Authors: Katharine Nichole Holm, Anthony W. Herren, Sandra L. Taylor, Jamie L. Randol, Kyoungmi Kim, Glenda Espinal, Verónica Martiínez-Cerdeño, Isaac N. Pessah, Randi J. Hagerman and Paul J. Hagerman
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Abstract: Background: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder associated with premutation CGG-repeat expansions (55–200 repeats) in the 5′ non-coding portion of the fragile X mental retardation 1 (FMR1) gene. Core features of FXTAS include progressive tremor/ataxia, cognitive decline, variable brain volume loss, and white matter disease. The principal histopathological feature of FXTAS is the presence of central nervous system (CNS) and non-CNS intranuclear inclusions. Objective: To further elucidate the molecular underpinnings of FXTAS through the proteomic characterization of human FXTAS cortexes. Results: Proteomic analysis of FXTAS brain cortical tissue (n = 8) identified minor differences in protein abundance compared to control brains (n = 6). Significant differences in FXTAS relative to control brain predominantly involved decreased abundance of proteins, with the greatest decreases observed for tenascin-C (TNC), cluster of differentiation 38 (CD38), and phosphoserine aminotransferase 1 (PSAT1); proteins typically increased in other neurodegenerative diseases. Proteins with the greatest increased abundance include potentially novel neurodegeneration-related proteins and small ubiquitin-like modifier 1/2 (SUMO1/2). The FMRpolyG peptide, proposed in models of FXTAS pathogenesis but only identified in trace amounts in the earlier study of FXTAS inclusions, was not identified in any of the FXTAS or control brains in the current study. Discussion: The observed proteomic shifts, while generally relatively modest, do show a bias toward decreased protein abundance with FXTAS. Such shifts in protein abundance also suggest altered RNA binding as well as loss of cell–cell adhesion/structural integrity. Unlike other neurodegenerative diseases, the proteome of end-stage FXTAS does not suggest a strong inflammation-mediated degenerative response.
Authors: Elisa Penna, Jon M. Mangum, Hunter Shepherd, Veronica Martínez-Cerdeño and Stephen C. Noctor
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Abstract: Microglial cells make extensive contacts with neural precursor cells (NPCs) and affiliate with vasculature in the developing cerebral cortex. But how vasculature contributes to cortical histogenesis is not yet fully understood. To better understand functional roles of developing vasculature in the embryonic rat cerebral cortex, we investigated the temporal and spatial relationships between vessels, microglia, and NPCs in the ventricular zone. Our results show that endothelial cells in developing cortical vessels extend numerous fine processes that directly contact mitotic NPCs and microglia; that these processes protrude from vessel walls and are distinct from tip cell processes; and that microglia, NPCs, and vessels are highly interconnected near the ventricle. These findings demonstrate the complex environment in which NPCs are embedded in cortical proliferative zones and suggest that developing vasculature represents a source of signaling with the potential to broadly influence cortical development. In summary, cortical histogenesis arises from the interplay among NPCs, microglia, and developing vasculature. Thus, factors that impinge on any single component have the potential to change the trajectory of cortical development and increase susceptibility for altered neurodevelopmental outcomes.
Authors: Nisha Raj, Zachary T McEachin, William Harousseau, Ying Zhou, Feiran Zhang, Megan E Merritt-Garza, J Matthew Taliaferro, Magdalena Kalinowska, Samuele G Marro, Chadwick M Hales, Elizabeth Berry-Kravis, Marisol W Wolf-Ochoa, Veronica Martinez-Cerdeño, Marius Wernig, Lu Chen, Eric Klann, Stephen T Warren, Peng Jin, Zhexing Wen, Gary J Bassell
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Abstract: Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.
Authors: Zachary Rabow, Taryn Morningstar, Megan Showalter, Hailey Heil, Krista Thongphanh, Sili Fan, Joanne Chan, Verónica Martínez-Cerdeño, Robert Berman, David Zagzag, Evgeny Nudler, Oliver Fiehn, Mirna Lechpammer
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Abstract: Introduction: Dimethyl sulfoxide (DMSO) is a widely used solvent to dissolve hydro- phobic substances for clinical uses and experimental in vivo purposes. While usually regarded safe, our prior studies suggest changes to behavior following DMSO expo- sure. We therefore evaluated the effects of a five-day, short-term exposure to DMSO on postnatal infant rats (P6-10).
Methods: DMSO was intraperitoneally injected for five days at 0.2, 2.0, and 4.0 ml/ kg body mass. One cohort of animals was sacrificed 24 hr after DMSO exposure to analyze the neurometabolic changes in four brain regions (cortex, hippocampus, basal ganglia, and cerebellum) by hydrophilic interaction liquid chromatography. A second cohort of animals was used to analyze chronic alterations to behavior and pathological changes to glia and neuronal cells later in life (P21-P40).
Results: 164 metabolites, including key regulatory molecules (retinoic acid, orotic acid, adrenic acid, and hypotaurine), were found significantly altered by DMSO exposure in at least one of the brain regions at P11 (p < .05). Behavioral tests showed significant hypoactive behavior and decreased social habits to the 2.0 and 4.0 ml DMSO/kg groups (p < .01). Significant increases in number of microglia and astrocytes at P40 were observed in the 4.0 ml DMSO/kg group (at p < .015.)
