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1.
Elife ; 122023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37249575

RESUMO

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near-complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are likely due to a combination of increased activity level and sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle and hyperactivity. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.


Assuntos
Músculo Esquelético , Termogênese , Camundongos , Humanos , Animais , Músculo Esquelético/metabolismo , Termogênese/genética , Metabolismo Energético/fisiologia , Proteolipídeos/metabolismo , Citoplasma/metabolismo , Cromossomos Humanos/metabolismo , Cálcio/metabolismo
2.
bioRxiv ; 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36778465

RESUMO

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are due to sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

3.
Pharmaceutics ; 14(2)2022 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-35214105

RESUMO

The inherent instability of nucleic acids within serum and the tumor microenvironment necessitates a suitable vehicle for non-viral gene delivery to malignant lesions. A specificity-conferring mechanism is also often needed to mitigate off-target toxicity. In the present study, we report a stable and efficient redox-sensitive nanoparticle system with a unique core-shell structure as a DNA carrier for cancer theranostics. Thiolated polyethylenimine (PEI-SH) is complexed with DNA through electrostatic interactions to form the core, and glycol chitosan-modified with succinimidyl 3-(2-pyridyldithio)propionate (GCS-PDP) is grafted on the surface through a thiolate-disulfide interchange reaction to form the shell. The resulting nanoparticles, GCS-PDP/PEI-SH/DNA nanoparticles (GNPs), exhibit high colloid stability in a simulated physiological environment and redox-responsive DNA release. GNPs not only show a high and redox-responsive cellular uptake, high transfection efficiency, and low cytotoxicity in vitro, but also exhibit selective tumor targeting, with minimal toxicity, in vivo, upon systemic administration. Such a performance positions GNPs as viable candidates for molecular-genetic imaging and theranostic applications.

4.
Am J Hum Genet ; 109(2): 328-344, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35077668

RESUMO

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.


Assuntos
Ansiedade/genética , Cromossomos Humanos Par 21 , Síndrome de Down/genética , Efeito Fundador , Hipercinese/genética , Animais , Ansiedade/metabolismo , Ansiedade/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipercinese/metabolismo , Hipercinese/patologia , Cariótipo , Aprendizagem , Masculino , Mutagênese Insercional , Tamanho do Órgão , Postura , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Ratos , Ratos Transgênicos
5.
Acta Neuropathol Commun ; 9(1): 137, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399854

RESUMO

Down syndrome (DS) is the leading genetic cause of intellectual disability and causes early-onset dementia and cerebellar hypoplasia. The prevalence of attention deficit hyperactivity disorder is elevated in children with DS. The aneuploid DS mouse model "Ts65Dn" shows prominent brain phenotypes, including learning and memory deficits, cerebellar hypoplasia, and locomotor hyperactivity. Previous studies indicate that impaired Sonic hedgehog (Shh) signaling contributes to neurological phenotypes associated with DS and neurodegenerative diseases. However, because of a lack of working inducible Shh knock-in mice, brain region-specific Shh overexpression and its effects on cognitive function have not been studied in vivo. Here, with Gli1-LacZ reporter mice, we demonstrated that Ts65Dn had reduced levels of Gli1, a sensitive readout of Shh signaling, in both hippocampus and cerebellum at postnatal day 6. Through site-specific transgenesis, we generated an inducible human Shh knock-in mouse, TRE-bi-hShh-Zsgreen1 (TRE-hShh), simultaneously expressing dually-lipidated Shh-Np and Zsgreen1 marker in the presence of transactivator (tTA). Double transgenic mice "Camk2a-tTA;TRE-hShh" and "Pcp2-tTA;TRE-hShh" induced Shh overexpression and activated Shh signaling in a forebrain and cerebellum, respectively, specific manner from the perinatal period. Camk2a-tTA;TRE-hShh normalized locomotor hyperactivity and improved learning and memory in 3-month-old Ts65Dn, mitigated early-onset severe cognitive impairment in 7-month-old Ts65Dn, and enhanced spatial cognition in euploid mice. Camk2a-tTA;TRE-hShh cohort maintained until 600days old showed that chronic overexpression of Shh in forebrain from the perinatal period had no effect on longevity of euploid or Ts65Dn. Pcp2-tTA;TRE-hShh did not affect cognition but mitigated the phenotype of cerebellar hypoplasia in Ts65Dn. Our study provides the first in vivo evidence that Shh overexpression from the perinatal period protects DS brain integrity and enhances learning and memory in normal mice, indicating the broad therapeutic potential of Shh ligand for other neurological conditions. Moreover, the first inducible hShh site-specific knock-in mouse could be widely used for spatiotemporal Shh signaling regulation.


