Fibrillin-1 Mutation Promotes Cerebrovascular Aging, Neuropathology, and Vulnerability to Traumatic Brain Injury in Mice

Abstract

Background: Aging-associated vascular and cerebrovascular dysfunction is prevalent in many connective tissue disorders. Marfan syndrome (MFS) is the most common monogenetic autosomal dominant disorder of connective tissue, characterized by mutations in the gene encoding for fibrillin-1 (Fbn1), with no biologic sex or ethnic bias. Fbn1 protein provides structural support for muscles, bones, and blood vessels as well as a scaffold for elastin fiber maturation. It also can bind cytokines and growth factors, therefore controlling the downstream activation of many signaling pathways. MFS-associated Fbn1 mutation results in an increased risk of life-threatening problems involved in weakening of blood vessel walls that can lead to dilation, dissection, and rupture. The role of Fbn1 mutation on cerebrovascular function has barely begun to be addressed though MFS patients report higher incidence of neurological deficits including headaches, migraines, cerebral aneurysms, stroke, and attention deficit hyperactivity disorder. Many hallmarks of cerebrovascular aging, such as extracellular matrix (ECM) impairment, vascular wall weakening, and stiffening, blood brain barrier (BBB) permeability, and exacerbated cytokine production and inflammation are also prevalent in MFS. These manifestations in aging and MFS are mainly linked to increased transforming growth factor-beta (TGF-β) signaling. In mice, Fbn1 mutation induces vascular dysfunction by 6-month (6M) of age. The well-established mouse model of MFS has been used in over 290 published scientific reports, yet only two have focused on the cerebral vasculature. These studies have demonstrated significant increase in wall/lumen hypertrophy, matrix metalloproteinases (MMPs), and blood brain barrier permeability in the middle cerebral artery (MCA) and in TGF-β cytokine and MMP production in the choroid plexus. These findings suggest that cerebrovascular aging in MFS is not only mirroring the process of normal aging but is also happening at an accelerated rate. Consequently, this has given rise to our hypothesis that the Fbn1 mutation accelerates the decline in cerebrovascular function and neuropathology that are commonly associated with aging, leaving the brain more susceptible to the adverse effects of mild traumatic brain injury.Methods: To test this hypothesis, we evaluated cerebrovascular alterations and neuropathology in 6-month-old male and female Fbn1+/- mice (MFS), and 6- and 12-month-old C57BL/6 (CTRL) mice through these Aims: 1) To measure aging-associated cerebrovascular function and neuropathology and in CTRL and MFS mice, and 2) to test the influence of mild traumatic brain injury on neuropathology in a well-established CTRL and MFS mice. The goals of the study were achieved through high-resolution ultrasound imaging, immunohistochemical staining, immunofluorescence imaging, in vivo electrochemistry recordings, neurobehavioral severity scale assay, and midline fluid percussion injury. Results: In this project we verified central and peripheral vascular dysfunction and demonstrated, for the first time, that this dysfunction extends to the cerebral vasculature in MFS mice. In the vasculature, 6-month-old MFS mice displayed aortic root dilation and loss of wall elasticity with no change in diastolic coronary blood flow velocity, a reduction in peak blood flow velocity in the left pulmonary artery similarly to 12-month-old control mice, and a decrease in peak blood flow velocity in the posterior cerebral artery (PCA) in 6-month-old MFS male mice comparable to 12-month-old healthy control male mice. We also observed left ventricular hypertrophy in 6-month-old MFS male mice with a decrease in mitral valve early-filling velocities evident in both male and female MFS mice. In addition, we performed the first known evaluation of neuropathology in the PCA-perfused hippocampus in the MFS mouse model. 6-month-old MFS and 12-month-old control mice demonstrated increased BBB permeability measured via Evan’s blue extravasation in the dentate gyrus (DG), increased BBB permeability measured via IgG staining throughout the hippocampus, and neuroinflammation as evident by Ionized calcium binding adaptor molecule 1 (Iba1) positive staining of microglia. 6-month-old MFS mice showed elevated levels of tonic glutamate and reduced evoked glutamate release in the DG of the hippocampus, mirroring the findings in 12-month-old control mice. Furthermore, at 6 months of age, MFS male mice had faster T100 glutamate clearance times in the DG of the hippocampus. Neurobehavioral severity scale testing demonstrated increased scores in 6-month-old MFS and 12-month-old control mice, indicative of neurobehavioral alterations. To test vulnerability to mild traumatic brain injury (mTBI), we utilized fluid percussion injury, where 6-month-old MFS mice, both male and female, and 6M-control female mice needed a 15% reduction in pulse pressure compared to age-matched healthy control male mice to achieve similar mTBI-induced righting reflex times. At one day post TBI, both MFS and control mice demonstrated increased hippocampal BBB permeability and neuroinflammation as compared to non-injured control subjects, with no difference observed between injured MFS and injured controls. Glutamate neurotransmission responses to injury were not different in MFS mice at 1DPI. Additionally, one day after mild TBI, female MFS mice showed a significant increase in NSS scores compared to female MFS mice subjected to sham procedures. Conclusion: Fbn1 mutation plays a critical role in accelerated cerebrovascular aging and neuropathology that increases the risk of vulnerability for more severe outcomes after neurological insult such as mild traumatic brain injury. This study represents the initial exploration into the Fbn1 mutation as a factor in accelerated and premature cerebrovascular aging and dysfunction. Identifying that the downstream effects of the Fbn1 mutation could uncover mechanisms and potential therapeutic targets aimed at preventing and mitigating increasing vascular dysfunction and neuropathology in MFS, related connective tissue disorders, and the aging population.