A 10-year follow-up of high-dose ambroxol treatment combined with enzyme replacement therapy for neuropathic Gaucher disease.

High-dose ambroxol therapy combined with ERT in patients with neuropathic Gaucher disease.

Clinical and genetic characteristics of three patients with congenital insensitivity to pain with anhidrosis: Case reports and a review of the literature.

BACKGROUND: Congenital insensitivity to pain with anhidrosis (CIPA) is an extremely rare autosomal recessive disorder caused by loss-of-function mutations of the NTRK1 gene, affecting the autonomic and sensory nervous system. Clinical manifestation is varied and includes recurrent fever, pain insensitivity, anhidrosis, self-mutilating behavior, and intellectual disability. METHODS: Clinical and genetic features were assessed in two males and one female with genetically confirmed CIPA using exome or genome sequencing. RESULTS: CIPA symptoms including recurrent fever, pain insensitivity, and anhidrosis manifested at the age of 1 year (age range: 0.3-8 years). Two patients exhibited self-mutilation tendencies, intellectual disability, and developmental delay. Four NTRK1 (NM_002529.3) mutations, c.851-33T>A (p.?), c.2020G>T (p.Asp674Tyr), c.2303C>T (p.Pro768Leu), and c.574-156_850+1113del (exons 5-7 del) were identified. Two patients exhibited early onset and severe phenotype, being homozygous for c.851-33T>A (p.?) mutations and compound heterozygous for c.851-33T>A (p.?) and c.2020G>T (p.Asp674Tyr) mutation of NTRK1. The third patient with compound heterozygous mutations of c.2303C>T (p.Pro768Leu) and c.574-156_850+1113del (exons 5-7 del) displayed a late onset and milder clinical manifestation. CONCLUSION: All three patients exhibited variable phenotypes and disease severity. This research enriches our understanding of clinical and genetic aspects of CIPA, highlighting variable phenotypes and disease severity.

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency through molecular genetic analysis of the CYP21A2 gene.

PURPOSE: Deficiency of 21-hydroxylase (21-OHD) is an autosomal recessively inherited disorder that is characterized by adrenal insufficiency and androgen excess. This study was performed to investigate the clinical utility of prenatal diagnosis of 21-OHD using molecular genetic testing in families at risk. METHODS: This study included 27 pregnant women who had previously borne a child with 21-OHD. Fetal tissues were obtained using chorionic villus sampling (CVS) or amniocentesis. After the genomic DNA was isolated, Sanger sequencing of CYP21A2 and multiplex ligation-dependent probe amplification were performed. The clinical and endocrinological findings were reviewed retrospectively. RESULTS: A total of 39 prenatal genetic tests was performed on 27 pregnant women and their fetal tissues. The mean gestational age at the time of testing was 11.7 weeks for CVS and 17.5 weeks for amniocentesis. Eleven fetuses (28.2%) were diagnosed with 21-OHD. Among them, 10 fetuses (90.9%) harbored the same mutation as siblings who were previously diagnosed with 21-OHD. Among these, 4 fetuses (3 males and 1 female) identified as affected were born alive. All 4 patients have been treated with hydrocortisone, 9α-fludrocortisone, and sodium chloride since a mean of 3.7 days of life. The male patients did not show hyponatremia and dehydration, although they harbored pathogenic variants associated with the salt-wasting type of 21-OHD. CONCLUSION: This study demonstrated the diagnostic efficacy and clinical consequences of diagnosis by prenatal genetic testing in families at risk for 21-OHD. All patients identified as affected were treated with hydrocortisone and 9α-fludrocortisone early after birth, which can prevent a life-threatening adrenal crisis.

Long-term endocrine sequelae after hematopoietic stem cell transplantation in children and adolescents.

