Immunotherapy and improved diagnosis and prognosis of the small vessel disease CADASIL

Awardee: Helena Karlström

Institution: Karolinska Institutet

Grant Amount: $109,856.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

Cerebral small vessel diseases (SVD) are medical conditions that cause great human suffering and costs for society. SVD cause approximately 20% of all strokes and more than 40% of dementia cases in elderly and are on the rise in an ageing population. SVD are difficult to diagnose, as current imaging modalities only recognize vessels, which are approximately ten times larger than those primarily affected by SVD. It is an emerging notion that the brain vasculature is affected in SVD which contributes to the neurodegenerative process. CADASIL is the most common hereditary form of SVD and to investigate this monogenic variant will be of importance for providing valuable insights into the molecular mechanisms underpinning idiopathic SVD and for development of therapeutic treatment. In this proposal we want to explore ways to restore the brain vascular system in a CADASIL mouse model which has an ongoing pathology (aggregated NOTCH3) by two immunization treatment strategies i.e an active and a passive vaccination approach against aggregated NOTCH3. We have recently shown promising in vivo results with an active immunization study in a CADASIL mouse model where we immunized aggregated NOTCH3 just before pathology onset for four months (preventive study). Our results are very encouraging since we observe that NOTCH3 accumulation around the small vessels in the brain is reduced, but without any Notch3 related side effects (coverage of the vascular tree in the retina, kidney morphology and inflammation status is unaffected). We also observed reduced levels of NOTCH3 ECD in the blood after immunization, which could be of great importance for diagnosis, monitor prognosis and therapeutic efficacy (Oliveira et al EMBO Mol Med, 2023). The results from these will be important novel step towards future therapy development for the disease.

Advance in PIP2 treatment to restore capillary blood flow

Awardee: Fabrice Dabertrand

Institution: University of Colorado Anschutz Medical Campus

Grant Amount: $117,734

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Neurons in the brain have few energy reserves and therefore depend on local blood flow through arterioles and capillaries for a continuous supply of nutrients. A deficit in cerebral blood flow hemodynamics is an early feature of CADASIL, which suggests that cerebrovascular dysfunction has a key role in the pathogenesis leading to dementia. We recently discovered how to rescue the coupling between neuronal activity and local blood flow regulation using a phospholipid-based treatment with PIP2. However, PIP2 can act on several targets, including as an inhibitor of TRPV4 channels, an important player in capillary blood flow regulation. The proposed work aims at characterizing the impact of CADASIL on this pathway and further develop the PIP2 treatment to improve cerebral blood flow in CADASIL mouse model.

Single-nuclei RNA-seq for CADASIL understanding and therapeutic target discovery

Awardee: Israel Fernández Cadenas

Institution: Fundació Privada Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau

Grant Amount: $60,228

Funding Period: February 1, 2022 - January 31, 2023

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Summary:

The aim of this project is to understand the reason why CADASIL is produced, and possible factors associated with the severity of the disease. To do this, we will use a new and innovative strategy with omic technology (single-nuclei RNA-seq analysis) to obtain transcripts and pathways associated with the disease and its severity. Based on this information, we purpose to find therapeutic targets overexpressing/inhibiting the molecules found to be significant in the single-nuclei RNA-seq study and those found significant in other omic studies of CADASIL already published, to evaluate later the benefits in our human cellular model (pattern of aggregation of Notch3).

Modelling CADASIL with patient iPSC-based 3D vessel-on-chip

Awardee: Saskia Lesnik-Oberstein

Institution: Leiden University Medical Center

Grant Amount: $60,228

Funding Period: February 1, 2022 - January 31, 2023

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Summary:

CADASIL is an hereditary small vessel disease caused by mutations in the NOTCH3 gene. These mutations lead to progressive changes in small brain arteries and reduced blood flow to the brain. Patients with CADASIL suffer from strokes and vascular dementia from mid-adulthood. It has recently been shown by our research group, that some NOTCH3 mutations lead to a much earlier onset of CADASIL than other mutations, but why this is the case is not yet understood. CADASIL vessel models representing both severe and mild mutations will enable us to study the molecular mechanisms underlying these differences and will teach us about CADASIL disease pathomechanisms in general. Our university medical center is a CADASIL expert center and for this project we will collaborate with the internationally leading vessel model group in our research center. Together, we will develop 3D CADASIL vessels-on-chip, built up of CADASIL vascular cells. These cells are obtained by harvesting pluripotent stem cells from blood samples of CADASIL patients with different mutations. The stem cells are then differentiated into vascular cells and incorporated into the chips. We will examine structural and functional abnormalities of the vessel wall and the differences between vessels with severe and mild mutations. We aim to share these CADASIL vessel-on-chip with the international CADASIL research community to promote CADASIL research.

