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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]

Secreted amyloid precursor protein alpha (sAPPα) is a potent neurotrophin in the CNS but a dedicated receptor has not been found. However, protein interactions involving amyloid beta (Aβ), a peptide cleaved from the same parent peptide as sAPPα, indicate that insulin receptors (IRs) could be a target of amyloid peptides. In this study, in vitro analysis of cortical neuronal cultures revealed that exogenous sAPPα increased IR phosphorylation in the absence of insulin. Furthermore, in an APP overexpressing mouse model, sAPPα bound IRs in the cortex with significantly greater binding in hypoinsulinemic animals. To further examine the effects of sAPPα on the diabetic brain, we next rendered sAPPα overexpressing mice insulin depleted and found that sAPPα blocked aberrant tau phosphorylation (T231) in cortical tissue after 16 weeks diabetes. sAPPα overexpression also prevented hyperphosphorylation of AKT/GSK3 and activation of the unfolded protein response (UPR). In total, these data show sAPPα binds and activates neuronal IRs and that sAPPα has a protective effect on diabetic brain tissue. [ABSTRACT FROM AUTHOR]

Phosphorylation of α-synuclein at the serine-129 site (α-syn Ser129P) is an established pathologic hallmark of synucleinopathies and a therapeutic target. In physiologic states, only a fraction of α-syn is phosphorylated at this site, and most studies have focused on the pathologic roles of this post-translational modification. We found that unlike wild-type (WT) α-syn, which is widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation. Surprisingly, preventing Ser129P blocked activity-dependent synaptic attenuation by α-syn—thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments Ser129P, which is a trigger for protein-protein interactions that are necessary for mediating α-syn function at the synapse. AlphaFold2-driven modeling and membrane-binding simulations suggest a scenario where Ser129P induces conformational changes that facilitate interactions with binding partners. Our experiments offer a new conceptual platform for investigating the role of Ser129 in synucleinopathies, with implications for drug development. • Native α-syn serine-129 phosphorylation is seen in discrete brain regions • Preventing α-syn serine-129 phosphorylation blocks activity-dependent α-syn functions • Neuronal activity augments α-syn serine-129 phosphorylation in cells and in vivo • α-syn serine-129 phosphorylation triggers protein-protein interactions at synapses Phosphorylation of α-synuclein at the serine-129 site is an established pathologic hallmark of Parkinson's disease and related "synucleinopathies," but new findings from Parra-Rivas et al. suggest a physiologic role for this post-translational modification in triggering α-synuclein function by facilitating protein-protein interactions at synapses. [ABSTRACT FROM AUTHOR]

Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein ( App ), relevant for Alzheimer's disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway - inhibiting toxic APP-β-cleavage fragments (including Aβ) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App -editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App -KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD., Competing Interests: Competing interest declaration: S.R. is the scientific co-founder and owns equity in CRISPRAlz.