The Roles of Cystatin B in the Brain and Pathophysiological Mechanisms of Progressive Myoclonic Epilepsy Type 1.

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder, also known as Unverricht-Lundborg disease (ULD). EPM1 patients suffer from photo-sensitive seizures, stimulus-sensitive myoclonus, nocturnal myoclonic seizures, ataxia and dysarthria. In addition, cerebral ataxia and impaired GABAergic inhibition are typically present. EPM1 is caused by mutations in the Cystatin B gene (CSTB). The CSTB protein functions as an intracellular thiol protease inhibitor and inhibits Cathepsin function. It also plays a crucial role in brain development and regulates various functions in neurons beyond maintaining cellular proteostasis. These include controlling cell proliferation and differentiation, synaptic functions and protection against oxidative stress, likely through regulation of mitochondrial function. Depending on the differentiation stage and status of neurons, the protein localizes either to the cytoplasm, nucleus, lysosomes or mitochondria. Further, CSTB can also be secreted to the extracellular matrix for interneuron rearrangement and migration. In this review, we will review the various functions of CSTB in the brain and discuss the putative pathophysiological mechanism underlying EPM1.

Generation of a human induced pluripotent stem cell line (UEFi004-A) from a patient with progressive myoclonic epilepsy type 1 (EPM1).

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder caused by mutations in the cystatin B gene (CSTB). Affected individual's manifest stimulus-sensitive and action myoclonus and tonic-clonic epileptic seizures. In this study, we have generated iPSCs from an EPM1 patient's skin fibroblasts with Sendai virus mediated transgene delivery. The iPSCs retained the patient specific promoter region expansion mutation, expressed pluripotency markers, differentiated into all three germ layers, and presented a normal karyotype. The line can in future be used to develop an in-vitro model for EPM1 and may help in understanding disease mechanisms at cellular and molecular level.

Oleuropein confers neuroprotection against rotenone-induced model of Parkinson's disease via BDNF/CREB/Akt pathway.

Major pathological features of Parkinson's disease (PD) include increase in oxidative stress leading to the aggregation of α-synuclein, mitochondrial dysfunction and apoptosis of dopaminergic neurons. In addition, downregulation of the expression of neurotrophic factors like-Brain Derived Neurotrophic Factor (BDNF) is also involved in PD progression. There has been a lot of interest in trophic factor-based neuroprotective medicines over the past few decades to treat PD symptoms. Rotenone, an insecticide, inhibits the mitochondrial complex I causing overproduction of ROS, oxidative stress, and aggregation of α-synuclein. It has been shown that BDNF and Tropomyosin receptor kinase B (TrkB) interaction initiates the regulation of neuronal cell development and differentiation by the serine/threonine protein kinases like Akt and GSK-3ß. Additionally, Transcription factor CREB (cAMP Response Element-binding protein) also determines the gene expression of BDNF. The homeostasis of these signalling cascades is compromised with the progression of PD. Therefore, maintaining the equilibrium of these signalling cascades will delay the onset of PD. Oleuropein (OLE), a polyphenolic compound present in olive leaves has been documented to cross blood brain barrier and shows potent antioxidative property. In the present study, the dose of 8, 16 and 32 mg/kg body weight (bwt) OLE was taken for dose standardisation. The optimised doses of 16 and 32 mg/kg bwt was found to be neuroprotective in Rotenone induced PD mouse model. OLE improves motor impairment and upregulate CREB regulation along with phosphorylation of Akt and GSK-3ß in PD mouse. In addition, OLE also reduces the mitochondrial dysfunction by activation of enzyme complexes and downregulates the proapoptotic markers in Rotenone intoxicated mouse model. Overall, our study suggests that OLE may be used as a therapeutic agent for treatment of PD by regulating BDNF/CREB/Akt signalling pathway.

Caspase-4/11 is critical for angiogenesis by repressing Notch1 signalling via inhibiting γ-secretase activity.

BACKGROUND AND PURPOSE: Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis. EXPERIMENTAL APPROACH: In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signalling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation. KEY RESULTS: Casp11 deficiency impaired adult angiogenesis in ischaemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-haematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signalling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. The γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD). CONCLUSIONS AND IMPLICATIONS: These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.

A novel peptide inhibitor of Dll4-Notch1 signalling and its pro-angiogenic functions.