Conclusions: Despite short-term exposure at low, putatively nontoxic concentrations, DMSO led to changes in behavior and social preferences, chronic alterations in glial cells, and changes in essential regulatory brain metabolites. The chronic neurological effects of DMSO exposure reported here raise concerns about its neurotoxicity and consequent safety in human medical applications and clinical trials.
Authors: Maria Jimena Salcedo-Arellanoa, Brett Dufourc, Yingratana McLennana, Verónica Martínez-Cerdeño, Randi Hagerman
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Abstract: This review aims to assemble many years of research and clinical experience in the fields of neurodevelopment and neuroscience to present an up-to-date understanding of the clinical presentation, molecular and brain pathology associated with Fragile X syndrome, a neurodevelopmental condition that develops with the full mutation of the FMR1 gene, located in the q27.3 loci of the X chromosome, and Fragile X-associated tremor/ataxia syndrome a neurodegenerative disease experienced by aging premutation carriers of the FMR1 gene. It is important to understand that these two syndromes have a very distinct clinical and pathological presentation while sharing the same origin: the mutation of the FMR1 gene; revealing the complexity of expansion genetics.
Authors: Yujing Gao, PhD, Verónica Martínez-Cerdeño, PhD, Kirk J. Hogan, MD, JD, Catriona A. McLean, MD, MBBS, FRCPA, FFSc (RCPA), and Paul J. Lockhart, PhD
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Abstract: Background: Pathogenic variants in the small GTPase Ras Analogue in Brain 39b (RAB39B) have been linked to the development of early-onset parkinsonism. The study was aimed at delineating the clinical and neuropathological features associated with a previously reported pathogenic variant in RAB39B (c.503C>A p.T168K) and testing for dysregulation of RAB39B in idiopathic PD.
Methods: Clinical details of a male individual hemizygous for the T168K variant were collected by systematic review of medical records. Neuropathological studies of fixed brain tissue were performed and steady-state RAB39B levels were determined by western blot analysis.
Results: Neuropathological examination showed extensive dopaminergic neuron loss, widespread Lewy pathology, and iron accumulation in the substantia nigra. Additional pathology was observed in the hippocampus and thalamus. Western blot analysis demonstrated that the T168K variant results in loss of RAB39B. In individuals with idiopathic PD (n = 10, 6 male/4 female), steady-state RAB39B was significantly reduced in the prefrontal cortex and substantia nigra.
Conclusions: T168K RAB39B is unstable in vivo and associated with dopaminergic neuron loss and Lewy pathology. Dysregulation of RAB39B in the prefrontal cortex and substantia nigra of individuals with idiopathic PD potentially implicates the protein more broadly in the pathological mechanisms underlying PD and related Lewy body disorders. © 2020 International Parkinson and Movement Disorder Society
Authors: Stephen. Tran, Hyun-Ik Jun, Jae Hoon Bahn, Adel Azghadi, Gokul Ramaswami, Eric L. Van Nostrand, Thai B. Nguyen, Yun-Hua E. Hsiao, Changhoon Lee, Gabriel A. Pratt, Verónica Martínez-Cerdeño, Randi J. Hagerman, Gene W. Yeo, Daniel H. Geschwind and Xinshu Xiao
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Abstract: Transcriptomic analyses of postmortem brains have begun to elucidate molecular abnormalities in autism spectrum disorder (ASD). However, a crucial pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here we profiled global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of postmortem brains of people with ASD. We observed a global bias for hypoediting in ASD brains, which was shared across brain regions and involved many synaptic genes. We show that the Fragile X proteins FMRP and FXR1P interact with RNA-editing enzymes (ADAR proteins) and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA-editing alterations in ASD and Fragile X syndrome, establishing this as a molecular link between these related diseases. Our findings, which are corroborated across multiple data sets, including dup15q (genomic duplication of 15q11.2-13.1) cases associated with intellectual disability, high- light RNA-editing dysregulation in ASD and reveal new mechanisms underlying this disorder.
Authors: Verónica Martínez-Cerdeño and Stephen C. Noctor
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Authors: Daniel García-Rincón, Javier Díaz-Alonso, Juan Paraíso-Luna, Zaira Ortega, José Aguareles, Adán de Salas-Quiroga, Cristina Jou, Inmaculada de Prada, Verónica Martínez-Cerdeño, Eleonora Aronica, Manuel Guzmán, María Ángeles Pérez-Jiménez and Ismael Galve-Roperh
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Abstract: Alterations of the PI3K/Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway are causally involved in a subset of malformations of cortical development (MCDs) ranging from focal cortical dysplasia (FCD) to hemimegalencephaly and megalencephaly. These MCDs represent a frequent cause of refractory pediatric epilepsy. The endocannabinoid system -especially cannabinoid CB1 receptor- exerts a neurodevelopmental regulatory role at least in part via activation of mTORC1 signaling. Therefore, we sought to characterize the possible contribution of endocannabinoid system signaling to FCD. Confocal microscopy characterization of the CB1 receptor expression and mTORC1 activation was conducted in FCD Type II resection samples. FCD samples were subjected to single nucleotide polymorphism screening for endocannabinoid system elements, as well as CB1 receptor gene sequencing. Cannabinoid CB1 receptor levels were increased in FCD with overactive mTORC1 signaling. CB1 receptors were enriched in phospho-S6-positive cells including balloon cells (BCs) that co-express aberrant markers of undifferentiated cells and dysplastic neurons. Pharmacological regulation of CB1 receptors and the mTORC1 pathway was performed in fresh FCD-derived organotypic cultures. HU-210-evoked activation of CB1 receptors was unable to further activate mTORC1 signaling, whereas CB1 receptor blockade with rimonabant attenuated mTORC1 overactivation. Alterations of the endocannabinoid system may thus contribute to FCD pathological features, and blockade of cannabinoid signaling might be a new therapeutic intervention in FCD.