Purpose: As the survival rate for pediatric cancers increases significantly with advances in treatment modalities, long-term endocrine complications have also risen. This study investigated the frequencies and risks of endocrine sequelae of childhood cancer survivors after hematopoietic stem cell transplantation (HSCT). Methods: This study included 200 pediatric patients who underwent HSCT. Clinical and endocrinological findings were collected retrospectively. Median follow-up duration after HSCT was 14 years. Results: Endocrine complications occurred in 135 patients (67.5%). Children who underwent HSCT at pubertal age (n = 100) were at higher risk of endocrine complications than prepubertal age (79% vs. 56%, P = 0.001). The most common complication was hypogonadism (40%), followed by dyslipidemia (22%). Short stature and diabetes mellitus were more prevalent in the prepubertal group, whereas hypogonadism and osteoporosis were more common in the pubertal group. Female, pubertal age at HSCT, and glucocorticoid use were predictors of increased risk for any complication. Radiation exposure increased the risk of short stature and hypothyroidism. Hypogonadism was significantly associated with female, pubertal age at HSCT, and high-dose radiation. Pubertal age at HSCT also increased the risks of osteoporosis and dyslipidemia. Conclusion: This study demonstrated that long-term endocrine complications are common after HSCT in children and adolescents. Age at HSCT is a critical factor for endocrine complications after HSCT. These findings suggest that surveillance strategies for endocrine complications in childhood cancer survivors should be specified according to age at HSCT.

Impact of Early Diagnostic and Therapeutic Interventions and Clinical Course in Children and Adolescents with Multiple Endocrine Neoplasia Types 1 and 2.

PURPOSE: Multiple endocrine neoplasia types 1 (MEN1) and 2 (MEN2) are inherited endocrine tumor syndromes caused by mutations in the MEN1 or RET genes. This study aimed to investigate clinical outcomes and molecular characteristics among children with MEN. METHODS: This study included eight patients from seven unrelated families. Data on clinical course, biochemical findings, and radiologic studies were collected by retrospective chart review. All diagnoses were genetically confirmed by Sanger sequencing of MEN1 in three MEN1 patients and RET in four patients with MEN2A and one patient with MEN2B. RESULTS: Three patients with MEN1 from two families presented with hypoglycemia at a mean age of 11±2.6 years. Four patients with MEN2A were genetically diagnosed at a mean of 3.0±2.2 years of age by family screening; one of them was prenatally diagnosed by chorionic villus sampling. Three patients with MEN2A underwent prophylactic thyroidectomy from 5 to 6 years of age, whereas one patient refused surgery. The patient with MEN2B presented with a tongue neuroma and medullary thyroid carcinoma at 6 years of age. Subsequently, he underwent a subtotal colectomy because of bowel perforation and submucosal ganglioneuromatosis at 18 years of age. CONCLUSION: This study described the relatively long clinical course of pediatric MEN with a mean follow-up duration of 7.5±3.8 years. Insulinoma was the first manifestation in children with MEN1. Early diagnosis by family screening during the asymptomatic period enabled early intervention. The patient with MEN2B exhibited the most aggressive clinical course.

Clinical Characteristics and Long-Term Outcomes of Adrenal Tumors in Children and Adolescents.

OBJECTIVE: Adrenal tumors are generally rare in children and can be a part of familial cancer syndrome. This research was conducted to examine the clinical outcomes, histopathological results, and genetic etiologies of adrenal tumors in children and adolescents. METHODS: Thirty-one children and adolescents with adrenal tumors were included. Data on clinical outcomes and endocrine and radiologic results were retrospectively analyzed. Molecular analysis was conducted in select patients according to their phenotype and family history. RESULTS: The median age at diagnosis was 7.9 years (range: 0.8-17.8 years) with 5.1±1.8 cm of maximum tumor diameter. Adrenal adenoma (n=7), carcinoma (n=5), borderline (n=2), isolated micronodular adrenocortical disease (n=2), pheochromocytoma (n=8), paraganglioma (n=3), and ganglioneuroma (n=4) are all pathological diagnoses. The most common presenting symptom was excess production of adrenocortical hormones (n=15), including virilization and Cushing syndrome. Non-functioning adrenocortical tumors were found in a patient with congenital adrenal hyperplasia. Genetic etiologies were identified in TP53 (n=5), VHL (n=4), and PRKACA (n=1). Patients with mutations in TP53 were young (1.5±0.5 years) and had large masses (6.1±2.3 cm). CONCLUSIONS: This study describes clinical outcomes and the pathological spectrum of adrenal tumors in children and adolescents. Adrenocortical tumors mostly presented with an excess of the adrenocortical hormone. Patients with genetic defects presented at a young age and large size of tumors, necessitating genetic testing in patients at a young age.

Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing.

Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.

Mutation spectrum and frequency of copy number variations of the ANOS1 gene in patients with Kallmann syndrome or normosmic isolated hypogonadotropic hypogonadism.

Objective: This study was performed to investigate the molecular characteristics and frequency of copy number variations (CNVs) of ANOS1 in patients with Kallmann syndrome (KS) or normosmic isolated hypogonadotropic hypogonadism (nIHH) using multiplex ligation-dependent probe amplification (MLPA) analysis and sequencing. Methods: Among 45 patients from 43 independent families, Sanger sequencing, next-generation sequencing (NGS), or microarray was performed in 24 patients from 23 families, and MLPA was performed in 19 patients who did not show rare sequence variants (n = 18) or ANOS1 amplification by PCR (n = 1). Results: Seven patients (four patients with KS, one patient with nIHH, one prepubertal boy with anosmia, and one newborn patient) from six families (6/43, 14%) harbored molecular defects in ANOS1 including a nonsense mutation (c.1140G>A (p.W380*)), a frameshift mutation (c.1260del (p.Q421Kfs*61)), a splice site mutation (c.1449+1G>A), an exon 7 deletion, a complete deletion, and 7.9 Mb-sized inversion encompassing ANOS1. The complete deletion of ANOS1 was identified in a neonate with a micropenis and cryptorchidism. Unilateral renal agenesis was found in three patients, whereas only one patient displayed both synkinesia and sensorineural hearing loss. There was no reversal of hypogonadotropic hypogonadism in any patient during 9.1 ± 2.9 years of treatment with testosterone enanthate. Conclusions: Molecular defects in the ANOS1 gene could be identified in 14% of probands including various types of CNVs (3/43, 7.0%). Comprehensive analysis using sequencing and analysis for CNVs is required to detect molecular defects in ANOS1.

Clinical outcomes and genotype-phenotype correlations in patients with complete and partial androgen insensitivity syndromes.

PURPOSE: Androgen insensitivity syndrome (AIS) is a rare X-linked recessive disorder caused by unresponsiveness to androgens because of mutations in the AR gene. Here, we investigated the clinical outcomes and molecular spectrum of AR variants in patients with AIS attending a single academic center. METHODS: This study included 19 patients with AIS who were confirmed by molecular analysis of AR. Clinical features and endocrinological findings were retrospectively collected, including presenting features, external genitalia, sex of rearing, timing of gonadectomy, pubertal outcomes, and sex hormone levels. Molecular analysis of AR was performed using Sanger, targeted gene panel, or whole-exome sequencing. RESULTS: Among all 19 patients, 14 (74%) were classified as having complete AIS (CAIS), whereas 5 (26%) had partial AIS (PAIS). All patients with CAIS, and 3 patients with PAIS were reared as female. One patient with CAIS manifested a mixed germ cell tumor at the age of 30 years. Molecular analysis of AR identified 19 sequence variants; 12 (63%) were previously reported, and the remaining 7 (37%) were novel. Missense mutations were the most common type (12 of 19, 63%), followed by small deletions, nonsense mutations, an insertion, and a splice site mutation. CONCLUSION: Here, we describe the clinical outcomes and molecular characteristics of 19 Korean patients with AIS. Patients with PAIS manifested various degrees of masculinization of the external genitalia. Nonsense and frameshift mutations were frequent in patients with CAIS, whereas patients with PAIS harbored exclusively missense mutations.

