Oxidative-Stress-Associated Proteostasis Disturbances and Increased DNA Damage in the Hippocampal Granule Cells of the Ts65Dn Model of Down Syndrome

Oxidative stress (OS) is one of the neuropathological mechanisms responsible for the deficits in cognition and neuronal function in Down syndrome (DS). The Ts65Dn (TS) mouse replicates multiple DS phenotypes including hippocampal-dependent learning and memory deficits and similar brain oxidative status. To better understand the hippocampal oxidative profile in the adult TS mouse, we analyzed cellular OS-associated alterations in hippocampal granule cells (GCs), a neuronal population that plays an important role in memory formation and that is particularly affected in DS. For this purpose, we used biochemical, molecular, immunohistochemical, and electron microscopy techniques. Our results indicate that TS GCs show important OS-associated alterations in the systems essential for neuronal homeostasis: DNA damage response and proteostasis, particu larly of the proteasome and lysosomal system. Specifically, TS GCs showed: (i) increased DNA damage, (ii) reorganization of nuclear proteolytic factories accompanied by a decline in proteasome activity and cytoplasmic aggregation of ubiquitinated proteins, (iii) formation of lysosomal-related structures containing lipid droplets of cytotoxic peroxidation products, and (iv) mitochondrial ultrastructural defects. These alterations could be implicated in enhanced cellular senescence, accelerated aging and neurodegeneration, and the early development of Alzheimer?s disease neuropathology present in TS mice and the DS population.Funding: This work was supported by the following grants: “Instituto de Investigación Valdecilla” (IDIVAL; NVAL 19/23), Santander, Spain; “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas” (CIBERNED; CB06/05/0037) Spain; and “Agencia Estatal de Investicación, MICIN” (grant number: PID2020-117601RB-I00). Acknowledgments: The authors would like to thank Raquel García-Ceballos and Eva García Iglesias for their technical assistance

Nuclear Reorganization in Hippocampal Granule Cell Neurons from a Mouse Model of Down Syndrome: Changes in Chromatin Configuration, Nucleoli and Cajal Bodies

Down syndrome (DS) or trisomy of chromosome 21 (Hsa21) is characterized by impaired hippocampal-dependent learning and memory. These alterations are due to defective neurogenesis and to neuromorphological and functional anomalies of numerous neuronal populations, including hippocampal granular cells (GCs). It has been proposed that the additional gene dose in trisomic cells induces modifications in nuclear compartments and on the chromatin landscape, which could contribute to some DS phenotypes. The Ts65Dn (TS) mouse model of DS carries a triplication of 92 genes orthologous to those found in Hsa21, and shares many phenotypes with DS individuals, including cognitive and neuromorphological alterations. Considering its essential role in hippocampal memory formation, we investigated whether the triplication of this set of Hsa21 orthologous genes in TS mice modifies the nuclear architecture of their GCs. Our results show that the TS mouse presents alterations in the nuclear architecture of its GCs, affecting nuclear compartments involved in transcription and pre-rRNA and pre-mRNA processing. In particular, the GCs of the TS mouse show alterations in the nucleolar fusion pattern and the molecular assembly of Cajal bodies (CBs). Furthermore, hippocampal GCs of TS mice present an epigenetic dysregulation of chromatin that results in an increased heterochromatinization and reduced global transcriptional activity. These nuclear alterations could play an important role in the neuromorphological and/or functional alterations of the hippocampal GCs implicated in the cognitive dysfunction characteristic of TS mice

Bexarotene Impairs Cognition and Produces Hypothyroidism in a Mouse Model of Down Syndrome and Alzheimer’s Disease

