Multiorgan manifestations in spinal muscular atrophy treated with nusinersen

Die Spinale Muskelatrophie (SMA) ist eine autosomal-rezessiv vererbte neurodegenerative Erkrankung, die meist durch biallele Deletionen im Survival of Motor Neuron 1 (SMN1) Gen verursacht wird. Die daraus resultierende Degeneration der α-Motoneurone im Spinaltrakt führt zu einer progredienten Muskelschwäche, welche unbehandelt im schweren SMA Typ 1 meist innerhalb der ersten 2 Lebensjahre letal verläuft. Ein Krankheitsmodifikator stellt das SMN1-Homolog SMN2 dar, das sich in einer Base vom SMN1-Gen unterscheidet und dadurch in 80 % zu einem nicht-funktionalen Protein translatiert wird. Neue Therapieoptionen mit den Spleißmodifikatoren Nusinersen (seit 2017) und Risdiplam (seit 2021), sowie die Gentherapie Onsasemnogene abeparvovec (seit 2020) ermöglichen ein längeres Überleben der Betroffenen bei aktuell limitierten Erfahrungen zu Langzeiteffekten. Das Ziel dieser Arbeit war es, die systemischen Effekte unter Nusinersen-Behandlung zu evaluieren. Im ersten Teil wurden mögliche endokrinologische, metabolische und anthropometrische Effekte der Nusinersentherapie und der SMA selber bei 81 Kindern, Jugendlichen und Erwachsenen in einer multizentrischen, retrospektiven Studie analysiert. Hierbei fand sich ein signifikanter Anstieg der Body-Mass-Index-Perzentilen besonders bei Patient:innen mit SMA Typ 2 sowie zwischen 3,1 und 12 Jahren ohne Korrelation zur SMN2-Kopienanzahl oder Motorfunktion. Die endokrinologischen Laborparameter waren meist unauffällig, drei Betroffene wiesen aber Zeichen einer diabetogenen Stoffwechsellage auf. Diese Daten zeigen einen möglichen positiven Effekt der Therapie auf die sonst erschwerte Gewichtszunahme bei SMA-Erkrankten, wodurch Betroffene unabhängig von einem motorischen Zugewinn profitieren. Auf Grund des Rote-Hand-Briefs von 2017, welcher einen Hydrozephalus unter Nusinersentherapie beschreibt, wurde im zweiten Teil diese potenzielle Nebenwirkung bei 34 Patient:innen in einer monozentrischen, retrospektiven Studie untersucht. Der Liquoreröffnungsdruck (LD) war aber bei 70,5 % der Kinder mit ≥ 21 cm H2O leicht und bei 35,3 % mit ≥ 28 cm H2O deutlich pathologisch erhöht, bei zwei Kindern bereits vor Therapiebeginn. Keines der Kinder hatte klinische oder ophthalmologische Zeichen eines erhöhten intrakraniellen Drucks, sodass der erhöhte LD nicht therapiert wurde. Unsere Daten deuten auf einen erhöhten LD bei mit Nusinersen-behandelten Patient:innen hin, welcher bei Nachweis erhöhter Werte bereits vor Therapiebeginn aber auch Teil der Grunderkrankung sein könnte. Zusammenfassend unterstützt diese Arbeit die Hypothese, dass die SMA keine reine α- Motoneuronen Erkrankung darstellt, sondern systemische und zerebrale Manifestationen vorliegen können. Durch das längere Überleben der an SMA Erkrankten wird die Lebensqualität ein zunehmend wichtiger Faktor und damit auch die potenziell negativen Auswirkungen medikamentöser Therapien. In Folgestudien untersuchen wir aktuell die Folgen der Gentherapie bei SMA- Patient:innen.Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease caused in the majority by biallelic deletions in the survival of motor neuron 1 (SMN1) gene. In SMA, the progressive muscular atrophy results from a degeneration of the α-motor neurons in the spinal tract. Without treatment patients with the most severe SMA type 1 die within the first 2 years-of-life. The key disease modifier SMN1-homolog survival of motor neuron 2 (SMN2) gene, which differs from the SMN1 gene in only one base and is translated in 80% to a non-functional protein. New therapeutic options with the splicing modifiers nusinersen (since 2017) and risdiplam (since 2021) and the gene replacement therapy onsasemnogene abeparvovec (since 2020) result in an improved survival of patients with SMA. However, there is currently limited data on long-term systemic effects of these treatments. Therefore, the aim of this study was to analyze these effects in nusinersen-treated patients. In the first study, potential endocrinologic, metabolic and anthropometric effects of a treatment with nusinersen and of SMA itself were assessed in 81 children, adolescents and adults in a multicentric, retrospective study. We found a significant increase in the body-mass-index-centiles especially in patients with SMA type 2 and between 3.1 and 12 years without a correlation to the SMN2 copy number or motor function. The endocrinologic laboratory parameters were normal, except in three patients with signs of a diabetic metabolic state. Our data show a possible effect of nusinersen on body weight in some SMA patients, indicating a positive influence independent of motor function improvement. In the second study, we analyzed the lumbar puncture opening pressure (LD) in 34 patients with SMA treated with nusinersen in a monocentric, retrospective study. This study was initiated in response to the red-hand letter published in 2017 that warned of the development of a hydrocephalus under nusinersen therapy. The LD was mildly elevated ≥ 21 cm H2O in 70.5% of patients and severely elevated ≥ 28 cm H2O in 35.3%, in two patients LD was elevated already prior to treatment. Because none of the patients had clinical or ophthalmological signs of an increased intracranial pressure, we did not treat the increased LD. This study indicates a high proportion of patients under nusinersen treatment with an elevated LD, which could also be a so far unknown phenotype element of SMA given the elevated LD values prior to treatment. In conclusion, our work supports the hypothesis, that SMA is not only an α-motor neuron disease, but systemic and cerebral manifestations can be present. Due to the prolonged survival of SMA patients the long-term focus shifts towards quality of life and potential negative effects of the new drug treatments. Following studies will therefore work on the potential side effects of the gene replacement therapy in patients with SMA

