Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

Building the Future Therapies for Down Syndrome: The Third International Conference of the T21 Research Society.

Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6-9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer's disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar-ma-cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21.

Opportunities, barriers, and recommendations in down syndrome research.

BACKGROUND: Recent advances in medical care have increased life expectancy and improved the quality of life for people with Down syndrome (DS). These advances are the result of both pre-clinical and clinical research but much about DS is still poorly understood. In 2020, the NIH announced their plan to update their DS research plan and requested input from the scientific and advocacy community. OBJECTIVE: The National Down Syndrome Society (NDSS) and the LuMind IDSC Foundation worked together with scientific and medical experts to develop recommendations for the NIH research plan. METHODS: NDSS and LuMind IDSC assembled over 50 experts across multiple disciplines and organized them in eleven working groups focused on specific issues for people with DS. RESULTS: This review article summarizes the research gaps and recommendations that have the potential to improve the health and quality of life for people with DS within the next decade. CONCLUSIONS: This review highlights many of the scientific gaps that exist in DS research. Based on these gaps, a multidisciplinary group of DS experts has made recommendations to advance DS research. This paper may also aid policymakers and the DS community to build a comprehensive national DS research strategy.

Physiological Characterization and Transcriptomic Properties of GnRH Neurons Derived From Human Stem Cells.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play a key role in the regulation of reproductive function. In this study, we sought an efficient method for generating GnRH neurons from human embryonic and induced pluripotent stem cells (hESC and hiPSC, respectively). First, we found that exposure of primitive neuroepithelial cells, rather than neuroprogenitor cells, to fibroblast growth factor 8 (FGF8), was more effective in generating GnRH neurons. Second, addition of kisspeptin to FGF8 further increased the efficiency rates of GnRH neurogeneration. Third, we generated a fluorescent marker mCherry labeled human embryonic GnRH cell line (mCh-hESC) using a CRISPR-Cas9 targeting approach. Fourth, we examined physiological characteristics of GnRH (mCh-hESC) neurons: similar to GnRH neurons in vivo, they released the GnRH peptide in a pulsatile manner at ~60 min intervals; GnRH release increased in response to high potassium, kisspeptin, estradiol, and neurokinin B challenges; and injection of depolarizing current induced action potentials. Finally, we characterized developmental changes in transcriptomes of GnRH neurons using hESC, hiPSC, and mCh-hESC. The developmental pattern of transcriptomes was remarkably similar among the 3 cell lines. Collectively, human stem cell-derived GnRH neurons will be an important tool for establishing disease models to understand diseases, such as idiopathic hypothalamic hypogonadism, and testing contraceptive drugs.

CRISPR/Cas9 editing of APP C-terminus attenuates ß-cleavage and promotes α-cleavage.

CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-ß-cleavage and Aß production, while up-regulating neuroprotective APP-α-cleavage. APP N-terminus and compensatory APP-homologues remain intact, with no apparent effects on neurophysiology in vitro. Robust APP-editing is seen in human iPSC-derived neurons and mouse brains with no detectable off-target effects. Our strategy likely works by limiting APP and BACE-1 approximation, and we also delineate mechanistic events that abrogates APP/BACE-1 convergence in this setting. Our work offers conceptual proof for a selective APP silencing strategy.

Mutations in GFAP Disrupt the Distribution and Function of Organelles in Human Astrocytes.

How mutations in glial fibrillary acidic protein (GFAP) cause Alexander disease (AxD) remains elusive. We generated iPSCs from two AxD patients and corrected the GFAP mutations to examine the effects of mutant GFAP on human astrocytes. AxD astrocytes displayed GFAP aggregates, recapitulating the pathological hallmark of AxD. RNA sequencing implicated the endoplasmic reticulum, vesicle regulation, and cellular metabolism. Corroborating this analysis, we observed enlarged and heterogeneous morphology coupled with perinuclear localization of endoplasmic reticulum and lysosomes in AxD astrocytes. Functionally, AxD astrocytes showed impaired extracellular ATP release, which is responsible for attenuated calcium wave propagation. These results reveal that AxD-causing mutations in GFAP disrupt intracellular vesicle regulation and impair astrocyte secretion, resulting in astrocyte dysfunction and AxD pathogenesis.

Aberrant Calcium Signaling in Astrocytes Inhibits Neuronal Excitability in a Human Down Syndrome Stem Cell Model.

Down syndrome (DS) is a genetic disorder that causes cognitive impairment. The staggering effects associated with an extra copy of human chromosome 21 (HSA21) complicates mechanistic understanding of DS pathophysiology. We examined the neuron-astrocyte interplay in a fully recapitulated HSA21 trisomy cellular model differentiated from DS-patient-derived induced pluripotent stem cells (iPSCs). By combining calcium imaging with genetic approaches, we discovered the functional defects of DS astroglia and their effects on neuronal excitability. Compared with control isogenic astroglia, DS astroglia exhibited more-frequent spontaneous calcium fluctuations, which reduced the excitability of co-cultured neurons. Furthermore, suppressed neuronal activity could be rescued by abolishing astrocytic spontaneous calcium activity either chemically by blocking adenosine-mediated signaling or genetically by knockdown of inositol triphosphate (IP3) receptors or S100B, a calcium binding protein coded on HSA21. Our results suggest a mechanism by which DS alters the function of astrocytes, which subsequently disturbs neuronal excitability.

Modeling Down Syndrome with Patient iPSCs Reveals Cellular and Migration Deficits of GABAergic Neurons.