Assuntos
Cognição/fisiologia , Síndrome de Down/genética , Proteínas Hedgehog/genética , Locomoção/genética , Prosencéfalo/metabolismo , Animais , Cerebelo/metabolismo , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Síndrome de Down/fisiopatologia , Técnicas de Introdução de Genes , Hipocampo/metabolismo , Humanos , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Camundongos Transgênicos , Processamento Espacial/fisiologia , Proteína GLI1 em Dedos de Zinco/metabolismo
6.
Brain Commun ; 3(2): fcab062, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937769

RESUMO

The cerebellum is a complex system with distinct cortical laminar organization. Alterations in cerebellar microstructure are common and associated with many factors such as genetics, cancer and ageing. Diffusion MRI (dMRI) provides a non-invasive tool to map the brain structural organization, and the recently proposed diffusion-time (td )-dependent dMRI further improves its capability to probe the cellular and axonal/dendritic microstructures by measuring water diffusion at multiple spatial scales. The td -dependent diffusion profile in the cerebellum and its utility in detecting cerebellar disorders, however, are not yet elucidated. Here, we first deciphered the spatial correspondence between dMRI contrast and cerebellar layers, based on which the cerebellar layer-specific td -dependent dMRI patterns were characterized in both euploid and Ts65Dn mice, a mouse model of Down syndrome. Using oscillating gradient dMRI, which accesses diffusion at short td 's by modulating the oscillating frequency, we detected subtle changes in the apparent diffusivity coefficient of the cerebellar internal granular layer and Purkinje cell layer of Ts65Dn mice that were not detectable by conventional pulsed gradient dMRI. The detection sensitivity of oscillating gradient dMRI increased with the oscillating frequency at both the neonatal and adult stages. The td -dependence, quantified by ΔADC map, was reduced in Ts65Dn mice, likely associated with the reduced granule cell density and abnormal dendritic arborization of Purkinje cells as revealed from histological evidence. Our study demonstrates superior sensitivity of short-td diffusion using oscillating gradient dMRI to detect cerebellar microstructural changes in Down syndrome, suggesting the potential application of this technique in cerebellar disorders.

7.
Elife ; 92020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32597754

RESUMO

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.


Assuntos
Cromossomos Humanos Par 21/genética , Síndrome de Down/genética , Camundongos Transgênicos/genética , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Cardiopatias Congênitas/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Trissomia/genética , Sequenciamento Completo do Genoma
8.
Mol Biol Cell ; 28(5): 587-599, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28057765

RESUMO

Diabetes is linked to an increased risk for colorectal cancer, but the mechanistic underpinnings of this clinically important effect are unclear. Here we describe an interaction between the microtubule motor cytoplasmic dynein, the adenomatous polyposis coli tumor suppressor protein (APC), and glycogen synthase kinase-3ß (GSK-3ß), which could shed light on this issue. GSK-3ß is perhaps best known for glycogen regulation, being inhibited downstream in an insulin-signaling pathway. However, the kinase is also important in many other processes. Mutations in APC that disrupt the regulation of ß-catenin by GSK-3ß cause colorectal cancer in humans. Of interest, both APC and GSK-3ß interact with microtubules and cellular membranes. We recently demonstrated that dynein is a GSK-3ß substrate and that inhibition of GSK-3ß promotes dynein-dependent transport. We now report that dynein stimulation in intestinal cells in response to acute insulin exposure (or GSK-3ß inhibition) is blocked by tumor-promoting isoforms of APC that reduce an interaction between wild-type APC and dynein. We propose that under normal conditions, insulin decreases dynein binding to APC to stimulate minus end-directed transport, which could modulate endocytic and secretory systems in intestinal cells. Mutations in APC likely impair the ability to respond appropriately to insulin signaling. This is exciting because it has the potential to be a contributing factor in the development of colorectal cancer in patients with diabetes.


Assuntos
Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Dineínas do Citoplasma/metabolismo , Insulina/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Complicações do Diabetes/metabolismo , Feminino , Genes APC/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Ligação Proteica , Transdução de Sinais , beta Catenina/metabolismo
9.
Traffic ; 16(9): 941-61, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26010407

RESUMO

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.


Assuntos
Dineínas/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Citoplasma/metabolismo , Dineínas/química , Dineínas/genética , Quinase 3 da Glicogênio Sintase/genética , Humanos , Insulina/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Sistemas do Segundo Mensageiro
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