Alzheimer’s Disease

Alzheimer’s Disease (AD) Neurodegenerative disorder defined by pathological brain lesions and a decline in cognitive function. Widespread loss of synapse density and continuous degeneration of cholinergic and glutamatergic pathways Effects 30% of the population over 80 years old. The production and deposition of oligomeric and aggregated forms of β-amyloid peptides (Aβ), amyloid plaques, are associated with neuronal dysfunction and cell death. Hyper-phosphorylated tau protein aggregates into neurofibrillary tangles in nerve cell bodies, and as dystrophic neurites associated with amyloid plaques

Aβ production Sequential action of two aspartic proteases β-secretase 1st step of Aβ production cleaving APP Generates the N-terminus of Aβ and a membrane tethered C-terminal fragment 99 Amino Acids in length (C99) Γ – secretase Cleaves the C99 within the transmembrane region releasing the Aβ from the cell Imprecise cleavage results in varied lengths 38-42 amino acids Aβ40 and Aβ42 are the most common fragments in amyloid

Β-Site APP Cleaving Enzyme 1 (BACE1) Transmembrane aspartyl protease Initiator enzyme for Aβ production Axonally transported in neurons and accumulates near amyloid deposits in AD patients C sites represent juxta- membrane cysteine residues which are the sites for S-palmitoylation Colored regions depict BACE1 domains with the corresponding amino acid numbers. The BACE1 catalytic domain contains two aspartic protease active site motifs, DTGS and DSGT, at positions 92–95 and 289–292, respectively (red bars). Acetylation (R), glycosylation (N), S-palmitoylation (C), phosphorylation (P) and ubiquitination (Ub) sites are indicated where known. Three disulfide bonds (S--S) connect amino acids 216–420, 278–443 and 330–380.

S-palmitoylation Reversible posttranslational modification that occurs at four juxta-membrane cysteine residues on BACE1 Responsible for targeting a variety of integral membrane proteins to lipid rafts in both nonneuronal cells (e g. and neurons Important for trafficking and function of several synaptic proteins This study proves that S-palmitoylation of BACE1 plays a major role in amyloid pathogenesis

Lipid Rafts Located in the plasma membranes of cells Consist of glycosphingolipids and protein receptors organized in glycoplipoprotein microdomains Compartmentalize processes regulating neurotransmission and receptor trafficking BACE1 anchors to lipid rafts when present in the membrane BACE1 can break down APP bound or unbound, but it is thought to be more potent when bound 4CA-BACE1 showed impaired lipid raft association in mouse brains

Knock-in Mice (4CA) The mice used in this experiment were engineered with a cysteine-to-alanine substitutions at all four S- palmitoylation sites preventing the BACE1 from undergoing S-palmitoylation A vector containing flanking loxP sites and point mutation Cysteine/Alanine is inserted into mouse embryonic stem (ES) cells to undergo homologous recombination forming 4CA mice EThose 4CA mice were then bred to 5XFAD mice These mice have been engineered to exhibit symptoms of AD relatively quickly

Synaptic Activity-Dependent Release of Aβ is Reduced Synaptic activity dynamically modulates brain interstitial fluid (ISF) Aβ levels in vivo Aβ levels were measured using an ELISA test Picrotoxin (PTX) , a non-competitive inhibitor of GABAa receptor was delivered to the hippocampus of wild type (WT) and 4CA mice to stimulate an ISF Aβ response There was significant increase from baseline in Aβ levels in WT mice There was no significant change from baseline in the 4CA mice

Mitigated Memory Deficits in 5XFAD Mice Y-maze testing and fear conditioning data indicate that reduced amyloid burden from the absence of BACE1 S-palmitoylation improves behavior in spatial working memory and associative learning Y maze is a behavioral test for measuring the willingness of rodents to explore new environments based on how many times they enter each arm 5X4CA mice showed more alternation The fear conditioning was in the form of electric shock to the foot over a 2 minutes period

Y-Maze

When BACE1 S-palmitoylation is absent There is no loss of enzymatic ability to process endogenous APP or the several other neuronal substrates it reacts with Aβ release significantly reduced Reduced cerebral amyloid burden Reduced dystrophic neurite accumulation of S- palmitoylation deficient BACE1 Mitigates memory deficits in 5XFAD mice