RESUMO
BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs) have intestinal barrier dysfunction. Creatine regulates energy distribution within cells and reduces the severity of colitis in mice. We studied the functions of the creatine transporter solute carrier family 6 member 8 (SLC6A8, also called CRT) in intestinal epithelial cells (IECs) and mice, and we measured levels in mucosal biopsies from patients with IBD. METHODS: Colon biopsy specimens from patients with IBD (30 with Crohn's disease and 27 with ulcerative colitis) and 30 patients without IBD (control individuals) and colon tissues from mice (with and without disruption of Crt) were analyzed by immunofluorescence, immunoblots, and/or quantitative reverse-transcription polymerase chain reaction (qRT-PCR). CRT was knocked down or overexpressed in T84 cells, which were analyzed by immunofluorescence, immunoblots, high-performance liquid chromatography (to measure creatine levels), qRT-PCR, transepithelial electrical resistance, barrier function, actin localization, wound healing, mitochondrial oxygen consumption, and glycolysis extracellular acidification rate assays. Organoids from colon cells of CRT-knockout mice and control mice were analyzed by qRT-PCR, immunoblot, and transepithelial electrical resistance. RESULTS: CRT localized around tight junctions (TJs) of T84 IECs. In analyses of IECs with CRT knockdown or overexpression, we found that CRT regulates intracellular creatine, barrier formation, and wound healing. CRT-knockout organoids also had diminished barrier formation. In the absence of adequate creatine, IECs transition toward a stressed, glycolysis-predominant form of metabolism; this resulted in leaky TJs and mislocalization of actin and TJ proteins. Colon tissues from patients with IBD had reduced levels of CRT messenger RNA compared with those from control individuals. CONCLUSIONS: In an analysis of IEC cell lines and colonoids derived from CRT-knockout mice, we found that CRT regulates energy balance in IECs and thereby epithelial integrity and barrier function. Mucosal biopsy specimens from patients with ulcerative colitis and inactive Crohn's disease have lower levels of CRT, which might contribute to the reduced barrier function observed in patients with IBD.
Assuntos
Colite Ulcerativa/patologia , Colo/patologia , Doença de Crohn/patologia , Mucosa Intestinal/patologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Adulto , Animais , Biópsia , Estudos de Casos e Controles , Linhagem Celular , Metabolismo Energético , Células Epiteliais/citologia , Células Epiteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Junções Íntimas/patologiaRESUMO
Creatine (Cr) is a guanidino compound that provides readily available phosphate pools for the regeneration of spent adenosine triphosphate (ATP). The lack of brain Cr causes moderate to severe intellectual disability, language impairment, and epilepsy. The most prevalent cause of Cr deficiency are mutations in the X-linked SLC6A8 (Creatine transporter; CrT) gene, known as CrT deficiency (CTD). One of the most critical areas that need to be addressed is whether Cr is necessary for brain development. To address this concern, the Slc6a8 gene was knocked out in either neonatal (postnatal day (P)5) or adult (P60) mice using a tamoxifen-inducible Cre recombinase driven by the human ubiquitin C (UBC) promoter. Mice were tested in the Morris water maze, novel, object recognition, and conditioned fear 60 days after Slc6a8 deletion. In addition, overnight locomotor activity was analyzed. Mice that had the gene deleted on P5 showed deficits in the Morris water maze and novel object recognition, while there were no deficits in P60 knockout mice. Interestingly, the P5 knockout mice showed hyperactivity during the dark phase; however, when examining control mice, the effect was due to the administration of tamoxifen from P5 to 10. Taken together, the results of this study show that Cr is necessary during periods of brain development involved in spatial and object learning. This study also highlights the continued importance of using proper control groups for behavioral testing.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Disfunção Cognitiva/genética , Creatina/deficiência , Proteínas de Membrana Transportadoras/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Animais , Encéfalo/metabolismo , Medo , Feminino , Aprendizagem em Labirinto , Transtornos da Memória/genética , Camundongos , Camundongos Knockout , Deleção de SequênciaRESUMO
Mitochondrial dysfunction plays a central role in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). This study was designed to determine whether the dipeptide carnosine, which has been shown to protect against oxidative stress and mitochondrial dysfunction, would provide a beneficial effect on mitochondrial function in the Thy1-aSyn mouse model of PD. Thy1-aSyn mice, which overexpress wild-type human alpha-synuclein (aSyn), exhibit progressive non-motor and motor deficits as early as 2â¯months of age. Two-month old Thy1-aSyn mice and wild-type littermates were randomly assigned to treatment groups with intranasal (IN) and drinking water carnosine, with controls receiving 10⯵l of sterile waster intranasally or carnosine-free drinking water, respectively. After two months of treatment, mice were euthanized, and the midbrain was dissected for the evaluation of the gene expression and mitochondrial function. Transcriptional deficiencies associated with the aSyn overexpression in Thy1-aSyn mice were related to ribosomal and mitochondrial function. These deficiencies were attenuated by IN carnosine administration, which increased the expression of mitochondrial genes and enhanced mitochondrial function. These results suggest a potential neuroprotective role for IN-carnosine in PD patients.