BACKGROUND AND PURPOSE: The Dll4-Notch1 signalling pathway plays an important role in sprouting angiogenesis, vascular remodelling and arterial or venous specificity. Genetic or pharmacological inhibition of Dll4-Notch1 signalling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4-Notch1 signalling have not been described. EXPERIMENTAL APPROACH: We designed a new peptide targeting Notch signalling, referred to as TAT-ANK, and assessed its effects on angiogenesis. In vitro, tube formation and fibrin gel bead assay were carried out, using human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, a developmental retinal model and tumour models in mice were used. The mechanisms underlying TAT-ANK activity were investigated by immunochemistry, western blotting, immunoprecipitation, RT-qPCR and luciferase reporter assays. KEY RESULTS: The amino acid residues 179-191 in the G-protein-coupled receptor-kinase-interacting protein-1 (GIT1-ankyrin domain) are crucial for GIT1 binding to the Notch transcription repressor, RBP-J. We designed the peptide TAT-ANK, based on residues 179-191 in GIT1. TAT-ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP-J. The analyses of biological functions showed that TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signalling. CONCLUSIONS AND IMPLICATIONS: We synthesized and investigated the biological actions of TAT-ANK peptide, a new inhibitor of Notch signalling. This peptide will be of significant interest to research on Dll4-Notch1 signalling and to clinicians carrying out clinical trials using Notch signalling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications for angiogenesis-related diseases.

Safety and Efficacy of Therapeutic Erythrocytapheresis Treatment in Chronic Mountain Sickness Patients in Shigatse, Tibet, China.

BACKGROUND Therapeutic erythrocytapheresis (TEA) is a medical technology that separates erythrocytes from whole blood and has been used in various hematological conditions. However, reports on the use of TEA to treat chronic mountain sickness (CMS) are lacking. The aim of the present study was to evaluate the efficacy, safety, and use of TEA in treatment of CMS. MATERIAL AND METHODS A total of 32 patients living in the Shigatse area of Tibet (altitude 4000 m) who had CMS were treated with TEA. Clinical data, CMS score, Borg dyspnea score, 6-min walking test score, and NYHA classification values were collected prior to and after TEA therapy. RESULTS TEA treatment significantly increased SpO2 (93.8±2.6 vs. 80.5±5.8%, P<0.001) and decreased red blood cell (5.77±0.70 vs. 7.48±0.67×10¹²/L, P<0.001), hematocrit (53.8±5.6 vs. 69.2±4.8%, P<0.001) and hemoglobin (178±16 vs. 236±14 g/L, P<0.001). Significantly lower systolic and diastolic blood pressure were also noted (P<0.001). Echocardiography showed higher left ventricle diameter (4.6±0.4 vs. 4.4±0.5 cm, P<0.01). TEA markedly decreased CMS scores (0.45±0.85 vs. 7.58±2.31, P<0.001), Borg dyspnea scale scores (0.48±0.73 vs. 0.88±0.81, P<0.001), and NYHA classification scores (P<0.05). Additionally, there was marked improvement in the 6-min walking test scores (578.5±83.1 vs. 550.4±79.0 m, P<0.001). The procedure was well tolerated, with no complications. CONCLUSIONS Our novel approach of treating CMS patients with TEA safely and effectively reduced erythrocytosis, which remains a fundamental challenge in CMS patients.

Novel COL11A2 Pathogenic Variants in a Child with Autosomal Recessive Otospondylomegaepiphyseal Dysplasia: A Review of the Literature.

Otospondylomegaepiphyseal dysplasia (OSMED) is an inherited autosomal dominant and recessive skeletal dysplasia caused by both heterozygous and homozygous pathogenic variants in COL11A2 encoding the α2(XI) collagen chains, a part of type XI collagen. Here, we describe a 2-year-old girl presenting from birth with a phenotype suggestive of OSMED. On whole exome sequence analysis of the family via commercially available methods, we detected two novel heterozygous pathogenic variants in the proband. In addition, we reviewed the phenotype of autosomal recessive OSMED cases with COL11A2 pathogenic variants reported to date and quantitatively highlighted the phenotypic spectrum.

Dll4-Notch1 signaling but not VEGF-A is essential for hyperoxia induced vessel regression in retina.

