Background: Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease affecting multiple body systems with wide variability in presentation. In 2013, Pediatric Neurology published articles outlining updated diagnostic criteria and recommendations for surveillance and management of disease manifestations. Advances in knowledge and approvals of new therapies necessitated a revision of those criteria and recommendations. Methods: Chairs and working group cochairs from the 2012 International TSC Consensus Group were invited to meet face-to-face over two days at the 2018 World TSC Conference on July 25 and 26 in Dallas, TX, USA. Before the meeting, working group cochairs worked with group members via e-mail and telephone to (1) review TSC literature since the 2013 publication, (2) confirm or amend prior recommendations, and (3) provide new recommendations as required. Results: Only two changes were made to clinical diagnostic criteria reported in 2013: "multiple cortical tubers and/or radial migration lines" replaced the more general term "cortical dysplasias," and sclerotic bone lesions were reinstated as a minor criterion. Genetic diagnostic criteria were reaffirmed, including highlighting recent findings that some individuals with TSC are genetically mosaic for variants in TSC1 or TSC2. Changes to surveillance and management criteria largely reflected increased emphasis on early screening for electroencephalographic abnormalities, enhanced surveillance and management of TSCassociated neuropsychiatric disorders, and new medication approvals. Conclusions: Updated TSC diagnostic criteria and surveillance and management recommendations presented here should provide an improved framework for optimal care of those living with TSC and their families.

Tuberous sclerosis complex (TSC)1 is an autosomal dominant disorder characterized by benign hamartomas in brain, kidney, heart, lung, and skin. It is a developmental disorder with abnormalities in cell migration, differentiation, and proliferation. Neurological complications include seizures, mental retardation, and autism (1). The disease is caused by mutations in tumor suppressor genes TSC1 or TSC2 encoding hamartin and tuberin, respectively (2, 3). Hamartin and tuberin associate in vivo forming a complex with other proteins (4). Rodent models of Tsc1 and Tsc2 develop renal cyst adenomas with liver hemangiomas observed in mouse models (5-8). Mutations in either Drosophila Tsc1 or Tsc2 show increase in cell and organ size, whereas co-expression of dTsc1 and dTsc2 inhibits growth and reduces cell size (9 -12). Studies from fly also suggest that dS6 kinase may be an inhibitory target of dTsc1 and dTsc2 in growth control (10 -12). Recent studies have confirmed that mammalian TSC1 and TSC2 inhibit S6 kinase (S6K). Cells harboring mutations in either TSC1 or TSC2 have constitutively high phosphorylation of both S6K and its substrate S6 (8, 13-18). Furthermore, tuberin and hamartin function together to inhibit target of rapamycin-mediated signaling to S6K in mammals as well as in fly (15,16,18,19). Akt phosphorylates tuberin and inhibits tuberin-hamartin function (14,16,20,21). Furthermore, it is evident from the most recent reports that the small GTPase Rheb is a direct target of tuberin and hamartin in both the Drosophila and mammalian systems (22)(23)(24). These studies demonstrate Rheb GTPase-activating protein activity for tuberin, and the loss of tuberin and/or hamartin lead to an increase in GTP-bound Rheb, which in turn leads to activation of the mammalian target of rapamycin/S6K/4E-binding protein signaling pathway.Thus, recent studies have placed tuberin and hamartin in growth signaling pathways; however, critical CNS functions of these proteins remain unknown. Our recent work demonstrates that unlike kidney lesions, CNS lesions such as cortical tubers do not display a second somatic mutation and other mechanisms might play a role during tumorigenesis in the CNS (25). Furthermore, we have shown that the tuberinhamartin complex exists with neurofilament light chain and ezrin, radixin, and moesin (ERM) proteins in the growth cone and could play a role in neuronal migration (26). In our efforts to understand the tuberin-hamartin complex in the CNS, we have identified Pam (protein-associated with Myc) (27) as a physiologically relevant tuberin interactor and have shown that Pam exists in complex with tuberin-hamartin in the CNS. Our results also demonstrate that HIW, the Drosophila homolog of Pam, can genetically interact with dTsc1-dTsc2 in the fly. The evidence that the Pam homologs in worm (RPM-1) (28, 29) and in Drosophila (HIW) (30) are important regulators of synaptic growth suggests that the interaction of Pam with tuberin-hamartin will be highly relevant within the CNS. EXPERIMENTAL PROCEDUREScDNA Const...