What matters when? Social and dimensional comparisons in the context of university major choice

Students compare their achievement to different standards in order to evaluate their ability. We built on the theoretical frameworks of situated expectancy-value theory, dimensional comparison theory, and the big-fish-little-pond effect literature to examine the role of social and dimensional comparisons for ability self-concept and subjective task value (STV) in secondary school and university major choice. We used two German longitudinal data sets from different cohorts with data collection in 12th grade and 2 years after high school graduation (Study 1: N = 2,207, Study 2: N = 1,710). Dimensional and social comparisons predicted students\u27 self-concept and domain-specific STV in school: Individual achievement was positively related to ability self-concept and STV in the corresponding domain and negatively related in the noncorresponding domain. School-level mean achievement was negatively related to ability self-concept and STV in the corresponding domain. Dimensional comparisons were directly related to university major choice, social comparisons were only indirectly related. (DIPF/Orig.

Immunofluorescence Staining of Paraffin Sections Step by Step.

Immunofluorescence staining is the most frequently applied technique to detect and visualize various molecules in biological samples. Many protocols can be found in the literature and the websites of commercial antibody producers. This can result in a time-consuming and costly methodical work to establish "simple" antibody staining. We here summarize in a stepwise manner an easy-to-follow immunofluorescence staining protocol with an improved specific fluorescent signal and a reduced background and non-specific binding signal. This will help scientists to save time, effort, and antibody costs during the application of such a valuable technique.We thank Jutta Schüler, Susanne Kosanke, and Magdalena John for technical assistance as well as Pierre Gressens and his lab members for an initial protocol that served as a basis. We acknowledge support from the German Research Foundation (DFG, SFB665, SFB1315), the Open Access Publication Fund of Charité—Universitätsmedizin Berlin, and the German Academic Exchange Service (DAAD)

Proteome changes in autosomal recessive primary microcephaly

Background/aim: : Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. Material and methods: : We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. Result: : We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. Conclusion: : Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

The Mim1 complex imports α-helical mitochondrial outer membrane proteins with multiple transmembrane segments

Role of Cdk5rap2 in neocortical inhibition and excitation balance

Autosomal recessive primary microcephaly type 3 (MCPH3) is characterized by congenital microcephaly and intellectual disability. Further features include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similarly result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons using a combined morphological and electrophysiological approach

Altered inhibition and excitation in neocortical circuits in congenital microcephaly

Congenital microcephaly is highly associated with intellectual disability. Features of autosomal recessive primary microcephaly subtype 3 (MCPH3) also include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similar to the human condition result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons. Cdk5rap2-deficient murine neurons show poorly branched dendritic arbors and an increased density of immature thin spines and glutamatergic synapses in vivo. Moreover, the excitatory drive is enhanced in ex vivo brain slice preparations of Cdk5rap2 mutant mice. Concurrently, we show that pyramidal neurons receive fewer inhibitory inputs. Together, these findings point towards a shift in the excitation - inhibition balance towards excitation in Cdk5rap2 mutant mice. Thus, MCPH3 is associated not only with a neural progenitor proliferation defect but also with altered function of postmitotic neurons and hence with altered connectivity

Monoallelic CRMP1 gene variants cause neurodevelopmental disorder

Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual disability, and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human CRMP1 variants impact the oligomerization of CRMP1 proteins. Moreover, overexpression of the CRMP1 variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic variants in CRMP1 gene have never been linked to human disease. We report for the first-time variants in the CRMP1 gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease