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HTRA1 Mutation and Expression Regulation in Cerebral Small Vessel Disease

ABSTRACT: Cerebral small vessel disease (CSVD) refers to a series of clinical, radiological, and pathological syndromes caused by various etiologies affecting small arteries, arterioles, venules, capillaries, and small veins in the brain. It can lead to cognitive impairment, stroke, gait abnormalities, and other neurological symptoms and signs. Globally, approximately 25% to 30% of strokes are caused by CSVD. The underlying mechanisms of CSVD are multifaceted, involving endothelial dysfunction, blood-brain barrier (BBB) inflammation, neuronal apoptosis, chronic cerebral hypoperfusion, and their complex interactions. Current treatments often fail to achieve satisfactory outcomes. Therefore, understanding the pathogenic mechanisms of CSVD is crucial for developing effective therapeutic strategies to mitigate its detrimental effects. The high-temperature requirement protease A-1 (HTRA1) mutations can cause hereditary CSVD. CSVD associated with HTRA1 mutations is referred to as HTRA1-associated CSVD. Homozygous HTRA1 mutations cause Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CARASIL), a rare hereditary CSVD that is inherited in an autosomal recessive manner. Heterozygous HTRA1 mutations can lead to Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy type 2 (CADASIL 2), also known as heterozygous HTRA1 mutation carriers. This condition is characterized by clinical manifestations such as stroke, cognitive impairment, gait abnormalities, alopecia, and spondylosis. To date, 35 pathogenic mutations in the HTRA1 gene have been reported, primarily exhibiting autosomal dominant inheritance. Current research mainly focuses on case reports and the potential pathogenic mechanisms associated with different mutation sites. HTRA1 mutations can lead to downregulation of HTRA1 mRNA and protein expression, thereby reducing HTRA1 protease activity. However, the specific morphological and functional changes associated with CSVD, especially those in endothelial cells, remain unclear. This study aims to explore the mutation spectrum and clinical phenotypes of heterozygous HTRA1 mutation carriers. By comparing whole-blood RNA sequencing (RNA-seq) analysis between heterozygous HTRA1 mutation carriers and healthy controls, we identify differentially expressed genes. Based on the RNA-seq results, we further investigate the effects of abnormal HtrA1 expression on the biological functions of mouse brain microvascular endothelial cells and mouse cognitive behavior. This study elucidates the role of HTRA1 in CSVD, providing insights into the pathogenesis and new therapeutic targets for patients with heterozygous HTRA1 mutations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE324041 | GEO | 2026/03/11

REPOSITORIES: GEO

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Project description:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a small vessel disease caused by NOTCH3 gene mutations, leading to vascular smooth muscle cell degeneration, arteriopathy, and subcortical ischemic infarcts. Many CADASIL patients, however, also develop cognitive impairment, indicating that neuronal functions are disturbed, but less is known about the cellular and molecular basis of these aspects in CADASIL. In this study, we used a humanized CADASIL mouse model harbouring the R182C mutation (R182C-TgN3), post-mortem human CADASIL brain sections with three different NOTCH3 gene mutations and primary human vascular smooth muscle cells harbouring the R133C NOTCH3 mutation as primary cellular models to characterise the neurovascular contribution to cognitive impairment. To specifically evaluate neuronal, mitochondrial and neurovascular function, we performed ex vivo electrophysiology, immunohistochemistry (confocal and iDISCO+ methods), western blotting, Seahorse assay, quantitative polymerase chain reaction (qPCR), and single-cell RNA sequencing. In CADASIL mice, hippocampal gamma oscillation patterns were impaired along with significant decreases in neuronal fiber length and aberrant neuronal morphology. The latter two phenotypes were also observed in post-mortem human brain tissue from CADASIL patients. Consistent with these findings, we noted significantly lower levels of mitochondrial respiratory complexes in the CADASIL mouse hippocampus, isolated mouse brain vessels and primary human vascular smooth muscle cells. Human vascular smooth muscle cells exhibited reduced oxygen consumption rates leading to reduced ATP production as well as decreased glycolytic capacity in conjunction with increased pro-inflammatory gene expression, suggesting a broader impact on cellular energy metabolism and a neuroinflammatory process. In the CADASIL mice, we also observed extensive accumulation of the NOTCH3 extracellular domain on hippocampal vessels. Light sheet imaging with iDISCO+ clearing demonstrated substantial vascular smooth muscle cell loss and reduced vessel density in the hippocampus at 9 months of age. Additionally, 3D imaging showed increased microglial attachment to vessels and enlargement of the size of the vessel-associated microglia in CADASIL mice. Single-cell RNA sequencing revealed a microglial subcluster expressing genes involved in mitochondria respiration and inflammation. Collectively, our results reveal that exacerbated vascular network damage may mediate cognitive decline observed in the later stages of CADASIL and highlight the critical role of the neurovascular unit. Our findings provide valuable insights into the underlying mechanisms of neuronal dysfunction and pave the way for future research and potential therapeutic strategies.

Project description:While most cases of vascular dementia represent complex interactions between host genetics and environmental factors, mendelian forms of vascular dementia also exist. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian disease characterized by progressive vascular deterioration, cognitive deficits, and strokes. Mutations in the NOTCH3 receptor underlies the pathologies in CADASIL. NOTCH3 is primarily expressed in vascular smooth muscle cell (vSMC) and its’ expression is critical for differentiation and functional integrity of arterial vSMCs, albeit through unclear mechanism(s). To elucidate the contribution of NOTCH3 in the maintenance of cerebral vascular architecture and function, we performed micro-computed tomography (micro-CT) on the brains of aged Notch3-deficient animals. Micro-CT assessment of the cerebral vasculature architecture showed significant abnormalities including severe vessel dilation and tortuosity (dolicoectasia) of the middle cerebral artery and its branches in the Notch3-/- compared to aged-match controls. To identify the molecular pathway from NOTCH3 dysregulation to the observed cerebral vascular dysfunction, we performed single-cell RNASeq on cerebral arteries isolated from young (4w) and old (104w) Notch3-/- animals. Evaluation of the vSMC-specific transcriptomes indicated significant loss of proteins associated with muscle contraction and increased extracellular matrix production in animals that lack NOTCH3. Using a combination of immunofluorescence microscopy and in vitro functional assays, we confirmed that continued expression of Notch3 is a critical requirement for maintenance of vSMC contractile function. Impaired contractility also affected flow of cerebrospinal fluid in the parenchyma of Notch3-/- . MRI and behavioral assessments were performed in the Notch3-/- animals to elucidate the relationship between impaired vascular contractility to cognitive function. Taken together these findings link the molecular dysfunction of NOTCH3 through its regulation of vascular contractility and cerebral vessel architecture to altered neurological function and clarify the molecular pathways to cellular pathology of Notch3 driven dementias.

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HTRA1 Mutation and Expression Regulation in Cerebral Small Vessel Disease

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