All individuals with Down syndrome (DS) eventually develop Alzheimer's disease (AD) neuropathology, including neurodegeneration, increases in ?-amyloid (A?) expression, and aggregation and neurofibrillary tangles, between the third and fourth decade of their lives. There is currently no effective treatment to prevent AD neuropathology and the associated cognitive degeneration in DS patients. Due to evidence that the accumulation of A? aggregates in the brain produces the neurodegenerative cascade characteristic of AD, many strategies which promote the clearance of A? peptides have been assessed as potential therapeutics for this disease. Bexarotene, a member of a subclass of retinoids that selectively activates retinoid receptors, modulates several pathways essential for cognitive performance and A? clearance. Consequently, bexarotene might be a good candidate to treat AD-associated neuropathology. However, the effects of bexarotene treatment in AD remain controversial. In the present study, we aimed to elucidate whether chronic bexarotene treatment administered to the most commonly used murine model of DS, the Ts65Dn (TS) mouse could reduce A? expression in their brains and improve their cognitive abilities. Chronic administration of bexarotene to aged TS mice and their CO littermates for 9 weeks diminished the reference, working, and spatial learning and memory of TS mice, and the spatial memory of CO mice in the Morris water maze. This treatment also produced marked hypoactivity in the plus maze, open field, and hole board tests in TS mice, and in the open field and hole board tests in CO mice. Administration of bexarotene reduced the expression of A?1-40, but not of A?1-42, in the hippocampi of TS mice. Finally, bexarotene increased Thyroid-stimulating hormone levels in TS mice and reduced Thyroid-stimulating hormone levels in CO mice, while animals of both karyotypes displayed reduced thyroxine levels after bexarotene administration. The bexarotene-induced hypothyroidism could be responsible for the hypoactivity of TS and CO mice and their diminished performance in the Morris water maze. Together, these results do not provide support for the use of bexarotene as a potential treatment of AD neuropathology in the DS population.FUNDING: This study was supported by the Institute of Research Valdecilla (IDIVAL) (NVAL 16/21 and NVAL 19/23) and the Consejería de Universidades, Igualdad, Cultura y Deporte del Gobierno de Cantabria (16. VP39.64662)

Nusinersen ameliorates motor function and prevents motoneuron Cajal body disassembly and abnormal poly(A) RNA distribution in a SMA mouse model

Spinal muscular atrophy (SMA) is a devastating autosomal recessive neuromuscular disease characterized by degeneration of spinal cord alpha motor neurons (αMNs). SMA is caused by the homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene, resulting in reduced expression of SMN protein, which leads to αMN degeneration and muscle atrophy. The majority of transcripts of a second gene (SMN2) generate an alternative spliced isoform that lacks exon 7 and produces a truncated nonfunctional form of SMN. A major function of SMN is the biogenesis of spliceosomal snRNPs, which are essential components of the pre-mRNA splicing machinery, the spliceosome. In recent years, new potential therapies have been developed to increase SMN levels, including treatment with antisense oligonucleotides (ASOs). The ASO-nusinersen (Spinraza) promotes the inclusion of exon 7 in SMN2 transcripts and notably enhances the production of full-length SMN in mouse models of SMA. In this work, we used the intracerebroventricular injection of nusinersen in the SMN∆7 mouse model of SMA to evaluate the effects of this ASO on the behavior of Cajal bodies (CBs), nuclear structures involved in spliceosomal snRNP biogenesis, and the cellular distribution of polyadenylated mRNAs in αMNs. The administration of nusinersen at postnatal day (P) 1 normalized SMN expression in the spinal cord but not in skeletal muscle, rescued the growth curve and improved motor behavior at P12 (late symptomatic stage). Importantly, this ASO recovered the number of canonical CBs in MNs, significantly reduced the abnormal accumulation of polyadenylated RNAs in nuclear granules, and normalized the expression of the pre-mRNAs encoding chondrolectin and choline acetyltransferase, two key factors for αMN homeostasis. We propose that the splicing modulatory function of nusinersen in SMA αMN is mediated by the rescue of CB biogenesis, resulting in enhanced polyadenylated pre-mRNA transcription and splicing and nuclear export of mature mRNAs for translation. Our results support that the selective restoration of SMN expression in the spinal cord has a beneficial impact not only on αMNs but also on skeletal myofibers. However, the rescue of SMN expression in muscle appears to be necessary for the complete recovery of motor function.The authors wish to thank Raquel García-Ceballos for technical assistance and Alfonso Nieva for video editing. We are also grateful to Prof. A. I. Lamond (University of Dundee, UK) for anti-coilin antibody (204.10) and Prof. Larry Gerace (The Scripps Research Institute, La Jolla, USA) for anti-LaminA/C antibody. The authors are also indebted to Prof. Josep E. Esquerda for critical reading of the manuscript and helpful suggestions. This work was supported by the following grants: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0037) Spain; Instituto de Investigación Sanitaria Valdecilla (IDIVAL, Next-Val 17/22), Santander, Spain; and Spanish Ministerio de Ciencia, Innovación y Universidades cofinanced by Fondo Europeo de Desarrollo Regional (FEDER) (RTI2018-099278-B-I00)

