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2.
Biomedicines ; 12(8)2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39200191

RESUMO

Alzheimer's disease (AD) is a severe neurodegenerative disorder and the most common form of dementia, causing the loss of cognitive function. Our previous study has shown, using a doubly mutated mouse model of AD (APP/PS1), that the neural adhesion molecule L1 directly binds amyloid peptides and decreases plaque load and gliosis when injected as an adeno-associated virus construct (AAV-L1) into APP/PS1 mice. In this study, we microinjected AAV-L1, using a Hamilton syringe, directly into the 3-month-old APP/PS1 mouse hippocampus and waited for a year until significant neurodegeneration developed. We stereologically counted the principal neurons and parvalbumin-positive interneurons in the hippocampus, estimated the density of inhibitory synapses around principal cells, and compared the AAV-L1 injection models with control injections of green fluorescent protein (AAV-GFP) and the wild-type hippocampus. Our results show that there is a significant loss of granule cells in the dentate gyrus of the APP/PS1 mice, which was improved by AAV-L1 injection, compared with the AAV-GFP controls (p < 0.05). There is also a generalized loss of parvalbumin-positive interneurons in the hippocampus of APP/PS1 mice, which is ameliorated by AAV-L1 injection, compared with the AAV-GFP controls (p < 0.05). Additionally, AAV-L1 injection promotes the survival of inhibitory synapses around the principal cells compared with AAV-GFP controls in all three hippocampal subfields (p < 0.01). Our results indicate that L1 promotes neuronal survival and protects the synapses in an AD mouse model, which could have therapeutic implications.

3.
Biomolecules ; 14(4)2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38672524

RESUMO

Neuronal plasticity is a crucial mechanism for an adapting nervous system to change. It is shown to be regulated by perineuronal nets (PNNs), the condensed forms of the extracellular matrix (ECM) around neuronal bodies. By assessing the changes in the number, intensity, and structure of PNNs, the ultrastructure of the PNN mesh, and the expression of inhibitory and excitatory synaptic inputs on these neurons, we aimed to clarify the role of an ECM glycoprotein, tenascin-C (TnC), in the dorsal hippocampus. To enhance neuronal plasticity, TnC-deficient (TnC-/-) and wild-type (TnC+/+) young adult male mice were reared in an enriched environment (EE) for 8 weeks. Deletion of TnC in TnC-/- mice showed an ultrastructural reduction of the PNN mesh and an increased inhibitory input in the dentate gyrus (DG), and an increase in the number of PNNs with a rise in the inhibitory input in the CA2 region. EE induced an increased inhibitory input in the CA2, CA3, and DG regions; in DG, the change was also followed by an increased intensity of PNNs. No changes in PNNs or synaptic expression were found in the CA1 region. We conclude that the DG and CA2 regions emerged as focal points of alterations in PNNs and synaptogenesis with EE as mediated by TnC.


Assuntos
Matriz Extracelular , Hipocampo , Plasticidade Neuronal , Sinapses , Tenascina , Animais , Tenascina/metabolismo , Tenascina/genética , Masculino , Camundongos , Hipocampo/metabolismo , Matriz Extracelular/metabolismo , Sinapses/metabolismo , Camundongos Knockout , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Giro Denteado/metabolismo
4.
Biomolecules ; 14(4)2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38672483

RESUMO

The X-chromosome-linked cell adhesion molecule L1 (L1CAM), a glycoprotein mainly expressed by neurons in the central and peripheral nervous systems, has been implicated in many neural processes, including neuronal migration and survival, neuritogenesis, synapse formation, synaptic plasticity and regeneration. L1 consists of extracellular, transmembrane and cytoplasmic domains. Proteolytic cleavage of L1's extracellular and transmembrane domains by different proteases generates several L1 fragments with different functions. We found that myelin basic protein (MBP) cleaves L1's extracellular domain, leading to enhanced neuritogenesis and neuronal survival in vitro. To investigate in vivo the importance of the MBP-generated 70 kDa fragment (L1-70), we generated mice with an arginine to alanine substitution at position 687 (L1/687), thereby disrupting L1's MBP cleavage site and obliterating L1-70. Young adult L1/687 males showed normal anxiety and circadian rhythm activities but enhanced locomotion, while females showed altered social interactions. Older L1/687 males were impaired in motor coordination. Furthermore, L1/687 male and female mice had a larger hippocampus, with more neurons in the dentate gyrus and more proliferating cells in the subgranular layer, while the thickness of the corpus callosum and the size of lateral ventricles were normal. In summary, subtle mutant morphological changes result in subtle behavioral changes.