Authors: Diana Le Duc, Cecilia Giulivi, Susan M. Hiatt, Eleonora Napoli, Alexios Panoutsopoulos, Angelo De Crescenzo, Urania Kotzaeridou, Steffen Syrbe, Evdokia Anagnostou, Meron Azage, Renee Bend, Amber Begtrup, Natasha J. Brown, Benjamin Büttner, Megan T. Cho, Gregory M. Cooper, Jan H. Doering, Christèle Dubourg, David B. Everman, Michael S. Hildebrand, Francis Jeshira Reynoso Santos, Barbara Kellam, Jennifer Keller-Ramey, Johannes R. Lemke, Shuxi Liu, Dmitriy Niyazov, Katelyn Payne, Richard Person, Chloé Quélin, Rhonda E. Schnur, Brooke T. Smith, Jonathan Strober, Susan Walker, Mathew Wallis, Laurence Walsh, Sandra Yang, Ryan Yuen, Andreas Ziegler, Heinrich Sticht, Michael C. Pride, Lori Orosco, Verónica Martínez-Cerdeño, Jill Silverman, Jacqueline N. Crawley, Stephen W. Scherer, Konstantinos S. Zarbalis, Rami Jamra
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Abstract: The underpinnings of mild to moderate neurodevelopmental delay (NDD) remain elusive often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of thirteen individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment p = 0.003) as a monogenic cause of mild and non-specific NDD. Nine variants were protein-truncating and four missense. Overlapping symptoms included NDD, intellectual disability (ID), macrocephaly, and psychiatric disorders (ASD/ADHD). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH- domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway up-regulation.
We previously reported that the autophagy scaffolding protein Wdfy3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt- pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via down-regulation of the Wnt-pathway.
In summary, we present WDFY3 as a novel gene linked to mild to moderate NDD and ID and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.
Authors: Lisa Ma, Anthony W. Herren, Glenda Espinal, Jamie Randol, Bridget McLaughlin, Veronica Martinez-Cerdeño, Isaac N. Pessah, Randi J. Hagerman and Paul J. Hagerman
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Abstract: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55–200 CGG repeats) in the 5′ noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.
Authors: Jessica L. Famula, Forrest McKenzie, Yingratana A. McLennan, James Grigsby, Flora Tassone, David Hessl, Susan M. Rivera, Verónica Martínez-Cerdeño and Randi J. Hagerman
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Abstract: Here we report five cases of male FMR1 premutation carriers who present without clinical symptoms of the fragile X-associated tremor/ataxia syndrome (FXTAS), but who on MRI demonstrate white matter hyperintensities in the middle cerebellar peduncles (MCP sign) and other brain regions, a rare finding. MCP sign is the major radiological feature of FXTAS; it is therefore remarkable to identify five cases in which this MRI finding is present in the absence of tremor and ataxia, the major clinical features of FXTAS. Subjects underwent a detailed neurological evaluation, neuropsychological testing, molecular testing, and MRI evaluation utilizing T2 imaging described here. Additional white matter disease was present in the corpus callosum in four of the five cases. However, all cases were asymptomatic for motor signs of FXTAS.
Authors: Nicole Barger, Janet Keiter, Anna Kreutz, Anjana Krishnamurthy, Cody Weidenthaler, Verónica Martínez-Cerdeño, Alice F. Tarantal and Stephen C. Noctor
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Abstract: Microglial cells are increasingly recognized as modulators of brain development. We previously showed that microglia colonize the cortical proliferative zones in the prenatal brain and regulate the number of precursor cells through phagocytosis. To better define cellular interactions between microglia and proliferative cells, we performed lentiviral vector- mediated intraventricular gene transfer to induce enhanced green fluorescent protein expression in fetal cerebrocortical cells. Tissues were collected and counterstained with cell-specific markers to label microglial cells and identify other cortical cell types. We found that microglial cells intimately interact with the radial glial scaffold and make extensive contacts with neural precursor cells throughout the proliferative zones, particularly in the rhesus monkey fetus when compared to rodents. We also identify a subtype of microglia, which we term ‘periventricular microglia’, that interact closely with mitotic precursor cells in the ventricular zone. Our data suggest that microglia are structural modulators that facilitate remodeling of the proliferative zones as precursor cells migrate away from the ventricle and may facilitate the delamination of precursor cells. Taken together, these results indicate that microglial cells are an integral component of cortical proliferative zones and contribute to the interactive milieu in which cortical precursor cells function.
