RESUMO
Methylmercury (MeHg) is a well-known neurotoxicant that induces various cellular functions depending on cellular- and developmental-specific vulnerabilities. MeHg has a high affinity for selenol and thiol groups, thus impairing the antioxidant system. Such affinity characteristics of MeHg led us to develop sensor vectors to assess MeHg toxicity. In this study, MeHg-mediated defects in selenocysteine (Sec) incorporation were demonstrated using thioredoxin reductase 1 cDNA fused with the hemagglutinin tag sequence at the C-terminus. Taking advantage of such MeHg-mediated defects in Sec incorporation, a cDNA encoding luciferase with a Sec substituted for cysteine-491 was constructed. This construct showed MeHg-induced decreases in signaling in a dose-dependent manner. To directly detect truncated luciferase under MeHg exposure, we further constructed a new sensor vector fused with a target for proteasomal degradation. However, this construct was inadequate because of the low rate of Sec insertion, even in the absence of MeHg. Finally, a Krab transcriptional suppressor fused with Sec was constructed and assessed to demonstrate MeHg-dependent increases in signal intensity. We confirmed that the vector responded specifically and in a dose-dependent manner to MeHg in cultured cerebellar granule cells. This vector is expected to allow monitoring of MeHg-specific toxicity via spatial and temporal imaging.
Assuntos
Compostos de Metilmercúrio , Compostos de Metilmercúrio/toxicidade , Animais , Humanos , Camundongos , Técnicas Biossensoriais/métodos , Luciferases/metabolismo , Luciferases/genética , Cerebelo/metabolismo , Cerebelo/efeitos dos fármacosRESUMO
LINE-1 and Alu retrotransposons are components of the human genome and have been implicated in many human diseases. These elements can influence human transcriptome plasticity in various mechanisms. Chimeric transcripts derived from LINE-1 and Alu can also impact the human transcriptome, such as exonization and post-transcriptional modification. However, its specific role in ASD neuropathology remains unclear, particularly in the cerebellum tissues. We performed RNA-sequencing of post-mortem cerebellum tissues from ASD and unaffected individuals for transposable elements profiling and chimeric transcript identification. The majority of free transcripts of transposable elements were not changed in the cerebellum tissues of ASD compared with unaffected individuals. Nevertheless, we observed that chimeric transcripts derived from LINE-1 and Alu were embedded in the transcripts of differentially expressed genes in the cerebellum of ASD, and these genes were related to developments and abnormalities of the cerebellum. In addition, the expression levels of these genes were correlated with the significantly decreased thickness of the molecular layer in the cerebellum of ASD. We also found that global methylation and expression of LINE-1 and Alu elements were not changed in ASD, but observed in the ASD sub-phenotypes. Our findings showed associations between transposable elements and cerebellar abnormalities in ASD, particularly in distinct phenotypic subgroups. Further investigations using appropriate models are warranted to elucidate the structural and functional implications of LINE-1 and Alu elements in ASD neuropathology.
Assuntos
Elementos Alu , Transtorno do Espectro Autista , Cerebelo , Elementos Nucleotídeos Longos e Dispersos , Humanos , Cerebelo/metabolismo , Cerebelo/patologia , Elementos Nucleotídeos Longos e Dispersos/genética , Elementos Alu/genética , Transtorno do Espectro Autista/genética , Masculino , Feminino , Retroelementos/genética , Metilação de DNA , Transcriptoma , AdultoRESUMO
The behavioral effects of α-synuclein oligomers were studied at various times after its chronic intranasal administration to 75-day-old C57BL/6J mice in comparison with the dynamics of changes in the transcriptional activity of caspases genes (Casp9, Casp8, and Casp3) in the hippocampus, frontal cortex, and cerebellum. The negative effects of α-synuclein oligomers on exploratory activity and short-term memory in the novel object recognition test were most pronounced after 90 days from the end of administration, while after 1 and 270 days, partial compensation of the studied cognitive functions was observed. Analysis of the expression of caspase genes suggests that early compensatory mechanisms are associated with suppression of the effector caspase-3 gene expression along with increased activity of the genes encoding initiator caspases-9 and -8. Late compensation processes are associated with a decrease in the activity of initiator caspases in the frontal cortex and cerebellum.