Pediatric hepatocellular carcinoma associated with Niemann-Pick disease type C: Case report and literature review.

Niemann-Pick disease type C (NPC) is a rare, autosomal recessive, lysosomal storage disease, resulting from mutations in the cholesterol trafficking proteins NPC1 or NPC2, which is characterized by progressive neurodegeneration and hepatic dysfunction. The hepatic involvement in NPC is usually neonatal cholestasis and hepatosplenomegaly. Only a few cases of severe hepatic complications were reported including acute liver failure, cirrhosis, and hepatocellular carcinoma (HCC). We described the case of a 6-year-old male with NPC with HCC. He had a history of neonatal cholestasis and motor delay. At the age of 6 months, he was diagnosed with NPC, which was confirmed by the detection of a compound heterozygous NPC1 mutation (p.C113Y/p.A927V). He presented recurrent hypoglycemia and abdominal distension. An ultrasound, computed tomography scan, and biopsy revealed that he had a stage IV HCC with pulmonary metastasis. With the literature review and this case, HCC can be a rare fatal comorbid condition in patients with NPC, particularly infantile-onset, male patients with a relatively long disease history, necessitating appropriate HCC surveillance.

KBG syndrome: Clinical features and molecular findings in seven unrelated Korean families with a review of the literature.

BACKGROUND: KBG syndrome is a rare genetic disorder involving macrodontia of the upper central incisors, craniofacial, skeletal, and neurologic symptoms, caused either by a heterozygous variant in ANKRD11 or deletion of 16q24.3, including ANKRD11. Diagnostic criteria were proposed in 2007 based on 50 cases, but KBG syndrome remains underdiagnosed. METHODS: Whole exome sequencing (WES) and array comparative genomic hybridization (array CGH) were conducted for genetic analysis and patient phenotypes were characterized based on medical records. RESULTS: Eight patients from seven unrelated families were confirmed with KBG syndrome. All patients (8/8, 100%) had some degree of craniofacial dysmorphism and developmental delay or intellectual disabilities. Triangular face, synophrys, anteverted nostril, prominent ears, long philtrum, and tented upper lip, which are typical facial dysmorphism findings in patients with KBG syndrome, were uniformly identified in the eight patients participating in this study, with co-occurrence rates of 4/8 (50%), 4/8 (50%), 4/8 (50%), 4/8 (50%), 5/8 (62.5%), and 5/8 (62.5%), respectively. Various clinical manifestations not included in the diagnostic criteria were observed. Six patients had point mutations in ANKRD11, one had an exonic deletion of ANKRD11, and one had a 16q24.3 microdeletion. According to the ACMG guidelines, all mutations were classified as pathogenic. The c.2454dup (p.Asn819fs*1) mutation in Pt 4 was reported previously. The remaining variants (c.397 + 1G>A, c.226 + 1G>A, c.2647del (p.Glu883Argfs*94), and c.4093C>T (p.Arg1365Ter)) were novel. CONCLUSION: The clinical and molecular features of eight patients from seven unrelated Korean families with KBG syndrome described here will assist physicians in understanding this rare genetic condition.

