Untersuchungen zum Einfluß von Methylenblau (Tetramethylthioninchlorid) auf das Verhalten adulter neuronaler Stammzellen der Maus

Über viele Jahrzehnte galt das zentrale Nervensystem als regenerationsunfähiges Gewebe. Heutzutage ist bekannt, dass umschriebene Areale des Gehirns von Säugetieren auch in postnatalen Stadien zur Neurogenese befähigt sind. Obwohl die adulte Neurogenese im Menschen limitiert zu sein scheint, wird sie zunehmend im Zusammenhang mit der Pathogenese neurodegenerativer Erkrankungen diskutiert. Sie gilt vor allem im Hinblick auf die steigende Prävalenz dieser Krankheiten in den Ländern der westlichen Welt als vielversprechendes und nachhaltiges therapeutisches Angriffsziel. Ziel dieser Arbeit war, einen möglichen Einfluss von Methylenblau (Tetramethythioninchlorid) auf das Verhalten adulter neuronaler Stammzellen (ANSC) der Maus zu untersuchen. Methylenblau (MB) ist ein therapeutisches Agenz, das bereits langjährig und sicher bei einer Vielzahl medizinischer Indikationen Anwendung findet. Basierend auf klinischen Studien durch Claude Wischik wird MB in den vergangenen Jahren zunehmend in der Therapie der Alzheimer Demenz diskutiert. Im Rahmen dieser Arbeit wurden verschiedene Aspekte des Verhaltens von ANSC untersucht. Hierfur wurden das Kurz- und Langzeitwachstum, die Expression ausgewählter Gene sowie die gerichtete Migration von ANSC unter dem Einfluss von MB erhoben. Die Versuche dieser Arbeit konnten zeigen, dass MB signifikant auf Prozesse der Neuroplastizität wirkt. Die Ergebnisse des Migrationsassays demonstrieren, dass MB eine Steigerung der gerichteten Migration der ANSC im Vergleich zur Kontrolle bewirkt. Nach Kenntnis der Autorin ist dies die erste Arbeit, die eine Beeinflussung des Migrationsverhaltens von ANSC durch MB demonstrieren kann. Diese Wirkungsweise MBs erscheint insbesondere vor dem Hintergrund von therapeutischen Überlegungen zur gezielten Lenkung neu generierter Neuronen in krankheitsbedingt geschädigte Hirnareale vielversprechend. Weiterhin konnte mit einem Ergebnis, das sich dem Signifikanzniveau annähert, eine mögliche Beeinflussung des Amyloidstoffwechsels durch MB angezeigt werden. Die Expressionsanalysen des Amyloid-produzierenden β-Amyloid-Cleaving-Enzyme-1 (BACE-1) deuten eine Erhöhung nach einer Kultivierung von ANSC in einem MBhaltigen Medium an. Der Trend, der anhand der Ergebnisse dieser Arbeit abgeleitet werden kann, könnte in Übereinstimmung mit alternativen Theorien interpretiert werden, die eine überwiegend pathologische Funktion des Enzyms zunehmend in Frage stellen. Die in dieser Arbeit propagierte, potentielle Beeinflussung adulter neuronaler Stammzellen bereichert die bisher bekannten Effekte MBs um eine vielversprechende Option. Möglicherweise könnte MB u.a. über eine Stimulation der adulten Neurogenese und nachfolgender Migration in der Zukunft das bisher enge Spektrum der AD-Therapien um eine Option bereichern.Recently an emerging interest into the mechanisms of neurogenesis in the adult (human) brain as relevant and possibly approachable process has grown on the way to finding sufficient treatment options for Alzheimer’s disease and other neurodegenerative conditions. The aim of this study was to investigate a possible influence of methylene blue (tetramethylthionin-chloride, MB) on the characteristics of murine adult neuronal stem cells. Based on clinical studies by Wischik et al. MB has increasingly been discussed in the treatment of Alzheimer’s disease. While no differences in the proliferative capacity could be identified, a general potential of MB in modulating the behavioral pattern of adult neuronal stem cells is indicated by the mobility assay. We believe that this is the first time that MB could be associated with the mobility of neuronal cell types. The results of this study insofar add to the spectrum of known target structures of MB within the central nervous system and may be relevant in regards to an interpretation of therapeutic effects of MB

ANK3 related neurodevelopmental disorders: expanding the spectrum of heterozygous loss-of-function variants