Assuntos
Caspase 3 , Caspase 8 , Caspase 9 , Cerebelo , Disfunção Cognitiva , Hipocampo , Camundongos Endogâmicos C57BL , alfa-Sinucleína , Animais , Camundongos , Caspase 3/genética , Caspase 3/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Hipocampo/metabolismo , Cerebelo/metabolismo , Lobo Frontal/metabolismo , Masculino , Modelos Animais de Doenças , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Memória de Curto Prazo/efeitos dos fármacosRESUMO
Neuronal development is a highly regulated process that is dependent on the correct coordination of cellular responses to extracellular cues. In response to semaphorin axon guidance proteins, the MICAL1 protein is stimulated to produce reactive oxygen species that oxidize actin on specific methionine residues, leading to filamentous actin depolymerization and consequent changes in neuronal growth cone dynamics. Crossing genetically modified mice homozygous for floxed Mical1 (Mical1fl/fl) alleles with transgenic mice expressing Cre recombinase under the control of a tyrosinase gene enhancer/promoter (Tyr::Cre) enabled conditional Mical1 deletion. Immunohistochemical analysis showed Mical1 expression in the cerebellum, which plays a prominent role in the coordination of motor movements, with reduced Mical1 expression in Mical1fl/fl mice co-expressing Tyr::Cre. Analysis of the gaits of mice running on a treadmill showed that both male and female Mical1fl/fl, Tyr::Cre mutant mice had significant alterations to their striding patterns relative to wild-type mice, although the specific aspects of their altered gaits differed between the sexes. Additional motor tests that involved movement on a rotating rod, descending a vertical pole, or crossing a balance beam did not show significant differences between the genotypes, suggesting that the effect of the Mical1fl/fl, Tyr::Cre genetic modifications was only manifested during specific highly coordinated movements that contribute to running. These findings indicate that there is a behavioral consequence in Mical1fl/fl, Tyr::Cre mutant mice that affects motor control as manifested by alterations in their gait.
Assuntos
Monofenol Mono-Oxigenase , Animais , Camundongos , Feminino , Masculino , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Marcha/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Camundongos Transgênicos , Cerebelo/metabolismo , Corrida/fisiologia , Camundongos Endogâmicos C57BLRESUMO
SNCA/PARK1 encodes α-synuclein, which is associated with familial Parkinson's disease. Despite its abundance in presynaptic terminals, the aggregation mechanism of α-synuclein and its relationship with Parkinson's disease have not yet been elucidated. Moreover, the ultrastructures of α-synuclein localization sites in neuronal presynaptic terminals remain unclear. Therefore, we herein generated transgenic mice expressing human α-synuclein tagged with mKate2 (hSNCA-mKate2 mice). These mice exhibited normal growth and fertility and had no motor dysfunction relative to their wild-type littermates, even at one year old. α-Synuclein-mKate2 accumulated in presynaptic terminals, particularly between Purkinje cells in the cerebellum and neurons in cerebellar nuclei. α-Synuclein-mKate2 was associated with the presynaptic marker, synaptophysin. In-resin CLEM and immunoelectron or electron microscopy revealed that α-synuclein-mKate2 localized on the surface of synaptic vesicles that were tightly arranged and assembled to form large synaptic pools in the cerebellum with negligible effects on the active zone. These results suggest that α-synuclein-associated ultrastructures in the presynaptic terminals of hSNCA-mKate2 mice reflect the structures of α-synuclein-assembled synaptic vesicle pools, and the size of vesicle pools increased. This transgenic mouse model will be a valuable tool for studying α-synuclein-associated synaptic vesicle pools.
Assuntos
Camundongos Transgênicos , Terminações Pré-Sinápticas , Vesículas Sinápticas , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Camundongos , Humanos , Células de Purkinje/metabolismo , Células de Purkinje/ultraestrutura , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Cerebelo/metabolismo , Cerebelo/ultraestrutura , Sinaptofisina/metabolismo , Sinaptofisina/genética , MasculinoRESUMO
BACKGROUND: The Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during postnatal CGN differentiation. RESULTS: We first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs. CONCLUSIONS: Our data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development.
