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ImportanceA few case studies have reported the APOE Christchurch (APOECh) variant to confer protective effects for Alzheimer’s disease (AD) and a higher risk of premature cardiovascular disease (CVD). However, these studies primarily focused on a single individual or siblings from the same family.ObjectiveWe sought to characterize the clinical characteristics of individuals with APOECh variation in 455,306 participants of the UK Biobank (UKB) to determine whether it is associated with AD protection or cardiovascular risk.Design, Setting, and ParticipantsA total of 37 individuals were identified as heterozygous carriers of APOECh in UKB sequencing data as of March 2022, resulting in allele frequency consistent with gnomAD (4.06×10−5). We limited our study to 36 European carriers and generated a noncarrier cohort matched on age, sex, and ancestry. Case-control analyses were performed to evaluate the frequency of 11 binary traits and differences in distributions of 80 quantitative traits and 10 polygenic risk scores (PRS) for lipid traits, CVD, and neurodegenerative diseases.Main Outcomes and MeasuresWe compared the frequencies of binary traits (binomial distribution probability) and distributions of quantitative traits and PRS (Kolmogorov-Smirnov test).ResultsAll 37 carriers are free of AD and only 4 have a parental history of AD. There are 22 out of 37 carriers with>1 cardiovascular (CV) condition in clinical and/or self-reported data, two of whom passed away due to heart disease. However, frequency of CVD, dyslipidemia, and hypertension is not enriched in carriers compared with matched non-carriers. Additionally, apolipoprotein B (apoB) is significantly lower in APOECh carriers before (p=0.004) and after statin adjustment (p=0.04). Comparisons of PRS show that carriers and non-carriers have a similar genetic burden of developing dyslipidemia and CVD, but carriers have lower PRS-based AD risk (p=0.02).Conclusions and RelevanceThis study demonstrates that APOECh carriers may have lower levels of apoB and a lower risk of AD. Cohorts with enriched cases are needed to further investigate whether the protective effect is linked to APOE genotypes or other factors.Key PointsQuestionHow does APOE Christchurch (APOECh) variant influence risk of Alzheimer’s disease (AD), cardiovascular disease, and dyslipidemia?FindingsAPOECh carriers do not have an elevated risk of dyslipidemia. However, carriers have lower levels of apolipoprotein B (apoB). Comparing polygenic risk scores of carriers vs. noncarriers of APOECh shows carriers are at a lower risk of developing AD before but not after adjusting for multiple comparisons.MeaningCarriers of APOECh may have a lower risk of developing AD due to lower apoB; however, further epidemiological and model organism studies are needed to validate these observations from the current study.

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools. ispartof: REPRODUCTIVE TOXICOLOGY vol:112 pages:23-35 ispartof: location:United States status: published

Alzheimer’s disease (AD) is the most common form of dementia, currently affecting 35 million people worldwide. Apolipoprotein E (APOE) ε4 allele is the major risk factor for sporadic, late-onset AD (LOAD), which comprises over 95% of AD cases, increasing the risk of AD 4-12 fold. Despite this, the role of APOE in AD pathogenesis is still a mystery. Aiming for a better understanding of APOE-specific effects, the ADAPTED consortium analyzed and integrated publicly available data of multiple OMICS technologies from both plasma and brain stratified by APOE haplotype (APOE2, APOE3 and APOE4). Combining genome-wide association studies (GWAS) with differential mRNA and protein expression analyses and single-nuclei transcriptomics, we identified genes and pathways contributing to AD in both APOE dependent and independent fashion. Interestingly, we characterized a set of biomarkers showing plasma and brain consistent protein profiles and opposite trends in APOE2 and APOE4 AD cases that could constitute screening tools for a disease that lacks specific blood biomarkers. Beside the identification of APOE-specific signatures, our findings advocate that this novel approach, based on the concordance across OMIC layers and tissues, is an effective strategy for overcoming the limitations of often underpowered single-OMICS studies.