Assuntos
Carnosina/administração & dosagem , Doença de Parkinson/tratamento farmacológico , Antígenos Thy-1/genética , alfa-Sinucleína/genética , Administração Intranasal , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Transcriptoma/efeitos dos fármacosRESUMO
Creatine (Cr) is a guanidino compound required for rapid replenishment of ATP in cells with a high-energy demand. In humans, mutations in the Cr transporter (CRT;SLC6A8) prevent Cr entry into tissue and result in a significant intellectual impairment, epilepsy, and aphasia. The lack of Cr on both the whole body and cellular metabolism was evaluated in Crt knockout (Crt (-/y) ) mice, a high-fidelity model of human CRT deficiency. Crt (-/y) mice have reduced body mass and, however, show a twofold increase in body fat. There was increased energy expenditure in a home cage environment and during treadmill running in Crt (-/y) mice. Consistent with the increases in the whole-body metabolic function, Crt (-/y) mice show increased cellular metabolism as well. Mitochondrial respiration increased in skeletal muscle fibers and hippocampal lysates from Crt (-/y) mice. In addition, Crt (-/y) mice had increased citrate synthase activity, suggesting a higher number of mitochondria instead of an increase in mitochondrial activity. To determine if the increase in respiration was due to increased mitochondrial numbers, we measured oxygen consumption in an equal number of mitochondria from Crt (+/y) and Crt (-/y) mice. There were no changes in mitochondrial respiration when normalized to mitochondrial number, suggesting that the increase in respiration observed could be to higher mitochondrial content in Crt (-/y) mice.
Assuntos
Adiposidade , Hipocampo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Animais , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Feminino , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Mutantes , Mitocôndrias Musculares/genética , Consumo de Oxigênio/genéticaRESUMO
Both egocentric route-based learning and spatial learning, as assessed by the Cincinnati water maze (CWM) and Morris water maze (MWM), respectively, are impaired following an 80% dopamine (DA) loss in the neostriatum after 6-hydroxydopamine (6-OHDA) administration in rats. The dorsolateral striatum (DLS) and the dorsomedial striatum (DMS) are implicated in different navigational learning types, namely the DLS is implicated in egocentric learning while the DMS is implicated in spatial learning. This experiment tested whether selective DA loss through 6-OHDA lesions in the DMS or DLS would impair one or both types of navigation. Both DLS and DMS DA loss significantly impaired route-based CWM learning, without affecting spatial or cued MWM performance. DLS 6-OHDA lesions produced a 75% DA loss in this region, with no changes in other monoamine levels in the DLS or DMS. DMS 6-OHDA lesions produced a 62% DA loss in this region, without affecting other monoamine levels in the DMS or DLS. The results indicate a role for DA in DLS and DMS regions in route-based egocentric but not spatial learning and memory. Spatial learning deficits may require more pervasive monoamine reductions within each region before deficits are exhibited. This is the first study to implicate DLS and DMS DA in route-based egocentric navigation.
Assuntos
Dopamina/fisiologia , Aprendizagem em Labirinto/fisiologia , Neostriado/fisiologia , Navegação Espacial/fisiologia , Animais , Monoaminas Biogênicas/análise , Monoaminas Biogênicas/síntese química , Dopamina/síntese química , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Neostriado/química , Oxidopamina/administração & dosagem , Ratos , Ratos Sprague-Dawley , Navegação Espacial/efeitos dos fármacosRESUMO
Creatine transporter (CrT) deficiency (CTD) is an X-linked disorder characterized by intellectual disability and speech delay. There have been reports that show female carriers have clinical symptoms. We have created CrT knockout (CrT(-/y)) mice in which males show severe cognitive deficits as a model of this disorder. The purpose of this study was to examine if the female carrier mice show cognitive deficits. Reductions in Cr levels as well as CrT transcript were observed in the brains of the female CrT(+/-) mice. CrT(+/-) mice show hyperactivity and increased latency to find the cued platform in the Morris water maze (MWM). CrT(+/-) female mice showed deficits in MWM hidden platform acquisition but not during reversal testing. Memory deficits on probe trials were observed during both phases. Novel object recognition memory and contextual fear memory were not affected in female CrT(+/-) mice. Female CrT(+/-) mice show moderate cognitive deficits, which is consistent with some of the human data. Female CrT(+/-) mice could prove to be beneficial in further understanding CTD and testing therapeutic approaches.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Transtornos Cognitivos/genética , Creatina/deficiência , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Animais , Comportamento Animal , Encéfalo/metabolismo , Transtornos Cognitivos/complicações , Creatina/genética , Modelos Animais de Doenças , Medo , Feminino , Regulação da Expressão Gênica , Heterozigoto , Locomoção , Aprendizagem em Labirinto , Memória , Transtornos da Memória/genética , Camundongos , Camundongos Knockout , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genéticaRESUMO
The creatine (Cr)-phosphocreatine shuttle is essential for ATP homeostasis. In humans, the absence of brain Cr causes significant intellectual disability, epilepsy, and language delay. Mutations of the creatine transporter (SLC6A8) are the most common cause of Cr deficiency. In rodents, Slc6a8 deletion causes deficits in spatial learning, novel object recognition (NOR), as well as in contextual and cued freezing. The mechanisms that underlie these cognitive deficits are not known. Due to the heterogeneous nature of the brain, it is important to determine which systems are affected by a loss of Cr. In this study, we generated mice lacking Slc6a8 in GABAergic neurons by crossing Slc6a8FL mice with Gad2-Cre mice. These Gad2-specific Slc6a8 knockout (cKO) mice, along with the ubiquitous Slc6a8 KO (Slc6a8-/y), Gad2-Cre+, and wild-type (WT) mice were tested in the Morris water maze, NOR, conditioned freezing, and the radial water maze. Similar to the Slc6a8-/y mice, cKO mice had reduced contextual and cued freezing compared with WT mice. The cKO mice had a mild spatial learning deficit during the reversal phase of the MWM, however they were not as pronounced as in Slc6a8-/y mice. In NOR, the Gad2-Cre mice spent less time with the novel object, similar to the reduced novel time in the cKO mice. There were no changes in radial water maze performance. Slc6a8 deletion in GABAergic neurons is sufficient to recapitulate the conditioned freezing deficits seen in Slc6a8-/y mice.
Assuntos
Transtornos Cognitivos , Disfunção Cognitiva , Humanos , Animais , Camundongos , Encéfalo , Disfunção Cognitiva/genética , Creatina , Fosfocreatina , Camundongos KnockoutRESUMO
Neonatal exposure to (+)-methamphetamine (Meth) results in long-term behavioural abnormalities but its developmental mechanisms are unknown. In a series of experiments, rats were treated from post-natal days (PD) 11-20 (stage that approximates human development from the second to third trimester) with Meth or saline and assessed using locomotor activity as the readout following pharmacological challenge doses with dopamine, serotonin and glutamate agonists or antagonists during adulthood. Exposure to Meth early in life resulted in an exaggerated adult locomotor hyperactivity response to the dopamine D1 agonist SKF-82958 at multiple doses, a high dose only under-response activating effect of the D2 agonist quinpirole, and an exaggerated under-response to the activating effect of the N-methyl-d-aspartic acid (NMDA) receptor antagonist, MK-801. No change in locomotor response was seen following challenge with the 5-HT releaser p-chloroamphetamine or the 5-HT2/3 receptor agonist, quipazine. These are the first data to show that PD 11-20 Meth exposure induces long-lasting alterations to dopamine D1, D2 and glutamate NMDA receptor function and may suggest how developmental Meth exposure leads to many of its long-term adverse effects.
Assuntos
Dopaminérgicos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Metanfetamina/farmacologia , Atividade Motora/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Masculino , Quipazina/farmacologia , Ratos , Ratos Sprague-Dawley , Agonistas do Receptor de Serotonina/farmacologia , Fatores de TempoRESUMO
We previously showed that developmental 3,4-methylenedioxymethamphetamine (MDMA) treatment induces long-term spatial and egocentric learning and memory deficits and serotonin (5-HT) reductions. During brain development, 5-HT is a neurotrophic factor influencing neurogenesis, synaptogenesis, migration, and target field organization. MDMA (10 mg/kg × 4/d at 2 h intervals) given on post-natal day (PD) 11-20 in rats (a period of limbic system development that approximates human third trimester brain development) induces 50% reductions in 5-HT during treatment and 20% reductions when assessed as adults. To determine whether the 5-HT reduction is responsible for the cognitive deficits, we used citalopram (Cit) pretreatment to inhibit the effects of MDMA on 5-HT reuptake in a companion study. Cit attenuated MDMA-induced 5-HT reductions by 50% (Schaefer et al., 2012). Here we tested whether Cit (5 or 7.5 mg/kg × 2/d) pretreatment attenuates the cognitive effects of MDMA. Within each litter, different offspring were treated on PD11-20 with saline (Sal) + MDMA, Cit + MDMA, Cit + Sal or Sal + Sal. Neither spatial nor egocentric learning/memory was improved by Cit pretreatment. Unexpectedly, Cit + Sal (at both doses) produced spatial and egocentric learning deficits as severe as those caused by Sal + MDMA. These are the first data showing cognitive deficits resulting from developmental exposure to a selective serotonin reuptake inhibitor. These data indicate the need for further research on the long-term safety of antidepressants during pregnancy.
Assuntos
Citalopram/toxicidade , Transtornos Cognitivos/induzido quimicamente , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Serotoninérgicos/toxicidade , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Feminino , Locomoção/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Natação/psicologiaRESUMO
Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.
RESUMO
Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.
Assuntos
Deficiência Intelectual , Deficiência Intelectual Ligada ao Cromossomo X , Humanos , Deficiência Intelectual/genética , Creatina/genética , Creatina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Encéfalo/metabolismo , Organoides/metabolismoRESUMO
Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.
Assuntos
Doença de Gaucher/enzimologia , Glucosilceramidase/genética , Glucosilceramidas/metabolismo , Proteínas Mutantes/metabolismo , Doenças do Sistema Nervoso/complicações , Psicosina/análogos & derivados , Saposinas/deficiência , Substituição de Aminoácidos/genética , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Cromatografia Líquida , Modelos Animais de Doenças , Doença de Gaucher/genética , Doença de Gaucher/patologia , Doença de Gaucher/fisiopatologia , Inflamação/complicações , Inflamação/patologia , Potenciação de Longa Duração/fisiologia , Longevidade , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Espectrometria de Massas , Camundongos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Fenótipo , Psicosina/metabolismo , Saposinas/metabolismoRESUMO
Saposins A, B, C and D are derived from a common precursor, prosaposin (psap). The few patients with saposin C deficiency develop a Gaucher disease-like central nervous system (CNS) phenotype attributed to diminished glucosylceramide (GC) cleavage activity by acid beta-glucosidase (GCase). The in vivo effects of saposin C were examined by creating mice with selective absence of saposin C (C-/-) using a knock-in point mutation (cysteine-to-proline) in exon 11 of the psap gene. In C-/- mice, prosaposin and saposins A, B and D proteins were present at near wild-type levels, but the saposin C protein was absent. By 1 year, the C-/- mice exhibited weakness of the hind limbs and progressive ataxia. Decreased neuromotor activity and impaired hippocampal long-term potentiation were evident. Foamy storage cells were observed in dorsal root ganglion and there was progressive loss of cerebellar Purkinje cells and atrophy of cerebellar granule cells. Ultrastructural analyses revealed inclusions in axonal processes in the spinal cord, sciatic nerve and brain, but no excess of multivesicular bodies. Activated microglial cells and astrocytes were present in thalamus, brain stem, cerebellum and spinal cord, indicating regional pro-inflammatory responses. No storage cells were found in visceral organs of these mice. The absence of saposin C led to moderate increases in GC and lactosylceramide (LacCer) and their deacylated analogues. These results support the view that saposin C has multiple roles in glycosphingolipid (GSL) catabolism as well as a prominent function in CNS and axonal integrity independent of its role as an optimizer/stabilizer of GCase.
Assuntos
Sistema Nervoso Central/metabolismo , Doença de Gaucher/metabolismo , Glucosilceramidase/metabolismo , Saposinas/deficiência , Animais , Comportamento Animal , Sistema Nervoso Central/citologia , Sistema Nervoso Central/enzimologia , Modelos Animais de Doenças , Feminino , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Doença de Gaucher/fisiopatologia , Glucosilceramidase/genética , Glicoesfingolipídeos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células de Purkinje/metabolismoRESUMO
Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0-P14) and adult (P22-P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K(+) cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine-phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.
Assuntos
Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Creatina Quinase Forma BB/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Creatina Quinase Forma BB/análise , Creatina Quinase Forma BB/biossíntese , Creatina Quinase Forma BB/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Masculino , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Distribuição TecidualRESUMO
Previous findings showed allocentric and egocentric learning deficits in rats after MDMA treatment from postnatal days (PD) 11-20 but not after treatment from PD 1-10. Shorter treatment periods (PD 1-5, 6-10, 11-15, or 16-20) resulted in allocentric learning deficits averaged across intervals but not for any interval individually and no egocentric learning deficits individually or collectively. Whether this difference was attributable to treatment length or age at the start of treatment was unclear. In the present experiment rat litters were treated on PD 1-10, 6-15, or 11-20 with 0, 10, or 15 mg/kg MDMA q.i.d. at 2-h intervals. Two male/female pairs/litter received each treatment. One pair/litter received acoustic startle with prepulse inhibition, straight channel swimming, Cincinnati water maze (CWM), and conditioned fear in a latent inhibition paradigm. The other pair/litter received locomotor activity, straight channel swimming, Morris water maze (MWM), and locomotor activity retest with MK-801 challenge. MDMA impaired CWM learning following PD 6-15 or 11-20 exposure. In MWM acquisition, all MDMA-treated groups showed impairment. During reversal and shift, the PD 6-15 and PD 11-20 MDMA-treated groups were significantly impaired. Reductions in locomotor activity were most evident after PD 6-15 treatment while increases in acoustic startle were most evident after PD 1-10 treatment. After MK-801 challenge, MDMA-treated offspring showed less locomotion compared to controls. Region-specific changes in brain monoamines were also observed but were not significantly correlated with behavioural changes. The results show that PD 11-20 exposure to MDMA caused the largest long-term cognitive deficits followed by PD 6-15 exposure with PD 1-10 exposure least affected. Other effects, such as those upon MK-801-stimulated locomotion showed greatest effects after PD 1-10 MDMA exposure. Hence, each effect has a different window of developmental susceptibility.
Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Comportamento Animal/efeitos dos fármacos , Monoaminas Biogênicas/metabolismo , Alucinógenos/farmacologia , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Estimulação Acústica , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Medo/efeitos dos fármacos , Feminino , Inibição Psicológica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Mortalidade , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Reflexo de Sobressalto/efeitos dos fármacos , Natação/psicologiaRESUMO
Prenatal maternal immune activation has been used to test the neurodevelopmental hypothesis of schizophrenia. Most of the data are in mouse models; far less is available for rats. We previously showed that maternal weight change in response to the immune activator polyinosinic-polycytidylic acid (Poly IC) in rats differentially affects offspring. Therefore, we treated gravid Harlan Sprague-Dawley rats i.p. on embryonic day 14 with 8 mg/kg of Poly IC or Saline. The Poly IC group was divided into those that lost or gained the least weight, Poly IC (L), versus those that gained the most weight, Poly IC (H), following treatment. The study design controlled for litter size, litter sampling, sex distribution, and test experience. We found no effects of Poly IC on elevated zero maze, open-field activity, object burying, light-dark test, straight channel swimming, Morris water maze spatial acquisition, reversal, or shift navigation or spatial working or reference memory, or conditioned contextual or cued fear or latent inhibition. The Poly IC (H) group showed a significant decrease in the rate of route-based learning when visible cues were unavailable in the Cincinnati water maze and reduced prepulse inhibition of acoustic startle in females, but not males. The Poly IC (L) group exhibited altered responses to acute pharmacological challenges: exaggerated hyperactivity in response to (+)-amphetamine and an attenuated hyperactivity in response to MK-801. This model did not exhibit the cognitive, or latent inhibition deficits reported in Poly IC-treated rats but showed changes in response to drugs acting on neurotransmitter systems implicated in the pathophysiology of schizophrenia (dopaminergic hyperfunction and glutamatergic hypofunction).
Assuntos
Dopaminérgicos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Exposição Materna , Poli I-C/farmacologia , Anfetamina/farmacologia , Animais , Maleato de Dizocilpina/farmacologia , Feminino , Aprendizagem/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Testes Neuropsicológicos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/psicologia , Ratos , Ratos Sprague-Dawley , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/fisiologia , Aumento de Peso/imunologia , Aumento de Peso/fisiologiaRESUMO
Late-life dementia typically develops over a period of many years beginning in midlife. Prevalence of metabolic disturbance also accelerates in middle age and is a prominent risk factor for dementia. Preliminary studies indicate that blueberry supplementation can improve cognitive performance and influence metabolism and brain function and therefore may have a role in early intervention to prevent neurodegeneration. In a randomized controlled trial, we investigated the effects of daily blueberry supplementation in a middle-aged sample of insulin-resistant participants with elevated risk for future dementia. We enrolled overweight men and women, aged 50 to 65 years, with subjective cognitive decline (SCD) and performed pre- and post-intervention assessments of cognition and metabolism and exploratory measures of peripheral mitochondrial function. We observed improved performances for the blueberry group on measures of lexical access, p = 0.003, and memory interference, p = 0.04, and blueberry-treated participants reported reduced memory encoding difficulty in daily life activities, p = 0.03. The blueberry-treated group also exhibited correction of peripheral hyperinsulinemia, p = 0.04, and a modest trend for increased mitochondrial uncoupling, p = 0.11. The cognitive findings indicated improved executive ability in this middle-aged sample. In addition, the changes in metabolic and bioenergetic measures imply potential mechanistic factors associated with anthocyanin and proanthocyanidin actions. The demonstration of these benefits in middle-aged individuals with insulin resistance and SCD suggests that ongoing blueberry supplementation may contribute to protection against cognitive decline when implemented early in at-risk individuals.
Assuntos
Mirtilos Azuis (Planta) , Disfunção Cognitiva , Demência , Cognição , Disfunção Cognitiva/epidemiologia , Disfunção Cognitiva/prevenção & controle , Demência/epidemiologia , Demência/prevenção & controle , Suplementos Nutricionais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Comportamento de Redução do RiscoRESUMO
(+)-Methamphetamine (MA), (±)-3,4-methylenedioxymethamphetamine (MDMA), (+)-amphetamine (AMPH), and (±)-fenfluramine (FEN) are phenylethylamines with CNS effects. At higher doses, each induces protracted reductions in brain dopamine (DA) and/or serotonin. Chronic MA and MDMA users show persistent monoamine reductions and cognitive impairments. In rats, similar neurochemical effects can be induced, yet cognitive impairments have been difficult to demonstrate. We recently showed that rats treated on a single day with MA (10 mg/kg x 4 at 2 h intervals) exhibit impaired egocentric learning (Cincinnati water maze [CWM]) without affecting spatial learning (Morris water maze [MWM]) (Herring et al., [2008] Psychopharmacology (Berl) 199:637650). Whether this effect is unique to MA or is a general characteristic of these drugs is unknown. Accordingly, this experiment compared these drugs on CWM performance. Drugs were given s.c. in four doses at 2 h intervals. MA doses were 10 or 12.5 mg/kg/dose, AMPH 25 mg/kg/dose (to match MA12.5-induced hyperthermia), MDMA 15 mg/kg/dose (previously established hyperthermia-inducing dose), and FEN 16.5 mg/kg/dose (equimolar to MA12.5). Two weeks later, rats were tested in the CWM (2 trials/day, 21 days). AMPH and MA (both doses) induced significant increases in CWM errors and latency to reach the goal with no differences in swim speed. MDMA and FEN did not significantly alter learning. Given that FEN selectively and MDMA preferentially affect serotonin whereas AMPH selectively and MA preferentially affect DA, the data suggest that egocentric learning may be predominantly dopaminergically mediated.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Serotoninérgicos/farmacologia , Comportamento Espacial/efeitos dos fármacos , Anfetamina/farmacologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Temperatura Baixa , Fenfluramina/farmacologia , Masculino , Metanfetamina/farmacologia , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Ratos , Ratos Sprague-Dawley , Natação , Fatores de TempoRESUMO