Assuntos
Neurônios , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Camundongos , Neurônios/metabolismo , Cerebelo/metabolismo , Diferenciação Celular/genética , Genoma , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Agathisflavone is a flavonoid that exhibits anti-inflammatory and anti-oxidative properties. Here, we investigated the neuroprotective effects of agathisflavone on central nervous system (CNS) neurons and glia in the cerebellar slice ex vivo model of neonatal ischemia. Cerebellar slices from neonatal mice, in which glial fibrillary acidic protein (GFAP) and SOX10 drive expression of enhanced green fluorescent protein (EGFP), were used to identify astrocytes and oligodendrocytes, respectively. Agathisflavone (10 µM) was administered preventively for 60 min before inducing ischemia by oxygen and glucose deprivation (OGD) for 60 min and compared to controls maintained in normal oxygen and glucose (OGN). The density of SOX-10+ oligodendrocyte lineage cells and NG2 immunopositive oligodendrocyte progenitor cells (OPCs) were not altered in OGD, but it resulted in significant oligodendroglial cell atrophy marked by the retraction of their processes, and this was prevented by agathisflavone. OGD caused marked axonal demyelination, determined by myelin basic protein (MBP) and neurofilament (NF70) immunofluorescence, and this was blocked by agathisflavone preventative treatment. OGD also resulted in astrocyte reactivity, exhibited by increased GFAP-EGFP fluorescence and decreased expression of glutamate synthetase (GS), and this was prevented by agathisflavone pretreatment. In addition, agathisflavone protected Purkinje neurons from ischemic damage, assessed by calbindin (CB) immunofluorescence. The results demonstrate that agathisflavone protects neuronal and myelin integrity in ischemia, which is associated with the modulation of glial responses in the face of ischemic damage.
Assuntos
Animais Recém-Nascidos , Cerebelo , Flavonoides , Fármacos Neuroprotetores , Animais , Fármacos Neuroprotetores/farmacologia , Camundongos , Cerebelo/metabolismo , Cerebelo/efeitos dos fármacos , Flavonoides/farmacologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Glucose/metabolismo , BiflavonoidesRESUMO
Neurotransmitter release consists of rapid synchronous release followed by longer-lasting asynchronous release (AR). Although the presynaptic proteins that trigger synchronous release are well understood, the mechanisms for AR remain unclear. AR is sustained by low concentrations of intracellular Ca2+ and Sr2+, suggesting the involvement of sensors with high affinities for both ions. Synaptotagmin 7 (SYT7) partly mediates AR, but substantial AR persists in the absence of SYT7. The closely related SYT3 binds Ca2+ and Sr2+ with high affinity, making it a promising candidate to mediate AR. Here, we use knockout mice to study the contribution of SYT3 and SYT7 to AR at cerebellar and hippocampal synapses. AR is dramatically reduced when both isoforms are absent, which alters the number and timing of postsynaptic action potentials. Our results confirm the long-standing prediction that SYT3 mediates AR and show that SYT3 and SYT7 act as dominant mechanisms for AR at three central synapses.
Assuntos
Cerebelo , Hipocampo , Camundongos Knockout , Sinapses , Sinaptotagminas , Animais , Cerebelo/metabolismo , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Hipocampo/metabolismo , Sinapses/metabolismo , Camundongos , Cálcio/metabolismo , Camundongos Endogâmicos C57BL , Transmissão SinápticaRESUMO
OBJECTIVE: Exploring the changes in cerebellar ferroptosis in hypertensive mice after lead exposure. METHODS: Twenty-five healthy C57 male mice were selected to construct a hypertensive model by intraperitoneal injection of angiotensin II(Ang II) at a concentration of 0.05 mg/kg for 7 consecutive days. After a systolic blood pressure of 140 mmHg, 20 hypertensive mice were randomly divided into a hypertensive control group and a hypertensive lead exposure group. Twenty C57 mice with normal blood pressure were randomly divided into a blood pressure normal control group and a blood pressure normal lead exposure group. The mice in the normal blood pressure control group and the hypertensive control group drank water freely. Mice in the lead exposure group with normal blood pressure and the lead exposure group with hypertension drank lead acetate water containing 250 mg/L. Ang II was injected intraperitoneally every two days in the hypertensive control group and hypertensive lead exposed group mice. Each group of mice was poisoned for 12 weeks. Using open field experiments and balance beam experiments to detect motor dysfunction in mice. Using a reagent kit to detect the levels of divalent iron(Fe~(2+)), malondialdehyde(MDA), and glutathione(GSH) in the cerebellum of different groups of mice. Western blot was used to determine the protein expression of member 11 of the solute carrier family 7(SLC7A11), glutathione peroxidase 4(GPX4), nuclear receptor coactivator 4(NCOA4), microtubule associated protein 1 light chain 3B(LC3B), and ferritin heavy chain 1(FTH1) in mouse cerebellar tissue. RESULTS: The result of the open field experiment showed that the activity distance(1013.04 cm) of mice in the hypertensive