Autografting is the gold-standard method for facial nerve repair with tissue loss. Its association with high-quality scaffolds and cell implants has disclosed distinct experimental outcomes. The aim of this study was to evaluate the functional and histological effects of bone marrow stem cells (BMSC) combined with polyglycolic acid tube (PGAt) in autografted rat facial nerves. After neurotmesis of the mandibular branch of the rat facial nerve, surgical repair consisted of nerve autografting (groups A-E) contained in pGAT (groups B-E), filled with basement membrane matrix (groups C-E) with undifferentiated BMSC (group D) or Schwann-like cells that had differentiated from BMSC (group E). Axon morphometrics and an objective compound muscle action potentials (CMAP) analysis were conducted. Immunofluorescence assays were carried out with Schwann cell marker S100 and anti-β-galactosidase to label exogenous cells. Six weeks after surgery, animals from either cell-containing group had mean CMAP amplitudes significantly higher than control groups. Differently from other groups, facial nerves with Schwann-like cell implants had mean axonal densities within reference values. This same group had the highest mean axonal diameter in distal segments. We observed expression of the reporter gene lacZ in nerve cells in the graft and distally from it in groups D and E. Group-E cells had lacZ coexpressed with S100. In conclusion, regeneration of the facial nerve was improved by BMSC within PGAt in rats, yet Schwann-like cells were associated with superior effects. Accordingly, groups D and E had BMSC integrated in neural tissue with maintenance of former cell phenotype for six weeks.

Tuberous sclerosis complex, an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. The proteins encoded by TSC1 and TSC2, hamartin and tuberin, respectively, associate with each other forming a tight complex. Here we show that hamartin binds the neurofilament light chain and it is possible to recover the hamartin-tuberin complex over the neurofilament light chain rod domain spanning amino acids 93-156 by affinity precipitation. Homologous rod domains in other intermediate filaments such as neurofilament medium chain, ␣-internexin, vimentin, and desmin are not able to bind hamartin. In cultured cortical neurons, hamartin and tuberin co-localize with neurofilament light chain preferentially in the proximal to central growth cone region. Interestingly, in the distal part of the growth cone hamartin overlaps with the ezrin-radixin-moesin family of actin binding proteins, and we have validated the interaction of hamartin with moesin. These results demonstrate that hamartin may anchor neuronal intermediate filaments to the actin cytoskeleton, which may be critical for some of the CNS functions of the hamartin-tuberin complex, and abolishing this through mutations in TSC1 or TSC2 may lead to certain neurological manifestations associated with the disease.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in multiple organs, notably the brain and kidneys. The disease is caused by mutations in TSC1 or TSC2 genes, coding hamartin and tuberin, respectively. Immunofluorescence analysis of tuberin and hamartin performed here demonstrates that both proteins are specifically expressed in the distal urinary tubule, comprising the distal tubules, connecting segment, and collecting ducts. Hamartin, distinct from tuberin, is expressed in the thick ascending limbs of Henle and in juxtaglomerular cells, where it colocalizes with renin. In positive epithelial cells, tuberin localizes to the cytoplasm as well as the apical membrane. Hamartin, however, preferentially localizes to the apical membrane. The two proteins colocalize at the apical membrane of type A intercalated cells and connecting tubule cells, whereas in type B intercalated cells they reveal a variable pattern of expression. The cell-specific expression of tuberin and hamartin described here will provide critical insight into the cell types that give rise to kidney lesions, and the tumor suppressor role of these proteins in TSC.

Regeneration of the facial nerve was improved by both uBMSC and dBMSC in rats, yet uBMSC was associated with superior functional results.

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)29 allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.

BackgroundCulturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.MethodsOrgans of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα). Immunofluorescence assays were conducted for phenotype characterization.ResultsThe TGFα group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells.ConclusionsDissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.