Defective heme transport in the development of congenital hydrocephalus

PROJECT SUMMARY/ABSTRACT
Congenital hydrocephalus (CH) is a debilitating neurologic condition with complex genetic and environmental
inputs, characterized by excessive accumulation of cerebro-spinal fluid (CSF) and enlarged ventricles. Emerging
research suggests that disrupted neuroprogenitor cell (NPC) proliferation/differentiation, abnormal brain
angiogenesis and hypoxia may be involved in CH pathogenesis. Despite these recent advances there remain
critical gaps in our knowledge of disease etiology due to the lack of informative models. We developed a mouse
model for Proliferative Vasculopathy and Hydranencephaly Hydrocephalus (PVHH), a genetic form of CH caused
by mutation in the heme transporter, Flvcr2. Similar to humans, mice with genetic deletion of Flvcr2 in vascular
endothelial cells (ECs) develop abnormal brain blood vessels, tissue hypoxia, disrupted NPC differentiation, and
CH. In preliminary studies, we also found that neural cells produce and export large amounts of heme, that NPCs
strongly express the heme exporter, Flvcr1a, and that NPC-specific deletion of Flvcr1a causes a hydrocephalus
phenotype similar to Flvcr2 mutant mice. Together, this work links abnormal angiogenesis to disrupted
brain development and CH, and uncovers a central role for heme in these pathologies. In this proposal,
we investigate how heme, a molecule important for carrying oxygen in the body, is involved in the pathogenesis
of PVHH. We hypothesize that heme released from NPCs regulates brain angiogenesis and the NPC
micro-environment, and that disrupted heme transport causes reduced brain vascularization, tissue
hypoxia and downstream hydrocephalus. We will test this hypothesis in three distinct but interrelated aims:
In Aim 1, we will determine how heme is trafficked in the brain. Using innovative heme reporters and new
proteomics approaches, we will determine the primary cellular source of heme, mechanisms of heme
transport/trafficking, and the proteins interacting with heme in the brain. In Aim 2, we will focus on how heme
regulates brain angiogenesis in PVHH. Our preliminary data indicate that heme directly regulates Dll4-Notch
signaling, a pathway known to suppress angiogenic sprouting and reduce vascular growth. Using pharmacologic
treatments and gene perturbations, we will modulate heme and Dll4-Notch signaling in vitro and in vivo, and
determine whether Dll4-Notch is sufficient and necessary to produce the PVHH phenotype. In Aim 3, we will
determine the specific role of hypoxia and HIF-VEGF signaling in PVHH. In our PVHH models, we observe
severe hypoxia, strong upregulation of hypoxic signaling factor HIF2a in NPCs, and associated increase in the
HIF target gene, VEGF. Hypoxia and increased VEGF is found in humans with hydrocephalus, and targeting
VEGF in mouse models of CH reduces hydrocephalus. Here, we will block HIF-VEGF signaling using genetic
and pharmacologic approaches, then determine the impact on the PVHH phenotype. Together, these three aims
will explore a new role for heme in the development of PVHH, with the broader goal of understanding and
identifying new treatment targets for other forms of CH.

Grant Number: 5R01NS123168-05
NIH Institute/Center: NIH
Principal Investigator: Thomas Arnold