Astrocytes, the main supportive cell type of the brain, show functional impairments upon ageing and in a broad spectrum of neurological disorders. Limited access to human astroglia for pre-clinical studies has been a major bottleneck delaying our understanding of their role in brain health and disease. We demonstrate here that functionally mature human astrocytes can be generated by SOX9 overexpression for 6 days in pluripotent stem cell (PSC)-derived neural progenitor cells. Inducible (i)SOX9-astrocytes display functional properties comparable to primary human astrocytes comprising glutamate uptake, induced calcium responses and cytokine/growth factor secretion. Importantly, electrophysiological properties of iNGN2-neurons co-cultured with iSOX9-astrocytes are indistinguishable from gold-standard murine primary cultures. The high yield, fast timing and the possibility to cryopreserve iSOX9-astrocytes without losing functional properties makes them suitable for scaled-up production for high-throughput analyses. Our findings represent a step forward to an all-human iPSC-derived neural model for drug development in neuroscience and towards the reduction of animal use in biomedical research. Graphical Abstract

Vacuolar H(+)-ATPase–dependent (V-ATPase–dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell–derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase–dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase–dependent signaling and protein degradation in the developing human central nervous system.

APOE genotype is the strongest genetic risk factor for Alzheimer’s Disease (AD). The low degree of homology between mouse and human APOE is a concerning issue in preclinical models currently used to study the role of this gene in AD pathophysiology. A key objective of ADAPTED (Alzheimer’s Disease Apolipoprotein Pathology for Treatment Elucidation and Development) project was to generate in vitro models that better recapitulate human APOE biology. We describe a new set of induced pluripotent stem cells (iPSC) lines carrying common APOE variants (Ɛ2, Ɛ3, and Ɛ3/Ɛ4) and a knock-out isogenic to the parental APOE Ɛ4/Ɛ4 line (UKBi011-A)., This study was funded by the ADAPTED (Alzheimer’s Disease Apolipoprotein Pathology for Treatment Elucidation and Development) consortium which has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement No 115975. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and the European Federation of Pharmaceutical Industries and Associations.

Neurog2 is the gene encoding the neuronal transcription factor NGN2, which can convert stem cells into functional neurons in a fast and efficient way. Here we report the generation of two iPS cell lines, where DOX inducible constructs of neurog2 either without or with T2A-eGFP were inserted into the safe-site locus AAVS1. These iPS cell lines, BIONi010-C-13 and BIONi010-C-15, respectively, stay pluripotent without DOX but differentiate to (GFP positive) neurons when DOX is added without the need of differentiation factors.

Background Lysophosphatidic acids (LPAs) are bioactive signaling phospholipids that have been implicated in Alzheimer’s disease (AD). It is largely unknown whether LPAs are associated with AD pathology and progression from mild cognitive impairment (MCI) to AD. Methods The current study was performed on cerebrospinal fluid (CSF) and plasma samples of 182 MCI patients from two independent cohorts. We profiled LPA-derived metabolites using liquid chromatography-mass spectrometry. We evaluated the association of LPAs with CSF biomarkers of AD, Aβ-42, p-tau, and total tau levels overall and stratified by APOE genotype and with MCI to AD progression. Results Five LPAs (C16:0, C16:1, C22:4, C22:6, and isomer-LPA C22:5) showed significant positive association with CSF biomarkers of AD, Aβ-42, p-tau, and total tau, while LPA C14:0 and C20:1 associated only with Aβ-42 and alkyl-LPA C18:1, and LPA C20:1 associated with tau pathology biomarkers. Association of cyclic-LPA C16:0 and two LPAs (C20:4, C22:4) with Aβ-42 levels was found only in APOE ε4 carriers. Furthermore, LPA C16:0 and C16:1 also showed association with MCI to AD dementia progression, but results did not replicate in an independent cohort. Conclusions Our findings provide evidence that LPAs may contribute to early AD pathogenesis. Future studies are needed to determine whether LPAs play a role in upstream of AD pathology or are downstream markers of neurodegeneration.