It is well recognized that decreased vascular endothelial growth factor A (VEGF-A) mRNA plays an important role in retinal vessel regression induced by hyperoxia. However, this concept has been challenged by increasing new evidence. Furthermore, VEGF-A strongly enhances Dll4 expression and inhibition of Dll4-Notch signaling leads to excessive sprouting angiogenesis. Recently, it is shown that inactivation of Dll4-Notch1 signaling reduce hyperoxia induced vessel regression. It is unknown whether sprouting angiogenesis contributes to the protective effect or not and further investigations are needed. Moreover, the expression of Dll4 or Notch1 activation in the regressing plexus remains elucidated. To determine the role of VEGF-A and Dll4-Notch1 signaling in hyperoxia induced vascular regression in the retina, we used mice at postnatal day 5 (P5) - P7. Hyperoxia induced massive vascular regression in the central plexus but not in the angiogenic plexus and had no effect on sprouting angiogenesis. Immunostaining showed that VEGF-A was significantly repressed in the angiogenic front region after hyperoxia exposure but not detectable in the central area of both normoxia and hyperoxia treated retinas. In contrast, Notch ligand Delta-like 4 (Dll4) and Notch1 intracellular domain (N1-ICD) expression were inhibited in the regressing capillaries of central retina but comparable in the angiogenic plexus after high oxygen treatment. Moreover, administration of Dll4 neutralizing antibody or γ-Secretase inhibitor DAPT significantly aggravated vessel regression induced by short-time hyperoxia administration. Our data show that repressed Dll4-Notch1 signaling pathway but not downregulation of VEGF-A expression are responsible for hyperoxia induced pervasive vessel regression.

XAV939 Inhibits Intima Formation by Decreasing Vascular Smooth Muscle Cell Proliferation and Migration Through Blocking Wnt Signaling.

BACKGROUND: Excessive proliferation, migration, and oxidative stress of vascular smooth muscle cells (VSMCs) are key mechanisms involved in intima formation, which is the basic pathological process of in stent restenosis. This study aims at exploring the role of XAV939 in proliferation, migration, and reactive oxygen species (ROS) generation of VSMCs, and hence evaluating its effects on intima formation. METHODS: Carotid artery ligation models for C57BL/6 mice were established and gave them different intervention: saline, XAV939, Axin2 overexpression adenovirus, and negative control adenovirus. The intima formation was assayed by intima area and intima/media ratio. To investigate the underlying mechanisms, primary rat VSMCs were cultured and treated with XAV939 and platelet-derived growth factor-BB. EdU, direct cell counting, cell wound-healing assay, and flow cytometry were used to measure proliferation, migration, cell cycle, apoptosis, and ROS generation of VSMCs, respectively. By Western blot, we examined proliferating cell nuclear antigen, Cyclin D1, Cyclin E, p21, ß-actin, JNK, phosphorylated JNK, Axin2 and ß-catenin expression. Immunofluorescence staining and confocal microscopy were conducted to detect translocation of ß-catenin. RESULTS: XAV939 inhibited intima formation, which was exhibited by the loss of intima area and I/M ratio and attenuated proliferation, migration, and ROS generation, as well as promoted cell cycle arrest of VSMCs. Specifically, XAV939 inhibited Wnt pathway. CONCLUSIONS: XAV939 attenuates intima formation because of its inhibition of proliferation, migration, and apoptosis of VSMCs through suppression of Wnt signaling pathway.

Bone marrow injection: A novel treatment for tennis elbow.

OBJECTIVE: The objective of this prospective study was assessment of efficacy of bone marrow aspirate (BMA) (containing plasma rich in growth factors and mesenchymal stem cells) injection in treatment of tennis elbow. MATERIALS AND METHODS: A total of 30 adult patients of previously untreated tennis elbow were administered single injection of BMA. This concentrate was made by centrifugation of iliac BMA at 2000 rpm for 20-30 min and only upper layer containing platelet rich plasma and mononuclear cells was injected. Assessment was performed at baseline, 2 weeks, 6 weeks and 12 weeks using Patient-rated Tennis Elbow Evaluation (PRTEE) score. RESULTS: Baseline pre-injection mean PRTEE score was 72.8 ± 6.97 which decreased to a mean PRTEE score of 40.93 ± 5.94 after 2 weeks of injection which was highly significant (P < 0.0001). The mean PRTEE score at 6 week and 12 week follow-up was 24.46 ± 4.58 and 14.86 ± 3.48 respectively showing a highly significant decrease from baseline scores (P < 0.0001). CONCLUSION: Treatment of tennis elbow patients with single injection of BMA showed a significant improvement in short to medium term follow-up. In future, such growth factors and/or stem cells based injection therapy can be developed as an alternative conservative treatment for patients of tennis elbow, especially who have failed non-operative treatment before surgical intervention is taken.