HB-EGF/EGFR signaling in capillary dysfunction in CADASIL

Awardee: Masayo Koide

Institution: University of Vermont Larner College of Medicine

Award Amount: $82,795

Funding Period: February 1, 2021 - January 31, 2022

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Summary:

CADASIL, short for Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarct and Leukoencephalopathy, is the most common genetic cause of a pathology known as small vessel disease (SVD) of the brain. During SVDs such as CADASIL, the structure and function of small blood vessels (arteries, arterioles, venules, and capillaries) within the brain become compromised. An early result of this vascular dysfunction is a decrease in blood flow to the brain (cerebral blood flow or CBF), which eventually leads to dementia and/or strokes. This study will elucidate the molecular mechanisms of compromised CBF increases in response to neural activity (“functional hyperemia”) using a clinically relevant CADASIL mouse model. We will specifically focus on examining the impact of CADASIL on capillaries, the smallest and most abundant vessels in the brain, which we have previously shown to be the molecular cornerstone in functional hyperemia responses in healthy animals. Considering that it is known that CADASIL causes an abnormal accumulation of specific proteins around the outside of small vessels in the brain, we propose to examine how two of these proteins, epidermal growth factor receptor (EGFR) and heparin-binding EGF-like growth factor (HB-EGF), contribute to capillary dysfunction in CADASIL. This project, by providing a greater understanding of the cellular pathways contributing to CADASIL pathologies, will create a firm footing for future therapeutic development.

Publications:

PIP2 corrects cerebral blood flow deficits in small vessel disease by rescuing capillary Kir2.1 activity

Final Report Lay Summary:

CADASIL, short for Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarct and Leukoencephalopathy, is the most common genetic cause of a pathology known as smal vessel disease (SVD) of the brain. During SVDs such as CADASIL, the structure and function of smal blood vessels (arteries, arterioles, venules, and capilaries) within the brain become compromised. An early result of this vascular dysfunction is a decrease in blood flow to the brain (cerebral blood flow or CBF), which eventua ly leads to dementia and/or strokes. This study examined the molecular mechanisms of compromised CBF increases in response to neural activity (“functional hyperemia”) using a clinica ly relevant CADASIL mouse model. We specifica ly focused on examining the impact of CADASIL on capilaries, the sma lest and most abundant vessels in the brain, which we have previously shown to be the molecular cornerstone in functional hyperemia responses in healthy animals. Considering that it is known that CADASIL causes an abnormal accumulation of specific proteins around the outside of sma l vessels in the brain, we examined how two of these proteins, epidermal growth factor receptor (EGFR) and heparin-binding EGF-like growth factor (HBEGF), contribute to capilary dysfunction in CADASIL. State-of-art techniques, including laser Doppler flowmetry, patch-clamp electrophysiology, two-photon microscopy and a newly developed capilary-arteriole continuum preparation were used to examine the role of capilary EGFR signaling in causing cerebral blood flow deficits in CADASIL model mice. Notably, we found that CADASIL-induced functional hyperemia deficits were caused by insufficient phosphatidylinositol 4,5-bisphosphate (PIP2), an endogenous activator of Kir2.1 potassium channels in capilary EC membranes. Furthermore, our data demonstrates that PIP2 content in capilary EC membranes can be modified by HB-EGF/EGFR signaling. In fact, the application of PIP2 or stimulation of EGFR restored capilary Kir2.1 channel activity and functional hyperemia in CADASIL mice. These results support the concept that HB-EGF/EGFR signaling modulates PIP2 content in capilary EC membrane, EC Kir2.1 channel activity, and functional hyperemia. In summary, we demonstrated a novel mechanism underlying functional hyperemia deficits in CADASIL. Our findings suggest that capilary HB-EGF/EGFR signaling and exogenous PIP2 administration may have potential as therapeutic targets as CADASIL treatments. This project, supported by the 2021 Milion Do lar Bike Ride Pilot Grant program, provides a wealth of new information to deepen our understanding of the pathologies involved in CADASIL and other smal vessel diseases of the brain and creates a foundation for future therapeutic studies.

Pericyte contractility in CADASIL

Awardee: Fabrice Dabertrand

Institution: University of Colorado Denver Anschutz Medical Campus

Award Amount: $81,951