The supernatants were collected as RIPA soluble fractions

The supernatants were collected as RIPA soluble fractions. vascular health may help stave off vascular and neurodegenerative pathologies underlying late-life dementia. Vascular risk factors including excessive salt consumption have long been associated with cerebrovascular diseases and cognitive impairment1C3. A diet rich in salt is an impartial risk factor for stroke and dementia3,8C10 and has been linked to the cerebral small vessel disease LY3000328 underlying vascular cognitive impairment11, a condition associated with endothelial dysfunction and reduced cerebral blood (CBF)12. In mice, a high salt diet (HSD) induces cognitive dysfunction by targeting the cerebral microvasculature through a gut-initiated adaptive immune response mediated by Th17 lymphocytes7. The producing increase in circulating IL17 prospects to inhibition of endothelial nitric oxide (NO) synthase (eNOS) and reduced vascular NO production, which, in turn, impairs endothelial vasoactivity and lowers cerebral blood flow (CBF) by LY3000328 25%7. However, it remains unclear how hypoperfusion, in HSD or other vascular risk factors, prospects to impaired cognition. The prevailing view is usually that hypoperfusion compromises the delivery of oxygen and glucose to energy-demanding brain regions involved in cognition12,13. But the relatively small CBF reduction associated with HSD in mice7 and vascular cognitive impairment in humans14 may not be sufficient to impair LY3000328 cognitive function15, implicating vascular factors beyond cerebral perfusion. Excessive phosphorylation of the microtubule associated protein tau promotes the formation of insoluble tau aggregates, thought to mediate neuronal dysfunction and cognitive impairment in AD and other tauopathies16. However, tau accumulation has progressively been detected also in cerebrovascular pathologies associated with endothelial dysfunction and cognitive impairment5,6. Therefore, we investigated whether tau accumulation rather than cerebral hypoperfusion contributes to the cognitive dysfunction induced by HSD. First, we established if HSD induces tau phosphorylation. Male C56Bl/6 mice were placed on a normal diet (ND) or HSD (4 or 8% NaCl), a commonly used model of excessive dietary salt corresponding to a 8C16 fold increase in the LY3000328 salt content in the regular mouse chow7,17. Phosphorylation of tau epitopes promoting tau aggregation and neuronal dysfunction16 were assessed over time by Western blotting. HSD (8%) induced a sustained increase in p-tau (AT8, RZ3) in neocortex and hippocampus without increasing total tau (Tau 46) (Fig. 1a). In the hippocampus, an increase in PHF13 and pSer199Ser202 was also observed (Extended Data Fig. 1a). The tau phosphorylation (AT8) was abolished by lambda protein phosphatase (Extended Data Fig. 1b). AT8 and RZ3 were also increased in neocortex of female mice fed a HSD (Extended Data Fig. 1c). HSD did not increase tau acetylation (K280), a post translational modification implicated in tau pathology18 (Extended Data Fig. 1a). AT8 and MC1 immunoreactivities were detected in the pyriform cortex, but neurofibrillary tangles were not observed (Fig. 1b, Extended Data LY3000328 Fig. 1d, ?,e).e). No neuronal or white matter damage was observed, nor significant changes in astrocytes, microglia/macrophages, or pericytes (Extended Data Fig. 2aCc). Increased AT8 was also observed in NIK neocortex with lower amounts of dietary salt (4%) (Extended Data Fig. 1f). Open in a separate windows Fig. 1: HSD increases tau phosphorylation and insoluble tau.a, HSD increases AT8 and RZ3 levels. (CTX: AT8, ND/HSD n=8/9, *p 0.0001 vs ND; RZ3, ND/HSD n=12/11, *p 0.0001 vs ND; HIPP: AT8, ND/HSD n=9/9, *p 0.0001 vs ND; RZ3, ND/HSD n=9/9, *p=0.0011 vs ND, two-tailed unpaired t-test). b, HSD increases neuronal AT8 immunoreactivity in the piriform cortex (size bar=500 m; 100 m in inset). Representative images from ND and HSD mice (n=5/group)..