Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases.

Accumulating evidence suggests that endothelial cells can be useful therapeutic targets. One of the potential targets is an endothelial cell-specific protein, Roundabout4 (ROBO4). ROBO4 has been shown to ameliorate multiple diseases in mice, including infectious diseases and sepsis. However, its mechanisms are not fully understood. In this study, using RNA-seq analysis, we found that ROBO4 downregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2. Mechanistic analysis reveals that ROBO4 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1) and TNF receptor-associated factor 7 (TRAF7), a ubiquitin E3 ligase. In this complex, ROBO4 enhances IQGAP1 ubiquitination through TRAF7, inhibits prolonged RAC1 activation, and decreases PTGS2 expression in inflammatory endothelial cells. In addition, Robo4-deficiency in mice exacerbates PTGS2-associated inflammatory diseases, including arthritis, edema, and pain. Thus, we reveal the molecular mechanism by which ROBO4 suppresses the inflammatory response and vascular hyperpermeability, highlighting its potential as a promising therapeutic target for inflammatory diseases.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin-mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2-induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2-induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Correction: PLA2G6-associated neurodegeneration presenting as a complicated form of hereditary spastic paraplegia.

The originally published version of this article contained an error in Fig. 1 and Table 2. The correct figure and table of this article should have read as below. This has now been corrected in the PDF and HTML versions of the article. The authors apologize for any inconvenience caused.

PLA2G6-associated neurodegeneration presenting as a complicated form of hereditary spastic paraplegia.

PLA2G6-associated neurodegeneration (PLAN) comprises heterogeneous neurodegenerative disorders, including infantile neuroaxonal dystrophy, neurodegeneration with brain iron accumulation 2B, and Parkinson disease 14 (PARK14). In addition, very recently, PLA2G6 mutations have been reported to represent a phenotype of hereditary spastic paraplegia (HSP). In this study, we screened 383 HSP families to clarify the frequency of PLA2G6 mutations in the Japan Spastic Paraplegia Research Consortium, and revealed the clinical characteristics of HSP with PLA2G6 mutations. We found three families with compound heterozygous mutations of the PLA2G6 gene, c.517 C > T/c.1634A > G, c.662 T > C/c.991 G > T, and c.1187-2 A > G/c.1933C > T, and one family with a homozygous mutation of the PLA2G6 gene, c.1904G > A/c.1904G > A. All three families with compound heterozygous mutations presented a uniform phenotype of a complicated form of HSP with infantile/child-onset spastic paraplegia, cerebellar ataxia, and mental retardation. On the other hand, the family with a homozygous mutation presented a late-onset complicated form of HSP with parkinsonism. This study may extend the clinical and genetic findings for PLAN.

Phosphorylation of drebrin by cyclin-dependent kinase 5 and its role in neuronal migration.

Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase which plays a key role in neuronal migration, neurite outgrowth, and spine formation during brain development. Dynamic remodeling of cytoskeletons is required for all of these processes. Cdk5-p35 phosphorylates many cytoskeletal proteins, but it is not fully understood how Cdk5-p35 regulates cytoskeletal reorganization associated with neuronal migration. Since actin filaments are critical for the neuronal movement and process formation, we aimed to find Cdk5 substrates among actin-binding proteins. In this study, we isolated actin gels from mouse brain extracts, which contain many actin-binding proteins, and phosphorylated them by Cdk5-p35 in vitro. Drebrin, a side binding protein of actin filaments and well known for spine formation, was identified as a phosphorylated protein. Drebrin has two isoforms, an embryonic form drebrin E and an adult type long isoform drebrin A. Ser142 was identified as a common phosphorylation site to drebrin E and A and Ser342 as a drebrin A-specific site. Phosphorylated drebrin is localized at the distal area of total drebrin in the growth cone of cultured primary neurons. By expressing nonphosphorylatable or phosphorylation mimicking mutants in developing neurons in utero, the reversible phosphorylation/dephosphorylation reaction of drebrin was shown to be involved in radial migration of cortical neurons. These results suggest that Cdk5-p35 regulates neuronal migration through phosphorylation of drebrin in growth cone processes.

Measurement of single low-density lipoprotein particles by atomic force microscopy.

BACKGROUND: The size of lipoprotein particles is relevant to the risk of coronary artery disease (CAD). METHODS: We investigated the feasibility of atomic force microscopy (AFM) for evaluating the size of large low-density lipoprotein (LDL) and small dense LDL (sd-LDL) separated by ultracentrifugation. The measurements by AFM in tapping mode were compared to those by electron microscopy (EM). RESULTS: There was a significant difference in particle sizes determined by AFM between large LDL (20.6 ± 1.9 nm, mean ± SD) and sd-LDL (16.2 ± 1.4 nm) obtained from six healthy volunteers (P < 0.05). The particle sizes determined by EM for the same samples were 23.2 ± 1.4 nm for large LDL and 20.4 ± 1.4 nm for sd-LDL. The difference between large LDL and sd-LDL detected by EM was also statistically significant (P < 0.05). In addition, the particle sizes of each lipoprotein fraction were significantly different between AFM and EM: P < 0.05 for large LDL and P < 0.05 for sd-LDL. CONCLUSIONS: AFM can differentiate between sd-LDL and large LDL particles by their size, and might be useful for evaluating risk for CAD.

[Prevalence of allergy in patients with severe motor and intellectual disabilities (SMID)].