Authors: Jarek Wegiel , W. Ted Brown, Giuseppe La Fauci, Tatyana Adayev, Richard Kascsak, Regina Kascsak, Michael Flory, Wojciech Kaczmarski, Izabela Kuchna, Krzysztof Nowicki, Verónica Martínez-Cerdeño , Thomas Wisniewski, and Jerzy Wegiel
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Abstract: Fragile X syndrome (FXS), caused by lack of fragile X mental retardation protein (FMRP), is associated with a high prevalence of autism. The deficit of FMRP reported in idiopathic autism suggests a mechanistic overlap between FXS and autism. The overall goal of this study is to detect neuropathological commonalities of FMRP deficits in the brains of people with idiopathic autism and with syndromic autism caused by dup15q11.2-q13 (dup15). This study tests the hypothesis based on our preliminary data that both idiopathic and syndromic autism are associated with brain region-specific deficits of neuronal FMRP and structural changes of the affected neurons. This immunocytochemical study revealed neuronal FMRP deficits and shrinkage of deficient neurons in the cerebral cortex, subcortical structures, and cerebellum in subjects with idiopathic and dup(15)/autism. Neuronal FMRP deficit coexists with surprising infiltration of the brains of autistic children and adults with FMRP-positive astrocytes known to be typical only for the fetal and short postnatal periods. In the examined autistic subjects, these astrocytes selectively infiltrate the border between white and gray matter in the cerebral and cerebellar cortex, the molecular layer of the cortex, part of the amygdala and thalamus, central cerebellar white matter, and dentate nucleus. Astrocyte pathology results in an additional local loss of FMRP in neurons and their shrinkage. Neuronal deficit of FMRP and shrinkage of affected neurons in structures free of FMRP-positive astrocytes and regions infiltrated with FMRP-expressing astrocytes appear to reflect mechanistic, neuropathological, and functional commonalities of FMRP abnormalities in FXS and autism spectrum disorder. Autism Res 2018. © 2018 International Society for Autism Research, Wiley Periodicals, Inc.
Authors: Jarek Wegiel, Wojciech Kaczmarski, Michael Flory, Veronica Martinez-Cerdeno, Thomas Wisniewski, Krzysztof Nowicki, Izabela Kuchna and Jerzy Wegiel
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Abstract: Introduction: In autism spectrum disorder, lack of coherence and of complex information processing, and narrowly focused interests and repetitive behaviors are considered a sign of long-range underconnectivity and short-range overconnectivity. The goal of this morphometric study of five anatomically and functionally different segments of the corpus callosum (CC) was to establish patterns of differences between long-range interhemispheric connections in nine neurotypical and nine autistic subjects.
Results: Electron microscopy revealed a significant reduction in average axon diameter and axon cross-sectional area in autistic subjects, and reduction in CC segment–specific diversification of connections of functionally different cortical regions. The study shows an increase in the percentage of small diameter axons (< 0.651 μm) and a decrease in the percentage of axons with large diameter (> 1.051 μm). The total number of small-diameter axons is reduced in segment I and III by 43% on average. The number of medium- and large-diameter axons is reduced in all five CC segments by an average of 49 and 72%, respectively.
Conclusions: The detected pattern of pathology suggests a failure of mechanisms controlling guidance of axons during development leading to axonal deficit, and failure of mechanisms controlling axon structure. A reduction in axon diameter may affect the velocity and volume of signal transmission, and distort functional specialization of CC segments. Significant deficits in axon number and reduction in axon size in all five CC segments appear to be substantial components of brain connectome integrity distortion which may contribute to the autism phenotype.
Authors: Chantal Sellier, Ronald A.M. Buijsen, Fang He, Sam Natla, Laura Jung, Philippe Tropel, Angeline Gaucherot, Hugues Jacobs, Hamid Meziane, Alexandre Vincent, Marie-France Champy, Tania Sorg, Guillaume Pavlovic, Marie Wattenhofer-Donze, Marie-Christine Birling, Mustapha Oulad-Abdelghani, Pascal Eberling, Frank Ruffenach, Mathilde Joint, Mathieu Anheim, Verónica Martínez-Cerdeño, Flora Tassone, Rob Willemsen, Renate K. Hukema, Stéphane Viville, Cecile Martinat, Peter K. Todd and Nicolas Charlet-Berguerand
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Abstract: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 50 UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed trans- genic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2b and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2b rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nu- clear lamina architecture and drives pathogenesis in FXTAS.
Authors: Mirna Lechpammer, Verónica Martínez Cerdeño, Michael Ryan Hunsaker, Mina Hah, Hilary Gonzales, Steve Tisch, Ronald Joffe, Roger Pamphlett, Flora Tassone, Paul J. Hagerman, Samuel J. Bolitho, Randi J. Hagerman
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Abstract: This report describes unique presentations of inclusion body myositis (IBM) in two unrelated patients, one male and one female, with genetically and histologically confirmed fragile X-associated tremor/ataxia syndrome (FXTAS). We summarize overlapping symptoms between two disorders, clinical course, and histopathological analyses of the two patients with FXTAS and sporadic IBM, clinically defined per diagnostic criteria of the European Neuromuscular Centre. In case 1, a post-mortem analysis of available brain and muscle tissues is also described. Histopathological features (rimmed vacuoles) consistent with clinically defined IBM were detected in both presented cases. Postmortem testing in case 1 revealed the presence of an FMR1 premutation allele of 60 CGG repeats in both brain and skeletal muscle samples. Case 2 was a premutation carrier with 71 CGG re- peats who had a son with FXS. Given that FXTAS is associated with immune-mediated disorders among premutation carriers, it is likely that the pathogeneses of IBM and FXTAS are linked. This is, to our knowledge, the first report of these two conditions presenting together, which expands our understanding of clinical symptoms and unusual presentations in patients with FXTAS. Following detection of a premutation allele of the FMR1 gene, FXTAS patients with severe muscle pain should be assessed for IBM.
