE1 accumulation and BACE1-mediated cleavage of Sez6 and Sez6L1 could lead to better understanding of their physiological role as well as mechanisms of AD pathology, pointing to potential novel targets of protective therapy. The proposed study is novel and so far there are no any data, to our knowledge, on the metabolic status of BACE1-substrates in AD.

To test this we will use a well-defined tg-mouse model od AD – 5xFAD mice that shows a profound AD brain pathology, including accumulation of Aβ plaques already at 2-months of age. Using western blotting and immunofluorescent confocal microscopy we will analyze Sez6 and Sez6L1 proteolytic cleavage and distribution in different brain regions, such as hippocampus and cortex that are primarily affected in AD and cerebellum which is spared, at 2-, 4- and 8-months of age. Since BACE1 accumulation in AD is most likely due to lysosomal dysfunction (Kandalepas et al. Acta Neuropathol 2013), we will compare the obtained data on Sez6/Sez6L1 with the levels and distribution of BACE1, pre- and post-synaptic markers (SYN and PSD95, respectively) as well as with the levels of lysosomal marker LAMP1 and LC3II/I-ratio, a marker of lysosomal (dys)function. By comparing the obtained data in hippocampus/cortex vs. cerebellum we may identify a potential protective mechanism against AD that is specific for the cerebellum and can not be defined in the affected brain regions, such as hippocampus and cerebellum, and vice versa. Different vulnerabilities of these brain regions may give us some clues to protective and/or vulnerable mechanism(s) of neurodegeneration and may also identify critical components in this process. Further studies of similarities and differences between these brain structures (hippocampus/cortex vs. cerebellum) are encouraged to increase our understanding of the molecular pathogenesis of AD, as well as our chances of finding disease-modifying and/or protective therapies.