The hypothalamic–pituitary–thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gα_q/11proteins. Activation of Gα_q/11pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gα_q/11proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gα_q/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.

Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at all time points.

Keeping neural stem cells under proliferation, followed by terminal differentiation, can substantially increase the number of neurons generated. With regard to the usability of proliferating neurospheres (NSPHs) cultures, adherent induction protocols have not yet been studied in comparison to embryoid body (EB)-based protocols. To compare these proctocols, neural induction of human induced pluripotent stem cells was performed by dual SMAD inhibition under both adherent and free-floating EB culture conditions. After 10 days, we transferred cells to low-attachment culture plates and proliferated them as free-floating neurospheres. RNA was collected, transcribed to cDNA and analyzed for sonic hedgehog expression that plays an important role during proliferation process. NSPHs were analyzed by immunofluorescence imaging directly and upon continued differentiation. The EB-based approach yielded in higher numbers of cells expressing the neural stem cell marker Nestin, and showed in contrast to the adherent induction protocol increased expression levels of sonic hedgehog. Although improvements to culture consistency and reliability are desirable, the EB-based protocol appears to be superior to the adherent protocol for both, the proliferation and differentiation capacity.

Mutations in the brain-specific β-tubulin 4A (TUBB4A) gene cause a broad spectrum of diseases, ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Currently, the mechanisms of how TUBB4A variants lead to this pleiotropic manifestation remain elusive. Here, we investigated whether TUBB4A mutations causing either DYT-TUBB4A (p.R2G and p.Q424H) or H-ABC (p.R2W and p.D249N) exhibit differential effects at the molecular and cellular levels. Using live-cell imaging of disease-relevant oligodendrocytes and total internal reflection fluorescence microscopy of whole-cell lysates, we observed divergent impact on microtubule polymerization and microtubule integration, partially reflecting the observed pleiotropy. Moreover, in silico simulations demonstrated that the mutants rarely adopted a straight heterodimer conformation in contrast to wild type. In conclusion, for most of the examined variants, we deciphered potential molecular disease mechanisms that may lead to the diverse clinical manifestations and phenotype severity across and within each TUBB4A-related disease.