Authors: Deborah A. Hall, Erin Robertson, Annie L. Shelton, Molly C. Losh, Montserrat Mila, Esther Granell Moreno, Beatriz Gomez-Anson, Verónica Martínez-Cerdeño, Jim Grigsby, Reymundo Lozano, Randi Hagerman, Lorena Santa Maria, Elizabeth Berry-Kravis & Joan A. O’Keefe
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Abstract: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by a repeat expansion in the fragile X mental retardation 1 (FMR1) gene. The disorder is characterized by kinetic tremor and cerebellar ataxia, shows age-dependent penetrance, and occurs more frequently in men. This paper summarizes the key emerging issues in FXTAS as presented at the Second International Conference on the FMR1 Premutation: Basic Mechanisms & Clinical Involvement in 2015. The topics discussed include phenotype-genotype relationships, neuro- behavioral function, and updates on FXTAS genetics and imaging.
Authors: Risa Kashima, Sougata Roy, Manuel Ascano, Veronica Martinez-Cerdeno, Jeanelle Ariza-Torres, Sunghwan Kim, Justin Louie, Yao Lu, Patricio Leyton, Kenneth D. Bloch, Thomas B. Kornberg, Paul J. Hagerman, Randi Hagerman, Giorgio Lagna, Akiko Hata
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Epigenetic silencing of fragile X mental retardation 1 (FMR1) causes fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. FXS correlates with abnormal synapse and dendritic spine development, but the molecular link between the absence of the FMR1 product FMRP, an RNA binding protein, and the neuropathology is unclear. We found that the messenger RNA encoding bone morpho- genetic protein type II receptor (BMPR2) is a target of FMRP. Depletion of FMRP increased BMPR2 abundance, especially that of the full-length isoform that bound and activated LIM domain kinase 1 (LIMK1), a component of the noncanonical BMP signal transduction pathway that stimulates actin re- organization to promote neurite outgrowth and synapse formation. Heterozygosity for BMPR2 rescued the morphological abnormalities in neurons both in Drosophila and in mouse models of FXS, as did the postnatal pharmacological inhibition of LIMK1 activity. Compared with postmortem prefrontal cortex tissue from healthy subjects, the amount of full-length BMPR2 and of a marker of LIMK1 activity was increased in this brain region from FXS patients. These findings suggest that increased BMPR2 signal transduction is linked to FXS and that the BMPR2-LIMK1 pathway is a putative therapeutic target in patients with FXS and possibly other forms of autism.
Authors: Mirna Lechpammer, Yen P. Tran, Pia Wintermark, Verónica Martínez-Cerdeño, Viswanathan V. Krishnan, Waseem Ahmed, Robert F. Berman, Frances E. Jensen, Evgeny Nudler, David Zagzag
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Abstract: Encephalopathy of prematurity (EOP) is a complex form of cerebral injury that occurs in the setting of hypoxia-ischemia (HI) in premature infants. Using a rat model of EOP, we investigated whether neonatal HI of the brain may alter the expression of cystathionine b-synthase (CBS) and the components of the mammalian target of rapamycin (mTOR) signaling. We performed unilateral carotid ligation and induced HI (UCL/HI) in Long-Evans rats at P6 and found increased CBS expression in white matter (i.e. corpus callosum, cingulum bundle and external capsule) as early as 24 h (P7) postprocedure. CBS remained elevated through P21, and, to a lesser extent, at P40. The mTOR downstream target 70 kDa ribosomal protein S6 kinase (p70S6K and phospho-p70S6K) and 40S ribosomal protein S6 (S6 and phospho-S6) were also overexpressed at the same time points in the UCL/HI rats compared to healthy controls. Overexpression of mTOR components was not observed in rats treated with the mTOR inhibitor everolimus. Behavioral assays performed on young rats (postnatal day 35–37) following UCL/HI at P6 indicated impaired preference for social novelty, a behavior relevant to autism spectrum disorder, and hyperactivity. Everolimus restored behavioral patterns to those observed in healthy controls. A gait analysis has shown that motor deficits in the hind paws of UCL/HI rats were also significantly reduced by everolimus. Our results suggest that neonatal HI brain injury may inflict long-term damage by upregulation of CBS and mTOR signaling. We propose this cascade as a possible new molecular target for EOP—a still untreatable cause of autism, hyperactivity and cerebral palsy.