Assuntos
Encéfalo , Molécula L1 de Adesão de Célula Nervosa , Animais , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Camundongos , Masculino , Feminino , Encéfalo/metabolismo , Fibronectinas/metabolismo , Fibronectinas/genética , Mutação , Comportamento Animal , Domínios Proteicos , Neurônios/metabolismo , Hipocampo/metabolismo , Camundongos Endogâmicos C57BL
5.
Front Neurosci ; 17: 1187758, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37434764

RESUMO

Introduction: There is an increasing evidence supporting the hypothesis that traumatic experiences during early developmental periods might be associated with psychopathology later in life. Maternal deprivation (MD) in rodents has been proposed as an animal model for certain aspects of neuropsychiatric disorders. Methods: To determine whether early-life stress leads to changes in GABAergic, inhibitory interneurons in the limbic system structures, specifically the amygdala and nucleus accumbens, 9-day-old Wistar rats were exposed to a 24 h MD. On postnatal day 60 (P60), the rats were sacrificed for morphometric analysis and their brains were compared to the control group. Results: Results show that MD affect GABAergic interneurons, leading to the decrease in density and size of the calcium-binding proteins parvalbumin-, calbindin-, and calretinin-expressing interneurons in the amygdala and nucleus accumbens. Discussion: This study indicates that early stress in life leads to changes in the number and morphology of the GABAergic, inhibitory interneurons in the amygdala and nucleus accumbens, most probably due to the loss of neurons during postnatal development and it further contributes to understanding the effects of maternal deprivation on brain development.

6.
Biomolecules ; 13(5)2023 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-37238646

RESUMO

Adhesion molecules play major roles in cell proliferation, migration, survival, neurite outgrowth and synapse formation during nervous system development and in adulthood. The neural cell adhesion molecule L1 contributes to these functions during development and in synapse formation and synaptic plasticity after trauma in adulthood. Mutations of L1 in humans result in L1 syndrome, which is associated with mild-to-severe brain malformations and mental disabilities. Furthermore, mutations in the extracellular domain were shown to cause a severe phenotype more often than mutations in the intracellular domain. To explore the outcome of a mutation in the extracellular domain, we generated mice with disruption of the dibasic sequences RK and KR that localize to position 858RKHSKR863 in the third fibronectin type III domain of murine L1. These mice exhibit alterations in exploratory behavior and enhanced marble burying activity. Mutant mice display higher numbers of caspase 3-positive neurons, a reduced number of principle neurons in the hippocampus, and an enhanced number of glial cells. Experiments suggest that disruption of the dibasic sequence in L1 results in subtle impairments in brain structure and functions leading to obsessive-like behavior in males and reduced anxiety in females.


Assuntos
Fibronectinas , Molécula L1 de Adesão de Célula Nervosa , Animais , Feminino , Masculino , Camundongos , Fibronectinas/genética , Fibronectinas/metabolismo , Gliose/metabolismo , Hipocampo/metabolismo , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo
8.
Front Cell Dev Biol ; 10: 982663, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36518543

RESUMO

Early life stress negatively impacts brain development and affects structure and function of parvalbumin immunopositive (PV+) inhibitory neurons. Main regulators of PV+ interneurons activity and plasticity are perineuronal nets (PNNs), an extracellular matrix formation that enwraps PV+ interneurons mainly in the neocortex and hippocampus. To experimentally address the impact of early life stress on the PNNs and PV+ interneurons in the medial prefrontal cortex and dorsal hippocampus in rats, we employed a 24 h maternal deprivation protocol. We show that maternal deprivation in the medial prefrontal cortex of adult rats caused a decrease in density of overall PNNs and PNNs that enwrap PV+ interneurons in the rostral cingulate cortex. Furthermore, a staining intensity decrease of overall PNNs and PNN+/PV+ cells was found in the prelimbic cortex. Finally, a decrease in both intensity and density of overall PNNs and PNNs surrounding PV+ cells was observed in the infralimbic cortex, together with increase in the intensity of VGAT inhibitory puncta. Surprisingly, maternal deprivation did not cause any changes in the density of PV+ interneurons in the mPFC, neither had it affected PNNs and PV+ interneurons in the hippocampus. Taken together, our findings indicate that PNNs, specifically the ones enwrapping PV+ interneurons in the medial prefrontal cortex, are affected by early life stress.

9.
Front Cell Dev Biol ; 10: 952208, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36092707

RESUMO

Understanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an in vitro injury model, the mixed culture of cortical astrocytes and microglia, and observed that without TnC microglial cells tend to populate gap area in greater numbers and proliferate more, whereas astrocytes build up in the border region to promote faster gap closure. Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-α and IL-1ß upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of ß1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area.

10.
Neural Regen Res ; 17(8): 1802-1808, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35017441

RESUMO

The role that the immune system plays after injury of the peripheral nervous system is still not completely understood. Perforin, a natural killer cell- and T-lymphocyte-derived enzyme that mediates cytotoxicity, plays important roles in autoimmune diseases, infections and central nervous system trauma, such as spinal cord injury. To dissect the roles of this single component of the immune response to injury, we tested regeneration after femoral nerve injury in perforin-deficient (Pfp-/-) and wild-type control mice. Single frame motion analysis showed better motor recovery in Pfp-/- mice compared with control mice at 4 and 8 weeks after injury. Retrograde tracing of the motoneuron axons regrown into the motor nerve branch demonstrated more correctly projecting motoneurons in the spinal cord of Pfp-/- mice compared with wild-types. Myelination of regrown axons measured by g-ratio was more extensive in Pfp-/- than in wild-type mice in the motor branch of the femoral nerve. Pfp-/- mice displayed more cholinergic synaptic terminals around cell bodies of spinal motoneurons after injury than the injured wild-types. We histologically analyzed lymphocyte infiltration 10 days after surgery and found that in Pfp-/- mice the number of lymphocytes in the regenerating nerves was lower than in wild-types, suggesting a closed blood-nerve barrier in Pfp-/- mice. We conclude that perforin restricts motor recovery after femoral nerve injury owing to decreased survival of motoneurons and reduced myelination.

11.
Int J Neurosci ; 132(1): 1-12, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32672480

RESUMO

MATERIALS AND METHODS: Locomotor outcomes in perforin-deficient (Pfp-/-) mice and wild-type littermate controls were measured after severe compression injury of the lower thoracic spinal cord up to six weeks after injury. RESULTS: According to both the Basso mouse scale score and single frame motion analysis, motor recovery of Pfp-/- mice was similar to wild-type controls. Interestingly, immunohistochemical analysis of cell types at six weeks after injury showed enhanced cholinergic reinnervation of spinal motor neurons caudal to the lesion site and neurofilament-positive structures at the injury site in Pfp-/- mice, whereas numbers of microglia/macrophages and astrocytes were decreased compared with controls. CONCLUSIONS: We conclude that, although, loss of perforin does not change the locomotor outcome after injury, it beneficially affects diverse cellular features, such as number of axons, cholinergic synapses, astrocytes and microglia/macrophages at or caudal to the lesion site. Perforin's ability to contribute to Rag2's influence on locomotion was observed in mice doubly deficient in perforin and Rag2 which recovered better than Rag2-/- or Pfp-/- mice, suggesting that natural killer cells can cooperate with T- and B-cells in spinal cord injury.


Assuntos
Locomoção/fisiologia , Regeneração Nervosa/fisiologia , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Citotóxicas Formadoras de Poros/deficiência
12.
Development ; 148(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34414407

RESUMO

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Interneurônios/citologia , Neocórtex/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Animais , Calbindina 2/metabolismo , Cálcio/metabolismo , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/farmacologia , Dendritos/efeitos dos fármacos , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/farmacologia , Hipertrofia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Camundongos , Camundongos Knockout , Neocórtex/citologia , Neocórtex/efeitos dos fármacos , Neocórtex/patologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/farmacologia , Neuropeptídeo Y/metabolismo , Parvalbuminas/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato/metabolismo , Proteína Reelina , Serina Endopeptidases/deficiência , Serina Endopeptidases/farmacologia
13.
Proc Natl Acad Sci U S A ; 118(35)2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34429357

RESUMO

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.


Assuntos
Proliferação de Células/fisiologia , Córtex Cerebral/embriologia , Canalopatias/etiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Microcefalia/etiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Animais , Ciclo Celular , Morte Celular , Células Cultivadas , Córtex Cerebral/citologia , Canalopatias/embriologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , Camundongos Transgênicos , Microcefalia/embriologia , Células-Tronco Neurais/metabolismo , Ratos
14.
Front Neuroanat ; 15: 670766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34168541

RESUMO

Early life stress has profound effects on the development of the central nervous system. We exposed 9-day-old rat pups to a 24 h maternal deprivation (MD) and sacrificed them as young adults (60-day-old), with the aim to study the effects of early stress on forebrain circuitry. We estimated numbers of various immunohistochemically defined interneuron subpopulations in several neocortical regions and in the hippocampus. MD rats showed reduced numbers of parvalbumin-expressing interneurons in the CA1 region of the hippocampus and in the prefrontal cortex, compared with controls. Numbers of reelin-expressing and calretinin-expressing interneurons were also reduced in the CA1 and CA3 hippocampal areas, but unaltered in the neocortex of MD rats. The number of calbinin-expressing interneurons in the neocortex was similar in the MD rats compared with controls. We analyzed cell death in 15-day-old rats after MD and found no difference compared to control rats. Thus, our results more likely reflect the downregulation of markers than the actual loss of interneurons. To investigate synaptic activity in the hippocampus we immunostained for glutamatergic and inhibitory vesicular transporters. The number of inhibitory synapses was decreased in the CA1 and CA3 regions of the hippocampus in MD rats, with the normal number of excitatory synapses. Our results indicate complex, cell type-specific, and region-specific alterations in the inhibitory circuitry induced by maternal deprivation. Such alterations may underlie symptoms of MD at the behavioral level and possibly contribute to mechanisms by which early life stress causes neuropsychiatric disorders, such as schizophrenia.

15.
16.
Int J Clin Pharmacol Ther ; 59(6): 447-462, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33624582

RESUMO

Peripheral nerve injuries are common and present with a broad spectrum of symptoms, some of which may be the cause of life-long disabilities. The peripheral nerves show a far greater capacity for regeneration than those in the central nervous system, and the process of nerve regeneration resembles developmental processes to a certain degree. The regeneration of peripheral nerves does not always lead to a full functional recovery. That is why surgical methods are still the most reliable therapeutic options after injuries of peripheral nerves. However, there is an array of potential pharmacological options that could enhance the repair processes after surgery. This review gives a summary of the recent literature relevant to different classes of pharmacologically active substances that are used either as supplements or off-label as potential enhancers of peripheral nerve repair. Antioxidants, vitamins, calcium channel blockers, immunosuppressive drugs, growth factors, and neuroactive glycans are among the most researched in this field. More research is necessary to understand their mechanisms of action at the cellular and molecular level, and randomized clinical trials in order to establish their efficacy and safety, as well as possible synergistic or adverse interactions among them.


Assuntos
Traumatismos dos Nervos Periféricos , Humanos , Imunossupressores , Regeneração Nervosa , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Nervos Periféricos
17.
Neuroscience ; 459: 129-141, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33588005

RESUMO

Microglia/macrophages play important functional roles in regeneration after central nervous system injury. Infiltration of circulating macrophages and proliferation of resident microglia occur within minutes following spinal cord injury. Activated microglia/macrophages clear tissue debris, but activation over time may hamper repair. To study the role of these cells in regeneration after spinal cord injury we used CD11b-herpes simplex virus thymidine kinase (HSVTK) (TK) transgenic mice, in which viral thymidine kinase activates ganciclovir toxicity in CD11b-expressing myeloid cells, including macrophages and microglia. A severe reduction in number of these cells was seen in TK versus wild-type littermate mice at 1 week and 5 weeks after injury, and numbers of Mac-2 expressing activated microglia/macrophages were almost completely reduced at these time points. One week after injury TK mice showed better locomotor recovery, but recovery was similar to wild-type mice as measured weekly up to 5 weeks thereafter. At 5 weeks after injury, numbers of axons at the lesion site and neurons in the lumbar spinal cord did not differ between groups. Also, catecholaminergic innervation of spinal motoneurons was similar. However, cholinergic innervation was lower and glial scarring was increased in TK mice compared to wild-type mice. We conclude that reducing numbers of CD11b-expressing cells improves locomotor recovery in the early phase after spinal cord injury, but does not affect recovery in the following 4 weeks. These observations point to differences in outcomes of astrocytic response and cholinergic innervation under CD11b cell ablation, which are, however, not reflected in the locomotor parameters analyzed at 5 weeks after injury.


