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Published on: June 14, 2020
Basic Science and Pathogenesis
Jolene Wei Ling Lee1, Adeline Su Lyn Ng2, Eng-King Tan1,2
1Duke-NUS Medical School, Singapore, Singapore.
Insights
This study models CADASIL using patient-derived cells, revealing NOTCH3 mutations impair blood-brain barrier function. Understanding these vascular effects is key to CADASIL pathophysiology.
Area of Science:
- Neuroscience
- Genetics
- Stem Cell Biology
Background:
- Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic dementia linked to NOTCH3 gene mutations.
- The NOTCH3 mutant (MT) variant is prevalent in Asia, but its pathogenicity requires functional validation.
- Blood-Brain-Barrier (BBB) dysfunction is implicated in CADASIL's synaptic dysfunction.
Purpose of the Study:
- To model NOTCH3 MT pathogenesis using patient-derived induced pluripotent stem cells (iPSCs).
- To investigate the impact of NOTCH3 MT on vascular function within BBB cell types.
- To establish a 3D in vitro BBB model for studying CADASIL.
Main Methods:
- Reprogramming patient PBMCs into iPSCs and correcting MT via CRISPR-Cas9 for isogenic controls.
- Differentiating iPSCs into endothelial cells (ECs), pericytes (PCs), and astrocytes (ACs).
- Conducting functional assays (angiogenesis, migration, TEER) and creating 3D BBB-like microtissues.
Main Results:
- NOTCH3 expression varied across MT cell types (ECs, ACs, PCs).
- MT ECs and PCs showed opposing occludin trends, indicating compensatory mechanisms.
- MT ECs exhibited reduced angiogenesis, migration, and barrier tightness; 3D microtissues formed perfusable vessels.
Conclusions:
- NOTCH3 MT has multifaceted effects on multiple cell types, necessitating heterogeneous models.
- Investigating vascular deregulation in CADASIL is crucial for understanding disease mechanisms.
- The developed 3D BBB model shows promise for studying vascular function in CADASIL.
Background:
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is the genetic form of vascular dementia leading to vascular impairments and cognitive decline. CADASIL is caused by mutations in the NOTCH3 gene which encodes a single-pass transmembrane receptor. The NOTCH3 mutant (MT) is reported to be the most common risk variant in Asia with almost 1% of community controls in Singaporean Chinese are carriers. Despite clinical observations, this variant has yet to be listed as a "pathogenic" variant due to the lack of functional studies done. Several reports indicated that Blood-Brain-Barrier (BBB) dysfunction can be the driving mechanism for synaptic dysfunction in CADASIL. Our work aims to model NOTCH3 MT pathogenesis and study its effects on vascular function with patient-derived induced pluripotent stem cells (iPSC)-derived BBB cell types.
Method:
Patient peripheral blood monocnuclear cells (PBMC) obtained were reprogrammed into iPSC. These MT iPSC were corrected via CRISPR-Cas9 to serve as an isogenic control (Corrected). After which, MT and corrected iPSCs were differentiated into endothelial cells (EC), pericytes (PC) and astrocytes (AC) with previously described protocols. Functional assays such as angiogenesis, migration and trans-endothelial electrical resistance (TEER) assays were carried out. Subsequently, these cell types were incorporated into a 3D in vitro platform, forming BBB-like microtissues.
Result:
In comparison with corrected cell types, expression of NOTCH3 in MT cell types were varied, with no significant differences in ECs and decreased expression in ACs and PCs. MT ECs and PCs also displayed opposite expression trend of tight junction marker, occludin, suggesting a compensatory mechanism. In addition, MT ECs displayed decreased angiogenesis, migration and barrier tightness. BBB-like microtissues were able to spontaneously form perfusable vascular structures after 7 days in culture which can serve as a promising tool to study vascular function and properties.
Conclusion:
Our findings show that the effects of NOTCH3 MT is multi-faceted, affecting multiple cell types, calling for the need to study them in a heterogenous model. Ultimately, unravelling effects of vascular deregulation in CADASIL will contribute to understanding its disease pathophysiology.
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