Authors: Christopher L. Cunningham, Verónica Martínez-Cerdeño, Stephen C. Noctor
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Abstract: The germinal zones of the embryonic macaque neocortex comprise the ventricular zone (VZ) and the subventricular zone (SVZ). The mammalian SVZ is subdivided into an inner SVZ and an outer SVZ, with the outer SVZ being particularly large in primates. The existence of distinct precursor cell types in the neocortical proliferative zones was inferred over 100 years ago and recent evidence supports this concept. Precursor cells exhibiting diverse morphologies, patterns of transcription factor expression, and fate potential have been identified in the neocortical proliferative zones. Neurogenic precursor cells are thought to exhibit characteristics of glial cells, but the existence of neurogenic precursor cells that do not share glial specific properties has also been proposed. Therefore, one question that remains is whether neural precursor cells in the prenatal neocortex belong within the astroglial cell class, as they do in neurogenic regions of the adult neocortex, or instead include a diverse collection of precursor cells belonging to distinct cell classes. We examined the expression of astroglial markers by mitotic precursor cells in the telencephalon of prenatal macaque and human. We show that in the dorsal neocortex all mitotic cells at the surface of the ventricle, and all Pax6+ and Tbr2+ mitotic cells in the proliferative zones, express the astroglial marker GFAP. The majority of mitotic cells undergoing division away from the ventricle express GFAP, and many of the GFAP-negative mitoses express markers of cells derived from the ventral telencephalon or extracortical sites. In contrast, a markedly lower proportion of precursor cells express GFAP in the ganglionic eminence. In conclusion, we propose that the heterogeneity of neural precursor cells in the dorsal cerebral cortex develops within the GFAP+ astroglial cell class.
Authors: Christopher L. Cunningham, Verónica Martínez-Cerdeño and Stephen C. Noctor
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Abstract: Neurogenesis must be properly regulated to ensure that cell production does not exceed the requirements of the growing cerebral cortex, yet our understanding of mechanisms that restrain neuron production remains incomplete. We investigated the function of microglial cells in the developing cerebral cortex of prenatal and postnatal macaques and rats and show that microglia limit the production of cortical neurons by phagocytosing neural precursor cells. We show that microglia selectively colonize the cortical proliferative zones and phagocytose neural precursor cells as neurogenesis nears completion. We found that deactivating microglia in utero with tetracyclines or eliminating microglia from the fetal cerebral cortex with liposomal clodronate significantly increased the number of neural precursor cells, while activating microglia in utero through maternal immune activation significantly decreased the number of neural precursor cells. These data demonstrate that microglia play a fundamental role in regulating the size of the precursor cell pool in the developing cerebral cortex, expanding our understanding of the mechanisms that regulate cortical development. Furthermore, our data suggest that any factor that alters the number or activation state of microglia in utero can profoundly affect neural development and affect behavioral outcomes.
Authors: Sharifia Wills, Christy C. Rossi, Jeffrey Bennett, Verónica Martínez-Cerdeño, Paul Ashwood, David G. Amaral and Judy Van de Water
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Abstract: Background: Autism is a neurodevelopmental disorder characterized by impairments in social interaction and deficits in verbal and nonverbal communication, together with the presence of repetitive behaviors or a limited repertoire of activities and interests. The causes of autism are currently unclear. In a previous study, we determined that 21% of children with autism have plasma autoantibodies that are immunoreactive with a population of neurons in the cerebellum that appear to be Golgi cells, which are GABAergic interneurons.
Methods: We have extended this analysis by examining plasma immunoreactivity in the remainder of the brain. To determine cell specificity, double-labeling studies that included one of the calcium-binding proteins that are commonly colocalized in GABAergic neurons (calbindin, parvalbumin or calretinin) were also carried out to determine which GABAergic neurons are immunoreactive. Coronal sections through the rostrocaudal extent of the macaque monkey brain were reacted with plasma from each of seven individuals with autism who had previously demonstrated positive Golgi cell staining, as well as six negative controls. In addition, brain sections from adult male mice were similarly examined.
Results: In each case, specific staining was observed for neurons that had the morphological appearance of interneurons. By double-labeling sections with plasma and with antibodies directed against g-aminobutyric acid (GABA), we determined that all autoantibody-positive neurons were GABAergic. However, not all GABAergic neurons were autoantibody-positive. Calbindin was colabeled in several of the autoantibody-labeled cells, while parvalbumin colabeling was less frequently observed. Autoantibody-positive cells rarely expressed calretinin. Sections from the mouse brain processed similarly to the primate sections also demonstrated immunoreactivity to interneurons distributed throughout the neocortex and many subcortical regions. Some cell populations stained in the primate (such as the Golgi neurons in the cerebellum) were not as robustly immunoreactive in the mouse brain.
Conclusions: These results suggest that the earlier report of autoantibody immunoreactivity to specific cells in the cerebellum extend to other regions of the brain. Further, these findings confirm the autoantibody-targeted cells to be a subpopulation of GABAergic interneurons. The potential impact of these autoantibodies on GABAergic disruption with respect to the etiology of autism is discussed herein.
Authors: Christopher L. Cunningham, Verónica Martínez-Cerdeño, Eliecer Navarro Porras, Anish N. Prakash, James M. Angelastro, Rob Willemsen, Paul J. Hagerman, Isaac N. Pessah, Robert F. Berman and Stephen C. Noctor
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Abstract: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder caused by a premutation CGG-trinucleotide repeat expansion (55–200 CGG repeats) within the 5′-untranslated region of the FMR1 gene. Although FXTAS generally affects premutation carriers over 50 years of age, cognitive and psychological symptoms can appear in carriers during childhood, suggesting that the FMR1 premutation affects brain function early in life. Recent work with cultured hippocampal neurons from a premutation (Fmr1 CGG knock-in) mouse model revealed impaired development of early postnatal neurons, consistent with the developmental clinical involvement of premutation carriers. In the current work, we show that the presence of premutation CGG-repeat expansions in the mouse Fmr1 gene alters embryonic neocortical development. Specifically, embryonic premutation mice display migration defects in the neocortex and altered expression of neuronal lineage markers. The current data demonstrate that premutation alleles of the Fmr1 gene are associated with defects in developmental programs operating during pre- natal stages of brain formation and provide further evidence that the FMR1 premutation has a neurodevelopmental component.