lead exposure group was significantly lower than that of the hypertensive control group(1351.18 cm) and the lead exposure group with normal blood pressure(1287.35 cm). And the lead exposure group with hypertension also extended the time through the balance beam, which was 29.40 seconds(P<0.05). In addition, the Fe~(2+)content in the cerebellum of mice in the hypertensive lead exposure group was 3.33 µmol/g prot, which was 1.54 times that of the hypertensive control group and 1.14 times that of the lead exposure group with normal blood pressure. The MDA content was 4.71 nmol/mg prot, higher than that of the hypertensive control group and the lead exposure group with normal blood pressure. The GSH content was 5.36 µmol/g prot, lower than that of the hypertensive control group and the lead exposure group with normal blood pressure(P<0.05). Western blot result showed that compared with the hypertensive control group and the lead exposure group with normal blood pressure, the protein expression of SLC7A11 and GPX4 in the hypertensive lead exposure group was significantly reduced(P<0.05). In addition, compared with the control group with normal blood pressure, the expression of NCOA4 and LC3B proteins in the cerebellum of mice in the hypertension control group and lead exposure group with normal blood pressure increased, while the expression of FTH1 protein decreased(P<0.05). The expression of NCOA4 and LC3B proteins in the hypertensive lead exposure group was higher than that in the hypertensive control group and the lead exposure group with normal blood pressure, while the expression of FTH1 protein decreased(P<0.05). CONCLUSION: Lead exposure can exacerbate iron death in the cerebellar tissue of hypertensive mice, and iron autophagy may be involved in its occurrence and development.
Assuntos
Angiotensina II , Cerebelo , Ferroptose , Hipertensão , Chumbo , Camundongos Endogâmicos C57BL , Animais , Ferroptose/efeitos dos fármacos , Camundongos , Masculino , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Chumbo/toxicidade , Cerebelo/metabolismo , Cerebelo/efeitos dos fármacos , Malondialdeído/metabolismo , Glutationa Peroxidase/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Ferro/metabolismo , Glutationa/metabolismoRESUMO
Our understanding of human fetal cerebellum development during the late second trimester, a critical period for the generation of astrocytes, oligodendrocytes, and unipolar brush cells (UBCs), remains limited. Here, we performed single-cell RNA sequencing (scRNA-seq) in human fetal cerebellum samples from gestational weeks (GWs) 18-25. We find that proliferating UBC progenitors distribute in the subventricular zone of the rhombic lip (RLSVZ) near white matter (WM), forming a layer structure. We also delineate two trajectories from astrogenic radial glia (ARGs) to Bergmann glial progenitors (BGPs) and recognize oligodendrogenic radial glia (ORGs) as one source of primitive oligodendrocyte progenitor cells (PriOPCs). Additionally, our scRNA-seq analysis of the trisomy 21 fetal cerebellum at this stage reveals abnormal upregulated genes in pathways such as the cell adhesion pathway and focal adhesion pathway, which potentially promote neuronal differentiation. Overall, our research provides valuable insights into normal and abnormal development of the human fetal cerebellum.
Assuntos
Cerebelo , Síndrome de Down , Feto , Segundo Trimestre da Gravidez , Humanos , Cerebelo/embriologia , Cerebelo/anormalidades , Cerebelo/metabolismo , Síndrome de Down/genética , Síndrome de Down/patologia , Gravidez , Feminino , Diferenciação Celular , Oligodendroglia/metabolismo , Oligodendroglia/citologia , Neuroglia/metabolismo , Neuroglia/patologia , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
3-Nitrotyrosine (3-NT), a byproduct of oxidative and nitrosative stress, is implicated in age-related neurodegenerative disorders. Current literature suggests that free 3-NT becomes integrated into the carboxy-terminal domain of α-tubulin via the tyrosination/detyrosination cycle. Independently of this integration, 3-NT has been associated with the cell death of dopaminergic neurons. Given the critical role of tyrosination/detyrosination in governing axonal morphology and function, the substitution of tyrosine with 3-NT in this process may potentially disrupt axonal homeostasis, although this aspect remains underexplored. In this study, we examined the impact of 3-NT on the axons of cerebellar granule neurons, which is used as a model for non-dopaminergic neurons. Our observations revealed axonal shortening, which correlated with the incorporation of 3-NT into α-tubulin. Importantly, this axonal effect was observed prior to the onset of cellular death. Furthermore, 3-NT was found to diminish mitochondrial motility within the axon, leading to a subsequent reduction in mitochondrial membrane potential. The suppression of syntaphilin, a protein responsible for anchoring mitochondria to microtubules, restored the mitochondrial motility and axonal elongation that were inhibited by 3-NT. These findings underscore the inhibitory role of 3-NT in axonal elongation by impeding mitochondrial movement, suggesting its potential involvement in axonal dysfunction within non-dopaminergic neurons.