A phase II, multicenter, open-label trial to evaluate the safety and efficacy of ISU303 (Agalsidase beta) in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is caused by a deficiency in the activity of the lysosomal enzyme, α-galactosidase A (α-Gal A), which leads to globotriaosylceramide (Gb3) deposition in multiple tissues. The current management of FD is enzyme replacement therapy (ERT). We report on the efficacy and safety of a new agalsidase beta, ISU303, in FD. METHODS: Ten patients (7 males, 3 females) were enrolled and administered a 1 mg/kg dose of ISU303, every other week for 6 months. The primary endpoint was the normalization of plasma Gb3 level. The secondary endpoints were the changes from baseline in urine Gb3 and the plasma and urine lyso-globotriaosylsphingosine (lyso-Gb3) level. Echocardiography, renal function test, and pain-related quality of life were also assessed before and after administration. Safety evaluation was performed including vital signs, laboratory tests, electrocardiograms, antibody screening tests, and adverse events at each visit. RESULTS: At 22 weeks of treatment, plasma and urine Gb3 level decreased by a mean of 4.01 ±â€…1.29 µg/mL (range 2.50-5.70) (P = .005) and 1.12 ±â€…1.98 µg/mg Cr. (range 0.04-5.65) (P = .017), respectively. However, no significant difference was observed in plasma and urine lyso-Gb3 levels. Echocardiography also was not changed. Renal function and pain-related quality of life showed improvements, but there was no clinical significance. No severe adverse events were observed. Only 1 patient developed an anti-drug antibody without neutralizing activity during the trial. CONCLUSION: This study showed the efficacy and safety of ISU303. Treatment with ISU303 significantly resulted in plasma and urine Gb3 decrease in patients with FD. These results suggest that ISU303 is safe and effective and can alternative ERT for FD.

Water-stable perovskite-loaded nanogels containing antioxidant property for highly sensitive and selective detection of roxithromycin in animal-derived food products.

Luminescent inorganic lead halide perovskite nanoparticles lack stability in aqueous solutions, limiting their application to optical sensors. Here, hybrid CsPbBr3-loaded MIP nanogels were developed with enhanced stability in aqueous media. Multifunctional MIP nanogels with antioxidant function and hydrophobic cavities were synthesized from HEMA derivatives in the presence of roxithromycin as a template. The CsPbBr3 nanoparticles were loaded into pre-synthesized MIP nanogels via in-situ synthesis with a size distribution of 200 nm. The developed CsPbBr3-nanogel exhibits excellent stability to air/moisture and enhanced stability toward an aqueous solvent. The developed CsPbBr3-loaded MIP nanogels showed a selective and sensitive detection of ROX with a limit of detection calculated to be 1.7 × 10-5 µg/mL (20.6 pM). The developed CsPbBr3-loaded MIP antioxidant-nanogels were evaluated on practical application for the quantitative determination of ROX antibiotic in animal-derived food products with excellent analytical performance. The detection of ROX in animal-derived food products showed good recovery results, making them an ideal candidate for sensing ROX.

Phenotypic spectrum of patients with mutations in CHD7: clinical implications of endocrinological findings.

Objective: Heterozygous CHD7 mutations cause a broad spectrum of clinical phenotypes ranging from typical CHARGE syndrome to self-limited delayed puberty. This study aimed to investigate the clinical characteristics of endocrine dysfunction in patients with CHD7 mutations. Methods: The clinical features and endocrine findings from 30 patients with CHD7 variants were retrospectively reviewed. A diagnosis of CHARGE syndrome was based on the Verloes diagnostic criteria. Results: Seventeen patients fulfilled the criteria for typical CHARGE syndrome, one patient for partial/incomplete CHARGE, and the remaining eleven patients had atypical CHARGE syndrome. One patient was diagnosed with Kallmann syndrome and unilateral deafness. The most frequently observed features were inner ear anomalies (80.0%), intellectual disability (76.7%), and external ear anomalies (73.3%). The mean height and weight SDSs at diagnosis were -2.6 ± 1.3 and -2.2 ± 1.8, respectively. Short stature was apparent in 18 patients (60%), and 1 patient was diagnosed with growth hormone deficiency. Seventeen males showed genital hypoplasia, including micropenis, cryptorchidism, or both. Seven patients after pubertal age had hypogonadotropic hypogonadism with hyposmia/anosmia and olfactory bulb hypoplasia. Truncating CHD7 mutations were the most common (n = 22), followed by missense variants (n = 3), splice-site variants (n = 2), and large deletion (n = 2). Conclusions: A diverse phenotypic spectrum was observed in patients with CHD7 variants, and endocrine defects such as short stature and delayed puberty occurred in most patients. Endocrine evaluation, especially for growth and pubertal impairment, should be performed during diagnosis and follow-up to improve the patient's quality of life.

