Heme-Mediated Tau Phosphorylation Is Critical for Neuroaxonal Damage and Cognitive Impairment in Sickle Cell Disease

A large proportion of patients with sickle cell disease (SCD) suffer from deficits in learning and memory function, and magnetic resonance imaging (MRI) evidence of cerebrovascular disease and neuroaxonal damage. Chronic and acute intravascular hemolysis release toxic free heme from the hemolyzed red blood cells resulting in severe anemia, which is a risk factor for cerebrovascular disease and cognitive impairment in SCD. However, the mechanistic link between heme and cerebrovascular injury is poorly understood. Astrocytes, the modified glial cells, constitute the neurovascular coupling that maintain neuronal integrity and regulate cognitive function. We reported that transgenic SCD mice homozygous for human hemoglobin S (SS) had widespread neuroaxonal damage and astrocyte activation with poor cognitive responses compared to control mice with normal human hemoglobin A (AA) (PMID: 37215630). In this study, we tested whether heme alters endothelial signaling that impacts the adjacent astrocytic responses leading to neurocognitive dysfunction in SCD. We mimicked acute intravascular hemolysis in human SCD by intravenously injecting a modest dose of purified hemin into the SS mice. MRI-based diffusion tensor imaging (DTI) of the SS mouse brain indicated a significant drop (n=5; p<0.01) in fractional anisotropy (FA), a marker of neuroaxonal damage, in heme-challenged SS mice compared to vehicle-injected SS mice. Moreover, the SS mice had a higher number of glial fibrillary acidic protein (GFAP)-positive activated cerebral astrocytes (n=6; p<0.05) following the heme challenge. We then used the Y-maze and novel object recognition (NOR) tests to assess the neurocognitive ability of the heme challenged SS mice. We found that - i) the time spent in a new arm of the Y-maze was significantly higher (n=12; p<0.05), ii) the percent exploration time, indicating the time spent exploring the novel object in NOR testing, and iii) the discrimination index identifying the difference between the time spent exploring the familial object versus the new object, were significantly lower in the heme injected SS mice (n=12; p<0.001). Phosphorylation of Tau, an intracellular microtubular associated protein, is implicated in neuroaxonal damage and cognitive impairment. We discovered that heme induces the expression of phosphorylated Tau protein (pTau) in the cerebrovascular endothelium and the adjacent astrocytes in heme challenged SS mice (n=5; p<0.05). Increased expression of pTau was evident in the human primary brain microvascular endothelium as well as human primary astrocytes challenged with ferric heme in vitro (n=5 independent experiments; p<0.001). To test the role of pTau in neuroaxonal damage and cognitive impairment in the SS mice, we transplanted the bone marrow from the SS donor mice into a recipient Tau knockout mouse (Tau^-/-) to generate Tau-deficient sickle bone marrow chimeras (SS^Tau-/-) possessing the hematological characteristics of SS mice. Histopathological and DTI analysis showed that the SS^Tau-/- mice had enhanced neuroaxonal integrity compared to the wild-type control mice (SS^Tau+/+) following heme challenge. Moreover, the SS^Tau-/- mice showed significant improvement in Y-maze and NOR cognitive testing (n=10; p<0.05). In summary, our study shows that toxic extracellular heme contributes to neurocognitive dysfunction and identifies Tau as a molecular intermediate. Inhibition of Tau phosphorylation may be a potential therapeutic target to enhance neurocognitive function in SCD.