ANK3 encodes multiple isoforms of ankyrin-G, resulting in variegated tissue expression and function, especially regarding its role in neuronal development. Based on the zygosity, location, and type, ANK3 variants result in different neurodevelopmental phenotypes. Autism spectrum disorder has been associated with heterozygous missense variants in ANK3, whereas a more severe neurodevelopmental phenotype is caused by isoform-dependent, autosomal-dominant, or autosomal-recessive loss-of-function variants. Here, we present four individuals affected by a variable neurodevelopmental phenotype harboring a heterozygous frameshift or nonsense variant affecting all ANK3 transcripts. Thus, we provide further evidence of an isoform-based phenotypic continuum underlying ANK3-associated pathologies and expand its phenotypic spectrum

Modulating effects of FGF12 variants on NaV1.2 and NaV1.6 being associated with developmental and epileptic encephalopathy and Autism spectrum disorder: A case series

OBJECTIVE: Fibroblast Growth Factor 12 (FGF12) may represent an important modulator of neuronal network activity and has been associated with developmental and epileptic encephalopathy (DEE). We sought to identify the underlying pathomechanism of FGF12-related disorders. METHODS: Patients with pathogenic variants in FGF12 were identified through published case reports, GeneMatcher and whole exome sequencing of own case collections. The functional consequences of two missense and two copy number variants (CNVs) were studied by co-expression of wildtype and mutant FGF12 in neuronal-like cells (ND7/23) with the sodium channels NaV1.2 or NaV1.6, including their beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B). RESULTS: Four variants in FGF12 were identified for functional analysis: one novel FGF12 variant in a patient with autism spectrum disorder and three variants from previously published patients affected by DEE. We demonstrate the differential regulating effects of wildtype and mutant FGF12 on NaV1.2 and NaV1.6 channels. Here, FGF12 variants lead to a complex kinetic influence on NaV1.2 and NaV1.6, including loss- as well as gain-of function changes in fast and slow inactivation. INTERPRETATION: We could demonstrate the detailed regulating effect of FGF12 on NaV1.2 and NaV1.6 and confirmed the complex effect of FGF12 on neuronal network activity. Our findings expand the phenotypic spectrum related to FGF12 variants and elucidate the underlying pathomechanism. Specific variants in FGF12-associated disorders may be amenable to precision treatment with sodium channel blockers. FUNDING: DFG, BMBF, Hartwell Foundation, National Institute for Neurological Disorders and Stroke, IDDRC, ENGIN, NIH, ITMAT, ILAE, RES and GRIN

Modulating effects of FGF12 variants on NaV1.2 and NaV1.6 being associated with developmental and epileptic encephalopathy and Autism spectrum disorder:A case series

OBJECTIVE: Fibroblast Growth Factor 12 (FGF12) may represent an important modulator of neuronal network activity and has been associated with developmental and epileptic encephalopathy (DEE). We sought to identify the underlying pathomechanism of FGF12-related disorders. METHODS: Patients with pathogenic variants in FGF12 were identified through published case reports, GeneMatcher and whole exome sequencing of own case collections. The functional consequences of two missense and two copy number variants (CNVs) were studied by co-expression of wildtype and mutant FGF12 in neuronal-like cells (ND7/23) with the sodium channels Na(V)1.2 or Na(V)1.6, including their beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B). RESULTS: Four variants in FGF12 were identified for functional analysis: one novel FGF12 variant in a patient with autism spectrum disorder and three variants from previously published patients affected by DEE. We demonstrate the differential regulating effects of wildtype and mutant FGF12 on Na(V)1.2 and Na(V)1.6 channels. Here, FGF12 variants lead to a complex kinetic influence on Na(V)1.2 and Na(V)1.6, including loss- as well as gain-of function changes in fast and slow inactivation. INTERPRETATION: We could demonstrate the detailed regulating effect of FGF12 on Na(V)1.2 and Na(V)1.6 and confirmed the complex effect of FGF12 on neuronal network activity. Our findings expand the phenotypic spectrum related to FGF12 variants and elucidate the underlying pathomechanism. Specific variants in FGF12-associated disorders may be amenable to precision treatment with sodium channel blockers. FUNDING: DFG, BMBF, Hartwell Foundation, National Institute for Neurological Disorders and Stroke, IDDRC, ENGIN, NIH, ITMAT, ILAE, RES and GRIN

Exome Sequencing Discerns Syndromes in Patients from Consanguineous Families with Congenital Anomalies of the Kidneys and Urinary Tract

