Haptoglobin Is Present As Zonulin In The Brain And Overexpression Improves Intracerebral Hemorrhage Outcomes

Intracerebral hemorrhage (ICH) most commonly results from a hypertensive spontaneous bleed into the brain parenchyma and is a devastating stroke subtype. Hemolysis ensues and large quantities of hemoglobin (Hb) are released that initiate a toxic oxidative and neuroinflammatory cascade. Hp is an abundant plasma glycoprotein that binds and detoxifies extracorpuscular Hb with strong affinity, although very little Hp is made endogenously within the brain. Of note, Hp undergoes a complex biosynthesis into a single polypeptide prohaptoglobin that is co-translationally processed in the endoplasmic reticulum to form the α- and β-subunits, which subsequently dimerize. We hypothesized that overexpressing Hp locally in the brain would lead to improved outcomes following ICH. Overexpression was achieved using adeno-associated viral vectors and ICH was induced using the intrastriatal autologous whole blood injection model. Functional outcomes were assessed by a 24-point neurological deficit score. Western blotting under reducing and non-reducing conditions and mass spectrometry were utilized to confirm the identity of the overexpression product, which surprisingly was prohaptoglobin, also known as zonulin. At 72h post-ICH, zonulin-overexpressing mice have smaller lesion volumes with less blood accumulation and better neurologic function (p<0.05) when compared to controls (n=11-13/group). Zonulin-overexpressing mice also demonstrate less heme oxygenase-1 expression, lipid peroxidation, and peripheral neutrophil infiltration, and significantly increased microgliosis and astrogliosis. No differences in iron content were seen. Importantly, these results provide evidence that Hp in its fully processed form is not made endogenously within the brain, but instead transcription from this gene results in the formation of zonulin, which has very distinctly different functions. Notably, zonulin does not bind Hb or provide redox protection, but is a strong regulator of intercellular tight junctions, which has important implications for intracranial bleeding. Interestingly, we show that zonulin-overexpression improves ICH outcomes and thus this pathway represents a new potentially targetable avenue for the treatment of ICH.