Prenatal, but not Postnatal, Curcumin Administration Rescues Neuromorphological and Cognitive Alterations in Ts65Dn Down Syndrome Mice

Background: The cognitive dysfunction in Down syndrome (DS) is partially caused by deficient neurogenesis during fetal stages. Curcumin enhances neurogenesis and learning and memory. Objectives: We aimed to test the ability of curcumin to rescue the neuromorphological and cognitive alterations of the Ts65Dn (TS) mouse model of DS when administered prenatally or during early postnatal stages, and to evaluate whether these effects were maintained several weeks after the treatment. Methods: To evaluate the effects of prenatal curcumin administration, 65 pregnant TS females were subcutaneously treated with curcumin (300 mg/kg) or vehicle from ED (Embryonic Day) 10 to PD (Postnatal Day) 2. All the analyses were performed on their TS and Control (CO) male and female progeny. At PD2, the changes in neurogenesis, cellularity, and brain weight were analyzed in 30 TS and CO pups. The long-term effects of prenatal curcumin were evaluated in another cohort of 44 TS and CO mice between PD30 and PD45. The neuromorphological effects of the early postnatal administration of curcumin were assessed on PD15 in 30 male and female TS and CO pups treated with curcumin (300 mg/kg) or vehicle from PD2 to PD15. The long-term neuromorphological and cognitive effects were assessed from PD60 to PD90 in 45 mice. Data was compared by ANOVAs. Results: Prenatal administration of curcumin increased the brain weight (+45%, P < 0.001), the density of BrdU (bromodeoxyuridine)- (+150%, P < 0.001) and DAPI (4',6-diamidino-2-phenylindole)- (+38%, P = 0.005) positive cells, and produced a long-term improvement of cognition in TS (+35%, P = 0.007) mice with respect to vehicle-treated mice. Postnatal administration of curcumin did not rescue any of the short- or long-term altered phenotypes of TS mice. Conclusion: The beneficial effects of prenatal curcumin administration to TS mice suggest that it could be a therapeutic strategy to treat DS cognitive disabilities.This study was supported by the “Fondazione Generali e Assicurazioni Generali”, Italy; Fundación Tatiana Pérez de Guzmán el Bueno, IDIVAL (NVAL 19/23), and the Spanish Ministry of Economy and Competitiveness (PSI-2016-76194-R, AEI/FEDER, EU)

Prenatal Administration of Oleic Acid or Linolenic Acid Reduces Neuromorphological and Cognitive Alterations in Ts65dn Down Syndrome Mice