Many Alzheimer’s disease (AD) genes including Apolipoprotein E (APOE) are found to be expressed in blood-derived macrophages and thus may alter blood protein levels. We measured 91 neuro-proteins in plasma from 316 participants of the Rotterdam Study (incident AD = 161) using Proximity Extension Ligation assay. We studied the association of plasma proteins with AD in the overall sample and stratified by APOE. Findings from the Rotterdam study were replicated in 186 AD patients of the BioFINDER study. We further evaluated the correlation of these protein biomarkers with total tau (t-tau), phosphorylated tau (p-tau) and amyloid-beta (Aβ) 42 levels in cerebrospinal fluid (CSF) in the Amsterdam Dementia Cohort (N = 441). Finally, we conducted a genome-wide association study (GWAS) to identify the genetic variants determining the blood levels of AD-associated proteins. Plasma levels of the proteins, CDH6 (β = 0.638, P = 3.33 × 10−4) and HAGH (β = 0.481, P = 7.20 × 10−4), were significantly elevated in APOE ε4 carrier AD patients. The findings in the Rotterdam Study were replicated in the BioFINDER study for both CDH6 (β = 1.365, P = 3.97 × 10−3) and HAGH proteins (β = 0.506, P = 9.31 × 10−7) when comparing cases and controls in APOE ε4 carriers. In the CSF, CDH6 levels were positively correlated with t-tau and p-tau in the total sample as well as in APOE ε4 stratum (P −3). The HAGH protein was not detected in CSF. GWAS of plasma CDH6 protein levels showed significant association with a cis-regulatory locus (rs111283466, P = 1.92 × 10−9). CDH6 protein is implicated in cell adhesion and synaptogenesis while HAGH protein is related to the oxidative stress pathway. Our findings suggest that these pathways may be altered during presymptomatic AD and that CDH6 and HAGH may be new blood-based biomarkers.

INTRODUCTION: Tauopathies are neurodegenerative diseases characterized by TAU protein-related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. METHODS: To create a new in vitro tauopathy model, we generated a footprint-free triple MAPT-mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats-FokI and piggyBac transposase technology. RESULTS: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU-mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. DISCUSSION: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU-mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening. ispartof: ALZHEIMERS & DEMENTIA vol:14 issue:10 pages:1261-1280 ispartof: location:United States status: published

Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a "fully-humanized" set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/. ispartof: STEM CELL RESEARCH vol:54 ispartof: location:England status: published

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.eTOCThe 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.

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.

Summary Tauopathies 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., Highlights • Analysis of ZFN-engineered MAPT IVS10+16 with or without additional P301S mutation • Neurodevelopmental phenotypes in ZFN and patient-derived MAPT IVS10+16 neurons • Neurodegenerative phenotypes in MAPT IVS10+16/P301S double-mutant neurons, In this article, Verheyen and colleagues introduced 2 MAPT mutations (IVS10+16/P301S) into healthy donor iPSCs. Characterization of MAPT IVS10+16 neurons revealed neurodevelopmental neuron subtype differences, reduced neural progenitor proliferation, and aberrant WNT/SHH signaling, while the additional pro-aggregant P301S mutation revealed phenotypes more related to neurodegeneration. These iPSCs could help to unravel a potential link between pathogenic 4R tau and FTD.

Alzheimer's disease (AD) is the most frequent neurodegenerative disease amongst the elderly. The SNPs rs429358 and rs7412 in the APOE gene are the most common risk factor for sporadic AD, and there are three different alleles commonly referred to as APOE-epsilon 2, APOE-epsilon 3 and APOE-epsilon 4. Induced pluripotent stem cells (iPSCs) hold great promise to model AD as such cells can be differentiated in vitro to the required cell type. Here we report the use of CRISPR/Cas9 technology employed on iPSCs from a healthy individual with an APOE-epsilon 3/epsilon 4 genotype to obtain isogenic APOE-epsilon 2/epsilon 2, APOE-epsilon 3/epsilon 3, APOE-epsilon 4/epsilon 4 lines as well as an APOE-knock-out line.

Cajal-Retzius (CR) cells of the mammalian neocortex co-express the extracellular matrix protein Reelin and p73, a transcription factor involved in cell death and survival. Most neocortical CR cells derive from the cortical hem, with minor additional sources. We analyzed the distribution of Reelin and p73 immunoreactive (ir) neurons in the telencephalon of Lacerta galloti from early embryonic stages to hatching. Numerous Reelin-ir cells appeared in the pallial MZ from the preplate stage onward. Conversely, p73-ir cells were rare in the pallial preplate and not observed in the cortical plate. Subpallial p73-ir cells spread from the septum and the telencephalic-diencephalic boundary to the pial surface of the basal forebrain and amygdala, respectively, where they co-expressed Reelin and p73. A small group of Reelin/p73-ir CR cells appeared in a rudimentary cortical hem at the interface of the medial cortex and choroid plexus. Comparison with early embryonic stages of mice and humans showed similar foci of p73-ir cells in the septum and at the telencephalic-diencephalic boundary and revealed an increasing prominence of the cortical hem, in parallel with increasing numbers of neocortical Reelin/p73 positive CR cells, which attain highest differentiation in the human brain. Our data show that Reelin-expression in the pallium is evolutionarily conserved and independent of a cortical hem, and suggest that p73 in the cortical hem may be involved in the evolutionary increase in number and complexity of the mammalian neocortical CR cells.

