The expression of estrogen receptors (ERs) in the developing and adult human brain has not been clearly established, although estrogens are crucial for neuronal differentiation, synapse formation, and cognitive functions. By using immunohistochemistry, we have studied the distribution of ER alpha and ER beta in human cerebral cortex and hippocampus from early prenatal stages to adult life. ER alpha was detected in the cortex at 9 gestational weeks (GW), with a high expression in proliferating zones and the cortical plate. The staining intensity decreased gradually during prenatal development but increased again from birth to adulthood. In contrast, ER beta was first detected at 15 GW in proliferating zones, and at 16/17 GW, numerous ER beta immunopositive cells were also observed in the cortical plate. ER beta expression persisted in the adult cortex, being widely distributed throughout cortical layers II-VI. In addition, from around 15 GW to adulthood, ER alpha and ER beta were expressed in human hippocampus mainly in pyramidal cells of Ammon's horn and in the dentate gyrus. Western blotting and immunohistochemistry in the adult cerebral cortex and hippocampus revealed lower protein expression of ER alpha compared with ER beta. Double immunostaining showed that during fetal life both ERs are expressed in neurons as well as in radial glia, although only ER alpha is expressed in the Cajal-Retzius neurons of the marginal zone. These observations demonstrate that the expression of ER alpha and ER beta displays different spatial-temporal patterns during human cortical and hippocampal development and suggest that both ERs may play distinct roles in several processes related to prenatal brain development.

Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age = 33.1 +/- 2.0 yr, height = 175.9 +/- 1.7 cm, body mass = 81.2 +/- 3.8 kg, body fat = 22.5 +/- 1.9%; means +/- SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit anti-human leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum.

Eye formation is regulated by a complex network of eye field transcription factors (EFTFs) including LIM-homeodomain gene Lhx2. We disrupted Lhx2 function at different stages during this process using a conditional knockout strategy in mice. We find that Lhx2 function is required in an ongoing fashion to maintain optic identity across multiple stages, from the formation of the optic vesicle to the differentiation of the neuroretina. At each stage loss of Lhx2 led to upregulation of a set of molecular markers that are normally expressed in the thalamic eminence and in the anterodorsal hypothalamus in a portion of the optic vesicle or retina. Furthermore, the longer Lhx2 function was maintained, the further optic morphogenesis progressed. Early loss of function caused profound mispatterning of the entire telencephalic-optic-hypothalamic field, such that the optic vesicle became mispositioned and appeared to arise from the diencephalic-telencephalic boundary (DTB). At subsequent stages, loss of Lhx2 did not affect optic vesicle position, but caused arrest of optic cup formation. If Lhx2 was selectively disrupted in the neuroretina from E11.5, the neuroretina showed gross dysmorphology along with aberrant expression of markers specific to the thalamic eminence and anterodorsal hypothalamus. Our findings indicate a continual requirement for Lhx2 throughout the early stages of optic development, not only to maintain optic identity by suppressing alternative fates, but also to mediate multiple steps of optic morphogenesis. These findings provide new insight into the anophthalmic phenotype of the Lhx2 mutant and reveal novel roles for this transcription factor in eye development.

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Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer's disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

SummaryTauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

No abstract