Background: The cognitive impairments that characterize Down syndrome (DS) have been attributed to brain hypocellularity due to neurogenesis impairment during fetal stages. Thus, enhancing prenatal neurogenesis in DS could prevent or reduce some of the neuromorphological and cognitive defects found in postnatal stages. Objectives: As fatty acids play a fundamental role in morphogenesis and brain development during fetal stages, in this study, we aimed to enhance neurogenesis and the cognitive abilities of the Ts65Dn (TS) mouse model of DS by administering oleic or linolenic acid. Methods: In total, 85 pregnant TS females were subcutaneously treated from Embryonic Day (ED) 10 until Postnatal Day (PD) 2 with oleic acid (400 mg/kg), linolenic acid (500 mg/kg), or vehicle. All analyses were performed on their TS and Control (CO) male and female progeny. At PD2, we evaluated the short-term effects of the treatments on neurogenesis, cellularity, and brain weight, in 40 TS and CO pups. A total of 69 TS and CO mice were used to test the long-term effects of the prenatal treatments on cognition from PD30 to PD45, and on neurogenesis, cellularity, and synaptic markers, at PD45. Data were compared by ANOVAs. Results: Prenatal administration of oleic or linolenic acid increased the brain weight (+36.7% and +45%, P < 0.01), the density of BrdU (bromodeoxyuridine)- (+80% and +115%; P < 0.01), and DAPI (4',6-diamidino-2-phenylindole)-positive cells (+64% and +22%, P < 0.05) of PD2 TS mice with respect to the vehicle-treated TS mice. Between PD30 and PD45, TS mice prenatally treated with oleic or linolenic acid showed better cognitive abilities (+28% and +25%, P < 0.01) and a higher density of the postsynaptic marker PSD95 (postsynaptic density protein 95) (+65% and +44%, P < 0.05) than the vehicle-treated TS animals. Conclusion: The beneficial cognitive and neuromorphological effects induced by oleic or linolenic acid in TS mice suggest that they could be promising pharmacotherapies for DS-associated cognitive deficits.This study was supported by “Fondazione Generali e Assicurazioni Generali”, Italy; Fundación Tatiana Pérez de Guzmán el Bueno, IDIVAL (NVAL 19/23), and the Spanish Ministry of Economy and Competitiveness (PSI-2016- 76194-R, AEI/FEDER, EU)

Non-homogeneous dispersion of graphene in polyacrylonitrile substrates induces a migrastatic response and epithelial-like differentiation in MCF7 breast cancer cells

Background: Recent advances from studies of graphene and graphene-based derivatives have highlighted the great potential of these nanomaterials as migrastatic agents with the ability to modulate tumor microenvironments. Nevertheless, the administration of graphene nanomaterials in suspensions in vivo is controversial. As an alternative approach, herein, we report the immobilization of high concentrations of graphene nanoplatelets in polyacrylonitrile film substrates (named PAN/G10) and evaluate their potential use as migrastatic agents on cancer cells. Results: Breast cancer MCF7 cells cultured on PAN/G10 substrates presented features resembling mesenchymal-to-epithelial transition, e.g., (i) inhibition of migratory activity; (ii) activation of the expression of E-cadherin, cytokeratin 18, ZO-1 and EpCAM, four key molecular markers of epithelial differentiation; (iii) formation of adherens junctions with clustering and adhesion of cancer cells in aggregates or islets, and (iv) reorganization of the actin cytoskeleton resulting in a polygonal cell shape. Remarkably, assessment with Raman spectroscopy revealed that the above-mentioned events were produced when MCF7 cells were preferentially located on top of graphene-rich regions of the PAN/G10 substrates. Conclusions: The present data demonstrate the capacity of these composite substrates to induce an epithelial-like differentiation in MCF7 breast cancer cells, resulting in a migrastatic effect without any chemical agent-mediated signaling. Future works will aim to thoroughly evaluate the mechanisms of how PAN/G10 substrates trigger these responses in cancer cells and their potential use as antimetastatics for the treatment of solid cancers.This work was supported by Grants from the “Asociación Luchamos por la Vida” (Corrales de Buelna, Cantabria, Spain), Health Research Institute “Valdecilla” (INNVAL17/20, IDIVAL), MINECO/EIG-Concert Japan (X-MEM PCI2018-092929 project, International Joint Program 2018) and the Spanish Research Agency (PID2019-105827RB-I00 supported by MCIN/AEI/10.13039/501100011033)

Cellular alterations associated with excess gene dose or oxidative stress in hippocampal granule cells of a Down syndrome mouse model

