RESUMEN
The cytosolic proteins synucleins and synapsins are thought to play cooperative roles in regulating synaptic vesicle (SV) recycling, but mechanistic insight is lacking. Here, we identify the synapsin E-domain as an essential functional binding-partner of α-synuclein (α-syn). Synapsin E-domain allows α-syn functionality, binds to α-syn, and is necessary and sufficient for enabling effects of α-syn at synapses of cultured mouse hippocampal neurons. Together with previous studies implicating the E-domain in clustering SVs, our experiments advocate a cooperative role for these two proteins in maintaining physiologic SV clusters.
Asunto(s)
Hipocampo , Neuronas , Sinapsinas , alfa-Sinucleína , Animales , Humanos , Ratones , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/química , Células Cultivadas , Hipocampo/metabolismo , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos , Sinapsis/metabolismo , Sinapsinas/metabolismo , Sinapsinas/genética , Vesículas Sinápticas/metabolismoRESUMEN
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-ß-cleavage fragments (including Aß) 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.
RESUMEN
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.