Authors: Alice Wey & Verónica Martínez-Cerdeño & David Pleasure & Paul S. Knoepfler
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Abstract: Separate murine knockout (KO) of either c- or N- myc genes in neural stem and precursor cells (NSC) driven by nestin-cre causes microcephaly. The cerebellum is particularly affected in the N-myc KO, leading to a strong reduction in cerebellar granule neural progenitors (CGNP) and mature granule neurons. In humans, mutation of N-myc also causes microcephaly in Feingold Syndrome. We created a double KO (DKO) of c- and N-myc using nestin-cre, which strongly impairs brain growth, particularly that of the cerebellum. Granule neurons were almost absent from the Myc DKO cerebellum, and other cell types were relatively overrepresented, including astroglia, oligodendrocytes, and Purkinje neurons. These findings are indicative of a profound disruption of cell fate of cerebellar stem and precursors. DKO Purkinje neurons were strikingly lacking in normal arborization. Inhibitory neurons were ectopic and exhibited very abnormal GAD67 staining patterns. Also consistent with altered cell fate, the adult DKO cerebellum still retained a residual external germinal layer (EGL). CGNP in the DKO EGL were almost uniformly NeuN and p27KIP1 positive as well as negative for Math1 and BrdU at the peak of normal cerebellar proliferation at P6. The presence of some mitotic CGNP in the absence of S phase cells suggests a possible arrest in M phase. CGNP and NSC metabolism also was affected by loss of Myc as DKO cells exhibited weak nucleolin staining. Together these findings indicate that c- and N-Myc direct cerebellar development by maintaining CGNP and NSC populations through inhibiting differentiation as well as directing rapid cell cycling and active cellular metabolism.
Authors: Ana Milosevic, Stephen C. Noctor, Verónica Martínez-Cerdeño, Arnold R. Kriegstein, James E. Goldman
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Abstract: Forebrain subventricular zone (SVZ) progenitor cells give rise to glia and olfactory bulb interneurons during early postnatal life in rats. We investigated the potential of SVZ cells to alter their fate by transplanting them into a heterotypic neurogenic and gliogenic environment—the cerebellum. Transplanted cells were examined 1 to 7 weeks and 6 months post transplantation. Forebrain progenitors populated the cerebellum and differentiated into oligodendrocytes, cerebellar-specific Bergmann glia and velate astrocytes, and neurons. The transplanted cells that differentiated into neurons maintained an interneuronal fate: they were GABA-positive, expressed interneuronal markers, such as calretinin, and exhibited membrane properties that are characteristic of interneurons. However, the transplanted interneurons lost the expression of the olfactory bulb transcription factors Tbr2 and Dlx1, and acquired a cerebellar-like morphology. Forebrain SVZ progenitors thus have the potential to adapt to a new environment and integrate into diverse regions, and may be a useful tool in transplantation strategies.
Authors: Stephen C. Noctor, Verónica Martínez-Cerdeño, and Arnold R. Kriegstein
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Abstract: Neocortical precursor cells undergo symmetric and asymmetric divisions while producing large numbers of diverse cortical cell types. In Drosophila, cleavage plane orientation dictates the inheritance of fate-determinants and the symmetry of newborn daughter cells during neuroblast cell divisions. One model for predicting daughter cell fate in the mammalian neocortex is also based on cleavage plane orientation. Precursor cell divisions with a cleavage plane orientation that is perpendicular with respect to the ventricular surface (vertical) are predicted to be symmetric, while divisions with a cleavage plane orientation that is parallel to the surface (horizontal) are predicted to be asymmetric neurogenic divisions. However, analysis of cleavage plane orientation at the ventricle suggests that the number of predicted neurogenic divisions might be insufficient to produce large amounts of cortical neurons. To understand factors that correlate with the symmetry of cell divisions, we examined rat neocortical precursor cells in situ through real-time imaging, marker analysis, and electrophysiological recordings. We find that cleavage plane orientation is more closely associated with precursor cell type than with daughter cell fate, as commonly thought. Radial glia cells in the VZ primarily divide with a vertical orientation throughout cortical development and undergo symmetric or asymmetric self- renewing divisions depending on the stage of development. In contrast, most intermediate progenitor cells divide in the subventricular zone with a horizontal orientation and produce symmetric daughter cells. We propose a model for predicting daughter cell fate that considers precursor cell type, stage of development, and the planar segregation of fate determinants. J. Comp. Neurol. 508:28–44, 2008.