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.

p73, a homolog of the p53 tumor suppressor, codes for full-length transactivating (TA) and N-terminally truncated (DeltaN) isoforms, with pro- and anti-apoptotic activities, respectively. We examined the expression of the main p73 isoforms in adult human and mouse telencephalon and choroid plexus by immunohistochemistry on paraffin sections, and immunoblotting (IB) of tissue extracts and cerebrospinal fluid (CSF), using antibodies against different protein domains. Cortical neurons expressed TAp73 predominantly in the cytoplasm and DeltaNp73 mainly in the nucleus, with partial overlap in the cytoplasm. Highest expression was found in the hippocampus. IB showed an array of TAp73 variants in adult human cortex and hippocampus. IB of human choroid plexus and CSF using TAp73-specific antibodies revealed the presence of a approximately 90-kDa protein whose molecular weight was reduced after N-deglycosylation, suggesting that glycosylated TAp73 is exported into the CSF. In the mouse, high expression of TAp73 was also detected in the subcommissural organ (SCO), an ependymal gland absent in adult humans. TAp73 colocalized with anti-fibra-Reissner-antibody (AFRU), which is a marker of Reissner's fiber, the secreted SCO product. p73-deficient mice had generalized cortical hypoplasia and hydrocephalus; in addition, we observed a dramatic size reduction of the choroid plexus. However, the SCOs were apparently unaltered and continued to secrete Reissner's fiber. Our findings point to complex and widespread p73 activities in the maintenance of adult cortical neurons and in brain homeostasis. TAp73 in the CSF may play important roles in the maintenance of the adult ventricular wall as well as in the development of the proliferating neuroepithelium.

Epilepsy and mental retardation, originally of unknown cause, are now known to result from many defects including cortical malformations, neuronal circuitry disorders and perturbations of neuronal communication and synapse function. Genetic approaches in combination with MRI and related imaging techniques continually allow a re-evaluation and better classification of these disorders. Here we review our current understanding of some of the primary defects involved, with insight from recent molecular biology advances, the study of mouse models and the results of neuropathology analyses. Through these studies the molecular determinants involved in the control of neuron number, neuronal migration, generation of cortical laminations and convolutions, integrity of the basement membrane at the pial surface, and the establishment of neuronal circuitry are being elucidated. We have attempted to integrate these results with the available data concerning, in particular, human brain development, and to emphasize the limitations in some cases of extrapolating from rodent models. Taking such species differences into account is clearly critical for understanding the pathophysiological mechanisms associated with these disorders.

To examine the role of the p53 homolog p73 in brain development, we studiedp73-/-,p73+/-,E2F1-/-, andreelermutant mice. p73 in developing brain is expressed in Cajal-Retzius (CR) cells, the cortical hem, and the choroid plexus. p73-expressing CR cells are lost inp73-/-embryos, although Reelin is faintly expressed in the marginal zone. Ectopic neurons in thep73-/-preplate and cortical hem at embryonic day 12 implicate p73 in the early developmental program of the cortex; however, preplate partition and early cortical plate formation are not disturbed. Postnatalp73-/-mice show a mild hypoplasia of the rostral cortex and a severely disrupted architecture of the posterior telencephalon. In the developingp73-/-hippocampus, the most striking abnormality is the absence of the hippocampal fissure, suggesting a role of p73 in cortical folding.p73+/-mice have a less severe cortical phenotype; they display a dorsal shift of the entorhinal cortex and a reduced size of occipital and posterior temporal areas, which acquire entorhinal-like features such as Reelin-positive cells in layer II. CR cells appear unaffected by heterozygosity. We relate the malformations of the posterior pole in p73 mutant mice to alterations of p73 expression in the cortical hem and suggest that p73 forms part of an early signaling network that controls neocortical and archicortical regionalization. In mice deficient for the transcription factor E2F1, a main activator of the TAp73 (transactivating p73) isoform, we find a defect of the caudal cortical architecture resembling thep73+/-phenotype along with reduced TAp73 protein levels and propose that an E2F1-TAp73 dependent pathway is involved in cortical patterning.