The brain of Down syndrome (DS) patients exhibits fewer interneurons in the cerebral cortex, but its underlying mechanism remains unknown. By morphometric analysis of cortical interneurons generated from DS and euploid induced pluripotent stem cells (iPSCs), we found that DS GABA neurons are smaller and with fewer neuronal processes. The proportion of calretinin over calbindin GABA neurons is reduced, and the neuronal migration capacity is decreased. Such phenotypes were replicated following transplantation of the DS GABAergic progenitors into the mouse medial septum. Gene expression profiling revealed altered cell migratory pathways, and correction of the PAK1 pathway mitigated the cell migration deficit in vitro. These results suggest that impaired migration of DS GABAergic neurons may contribute to the reduced number of interneurons in the cerebral cortex and hippocampus in DS patients.

MDM2 inhibition rescues neurogenic and cognitive deficits in a mouse model of fragile X syndrome.

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein (FMRP). However, the mechanism remains unclear, and effective treatment is lacking. We show that loss of FMRP leads to activation of adult mouse neural stem cells (NSCs) and a subsequent reduction in the production of neurons. We identified the ubiquitin ligase mouse double minute 2 homolog (MDM2) as a target of FMRP. FMRP regulates Mdm2 mRNA stability, and loss of FMRP resulted in elevated MDM2 mRNA and protein. Further, we found that increased MDM2 expression led to reduced P53 expression in adult mouse NSCs, leading to alterations in NSC proliferation and differentiation. Treatment with Nutlin-3, a small molecule undergoing clinical trials for treating cancer, specifically inhibited the interaction of MDM2 with P53, and rescued neurogenic and cognitive deficits in FMRP-deficient mice. Our data reveal a potential regulatory role for FMRP in the balance between adult NSC activation and quiescence, and identify a potential new treatment for fragile X syndrome.

The amyloid precursor protein (APP) intracellular domain regulates translation of p44, a short isoform of p53, through an IRES-dependent mechanism.

p44 is a short isoform of the tumor suppressor protein p53 that is regulated in an age-dependent manner. When overexpressed in the mouse, it causes a progeroid phenotype that includes premature cognitive decline, synaptic defects, and hyperphosphorylation of tau. The hyperphosphorylation of tau has recently been linked to the ability of p44 to regulate transcription of relevant tau kinases. Here, we report that the amyloid precursor protein (APP) intracellular domain (AICD), which results from the processing of the APP, regulates translation of p44 through a cap-independent mechanism that requires direct binding to the second internal ribosome entry site (IRES) of the p53 mRNA. We also report that AICD associates with nucleolin, an already known IRES-specific trans-acting factor that binds with p53 IRES elements and regulates translation of p53 isoforms. The potential biological impact of our findings was assessed in a mouse model of Alzheimer's disease. In conclusion, our study reveals a novel aspect of AICD and p53/p44 biology and provides a possible molecular link between APP, p44, and tau.

The cyclic AMP phenotype of fragile X and autism.

Cyclic AMP (cAMP) is a second messenger involved in many processes including mnemonic processing and anxiety. Memory deficits and anxiety are noted in the phenotype of fragile X (FX), the most common heritable cause of mental retardation and autism. Here we review reported observations of altered cAMP cascade function in FX and autism. Cyclic AMP is a potentially useful biochemical marker to distinguish autism comorbid with FX from autism per se and the cAMP cascade may be a viable therapeutic target for both FX and autism.

Normal Neurogenesis but Abnormal Gene Expression in Human Fragile X Cortical Progenitor Cells.

Human stem and progenitor cells offer an innovative way to study early events in development. An exciting new opportunity for these cells is their application to study the underlying developmental consequences of genetic diseases. Because many diseases, ranging from leukemias to developmental disorders, are caused by single-gene defects, stem and progenitor cells that carry disease-causing genetic mutations are invaluable in understanding and treating disease. We have characterized human neural progenitor (hNPCs) cells that carry a single-gene defect that leads to the neurodevelopmental disorder Fragile X syndrome (FX). A loss-of-function mutation in the FMR1 gene leads to subtle changes in neural development and subsequent mental impairment characteristic of FX. hNPCs were isolated from fetal cortex carrying the FMR1 mutation to determine whether aberrations occur in their proliferation and differentiation. As expected, FX hNPCs have reduced expression of the FMR1 gene product Fragile X mental retardation protein (FMRP), and this decrease is maintained in culture and following differentiation. In contrast to a previously published report, the proliferation of FX hNPCs and their differentiation into neurons is not different from unaffected controls. Although the early development of FX hNPCs is essentially normal, microarray analysis reveals novel changes in the expression of signal transduction genes in FX hNPCs. Therefore, hNPCs have intrinsic characteristics that can be investigated to further our understanding and potential treatment of developmental disorders such as FX.

The cyclic AMP cascade is altered in the fragile X nervous system.

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.

High-resolution imaging demonstrates dynein-based vesicular transport of activated Trk receptors.

Target-derived neurotrophins signal from nerve endings to the cell body to influence cellular and nuclear responses. The retrograde signal is conveyed by neurotrophin receptors (Trks) themselves. To accomplish this, activated Trks may physically relocalize from nerve endings to the cell bodies. However, alternative signaling mechanisms may also be used. To identify the vehicle wherein the activated Trks are located and transported, and to identify associated motor proteins that would facilitate transport, we use activation-state specific antibodies in concert with immunoelectron microscopy and deconvolution microscopy. We show that the'activated Trks within rat sciatic nerve axons are preferentially localized to coated and uncoated vesicles. These vesicles are moving in a retrograde direction and so accumulate distal to a ligation site. The P-Trk containing vesicles, in turn, colocalize with dynein components, and not with kinesins. Collectively, these results indicate activated Trk within axons travel in vesicles and dynein is the motor that drives these vesicles towards the cell bodies.