BACKGROUND: Recent advances in medical care have drastically improved life prognosis of severe life-threatening diseases in newborns and young children. However, 'intact' survival without sequelae for all the children is still to be achieved and prevalence of severe motor and intellectual disabilities (SMID) secondary to central nervous system damages of any cause in childhood, especially in neonatal period, is increasing. Patients with SMID have complex problems involving multiple organs and multidisciplinary approach is mandatory. However, prevalence of allergic diseases in SMID patients is not known. METHODS: Forty one patients who were institutionalized and 26 outpatients at Mie National Hospital with SMID were enrolled. Diagnosis of allergic diseases was determined based on guidelines for the institutionalized patients and on a questionnaire asking if he/she had ever been diagnosed as the allergic diseases by any physician for outpatients. Serum total IgE, specific IgE to common allergens, eosinophil number, and exhaled nitric oxide by on-line tidal breathing method were measured. RESULTS: Lifetime prevalence of bronchial asthma, atopic dermatitis, food allergy, allergic rhinitis, allergic conjunctivitis, and drug allergy were 13.4%, 3.0%, 3.0%, 23.9%, 10.4%, and 6.0%, respectively. The sensitizations to Japanese cedar pollen, cypress, orchard grass, and ragweed were significantly more prevalent in outpatients compared with institutionalized patients. The prevalence of allergic rhinitis and allergic conjunctivitis are higher in outpatients compared with institutionalized patients (61.5% vs 0%, 23.1% vs 2.4%, respectively, p< 0.05). Exhaled nitric oxide levels in subjects who were diagnosed as asthma were significantly higher than in non-asthma. CONCLUSIONS: Allergic diseases are as common in SMID patients as general population. Further study is necessary to establish proper management for the patients.

Coumarins as novel 17beta-hydroxysteroid dehydrogenase type 3 inhibitors for potential treatment of prostate cancer.

The synthesis and SAR studies of 3- and 4-substituted 7-hydroxycoumarins as novel 17beta-HSD3 inhibitors are discussed. The most potent compounds from this series exhibited low nanomolar inhibitory activity with acceptable selectivity versus other 17beta-HSD isoenzymes and nuclear receptors.

Neuronal expression of two isoforms of mouse Septin 5.

Septin 5 (Sept5) is a member of the Septin GTPase family and is thought to be involved in exocytosis through interactions with syntaxin 1 in postmitotic neurons. In rats, Sept5 is alternatively spliced to produce a short (Sept5_v2) and long (Sept5_v1) isoform. We recently identified Sept5 in rat brain as a substrate for Cdk5/p35, which phosphorylates Ser17 of Sept5_v1. To date, however, only the short Sept5_v2 isoform has been reported in the mouse. To determine the general expression of the Sept5_v1 isoform in mammals, we isolated Sept5_v1 cDNA by PCR using mouse brain total RNA. Mouse Sept5_v1 cDNA showed a high degree of nucleotide and amino acid sequence homology to the corresponding isoform of rat and human Sept5. Both isoforms were expressed mainly in brain and testis at the mRNA level, but expression was restricted to brain at the protein level. Whereas Sept5_v1 mRNA was highly expressed in the cortex and hippocampus, Sept5_v2 mRNA was expressed at the similar extent across in various brain regions. The protein ratio of Sept5_v1 to Sept5_v2 was high in the hippocampus, roughly equivalent in the cortex and low in the cerebellum and medulla. Sept5_v2 expression increased gradually from E17 to P30, but expression of Sept5_v1 was delayed until P10. The two isoforms were distinguished by their pattern of N-terminal phosphorylation. Thus, these different expression and phosphorylation patterns suggest isoform-specific functions for Sept5 and that a phosphorylation-specific antibody will be useful to study this idea.

Abnormal glucose metabolism in aromatic L-amino acid decarboxylase deficiency.

We report sibling cases of aromatic L-amino acid decarboxylase (AADC) deficiency, which is a very rare congenital metabolic disorder. These patients were born to healthy and non-consanguineous parents, and presented oculogyric crises, paroxysmal dystonic attacks, and severe psychomotor retardation since early infancy. In cerebrospinal fluid the levels of homovanilic acid and 5-hydroxyindoleacetic acid were very low and the level of L-dopa was very high. The diagnosis was confirmed by the lack of AADC activity in plasma, and a point mutation in the AADC gene. MRI revealed a slightly small volume of the prefrontal areas and normal myelination in both patients. Positron emission tomography using 2-deoxy-2[(18)F] fluoro-D-glucose was performed in one patient, which revealed hypometabolism in the prefrontal cortex and bilateral basal ganglia with a little laterality. These findings suggested that the severe dystonic features were caused by abnormal function of bilateral basal ganglia and severe psychomotor retardation could be due to abnormalities in prefrontal cortical activity.

[Hypogeusia in a 9-year-old girl with multiple sclerosis].

We reported a case of hypogeusia in a 9-year-old girl with multiple sclerosis (MS). She had two episodes of neurological disturbances resembling those associated with MS, and diagnosed by McDonald's criteria. She complained of gustatory disturbance on the first attack. A quantitative test with four fundamental tastes (measurements with a filter paper disc) suggested decreased gustatory sensitivity in the right anterior part of the tongue. Magnetic resonance imaging (MRI) revealed a right thalamic lesion involving the ventral posteromedial nucleus parvocellular part (VPMpc). The patient was successfully treated with steroid pulse therapy, and improvement of neurological abnormalities involving gustatory disturbances was seen. Some studies of monkeys have demonstrated that the secondary neurons in the gustatory part of the solitary nucleus project ipsilaterally to VPMpc in the brainstem. The gustatory pathway in humans has not yet been demonstrated, but is speculated to be present based on that of monkeys. In the present case, we considered that the thalamic lesion involving VPMpc caused a decreased sense of taste in the ipsilateral part of the tongue. Several neurological abnormalities caused by multifocal demyelinating lesions can be observed in MS, but gustatory disturbance is rare. We quantitatively investigated unilateral gustatory disturbance in our patient, and concluded that the ipsilateral thalamic lesion detected by MRI could be causative.