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 knock-out 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 ofLhx2led 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. At subsequent stages, loss ofLhx2did not affect optic vesicle position but caused arrest of optic cup formation. IfLhx2was 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 theLhx2mutant and reveal novel roles for this transcription factor in eye development.

p63 and p73, family members of the tumor suppressor p53, are critically involved in the life and death of mammalian cells. They display high homology and may act in concert. The p73 gene is relevant for brain development, and p73-deficient mice display important malformations of the telencephalon. In turn, p63 is essential for the development of stratified epithelia and may also play a part in neuronal survival and aging. We show here that p63 and p73 are dynamically expressed in the embryonic and adult mouse and human telencephalon. During embryonic stages, Cajal-Retzius cells derived from the cortical hem co-express p73 and p63. Comparison of the brain phenotypes of p63- and p73- deficient mice shows that only the loss of p73 function leads to the loss of Cajal-Retzius cells, whereas p63 is apparently not essential for brain development and Cajal-Retzius cell formation. In postnatal mice, p53, p63, and p73 are present in cells of the subventricular zone (SVZ) of the lateral ventricle, a site of continued neurogenesis. The neurogenetic niche is reduced in size in p73-deficient mice, and the numbers of young neurons near the ventricular wall, marked with doublecortin, Tbr1 and calretinin, are dramatically decreased, suggesting that p73 is important for SVZ proliferation. In contrast to their restricted expression during brain development, p73 and p63 are widely detected in pyramidal neurons of the adult human cortex and hippocampus at protein and mRNA levels, pointing to a role of both genes in neuronal maintenance in adulthood. © 2010 Elsevier B.V.

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.

La Reserva Marina de La Graciosa y los Islotes del Norte de Lanzarote (RMLGINL) se creó en 1995 y, desde entonces, las actividades pesqueras de la población local y su cultura del mar han ido decreciendo progresivamente mientras que las actividades turísticas siguen emergiendo como la panacea en una pequeña isla, como La Graciosa, con no más de 600 habitantes. En este artículo tratamos de analizar cómo estos dos procesos están relacionados y qué papel está jugando la reserva marina en este caso. Usamos no sólo información cualitativa de los pescadores locales y de sus grupos domésticos, sino también cuantitativa contrastando algunos parámetros concretos como el número de pescadores y los barcos de pesca activos, los negocios relacionados con el sector servicios (restaurantes, apartamentos, etc.), antes y después de implementación de la reserva marina. En este sentido, un importante elemento es la percepción de esta institución desde la población local, y la evolución de su actitud a favor o en contra. Estos procesos están relacionados con el diseño institucional de las reservas marinas en las Islas Canarias, en donde la participación de las poblaciones pesqueras locales está severamente limitada. The Marine Reserve of La Graciosa Island and the islets of the North of Lanzarote was created in 1995 and, since then, the fishing activities of the local population and their local culture of the sea have been decreasing progressively while tourist activities are emerging as the panacea in a little island as La Graciosa with no more than 600 inhabitants. In this paper we try to analyse how these two processes are linked and what role is playing the local marine protected area in this case. We use not only qualitative information from fishers and their domestic units, but also quantitative data contrasting some objective parameters as the number of fishers and active fishing boats or service related business (restaurants, apartments, etc.), before and after the implementation of the marine reserve. In this sense, an important element is the perception of this institution from the local population, and the evolution of their attitude in favour or against it. These processes are related to the institutional design of the marine reserves in the Canary Island, where the participation of local fishing populations is severely limited.

Disease-relevant human induced pluripotent stem cells (iPSCs) are generated worldwide for research purposes; however, without robust and practical ethical, legal, and quality standards, there is a high risk that their true potential will not be realized. Best practices for tissue procurement, iPSC reprogramming, day-to-day cultivation, quality control, and data management aligned with an ethical and legal framework must be included into daily operations to ensure their promise is maximized. Here we discuss key learning experiences from 7 years of operating the European Bank for induced Pluripotent Stem Cells (EBiSC) and recommend how to incorporate solutions into a daily management framework., Highlights • Ethics for iPSCs must be explicit, GDPR compliant, and allow future research • iPSC use restrictions are linked to consent, reprogramming, and gene editing • Quality control must be implemented from primary tissue handling onward • Robust data management is essential to ensure privacy and enable data sharing, The European Bank for induced Pluripotent Stem Cells discusses common pitfalls during iPSC generation and recommends robust and practical solutions for ethical, legal, and quality frameworks. Through these recommendations, high-quality iPSC lines can be efficiently distributed, maximizing the use of tissue samples that have been gifted to researchers to accelerate disease research and the discovery of novel therapeutic solutions.