Cerebral small vessel disease (CSVD) is a leading cause of stroke and vascular cognitive impairment and dementia. Studying monogenic CSVD can reveal pathways that are dysregulated in common sporadic forms of the disease and may represent therapeutic targets. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause highly penetrant CSVD as part of a multisystem disorder referred to as Gould syndrome. COL4A1 and COL4A2 form heterotrimers [a1α1α2(IV)] that are fundamental constituents of basement membranes. However, their functions are poorly understood and the mechanism(s) by which COL4A1 and COL4A2 mutations cause CSVD are unknown. We used histological, molecular, genetic, pharmacological, and in vivo imaging approaches to characterize central nervous system (CNS) vascular pathologies in Col4a1 mutant mouse models of monogenic CSVD to provide insight into underlying pathogenic mechanisms. We describe developmental CNS angiogenesis abnormalities characterized by impaired retinal vascular outgrowth and patterning, increased numbers of mural cells with abnormal morphologies, altered contractile protein expression in vascular smooth muscle cells (VSMCs) and age-related loss of arteriolar VSMCs in Col4a1 mutant mice. Importantly, we identified elevated TGFβ signaling as a pathogenic consequence of Col4a1 mutations and show that genetically suppressing TGFβ signaling ameliorated CNS vascular pathologies, including partial rescue of retinal vascular patterning defects, prevention of VSMC loss, and significant reduction of intracerebral hemorrhages in Col4a1 mutant mice aged up to 8 months. This study identifies a novel biological role for collagen α1α1α2(IV) as a regulator of TGFβ signaling and demonstrates that elevated TGFβ signaling contributes to CNS vascular pathologies caused by Col4a1 mutations. Our findings suggest that pharmacologically suppressing TGFβ signaling could reduce the severity of CSVD, and potentially other manifestations associated with Gould syndrome and have important translational implications that could extend to idiopathic forms of CSVD.

Humans and mice with mutations in COL4A1 and COL4A2 manifest hallmarks of cerebral small vessel disease (cSVD). Mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1^+/G1344D) exhibit age-dependent intracerebral hemorrhages (ICHs) and brain lesions. Here, we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Furthermore, we found that dysregulation of membrane potential was associated with impaired Ca²⁺-dependent activation of large-conductance Ca²⁺-activated K⁺ (BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca²⁺ signaling. Col4a1 mutations impair protein folding, which can cause SR stress. Treating Col4a1^+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca²⁺ signaling, maintained BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICHs. We conclude that alterations in SR Ca²⁺ handling that impair ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone and contribute to age-related cSVD in Col4a1^+/G1344D mice.

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1^+/G394V mouse model, we found that vasoconstriction in response to internal pressure-the vascular myogenic response-is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP₂) is necessary for TRPM4 activity. Dialyzing SMCs with PIP₂ and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP₂ to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP₃), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-β) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-β signaling pathways stimulates PI3K to deplete PIP₂ and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1^+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP₂ depletion, revealing an age-dependent mechanism of cSVD.

Commercialization of the lithium-sulfur battery is hampered by bottlenecks like low sulfur loading, high cathode porosity, uncontrollable Li₂ S_x deposition and sluggish kinetics of Li₂ S activation. Herein, we developed a densely stacked redox-active hexaazatrinaphthylene (HATN) polymer with a surface area of 302 m²  g^-1 and a very high bulk density of ca. 1.60 g cm^-3 . Uniquely, HATN polymer has a similar redox potential window to S, which facilitates the binding of Li₂ S_x and its transformation chemistry within the bulky polymer host, leading to fast Li₂ S/S kinetics. The compact polymer/S electrode presents a high sulfur loading of ca. 15 mg_s  cm^-2 (200-μm thickness) with a low cathode porosity of 41 %. It delivers a high areal capacity of ca. 14 mAh cm^-2 and good cycling stability (200 cycles) at electrolyte-sulfur (E/S) ratio of 5 μL mg_s ^-1 . The assembled pouch cell delivers a cell-level high energy density of 303 Wh kg^-1 and 392 Wh L^-1 .

Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1β (IL-1β) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. β-amyloid (Aβ) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal Aβ was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, Aβ leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

Background

There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV⁺) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV⁺ OPSCC patients.

Methods

We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes.

Findings

A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10^-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation.

Interpretation

With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV⁺ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned.

Funding

CIHR, European Union, and the NIH.