C. elegans expressing D76N β 2 -microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

Funder: Ministero dell'Istruzione, dell'Università e della Ricerca Dipartimenti di Eccellenza 2018-2022 grant to the Molecular Medicine Department (University of Pavia)Abstract: The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype

Setleis Syndrome: Genetic and Clinical Findings in a New Case With Epilepsy

Focal facial dermal dysplasias are a group of inherited ectodermal disorders characterized by congenital bitemporal or periauricular scar-like depressions as well as other facial and nonfacial developmental defects. Four subtypes have been delineated, and mutations in the TWIST2 gene have been identified in type III focal facial dermal dysplasia (Setleis syndrome). PATIENTS: We describe a sporadic patient with the hallmark bitemporal scar-like lesions, severe intellectual disability, and focal epilepsy. RESULTS: The boy has typical features of Setleis syndrome, and he developed focal epilepsy, a previously unreported feature of this syndrome. No mutations in the TWIST2 gene were found, and there were no pathologic copy number abnormalities. CONCLUSIONS: Epilepsy could represent a new manifestation, and the patient described broadens the spectrum of clinical features associated with Setleis syndrome, including central nervous system involvement

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS

The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils

Effect of Caprylic triglyceride on food intake and lifespan of SOD1-G93A animals.

<p>(A) Food intake in SOD1-G93A animals treated with caprylic triglyceride (n = 18) or an isocaloric control diet (n = 17); (B) Mice in the two treatment groups (n = 11) were monitored daily and survival curve was plotted in GraphPad Prism.</p

Mitochondrial bioenergetic profile in the spinal cord of WT and SOD1 G93A mice on control or caprylic triglyceride diet.

<p>Mitochondria were isolated by differential centrifugation from the whole spinal cord of SOD1-G93A mice on control or caprylic triglyceride diet and oxygen consumption rates were analyzed using Seahorse XF24 extracellular flux analyzer. (A) A representative trace of OCR in the presence of pyruvate and malate. Adenosine diphosphate (ADP), oligomycin (O), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and a mixture of rotenone, antimycin A, N,N,N’,N’-tetramethylphenylenediamine and ascorbate (RATA) were injected at the indicated time points to measure basal, state 3, state 4o, maximal and complex IV OCR as indicated. OCR in the presence of pyruvate and malate in (B) SOD1-G93A (ALS) and (D) wild type (WT) mice. (C) Spare respiratory capacity of mitochondria from WT and ALS mice on control or caprylic triglyceride diet. Data are mean ± SEM, n = 3 for all groups, *p<0.05 as compared to control by two-tailed student t-test.</p

Nissl-stained motor neuron count in the lumbar spinal cord.

<p>(A) Representative photomicrographs of Nissl-stained sections at the ventral horn area of the lumbar spinal cord; (B) Motor neuron counts. Data are mean ± SEM, n = 3–4 per group, *p<0.05 by two-tailed t-test.</p

Glucose tolerance, ketone, triglyceride and corticosterone levels following caprylic triglyceride treatment.

<p>(A) Fasting serum glucose level and (B) glucose tolerance test in SOD1-G93A animals on control or caprylic triglyceride; (C) Blood ketone level at pre-symptomatic (10 weeks) or post-symptomatic (17 weeks) stage; (D) Plasma total triglyceride levels. (E) Plasma corticosterone level. All data are mean ± SEM, n = 4–5 for (A, B), n = 5 for (C) and n = 6–7 for (D, E) *p<0.05 by two-tailed t-test).</p

Loss-of-Function Mutations in TBC1D20 Cause Cataracts and Male Infertility in blind sterile Mice and Warburg Micro Syndrome in Humans.

blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology. Am J Hum Genet 2013 Dec 5; 93(6):1001-1014