RESUMEN: El síndrome de Down (SD) es la causa genética más común de discapacidad intelectual y se produce por la triplicación total o parcial del cromosoma 21 humano. En esta Tesis Doctoral se ha utilizado el modelo murino Ts65Dn (TS). Tanto el SD como el ratón TS presentan alteraciones en la memoria dependiente de hipocampo en la que las células granulares (CG) del giro dentado tienen un papel fundamental. Nuestros resultados muestran que el exceso de dosis génica produce cambios en la arquitectura nuclear, alteraciones en el patrón de fusión nucleolar y el ensamblaje de cuerpos nucleares de Cajal en las CG del TS. Además, el incremento de estrés oxidativo en el hipocampo del ratón TS se asocia con el aumento de daño oxidativo en lípidos y DNA, anomalías en la estructura mitocondrial y alteraciones en los sistemas de degradación intracelular. En su conjunto, estas alteraciones en las CG pueden contribuir a los déficits cognitivos presentes desde etapas tempranas y, posteriormente, a la neurodegeneración características del ratón TS y las personas con SD.ABSTRACT: Down syndrome (DS) or trisomy of chromosome 21 is the most frequent genetic cause of intellectual disability. In this Doctoral Thesis, we used the Ts65Dn (TS) mouse model. Both, SD individuals and TS mice present deficits in hippocampal-dependent memory in which dentate gyrus granular cells (GCs) play an essential role. The results of this study show that the excess of gene dose produced changes in nuclear architecture, alterations in the nucleolar fusion pattern and in the assembly of the nuclear Cajal bodies in TS GCs. Additionally, increased oxidative stress in the TS hippocampus is associated with alterations such as increased lipid peroxidation, structural mitochondrial anomalies, alterations of intracellular degradative systems and increased DNA damage. Collectively, these cellular alterations may contribute to the cognitive deficits characteristic of TS mice and DS individuals since early ages and, at later stages of the lifespan, to the neurodegeneration.Esta Tesis Doctoral ha sido financiada con ayudas a la investigación procedentes de: • Contrato predoctoral para la formación de doctores 2017 concedido a Alba Puente Bedia (BES-2017-080571 • Agencia Estatal de Investigación, MICIN (PS12016-76194-R). • Instituto de Investigación Valdecilla (IDIVAL; NVAL 19/23), Santander, España. • Consejería de Universidades, Igualdad, Cultura y Deporte del Gobierno de Cantabria (16.VP39.64662). • Agencia Estatal de Investigación, MICIN (PID2020-117601 RB-IOO)

Early postnatal oleic acid administration enhances synaptic development and cognitive abilities in the Ts65Dn mouse model of Down syndrome

Objectives: The brains of individuals with Down syndrome (DS) present defects in neurogenesis and synaptogenesis during prenatal and early postnatal stages that are partially responsible for their cognitive disabilities. Because oleic and linolenic fatty acids enhance neurogenesis, synaptogenesis, and cognitive abilities in rodents and humans, in this study we evaluated the ability of these compounds to restore these altered phenotypes in the Ts65Dn (TS) mouse model of DS during early postnatal stages. Methods: TS and euploid mice were treated with oleic or linolenic acid from PD3 to PD15, and the short- and long- term effects of these acids on neurogenesis and synaptogenesis were evaluated. The effects of these treatments on the cognitive abilities of TS mice during early adulthood were also evaluated. Results: Administration of oleic or linolenic acid did not modify cell proliferation immediately after treatment discontinuation or several weeks later. However, oleic acid increased the total number of DAPI+ cells (+ 26%), the percentage of BrdU+ cells that acquired a neural phenotype (+ 9.1%), the number of pre- (+ 29%) and post-synaptic (+ 32%) terminals and the cognitive abilities of TS mice (+ 18.1%). In contrast, linolenic acid only produced a slight cognitive improvement in TS mice. (+12.1%). Discussion: These results suggest that early postnatal administration of oleic acid could palliate the cognitive deficits of DS individuals.This study was supported by Fundazione Generali e Assicurazioni Generali (Italy), IDIVAL (NVAL 19/23), and the Spanish Ministry of Economy and Competitiveness (PSI-2016-76194-R, AEI/FEDER, EU)