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the leading cause of CKD in children, featuring a broad variety of malformations. A monogenic cause can be detected in around 12% of patients. However, the morphologic clinical phenotype of CAKUT frequently does not indicate specific genes to be examined. To determine the likelihood of detecting causative recessive mutations by whole-exome sequencing (WES), we analyzed individuals with CAKUT from 33 different consanguineous families. Using homozygosity mapping and WES, we identified the causative mutations in nine of the 33 families studied (27%). We detected recessive mutations in nine known disease-causing genes: ZBTB24, WFS1, HPSE2, ATRX, ASPH, AGXT, AQP2, CTNS, and PKHD1. Notably, when mutated, these genes cause multiorgan syndromes that may include CAKUT as a feature (syndromic CAKUT) or cause renal diseases that may manifest as phenocopies of CAKUT. None of the above monogenic disease-causing genes were suspected on clinical grounds before this study. Follow-up clinical characterization of those patients allowed us to revise and detect relevant new clinical features in a more appropriate pathogenetic context. Thus, applying WES to the diagnostic approach in CAKUT provides opportunities for an accurate and early etiology-based diagnosis and improved clinical management

Targeted sequencing of 96 renal developmental microRNAs in 1213 individuals from 980 families with congenital anomalies of the kidney and urinary tract

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney diseases in children and young adults, accounting for similar to 50% of cases. These anomalies represent maldevelopment of the genitourinary system and can be genetically explained in only 10-16% of cases by mutations or by copy number variations in protein coding sequences. Knock-out mouse models, lacking components of the microRNA (miRNA) processing machinery (i.e. Dicer, Drosha, Dgcr8), exhibit kidney malformations resembling human CAKUT. Given the Dicer-null mouse phenotype, which implicates a central role for miRNAs gene regulation during kidney development, we hypothesized that miRNAs expressed during kidney development may cause CAKUT in humans if mutated. To evaluate this possibility we carried out Next-Generation sequencing of 96 stem-loop regions of 73 renal developmental miRNA genes in 1248 individuals with non-syndromic CAKUT from 980 families. We sequenced 96 stem-loop regions encoded by 73 miRNA genes that are expressed during kidney development in humans, mice and rats. Overall, we identified in 31/1213 individuals from 26 families with 17 different single nucleotide variants. Two variants did not segregate with the disease and hence were not causative. Thirteen variants were likely benign variants because they occurred in control populations and/or they affected nucleotides of weak evolutionary conservation. Two out of 1213 unrelated individuals had potentially pathogenic variants with unknown biologic relevance affecting miRNAs MIR19B1 and MIR99A. Our results indicate that mutations affecting mature microRNAs in individuals with CAKUT are rare and thus most likely not a common cause of CAKUT in humans

Array-based molecular karyotyping in 115 VATER/VACTERL and VATER/VACTERL-like patients identifies disease-causing copy number variations

BackgroundThe acronym VATER/VACTERL refers to the rare nonrandom association of the following component features (CF): vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia, renal malformations (R), and limb defects (L). Patients presenting with at least three CFs are diagnosed as having VATER/VACTERL association while patients presenting with only two CFs are diagnosed as having VATER/VACTERL-like phenotypes. Recently, rare causative copy number variations (CNVs) have been identified in patients with VATER/VACTERL association and VATER/VACTERL-like phenotypes. MethodsTo detect further causative CNVs we performed array based molecular karyotyping in 75 VATER/VACTERL and 40 VATER/VACTERL-like patients. ResultsFollowing the application of stringent filter criteria, we identified 13 microdeletions and seven microduplications in 20 unrelated patients all of which were absent in 1,307 healthy inhouse controls (n < 0.0008). Among these, microdeletion at 17q12 was confirmed to be de novo. Three microdeletions at 5q23.1, 16q23.3, 22q11.21, and one microduplication at 10q11.21 were all absent in the available parent. Microdeletion of chromosomal region 22q11.21 was previously found in VATER/VACTERL patients rendering it to be causative in our patient. The remaining 15 CNVs were inherited from a healthy parent. ConclusionIn two of 115 patients' causative CNVs were found (2%). The remaining identified rare CNVs represent candidates for further evaluation. Rare inherited CNVs may constitute modifiers of, or contributors to, multifactorial VATER/VACTERL or VATER/VACTERL-like phenotypes. Birth Defects Research 109:1063-1069, 2017. (c) 2017 Wiley Periodicals, Inc