Assuntos
Axônios , Mitocôndrias , Tirosina , Tirosina/análogos & derivados , Tirosina/metabolismo , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Axônios/metabolismo , Tubulina (Proteína)/metabolismo , Células Cultivadas , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Cerebelo/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , CamundongosRESUMO
Compensatory changes in brain connectivity keep motor symptoms mild in prodromal Parkinson's disease. Studying compensation in patients is hampered by the steady progression of the disease and a lack of individual baseline controls. Furthermore, combining fMRI with walking is intricate. We therefore used a seed-based metabolic connectivity analysis based on 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake in a unilateral 6-OHDA rat model. At baseline and in the chronic phase 6-7 months after lesion, rats received an intraperitoneal injection of [18F]FDG and spent 50 min walking on a horizontal treadmill, followed by a brain PET-scan under anesthesia. High activity was found in the cerebellar anterior vermis in both conditions. At baseline, the anterior vermis showed hardly any stable connections to the rest of the brain. The (future) ipsilesional cerebellar hemisphere was not particularly active during walking but was extensively connected to many brain areas. After unilateral dopamine depletion, rats still walked normally without obvious impairments. The ipsilesional cerebellar hemisphere increased its activity, but narrowed its connections down to the vestibulocerebellum, probably aiding lateral stability. The anterior vermis established a network involving the motor cortex, hippocampus and thalamus. Adding those regions to the vermis network of (previously) automatic control of locomotion suggests that after unilateral dopamine depletion considerable conscious and cognitive effort has to be provided to achieve stable walking.
Assuntos
Cerebelo , Dopamina , Tomografia por Emissão de Pósitrons , Caminhada , Animais , Ratos , Dopamina/metabolismo , Caminhada/fisiologia , Cerebelo/metabolismo , Cerebelo/diagnóstico por imagem , Masculino , Fluordesoxiglucose F18/metabolismo , Imageamento por Ressonância Magnética/métodos , Oxidopamina , Teste de Esforço , Modelos Animais de Doenças , Córtex Motor/metabolismo , Córtex Motor/diagnóstico por imagem , Córtex Motor/fisiopatologiaRESUMO
Niemann-Pick disease type C (NPC) is a rare and fatal neurological disorder caused by mutations in Npc1 or Npc2, with Npc1 accounting for 95% of cases. These mutations result in the functional loss of their respective proteins, causing cellular abnormalities characterized by disrupted lipid dysregulation, calcium dysfunction, elevated damage associated molecular patterns (DAMPs), and a pro-inflammatory environment. This cellular pathology ultimately triggers neurodegeneration, with the cerebellum being the earliest and most affected region. We have recently shown atypical activation of interferon signaling in the presymptomatic Npc1-/- mouse cerebellum and, to a lesser extent, in the cerebral cortex. In addition, we reported that the Amyloid Precursor Protein (APP) is an NPC disease modifier. Loss of APP function leads to widespread neurodegeneration in the NPC brain, including exacerbated interferon signaling in the cerebellum. To better understand the role of APP as a disease modifier throughout the NPC brain, here we carried out a transcriptomic analysis of the cerebral cortex and cerebellum from 3-week-old Npc1-/- mice as well as age-matched controls in the presence and absence of APP. We report differential effects of APP loss of function in the cerebral cortex and cerebellum, including cholesterol and tau dysregulation, in both brain regions. Our findings demonstrate a novel link between APP loss and early pathogenic mechanisms in NPC.
Assuntos
Precursor de Proteína beta-Amiloide , Cerebelo , Colesterol , Homeostase , Interferons , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C , Transdução de Sinais , Proteínas tau , Animais , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Camundongos , Proteínas tau/metabolismo , Proteínas tau/genética , Colesterol/metabolismo , Fosforilação , Cerebelo/metabolismo , Cerebelo/patologia , Interferons/metabolismo , Interferons/genética , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Camundongos Knockout , Modelos Animais de DoençasRESUMO
Aging is characterized by a functional decline in several physiological systems. α-Klotho-hypomorphic mice (Kl-/-) exhibit accelerated aging and cognitive decline. We evaluated whether male and female α-Klotho-hypomorphic mice show changes in the expression of synaptic proteins, N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits, postsynaptic density protein 95 (PSD-95), synaptophysin and synapsin, and the activity of Na+, K+-ATPase (NaK) isoforms in the cerebellum and hippocampus. In this study, we demonstrated that in the cerebellum, Kl-/- male mice have reduced expression of GluA1 (AMPA) compared to wild-type (Kl+/+) males and Kl-/- females. Also, Kl-/- male and female mice show reduced É2/É3-NaK and Mg2+-ATPase activities in the cerebellum, respectively, and sex-based differences in NaK and Mg2+-ATPase activities in both the regions. Our findings suggest that α-Klotho could influence the expression of AMPAR and the activity of NaK isoforms in the cerebellum in a sex-dependent manner, and these changes may contribute, in part, to cognitive decline.
Assuntos
Cerebelo , Hipocampo , Proteínas Klotho , Receptores de AMPA , Caracteres Sexuais , ATPase Trocadora de Sódio-Potássio , Animais , Feminino , Masculino , Camundongos , Cerebelo/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Proteína 4 Homóloga a Disks-Large/genética , Hipocampo/metabolismo , Proteínas Klotho/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de AMPA/metabolismo , Receptores de AMPA/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Sinapsinas/metabolismo , Sinapsinas/genética , Sinaptofisina/metabolismoRESUMO
Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.
Assuntos
Transtorno do Espectro Autista , Cerebelo , Camundongos Knockout , Células de Purkinje , Animais , Camundongos , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Células de Purkinje/metabolismo , Cerebelo/metabolismo , Comportamento Animal/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Modelos Animais de Doenças , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , MasculinoRESUMO
Bromelain is a plant-based molecule with antioxidant, antithrombotic, anticancer, and anti-inflammatory properties. Bromelain has been shown to reduce the release of inflammatory cytokines. This study aimed to determine whether bromelain can prevent ataxia in rats caused by 3-acetylpyridine (3-AP). Thirty-six albino rats were divided into the control, 3-AP, and 3-AP + Brom groups. In the 3-AP + Brom group, bromelain was injected intraperitoneally at 40 mg/kg daily for 30 days. Various techniques such as rotarod, electromyography (EMG), elevated plus maze, IHC, and Sholl analysis were used to evaluate the possible effects of bromelain on cerebellar neurons and glial cells. The results demonstrated significant improvements in most of the 3-AP + Brom, including motor coordination, neuromuscular response, anxiety, oxidative capacity, microgliosis, astrogliosis, cell death, and morphological variables compared to the 3-AP group. The mechanism of action of bromelain in restoring cerebellar ataxia needs further investigation, but it may be a candidate to help restore degeneration in animals with ataxia.
Assuntos
Bromelaínas , Ataxia Cerebelar , Estresse Oxidativo , Animais , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ataxia Cerebelar/tratamento farmacológico , Bromelaínas/farmacologia , Bromelaínas/uso terapêutico , Ratos , Piridinas/farmacologia , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Antioxidantes/farmacologia , Atividade Motora/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Ratos WistarRESUMO
Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
Assuntos
Núcleo Celular , Dineínas , Cinesinas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Proteínas do Tecido Nervoso , Neurônios , Animais , Microtúbulos/metabolismo , Neurônios/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Dineínas/metabolismo , Núcleo Celular/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transporte Ativo do Núcleo Celular , Complexo Dinactina/metabolismo , Complexo Dinactina/genética , Movimento Celular , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Cerebelo/metabolismo , Cerebelo/citologia , Sítios de Ligação , HumanosRESUMO
Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While granule cell cilia are essential during early developmental stages, they become infrequent upon maturation. Here, we provide nanoscopic resolution of cilia in situ using large-scale electron microscopy volumes and immunostaining of mouse cerebella. In many granule cells, we found intracellular cilia, concealed from the external environment. Cilia were disassembled in differentiating granule cell neurons-in a process we call cilia deconstruction-distinct from premitotic cilia resorption in proliferating progenitors. In differentiating granule cells, cilia deconstruction involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Unlike ciliated neurons in other brain regions, our results show the deconstruction of concealed cilia in differentiating granule cells, which might prevent mitogenic hedgehog responsiveness. Ciliary deconstruction could be paradigmatic of cilia removal during differentiation in other tissues.
Assuntos
Diferenciação Celular , Cerebelo , Cílios , Proteínas Hedgehog , Neurônios , Cílios/metabolismo , Cílios/ultraestrutura , Animais , Neurônios/metabolismo , Neurônios/citologia , Neurônios/ultraestrutura , Camundongos , Cerebelo/metabolismo , Cerebelo/citologia , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neurogênese , Centríolos/metabolismo , Centríolos/ultraestrutura , Camundongos Endogâmicos C57BLRESUMO
To date, apart from moderate hypothermia, there are almost no adequate interventions available for neuroprotection in cases of brain damage due to cardiac arrest. Affected persons often have severe limitations in their quality of life. The aim of this study was to investigate protective properties of the active compound of dimethyl fumarate, monomethyl fumarate (MMF), on distinct regions of the central nervous system after ischemic events. Dimethyl fumarate is an already established drug in neurology with known anti-inflammatory and antioxidant properties. In this study, we chose organotypic slice cultures of rat cerebellum and hippocampus as an ex vivo model. To simulate cardiac arrest and return of spontaneous circulation we performed oxygen-glucose-deprivation (OGD) followed by treatments with different concentrations of MMF (1-30 µM in cerebellum and 5-30 µM in hippocampus). Immunofluorescence staining with propidium iodide (PI) and 4',6-diamidine-2-phenylindole (DAPI) was performed to analyze PI/DAPI ratio after imaging with a spinning disc confocal microscope. In the statistical analysis, the relative cell death of the different groups was compared. In both, the cerebellum and hippocampus, the MMF-treated group showed a significantly lower PI/DAPI ratio compared to the non-treated group after OGD. Thus, we showed for the first time that both cerebellar and hippocampal slice cultures treated with MMF after OGD are significantly less affected by cell death.
Assuntos
Cerebelo , Fumaratos , Glucose , Hipocampo , Fármacos Neuroprotetores , Animais , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Glucose/metabolismo , Ratos , Fármacos Neuroprotetores/farmacologia , Fumaratos/farmacologia , Técnicas de Cultura de Órgãos , Oxigênio/metabolismo , Ratos Wistar , Morte Celular/efeitos dos fármacosRESUMO
BACKGROUND: Aging is a progressive process characterized by weakness in brain function. Although metabolomics studies on the brain related with aging have been conducted, it is not yet fully understood. A systematic metabolomics study was performed to search for biomarkers and monitor altered metabolism in various brain tissues of the cortex, cerebellum, hypothalamus, and hippocampus of young (8 months old) and old rats (22 months old). METHODS: Simultaneous profiling analysis of amino acids (AAs), organic acids (OAs), and fatty acids (FAs) in the brain tissues of young and old rats were performed by gas chromatography-tandem mass spectrometry. RESULTS: Under optimal conditions, AA, OA, and FA profiling methods showed good linearity (r ≥0.995) with limit of detection of ≤30 and 73.2 ng and limit of quantification of ≤90.1 and 219.5 ng, respectively. Repeatability varied from 0.4 to 10.4 and 0.8 to 14.8% relative standard deviation and accuracy varied from -11.3 to 10.3 and -12.8 to 14.1% relative error, respectively. In the profiling analysis, total 32, 43, 45, and 30 metabolites were determined in cortex, cerebellum, hypothalamus, and hippocampus, respectively. In statistical analysis, eight AAs (alanine, valine, leucine, isoleucine, threonine, serine, proline, and phenylalanine) in the cortex and four metabolites (alanine, phenylalanine, 3-hydoxypropionic acid, and eicosadienoic acid) in the cerebellum were significantly evaluated (Q-value <0.05, variable importance in projection scores ≥1.0). In all brain tissues, the score plots of orthogonal partial least square discriminant analysis were clearly separated between the young and old groups. CONCLUSIONS: Metabolomics results indicate that mechanistic targets of rapamycin complex 1, branched chain-amino acid, and energy metabolism are related to inflammation and mitochondrial dysfunction in the brain during aging. Thus, these results may explain the characteristic metabolism of brain aging.