Title Page Title: Chronic Seizures Induce Sex-Specific Cognitive Deficits with Loss of Presenilin 2 Function Abbreviated title: Impact of Chronic Seizures with PSEN2 Knockout Authors:

(264 words) Patients with early-onset Alzheimer’s disease (EOAD) are at elevated risk for seizures, including patients with presenilin 2 (PSEN2) variants. Like people with epilepsy, uncontrolled seizures may worsen cognitive function in AD. While the relationship between seizures and amyloid beta accumulation has been more thoroughly investigated, the role of other drivers of seizure susceptibility in EOAD remain relatively understudied. We therefore sought to define the impact of loss of normal PSEN2 function and chronic seizures on cognitive function in the aged brain. Male and female PSEN2 KO and age- and sex-matched wild-type (WT) mice were sham or corneal kindled beginning at 6-months-old. Kindled and sham-kindled mice were then challenged up to 6 weeks later in a battery of cognitive tests: non-habituated open field (OF), T-maze spontaneous alternation (TM), and Barnes maze (BM), followed by immunohistochemistry for markers of neuroinflammation and neuroplasticity. PSEN2 KO mice required significantly more stimulations to kindle (males: p<0.02; females: p<0.02) versus WT. Across a range of behavioral tests, the cognitive performance of kindled female PSEN2 KO mice was most significantly impaired versus age-matched WT females. Male BM performance was generally worsened by seizures (p=0.038), but loss of PSEN2 function did not itself worsen cognitive performance. Conversely, kindled PSEN2 KO females made the most BM errors (p=0.007). Chronic seizures also significantly altered expression of hippocampal neuroinflammation and neuroplasticity markers in a sex-specific manner. Chronic seizures may thus significantly worsen hippocampus-dependent cognitive deficits in aged female, but not male, PSEN2 KO mice. Our work suggests that untreated focal seizures may worsen cognitive burden with loss of normal PSEN2 function in a sex-related manner. BM learning and memory test and euthanized 1 hour after the final testing day. The extent of reactive gliosis (as measured by GFAP, A-B; E-F) and neuronal density (as measured by NeuN; C-D: G-H) were evaluated in dorsal hippocampal structures (CA1 and Dentate Gyrus (DG)). There were no significant differences in area CA3, thus this region is not presented. A) There were significant kindling-induced increases in GFAP immunoreactivity in CA1 in both WT and PSEN2-KO mice (F (1, 67) = 20.65, p<0.0001). B) There were significant kindling-induced increases in GFAP immunoreactivity in DG in both WT and PSEN2-KO mice (F (1, 68) = 32.23, p<0.0001). C and D) There were no significant differences in NeuN immunoreactivity in CA1 and DG in male mice. E) There were significant kindling-induced increases in GFAP immunoreactivity in CA1 in both WT and PSEN2-KO female mice (F (1, 44) = 7.576, p=0.0086), as well as a main effect of genotype (F (1, 44) = 9.868, p=0.003) on GFAP immunoreactivity in area CA1. Post-hoc analysis demonstrated stimulation-induced increases in GFAP immunoreactivity in WT female mice only (p=0.030). F) There was only a main effect of genotype on DG astrogliosis (F (1, 45) = 7.057, p=0.0109; Figure 6F), with only WT female mice exhibiting kindling induced increased GFAP immunoreactivity (p=0.0157). G and H) There were no significant differences in NeuN immunoreactivity in CA1 and DG in female mice. I) Representative photomicrographs of immunohistochemical detection of GFAP and NeuN in mouse brain in WT and PSEN2 KO