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Distinct molecular mechanisms of HTRA1 mutants in manifesting heterozygotes with CARASIL
Hiroaki Nozaki1, Taisuke Kato1, Megumi Nihonmatsu1
1From the Department of Medical Technology, School of Health Sciences, Faculty of Medicine (H.N.), Department of Molecular Neuroscience, Resource Branch for Brain Disease, Brain Research Institute (T.K., A.Y., O.O.), Department of Neurology, Clinical Neuroscience Branch, Brain Research Institute (M. Nihonmatsu, Y. Saito, A.K., A.S., M.U., Y. Sekine, M. Nishizawa), Department of Regenerative and Transplant Medicine, Division of Orthopedic Surgery (N.E.), and Department of Molecular Genetics, Bioresource Science Branch, Brain Research Institute (R. Kuwano), Niigata University, Niigata City; Department of Neurology (I.M., T. Mizuno), Kyoto Prefectural University of Medicine; Department of Neurology (T.N.), Ichinomiya Municipal Hospital, Aichi; Department of Neurology (R. Koike), Nishi-Niigata Chuo National Hospital, Niigata; Department of Neurology (K.M.), Shiseikai-Daini Hospital, Tokyo; Department of Neurology (M.K.), Kanazawa Medical University, Ishikawa; Department of Neurology (S.I.), Chiba University; Department of Neurology (M.M.), Nantan General Hospital, Kyoto; Departments of Neurology (A.M.) and Advanced Diagnosis (S.M.), Nagoya Medical Center, Aichi; Department of Neurology (K.H.), Japanese Red Cross Akita Hospital; Department of Internal Medicine (T. Momotsu), Sado General Hospital, Niigata; and Institute for Medical Science of Aging (M.Y.), Aichi Medical University, Japan.
Insights
Mutant HTRA1 gene mutations cause cerebral small vessel disease in heterozygotes by impairing protein function or trimer formation. These findings shed light on the molecular mechanisms underlying this neurological condition.
Area of Science:
- Genetics
- Neurology
- Molecular Biology
Background:
- Cerebral small vessel disease (CSVD) is a significant cause of stroke and cognitive decline.
- Mutations in the HTRA1 gene are implicated in various cerebrovascular disorders.
- Understanding the precise molecular mechanisms of mutant HTRA1 in heterozygous individuals is crucial for therapeutic development.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying mutant HTRA1-dependent cerebral small vessel disease (CSVD) in heterozygous individuals.
- To investigate the impact of specific HTRA1 mutations on protease activity and protein complex formation.
- To differentiate the effects of mutations causing CSVD in heterozygotes from those associated with cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL).
Main Methods:
- Recruited 113 unrelated index patients with clinically diagnosed CSVD.
- Analyzed coding sequences of the HTRA1 gene for mutations.
- Evaluated HTRA1 protease activities using casein assays.
- Assessed oligomeric HTRA1 formation using gel filtration chromatography.
Main Results:
- Identified four heterozygous missense mutations in HTRA1 (p.G283E, p.P285L, p.R302Q, p.T319I) in 6 patients and 2 siblings.
- Mutant HTRA1 proteins exhibited decreased protease activity and inhibited wild-type HTRA1.
- Mutations p.G283E and p.T319I prevented trimer formation, while p.P285L and p.R302Q mutations affected domains critical for trimer-associated activation.
- In contrast, CARASIL-associated mutations (A252T, V297M) formed trimers with mutations outside critical activation domains.
Conclusions:
- Mutant HTRA1 proteins in manifesting heterozygotes may impair the HTRA1 activation cascade.
- Inability to form stable HTRA1 trimers is a potential mechanism for CSVD pathogenesis.
- These findings distinguish the molecular pathology of heterozygous HTRA1 mutations in CSVD from CARASIL.
Objective:
To elucidate the molecular mechanism of mutant HTRA1-dependent cerebral small vessel disease in heterozygous individuals.
Methods:
We recruited 113 unrelated index patients with clinically diagnosed cerebral small vessel disease. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography.
Results:
We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. The mean age at cognitive impairment onset was 51.1 years. Spondylosis deformans was observed in all cases, whereas alopecia was observed in 3 cases; an autopsied case with p.G283E showed arteriopathy in their cerebral small arteries. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (A252T and V297M) did not inhibit wild-type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer-associated HTRA1 activation.
Conclusions:
The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.
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