RATIONALE: Whether selective serotonin reuptake inhibitors (SSRIs) exposure during adolescent brain development causes lasting effects remains unresolved. OBJECTIVE: Assess the effects of fluoxetine and paroxetine 60 days after adolescent exposure compared with when on-drug. METHODS: Male Sprague-Dawley littermates (41 litters) were gavaged on postnatal days 33-53 with fluoxetine (3 or 10 mg/kg/day), paroxetine (3, 10 or, 17 mg/kg/day), or water; half were tested while on-drug (21 litters) and half after 60 days off-drug (20 litters). RESULTS: The highest dose of the drugs reduced body weight gain during treatment that rebounded 1 week post-treatment. On-drug, no significant group differences were found on elevated plus maze time-in-open, zone entries, or latency to first open entry; however, the high dose of paroxetine significantly reduced head-dips (N=20/group). No significant effects were found on-drug for acoustic startle response/prepulse inhibition (ASR/PPI) although a trend (p<0.10) was seen, which after combining dose levels, showed a significant increase in ASR amplitude for both fluoxetine and paroxetine (N=20-21/group). No differences on immobility time were seen in the Porsolt forced swim test or in plasma corticosterone at the end of forced swim (N-19-21/group). Off-drug, no effects were seen in the elevated plus maze (N=16/group), ASR/PPI (N=20/group), forced swim (N=19-20/group), or plasma corticosterone (N=19/group). At the doses tested, fluoxetine and paroxetine induced minor effects with drug on-board but no residual, long-term adverse effects in rats 60 days after drug discontinuation. CONCLUSIONS: The data provide no evidence that fluoxetine or paroxetine have long-term adverse effects on the behaviors measured here after adolescent to young adult exposure.
Assuntos
Encéfalo/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Fluoxetina/farmacologia , Paroxetina/farmacologia , Reflexo de Sobressalto/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Estimulação Acústica , Fatores Etários , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Corticosterona/sangue , Período Crítico Psicológico , Relação Dose-Resposta a Droga , Reação de Fuga/efeitos dos fármacos , Resposta de Imobilidade Tônica/efeitos dos fármacos , Estudos Longitudinais , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Ratos , Filtro Sensorial/efeitos dos fármacosRESUMO
Saposin B derives from the multi-functional precursor, prosaposin, and functions as an activity enhancer for several glycosphingolipid (GSL) hydrolases. Mutations in saposin B present in humans with phenotypes resembling metachromatic leukodystrophy. To gain insight into saposin B's physiological functions, a specific deficiency was created in mice by a knock-in mutation of an essential cysteine in exon 7 of the prosaposin locus. No saposin B protein was detected in the homozygotes (B-/-) mice, whereas prosaposin, and saposins A, C and D were at normal levels. B-/- mice exhibited slowly progressive neuromotor deterioration and minor head tremor by 15 months. Excess hydroxy and non-hydroxy fatty acid sulfatide levels were present in brain and kidney. Alcian blue positive (sulfatide) storage cells were found in the brain, spinal cord and kidney. Ultrastructural analyses showed lamellar inclusion material in the kidney, sciatic nerve, brain and spinal cord tissues. Lactosylceramide (LacCer) and globotriaosylceramide (TriCer) were increased in various tissues of B-/- mice supporting the in vivo role of saposin B in the degradation of these lipids. CD68 positive microglial cells and activated GFAP positive astrocytes showed a proinflammatory response in the brains of B-/- mice. These findings delineate the roles of saposin B for the in vivo degradation of several GSLs and its primary function in maintenance of CNS function. B-/- provide a useful model for understanding the contributions of this saposin to GSL metabolism and homeostasis.