Selective disruption of tau-SH3 interactions rescues seizure and sleep phenotypes.

Alzheimer's disease (AD) patients frequently experience seizures, sleep disturbances, and other forms of neural network dysfunction that accelerate cognitive decline. Although tau-lowering therapies may alleviate these features, they also risk disrupting essential physiological functions of tau, leading to motor impairments. We hypothesized that targeted mutations within tau's proline-rich domain-regions critical for binding SH3-containing proteins implicated in seizures and excitotoxicity-could selectively disrupt pathological interactions while preserving normal cognition and behaviour. To test this, we generated two tau knockin mouse lines: AxxA6, carrying proline-to-alanine mutations in the sixth PxxP motif, and R221A, containing an arginine-to-alanine substitution at residue 221. Tau-protein interactions were evaluated using proximity ligation assays in cultured hippocampal neurons and co-immunoprecipitation-mass spectrometry of cortical lysates. To model epilepsy, Kv1.1 heterozygous knockout mice were crossed with tau knockin mice. Mice underwent 24-hour cortical EEG recordings. Seizure susceptibility was assessed following intraperitoneal kainic acid (25 mg/kg). Hippocampal slice electrophysiology was used to measure epileptic bursting after picrotoxin/4-AP application. Comprehensive motor and cognitive testing were performed in AxxA6 and R221A lines at young and older ages. Both variants reduced tau's binding to the SH3-containing proteins BIN1, PLCγ1, and p85⍺/PI3K, with AxxA6 specifically decreasing Fyn interaction (P < 0.0001). Coimmunoprecipitation-mass spectrometry revealed variant-specific alterations in tau interactomes, including increased synaptotagmin-5 binding in both lines (P < 0.05). AxxA6 knockin mice displayed unique resistance to kainic-acid-induced seizures. AxxA6 knockin also reduced epileptic spike rates in Kv1.1-/- mice (P = 0.02), along with improved beta power during REM (P < 0.05), and rescued sleep disruptions (P < 0.002). Both AxxA6 and R221A prevented the increase in epileptiform bursting in Kv1.1-/- hippocampal slices after picrotoxin/4-AP (P < 0.05) and improved survival in Kv1.1-/- mice. Motor function, cognition, and body weight were preserved in both lines across ageing (3-7 and 14-18 months), in contrast to age-related weight gain and motor deficits in tau knockout mice. These findings demonstrate that precision targeting of tau's sixth PxxP motif can selectively disrupt pathological protein interactions while preserving physiological function, offering a promising therapeutic strategy to mitigate tau-driven neuronal and network dysfunction without compromising cognitive or motor health.