Assuntos
Microglia , Traumatismos da Medula Espinal , Animais , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Recuperação de Função Fisiológica , Medula Espinal
18.
J Neuroinflammation ; 17(1): 100, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32248813

RESUMO

BACKGROUND: Microglia are essential to maintain cell homeostasis in the healthy brain and are activated after brain injury. Upon activation, microglia polarize towards different phenotypes. The course of microglia activation is complex and depends on signals in the surrounding milieu. Recently, it has been suggested that microglia respond to ion currents, as a way of regulating their activity and function. METHODS AND RESULTS: Under the hypothesis that HCN and KCNQ/Kv7 channels impact on microglia, we studied primary rat microglia in the presence or absence of specific pharmacological blockade or RNA silencing. Primary microglia expressed the subunits HCN1-4, Kv7.2, Kv7.3, and Kv7.5. The expression of HCN2, as well as Kv7.2 and Kv7.3, varied among different microglia phenotypes. The pharmacological blockade of HCN channels by ZD7288 resulted in cell depolarization with slowly rising intracellular calcium levels, leading to enhanced survival and reduced proliferation rates of resting microglia. Furthermore, ZD7288 treatment, as well as knockdown of HCN2 RNA by small interfering RNA, resulted in an attenuation of later microglia activation-both towards the anti- and pro-inflammatory phenotype. However, HCN channel inhibition enhanced the phagocytic capacity of IL4-stimulated microglia. Blockade of Kv7/KCNQ channel by XE-991 exclusively inhibited the migratory capacity of resting microglia. CONCLUSION: These observations suggest that the HCN current contributes to various microglia functions and impacts on the course of microglia activation, while the Kv7/KCNQ channels affect microglia migration. Characterizing the role of HCN channels in microglial functioning may offer new therapeutic approaches for targeted modulation of neuroinflammation as a hallmark of various neurological disorders.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Microglia/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Pirimidinas/farmacologia , Interferência de RNA , Ratos , Ratos Wistar
19.
Front Immunol ; 11: 624612, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33679718

RESUMO

Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain.


Assuntos
Processamento Alternativo/imunologia , Astrócitos/imunologia , Cicatriz/imunologia , Traumatismos da Medula Espinal/imunologia , Tenascina/imunologia , Animais , Astrócitos/patologia , Cicatriz/genética , Cicatriz/patologia , Camundongos , Camundongos Knockout , Domínios Proteicos , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Tenascina/genética
20.
Restor Neurol Neurosci ; 37(4): 291-313, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31227672

RESUMO

BACKGROUND: We have shown that histone H1 is a binding partner for polysialic acid (PSA) and that it improves functional recovery, axon regrowth/sprouting, and target reinnervation after mouse femoral nerve injury. OBJECTIVE: Here, we analyzed whether histone H1 affects functional recovery, axon regrowth/sprouting, and target reinnervation after spinal cord injury of adult mice. Furthermore, we tested in vitro histone H1's effect on astrocytic gene expression, cell shape and migration as well as on cell survival of cultured motoneurons. METHODS: We applied histone H1 to compressed spinal cord and determined functional recovery and number of fibrillary acidic protein (GFAP)- and neuron-glial antigen 2 (NG2)- positive glial cells, which contribute to glial scarring. Histone H1's effect on migration of astrocytes, astrocytic gene expression and motoneuronal survival was determined using scratch-wounded astroglial monolayer cultures, astrocyte cultures for microarray analysis, and motoneuron cell culture under oxidative stress conditions, respectively. RESULTS: Histone H1 application improves locomotor functions and enhances monoaminergic and cholinergic reinnervation of the spinal cord. Expression levels of GFAP and NG2 around the lesion site were decreased in histone H1-treated mice relative to vehicle-treated mice six weeks after injury. Histone H1 reduced astrocytic migration, changed the shape of GFAP- and NG2-positive glial cells and altered gene expression. Gene ontology enrichment analysis indicated that in particular genes coding for proteins involved in proliferation, differentiation, migration and apoptosis are dysregulated. The up- and down-regulation of distinct genes was confirmed by qPCR and Western blot analysis. Moreover, histone H1 reduced hydrogen peroxide-induced cell death of cultured motoneurons. CONCLUSIONS: The combined observations indicate that histone H1 locally applied to the lesion site, improves regeneration after spinal cord injury. Some of these beneficial functions of histone H1 in vivo and in vitro can be attributed to its interaction with PSA-carrying neural cell adhesion molecule.


Assuntos
Astrócitos/fisiologia , Axônios/fisiologia , Movimento Celular/fisiologia , Expressão Gênica/fisiologia , Histonas/fisiologia , Locomoção/fisiologia , Neurônios Motores/fisiologia , Regeneração Nervosa/fisiologia , Neuroglia/fisiologia , Recuperação de Função Fisiológica/fisiologia , Ácidos Siálicos/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Animais , Astrócitos/efeitos dos fármacos , Axônios/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Histonas/farmacologia , Locomoção/efeitos dos fármacos , Camundongos , Neurônios Motores/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico
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