mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

Arabidopsis retromer subunit AtVPS29 is involved in SLY1-mediated gibberellin signaling.

KEY MESSAGE: Retromer protein AtVPS29 upregulates the SLY1 protein and downregulates the RGA protein, positively stimulating the development of the root meristematic zone, which indicates an important role of AtVPS29 in gibberellin signaling. In plants, the large retromer complex is known to play roles in multiple development processes, including cell polarity, programmed cell death, and root hair growth in Arabidopsis. However, many of its roles in plant development remain unknown. Here, we show that Arabidopsis trimeric retromer protein AtVPS29 (vacuolar protein sorting 29) modulates gibberellin signaling. The SLEEPY1 (SLY1) protein, known as a positive regulator of gibberellic acid (GA) signaling, exhibited lower abundance in vps29-3 mutants compared to wild-type (WT) plants. Conversely, the DELLA repressor protein, targeted by the E3 ubiquitin ligase SCF (Skp, Cullin, F-box) complex and acting as a negative regulator of GA signaling, showed increased abundance in vps29-3 mutants compared to WT. The vps29-3 mutants exhibited decreased sensitivity to exogenous GA supply in contrast to WT, despite an upregulation in the expression of GA receptor genes within the vps29-3 mutants. In addition, the expression of the GA synthesis genes was downregulated in vps29-3 mutants, implying that the loss of AtVPS29 causes the downregulation of GA synthesis and signaling. Furthermore, vps29-3 mutants exhibited a reduced meristematic zone accompanied by a decreased cell number. Together, these data indicate that AtVPS29 positively regulates SLY1-mediated GA signaling and plant growth.

Arabidopsis COP1 guides stomatal response in guard cells through pH regulation.

Plants rely on precise regulation of their stomatal pores to effectively carry out photosynthesis while managing water status. The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical light signaling repressor, is known to repress stomatal opening, but the exact cellular mechanisms remain unknown. Here, we show that COP1 regulates stomatal movement by controlling the pH levels in guard cells. cop1-4 mutants have larger stomatal apertures and disrupted pH dynamics within guard cells, characterized by increased vacuolar and cytosolic pH and reduced apoplastic pH, leading to abnormal stomatal responses. The altered pH profiles are attributed to the increased plasma membrane (PM) H+-ATPase activity of cop1-4 mutants. Moreover, cop1-4 mutants resist to growth defect caused by alkali stress posed on roots. Overall, our study highlights the crucial role of COP1 in maintaining pH homeostasis of guard cells by regulating PM H+-ATPase activity, and demonstrates how proton movement affects stomatal movement and plant growth.

SRF SUMOylation modulates smooth muscle phenotypic switch and vascular remodeling.

Serum response factor (SRF) controls gene transcription in vascular smooth muscle cells (VSMCs) and regulates VSMC phenotypic switch from a contractile to a synthetic state, which plays a key role in the pathogenesis of cardiovascular diseases (CVD). It is not known how post-translational SUMOylation regulates the SRF activity in CVD. Here we show that Senp1 deficiency in VSMCs increased SUMOylated SRF and the SRF-ELK complex, leading to augmented vascular remodeling and neointimal formation in mice. Mechanistically, SENP1 deficiency in VSMCs increases SRF SUMOylation at lysine 143, reducing SRF lysosomal localization concomitant with increased nuclear accumulation and switching a contractile phenotype-responsive SRF-myocardin complex to a synthetic phenotype-responsive SRF-ELK1 complex. SUMOylated SRF and phospho-ELK1 are increased in VSMCs from coronary arteries of CVD patients. Importantly, ELK inhibitor AZD6244 prevents the shift from SRF-myocardin to SRF-ELK complex, attenuating VSMC synthetic phenotypes and neointimal formation in Senp1-deficient mice. Therefore, targeting the SRF complex may have a therapeutic potential for the treatment of CVD.