Authors: Maria J. Galazo, Verónica Martínez-Cerdeño, César Porrero and Francisco Clascá
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Abstract: Inputs to the layer I apical dendritic tufts of pyramidal cells are crucial in ‘‘top-down’’ interactions in the cerebral cortex. A large population of thalamocortical cells, the ‘‘matrix’’ (M-type) cells, provides a direct robust input to layer I that is anatomically and functionally different from the thalamocortical input to layer VI. The developmental timecourse of M-type axons is examined here in rats aged E (embryonic day) 16 to P (postnatal day) 30. Anterograde techniques were used to label axons arising from 2 thalamic nuclei mainly made up of M-type cells, the Posterior and the Ventromedial. The primary growth cones of M-type axons rapidly reached the subplate of dorsally situated cortical areas. After this, interstitial branches would sprout from these axons under more lateral cortical regions to invade the overlying cortical plate forming secondary arbors. Moreover, retrograde labeling of M-type cell somata in the thalamus after tracer deposits confined to layer I revealed that large numbers of axons from multiple thalamic nuclei had already converged in a given spot of layer I by P3. Because of early ingrowth in such large numbers, interactions of M-type axons may significantly influence the early development of cortical circuits.
Authors: Stephen C. Noctor, Verónica Martínez-Cerdeño and Arnold R. Kriegstein
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Abstract: Recent work has begun to identify neural stem and progenitor cells in the embryonic and adult brain, and is unravelling the mechanisms whereby new nerve cells are created and delivered to their correct locations. Radial glial (RG) cells, which are present in the developing mammalian brain, have been proposed to be neural stem cells because they produce multiple cell types. Furthermore, time-lapse imaging demonstrates that RG cells undergo asymmetric self-renewing divisions to produce immature neurons that migrate along their parent radial fibre to reach the developing cerebral cortex. RG cells also produce intermediate progenitor (IP) cells that undergo symmetric division in the subventricular zone of the embryonic cortex to produce pairs of neurons. The symmetric IP divisions increase cell number within the same cortical layer. This two-step process of neurogenesis suggests new mechanisms for the generation of cell diversity and cell number in the developing cortex and supports a model similar to that proposed for the development of the fruit fly CNS. In this model, a temporal sequence of gene expression changes in asymmetrically dividing self-renewed RG cells could lead to the differential inheritance of cell identity genes in cortical cells generated at different cell cycles.
Authors: Stephen C. Noctor, PhD; Verónica Martínez-Cerdeño, PhD; Arnold R. Kriegstein, MD, PhD
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Abstract: The mammalian cerebral cortex is the most cellularly complex structure in the animal king- dom. Almost all cortical neurons are produced during a limited embryonic period by cortical progenitor cells in a proliferative region that surrounds the ventricular system of the developing brain. The proliferative region comprises 2 distinct zones, the ventricular zone, which is a neuroepithelial layer directly adjacent to the ventricular lumen, and the subventricular zone, which is positioned superficial to the ventricular zone. Recent advances in molecular and cell biology have made possible the study of specific cell populations, and 2 cortical progenitor cell types, radial glial cells in the ventricular zone and intermediate progenitor cells in the subventricular zone, have been shown to generate neurons in the embryonic cerebral cortex. These findings have refined our understanding of cortical neurogenesis, with implications for understanding the causes of neurodevelopmental disorders and for their potential treatment.
Authors: Tania Ramos-Moreno, Maria J. Galazo, Cesar Porrero, Verónica Martínez-Cerdeño and Francisco Clascá
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Abstract: Reelin, a large extracellular matrix glycoprotein, is secreted by several neuron populations in the developing and adult rodent brain. Secreted Reelin triggers a complex signaling pathway by binding lipoprotein and integrin membrane receptors in target cells. Reelin signaling regulates migration and dendritic growth in developing neurons, while it can modulate synaptic plasticity in adult neurons. To identify which adult neural circuits can be modulated by Reelin-mediated signaling, we systematically mapped the distribution of Reelin in adult rat brain using sensitive immunolabeling techniques. Results show that the distribution of intracellular and secreted Reelin is both very widespread and specific. Some interneuron and projection neuron populations in the cerebral cortex contain Reelin. Numerous striatal neurons are weakly immunoreactive for Reelin and these cells are preferentially located in striosomes. Some thalamic nuclei contain Reelin-immunoreactive cells. Double-immunolabeling for GABA and Reelin reveals that the Reelin-immunoreactive cells in the visual thalamus are the intrinsic thalamic interneurons. High local concentrations of extracellular Reelin selectively outline several dendrite spine-rich neuropils. Together with previous mRNA data, our observations suggest abundant axoplasmic transport and secretion in pathways such as the retino-collicular tract, the entorhino-hippocampal (‘perforant’) path, the lateral olfactory tract or the parallel fiber system of the cerebellum. A preferential secretion of Reelin in these neuropils is consistent with reports of rapid, activity-induced structural changes in adult brain circuits.
Authors: Stephen C. Noctor, Verónica Martínez-Cerdeño, Lidija Ivic and Arnold R. Kriegstein
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Abstract: Precise patterns of cell division and migration are crucial to transform the neuroepithelium of the embryonic forebrain into the adult cerebral cortex. Using time-lapse imaging of clonal cells in rat cortex over several generations, we show here that neurons are generated in two proliferative zones by distinct patterns of division. Neurons arise directly from radial glial cells in the ventricular zone (VZ) and indirectly from intermediate progenitor cells in the subventricular zone (SVZ). Furthermore, newborn neurons do not migrate directly to the cortex; instead, most exhibit four distinct phases of migration, including a phase of retrograde movement toward the ventricle before migration to the cortical plate. These findings provide a comprehensive and new view of the dynamics of cortical neurogenesis and migration.