Genotype-phenotype correlations and long-term efficacy of pamidronate therapy in patients with osteogenesis imperfecta.

PURPOSE: Osteogenesis imperfecta (OI) is a rare bone fragility disorder caused by defects in type 1 collagen biosynthesis. This study investigated the genotype-phenotype correlations and the efficacy of pamidronate therapy in patients with OI in a single academic center. METHODS: This study included 24 patients with OI. A clinical scoring system was used to evaluate disorder severity. COL1A1 and COL1A2 genes were analyzed in 13 patients using Sanger sequencing. Genotype-phenotype correlations and the efficacy of pamidronate therapy were analyzed through a retrospective medical chart review. RESULTS: Of the 24 patients, 18 (75%) were classified as type I (12 with type Ia and 6 with type Ib), 2 as type III (8.4%), and 4 as type IV (16.7%). Type Ia patients showed relatively higher lumbar bone mineral density (BMD) standard deviation scores (SDS) and lower clinical scores than those with other types. Seven patients with qualitative mutations had lower lumbar BMD-SDS (P=0.015) and higher clinical scores (P=0.008) than 6 patients with quantitative mutations. The annual fracture frequency and lumbar BMD-SDS improved in patients with qualitative mutations after pamidronate treatment. CONCLUSION: This study demonstrated that OI patients with qualitative mutations in COL1A1/2 had a more severe phenotype than those with quantitative mutations. Patients with qualitative mutations showed a significant reduction in fracture frequency and an increase in lumbar BMD-SDS after pamidronate treatment. Clinical score and genotype might be helpful for predicting phenotype and response to pamidronate therapy in OI patients.

Low-dose ionizing radiation alleviates Aß42-induced cell death via regulating AKT and p38 pathways in Drosophila Alzheimer's disease models.

Ionizing radiation is widely used in medicine and is valuable in both the diagnosis and treatment of many diseases. However, its health effects are ambiguous. Here, we report that low-dose ionizing radiation has beneficial effects in human amyloid-ß42 (Aß42)-expressing Drosophila Alzheimer's disease (AD) models. Ionizing radiation at a dose of 0.05 Gy suppressed AD-like phenotypes, including developmental defects and locomotive dysfunction, but did not alter the decreased survival rates and longevity of Aß42-expressing flies. The same dose of γ-irradiation reduced Aß42-induced cell death in Drosophila AD models through downregulation of head involution defective (hid), which encodes a protein that activates caspases. However, 4 Gy of γ-irradiation increased Aß42-induced cell death without modulating pro-apoptotic genes grim, reaper and hid The AKT signaling pathway, which was suppressed in Drosophila AD models, was activated by either 0.05 or 4 Gy γ-irradiation. Interestingly, p38 mitogen-activated protein-kinase (MAPK) activity was inhibited by exposure to 0.05 Gy γ-irradiation but enhanced by exposure to 4 Gy in Aß42-expressing flies. In addition, overexpression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT signaling pathway, or a null mutant of AKT strongly suppressed the beneficial effects of low-dose ionizing radiation in Aß42-expressing flies. These results indicate that low-dose ionizing radiation suppresses Aß42-induced cell death through regulation of the AKT and p38 MAPK signaling pathways, suggesting that low-dose ionizing radiation has hormetic effects on the pathogenesis of Aß42-associated AD.

Phenotypic differences between Drosophila Alzheimer's disease models expressing human Aß42 in the developing eye and brain.

