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Background: Our lab has developed a gene‐editing strategy to target the extreme C‐terminus (C‐term) of APP (amyloid precursor protein) – a gene with a central and indisputable role in AD. In physiologic states, the APP protein is cleaved by α‐secretases to generate neuroprotective sAPPα; but in AD (both sporadic and familial), the alternative β‐cleavage pathway is active, generating pathologic fragments (including Aβ). Specifically, our approach attenuates pathologic β‐cleavage products by disrupting a pentapeptide "YENPTY" domain at the APP C‐term, and this retains the edited APP (APP‐ΔC) at the cell‐surface, augmenting neuroprotective and neuroregenerative α‐cleavage. Method: In order to assess the safety and efficacy of our approach in vivo, we used two different CRISPR‐editing approaches. First, we generated germline edits in Wt and APP knockin (APPNL‐G‐F) mice by injecting embryos with APP C‐term targeting CRISPRs. We then selected founders to produce stable strains of WT and APP‐KI in which the APP C‐term was genomically deleted (referred to as WtΔC and KI‐ΔC mice respectively). In another set of experiments, we packaged APP C‐term CRISPRs into AAV vectors and systemically injected AAVs into APP‐KI mice. Result: WtΔC showed no cognitive deficits or histological abnormalities compared to Wt controls. KI‐ΔC showed a dramatic reduction of amyloid beta plaques and associated neuroinflammatory markers and similar results were observed in AAV‐treated KI mice. Moreover, both germline and somatic APP C‐term editing produced an increase in neuroprotective sAPPα. Conclusion: In total, these data demonstrate that our approach is safe and efficacious in vivo and validate the feasibility of our APP C‐terminus editing approach as a therapeutic for AD. [ABSTRACT FROM AUTHOR]

Background: α‐Synuclein (α‐syn) phosphorylation at the Serine‐129 (Ser129P) residue is a recognized pathologic hallmark of PD. Though only ∼4% of α‐syn is phosphorylated at this site under normal conditions, the tight endogenous regulation of α‐syn Ser129P suggests a physiologic role. Method: We have use immunocytochemistry in cultured hippocampal neurons and immunohistochemistry in mouse brains to localize α‐syn Ser‐129P, optical pHluorin assays to report SV‐recycling and trafficking, multielectrode arrays (MEA) to examine neurotransmitter release, immunoprecitations and GST Pulldown assays to evaluate protein‐protein interactions, and AlphaFold2‐driven modeling to visualize how Ser129P induces conformational changes. Result: We found that unlike native (total) α‐syn that is widely expressed throughout the brain, the overall pattern of α‐syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser‐129 site blocked the ability of α‐syn to attenuate activity‐dependent synaptic vesicle (SV) recycling – widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments α‐syn Ser‐129P, and this phosphorylation is required for α‐syn binding to VAMP2 and synapsin – two functional binding‐partners that are necessary for α‐syn function. AlphaFold2‐driven modeling suggests a scenario where Ser129P induces conformational changes in the C‐terminus that stabilizes this region and facilitates protein‐protein interactions. Conclusion: Our data from cellular, in vivo, cell‐free systems, and molecular simulations strongly suggest that the pathology‐ associated Ser129 modification has a physiologic role at the synapse, which has broad implications for α‐syn pathophysiology and drug development. [ABSTRACT FROM AUTHOR]