Drosophila melanogaster expressing amyloid-ß42 (Aß42) transgenes have been used as models to study Alzheimer's disease. Various Aß42 transgenes with different structures induce different phenotypes, which make it difficult to compare data among studies which use different transgenic lines. In this study, we compared the phenotypes of four frequently used Aß42 transgenic lines, UAS-Aß422X , UAS-Aß42BL33770 , UAS-Aß4211C39 , and UAS-Aß42H29.3 . Among the four transgenic lines, only UAS-Aß422X has two copies of the upstream activation sequence-amyloid-ß42 (UAS-Aß42) transgene, while remaining three have one copy. UAS-Aß42BL33770 has the 3' untranslated region of Drosophila α-tubulin, while the others have that of SV40. UAS-Aß4211C39 and UAS-Aß42H29.3 have the rat pre-proenkephalin signal peptide, while UAS-Aß422X and UAS-Aß42BL33770 have that of the fly argos protein. When the transgenes were expressed ectopically in the developing eyes of the flies, UAS-Aß422X transgene resulted in a strongly reduced and rough eye phenotype, while UAS-Aß42BL33770 only showed a strong rough eye phenotype; UAS-Aß42H29.3 and UAS-Aß4211C39 had mild rough eyes. The levels of cell death and reactive oxygen species (ROS) in the eye imaginal discs were consistently the highest in UAS-Aß422X , followed by UAS-Aß42BL33770 , UAS-Aß4211C39 , and UAS-Aß42H29.3 . Surprisingly, the reduction in survival during the development of these lines did not correlate with cell death or ROS levels. The flies which expressed UAS-Aß4211C39 or UAS-Aß42H29.3 experienced greatly reduced survival rates, although low levels of ROS or cell death were detected. Collectively, our results demonstrated that different Drosophila AD models show different phenotypic severity, and suggested that different transgenes may have different modes of cytotoxicity. Abbreviations: Aß42: amyloid-ß42; AD: Alzheimer's disease; UAS: upstream activation sequence.

In Vivo Screening of Traditional Medicinal Plants for Neuroprotective Activity against Aß42 Cytotoxicity by Using Drosophila Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by progressive neuronal loss with amyloid ß-peptide (Aß) plaques. Despite several drugs currently used to treat AD, their beneficial effects on AD progress remains under debate. Here, we established a rapid in vivo screening system using Drosophila AD models to assess the neuroprotective activities of medicinal plants that have been used in traditional Chinese medicine. Among 23 medicinal plants tested, the extracts from five plants, Coriandrum sativum, Nardostachys jatamansi, Polygonum multiflorum (P. multiflorum), Rehmannia glutinosa, and Sorbus commixta (S. commixta), showed protective effects against the Aß42 neurotoxicity. We further characterized the neuroprotective activity of ethanol extracts from P. multiflorum and S. commixta. Aß42-expressing flies that we used showed AD neurological phenotypes, such as decreased survival and motility and increased cell death and reactive oxygen species level. However, feeding these flies extracts from P. multiflorum or S. commixta showed strong suppression of such phenotypes. Similar results were observed in human cells, so that the treatment of P. multiflorum and S. commixta extracts increased the viability of Aß-treated SH-SY5Y cells. Moreover, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, one of the main constituents of P. multiflorum, also showed similar protective activity against Aß42 cytotoxicity in both Drosophila and human cells. Taken together, our results suggest that both P. multiflorum and S. commixta have therapeutic potential for the treatment of neurodegenerative diseases, such as AD.

CCN1 acutely increases nitric oxide production via integrin αvß3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.

Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser(1177) phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser(473) and S6K-Thr(389). However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser(1177) phosphorylation. Furthermore, neutralization of integrin αvß3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser(1177) phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvß3-Akt-S6K-eNOS-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.

CCN1 secreted by tonsil-derived mesenchymal stem cells promotes endothelial cell angiogenesis via integrin αv ß3 and AMPK.

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow-derived mesenchymal stem cells (BM-MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently-established tonsil-derived MSC (T-MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T-MSC, a higher level of CCN1 was secreted by T-MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T-MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T-MSC (CCM), and these stimulatory effects were reversed by neutralization with anti-CCN1 antibody. Treatment with recombinant human CCN1 (rh-CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh-CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh-CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well-known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin α(v) ß(3) with anti-integrin α(v) ß(3) antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T-MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin α(v) ß(3) and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration.