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1.
J Neurophysiol ; 132(2): 531-543, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38985935

RESUMO

Structural neuroplasticity such as neurite extension and dendritic spine dynamics is enhanced by brain-derived neurotrophic factor (BDNF) and impaired by types of inhibitory molecules that induce growth cone collapse and actin depolymerization, for example, myelin-associated inhibitors, chondroitin sulfate proteoglycans, and negative guidance molecules. These inhibitory molecules can activate RhoA/rho-associated coiled-coil containing protein kinase (ROCK) signaling (known to restrict structural plasticity). Intermittent hypoxia (IH) and high-intensity interval training (HIIT) are known to upregulate BDNF that is associated with improvements in learning and memory and greater functional recovery following neural insults. We investigated whether the RhoA/ROCK signaling pathway is also modulated by IH and HIIT in the hippocampus, cortex, and lumbar spinal cord of male Wistar rats. The gene expression of 25 RhoA/ROCK signaling pathway components was determined following IH, HIIT, or IH combined with HIIT (30 min/day, 5 days/wk, 6 wk). IH included 10 3-min bouts that alternated between hypoxia (15% O2) and normoxia. HIIT included 10 3-min bouts alternating between treadmill speeds of 50 cm·s-1 and 15 cm·s-1. In the hippocampus, IH and HIIT significantly downregulated Acan and NgR2 mRNA that are involved in the inhibition of neuroplasticity. However, IH and IH + HIIT significantly upregulated Lingo-1 and NgR3 in the cortex. This is the first time IH and HIIT have been linked to the modulation of plasticity-inhibiting pathways. These results provide a fundamental step toward elucidating the interplay between the neurotrophic and inhibitory mechanisms involved in experience-driven neural plasticity that will aid in optimizing physiological interventions for the treatment of cognitive decline or neurorehabilitation.NEW & NOTEWORTHY Intermittent hypoxia (IH) and high-intensity interval training (HIIT) enhance neuroplasticity and upregulate neurotrophic factors in the central nervous system (CNS). We provide evidence that IH and IH + HIIT also have the capacity to regulate genes involved in the RhoA/ROCK signaling pathway that is known to restrict structural plasticity in the CNS. This provides a new mechanistic insight into how these interventions may enhance hippocampal-related plasticity and facilitate learning, memory, and neuroregeneration.


Assuntos
Treinamento Intervalado de Alta Intensidade , Hipocampo , Ratos Wistar , Transdução de Sinais , Quinases Associadas a rho , Animais , Masculino , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Hipocampo/metabolismo , Transdução de Sinais/fisiologia , Ratos , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiologia , Plasticidade Neuronal/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Medula Espinal/metabolismo , Medula Espinal/fisiologia , Proteínas rho de Ligação ao GTP
2.
Mol Psychiatry ; 27(8): 3192-3203, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35760878

RESUMO

All components of the CNS are surrounded by a diffuse extracellular matrix (ECM) containing chondroitin sulphate proteoglycans (CSPGs), heparan sulphate proteoglycans (HSPGs), hyaluronan, various glycoproteins including tenascins and thrombospondin, and many other molecules that are secreted into the ECM and bind to ECM components. In addition, some neurons, particularly inhibitory GABAergic parvalbumin-positive (PV) interneurons, are surrounded by a more condensed cartilage-like ECM called perineuronal nets (PNNs). PNNs surround the soma and proximal dendrites as net-like structures that surround the synapses. Attention has focused on the role of PNNs in the control of plasticity, but it is now clear that PNNs also play an important part in the modulation of memory. In this review we summarize the role of the ECM, particularly the PNNs, in the control of various types of memory and their participation in memory pathology. PNNs are now being considered as a target for the treatment of impaired memory. There are many potential treatment targets in PNNs, mainly through modulation of the sulphation, binding, and production of the various CSPGs that they contain or through digestion of their sulphated glycosaminoglycans.


Assuntos
Proteoglicanas de Sulfatos de Condroitina , Matriz Extracelular , Matriz Extracelular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Dendritos/metabolismo , Plasticidade Neuronal/fisiologia
3.
Langmuir ; 39(50): 18410-18423, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38049433

RESUMO

The formation of surfaces decorated with biomacromolecules such as proteins, glycans, or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest and present effective guidelines toward optimizing incubation conditions for the quantitative control of surface densities. Focusing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.


Assuntos
Biotina , Biotina/química , Membrana Celular , Biopolímeros
4.
Int J Mol Sci ; 24(4)2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36835210

RESUMO

4-methylumbelliferone (4MU) has been suggested as a potential therapeutic agent for a wide range of neurological diseases. The current study aimed to evaluate the physiological changes and potential side effects after 10 weeks of 4MU treatment at a dose of 1.2 g/kg/day in healthy rats, and after 2 months of a wash-out period. Our findings revealed downregulation of hyaluronan (HA) and chondroitin sulphate proteoglycans throughout the body, significantly increased bile acids in blood samples in weeks 4 and 7 of the 4MU treatment, as well as increased blood sugars and proteins a few weeks after 4MU administration, and significantly increased interleukins IL10, IL12p70 and IFN gamma after 10 weeks of 4MU treatment. These effects, however, were reversed and no significant difference was observed between control treated and 4MU-treated animals after a 9-week wash-out period.


Assuntos
Ácido Hialurônico , Himecromona , Animais , Ratos , Ácido Hialurônico/metabolismo , Himecromona/efeitos adversos , Himecromona/uso terapêutico , Interleucina-12
5.
Mol Psychiatry ; 26(2): 556-567, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-31758091

RESUMO

Parkinson's disease (PD) is an α-synucleinopathy characterized by the progressive loss of specific neuronal populations. Here, we develop a novel approach to transvascularly deliver proteins of complex quaternary structures, including α-synuclein preformed fibrils (pff). We show that a single systemic administration of α-synuclein pff triggers pathological transformation of endogenous α-synuclein in non-transgenic rats, which leads to neurodegeneration in discrete brain regions. Specifically, pff-exposed animals displayed a progressive deterioration in gastrointestinal and olfactory functions, which corresponded with the presence of cellular pathology in the central and enteric nervous systems. The α-synuclein pathology generated was both time dependent and region specific. Interestingly, the most significant neuropathological changes were observed in those brain regions affected in the early stages of PD. Our data therefore demonstrate for the first time that a single, transvascular administration of α-synuclein pff can lead to selective regional neuropathology resembling the premotor stage of idiopathic PD. Furthermore, this novel delivery approach could also be used to deliver a range of other pathogenic, as well as therapeutic, protein cargos transvascularly to the brain.


Assuntos
Sistema Nervoso Entérico , Doença de Parkinson , Animais , Encéfalo/metabolismo , Sistema Nervoso Entérico/metabolismo , Humanos , Neurônios/metabolismo , alfa-Sinucleína/metabolismo
6.
Mol Psychiatry ; 26(10): 5658-5668, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34272488

RESUMO

Perineuronal nets (PNNs) are chondroitin sulphate proteoglycan-containing structures on the neuronal surface that have been implicated in the control of neuroplasticity and memory. Age-related reduction of chondroitin 6-sulphates (C6S) leads to PNNs becoming more inhibitory. Here, we investigated whether manipulation of the chondroitin sulphate (CS) composition of the PNNs could restore neuroplasticity and alleviate memory deficits in aged mice. We first confirmed that aged mice (20-months) showed memory and plasticity deficits. They were able to retain or regain their cognitive ability when CSs were digested or PNNs were attenuated. We then explored the role of C6S in memory and neuroplasticity. Transgenic deletion of chondroitin 6-sulfotransferase (chst3) led to a reduction of permissive C6S, simulating aged brains. These animals showed very early memory loss at 11 weeks old. Importantly, restoring C6S levels in aged animals rescued the memory deficits and restored cortical long-term potentiation, suggesting a strategy to improve age-related memory impairment.


Assuntos
Sulfatos de Condroitina , Plasticidade Neuronal , Envelhecimento , Animais , Encéfalo , Matriz Extracelular , Camundongos
7.
J Physiol ; 599(4): 1199-1224, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33146892

RESUMO

KEY POINTS: Spinal treatment can restore diaphragm function in all animals 1 month following C2 hemisection induced paralysis. Greater recovery occurs the longer after injury the treatment is applied. Through advanced assessment of muscle mechanics, innovative histology and oxygen tension modelling, we have comprehensively characterized in vivo diaphragm function and phenotype. Muscle work loops reveal a significant deficit in diaphragm functional properties following chronic injury and paralysis, which are normalized following restored muscle activity caused by plasticity-induced spinal reconnection. Injury causes global and local alterations in diaphragm muscle vascular supply, limiting oxygen diffusion and disturbing function. Restoration of muscle activity reverses these alterations, restoring oxygen supply to the tissue and enabling recovery of muscle functional properties. There remain metabolic deficits following restoration of diaphragm activity, probably explaining only partial functional recovery. We hypothesize that these deficits need to be resolved to restore complete respiratory motor function. ABSTRACT: Months after spinal cord injury (SCI), respiratory deficits remain the primary cause of morbidity and mortality for patients. It is possible to induce partial respiratory motor functional recovery in chronic SCI following 2 weeks of spinal neuroplasticity. However, the peripheral mechanisms underpinning this recovery are largely unknown, limiting development of new clinical treatments with potential for complete functional restoration. Utilizing a rat hemisection model, diaphragm function and paralysis was assessed and recovered at chronic time points following trauma through chondroitinase ABC induced neuroplasticity. We simulated the diaphragm's in vivo cyclical length change and activity patterns using the work loop technique at the same time as assessing global and local measures of the muscles histology to quantify changes in muscle phenotype, microvascular composition, and oxidative capacity following injury and recovery. These data were fed into a physiologically informed model of tissue oxygen transport. We demonstrate that hemidiaphragm paralysis causes muscle fibre hypertrophy, maintaining global oxygen supply, although it alters isolated muscle kinetics, limiting respiratory function. Treatment induced recovery of respiratory activity normalized these effects, increasing oxygen supply, restoring optimal diaphragm functional properties. However, metabolic demands of the diaphragm were significantly reduced following both injury and recovery, potentially limiting restoration of normal muscle performance. The mechanism of rapid respiratory muscle recovery following spinal trauma occurs through oxygen transport, metabolic demand and functional dynamics of striated muscle. Overall, these data support a systems-wide approach to the treatment of SCI, and identify new targets to mediate complete respiratory recovery.


Assuntos
Diafragma , Traumatismos da Medula Espinal , Animais , Humanos , Cinética , Oxigênio , Nervo Frênico , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Medula Espinal
8.
Int J Mol Sci ; 21(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339362

RESUMO

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).


Assuntos
Esclerose Lateral Amiotrófica/terapia , Células-Tronco Neurais/transplante , Plasticidade Neuronal , Transplante de Células-Tronco/métodos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Nervos Periféricos/fisiologia , Ratos , Ratos Sprague-Dawley , Tenascina/genética , Tenascina/metabolismo , Versicanas/genética , Versicanas/metabolismo
9.
Neurochem Res ; 44(6): 1367-1374, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30796750

RESUMO

Cell surface ß-Amyloid precursor protein (APP) is known to have a functional role in iron homeostasis through stabilising the iron export protein ferroportin (FPN). Mechanistic evidence of this role has previously only been provided through transcriptional or translational depletion of total APP levels. However, numerous post-translational modifications of APP are reported to regulate the location and trafficking of this protein to the cell surface. Stable overexpressing cell lines were generated that overexpressed APP with disrupted N-glycosylation (APPN467K and APPN496K) or ectodomain phosphorylation (APPS206A); sites selected for their proximity to the FPN binding site on the E2 domain of APP. We hypothesise that impaired N-glycosylation or phosphorylation of APP disrupts the functional location on the cell surface or binding to FPN to consequentially alter intracellular iron levels through impaired cell surface FPN stability. Outcomes confirm that these post-translational modifications are essential for the correct location of APP on the cell surface and highlight a novel mechanism by which the cell can modulate iron homeostasis. Further interrogation of other post-translational processes to APP is warranted in order to fully understand how each modification plays a role on regulating intracellular iron levels in health and disease.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Glicosilação , Camundongos , Fosforilação/genética , Mutação Puntual , Processamento de Proteína Pós-Traducional/genética , Transporte Proteico/genética
10.
Neural Plast ; 2019: 6804575, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31772567

RESUMO

The perineuronal net (PNN) is a mesh-like proteoglycan structure on the neuronal surface which is involved in regulating plasticity. The PNN regulates plasticity via multiple pathways, one of which is direct regulation of synapses through the control of AMPA receptor mobility. Since neuronal pentraxin 2 (Nptx2) is a known regulator of AMPA receptor mobility and Nptx2 can be removed from the neuronal surface by PNN removal, we investigated whether Nptx2 has a function in the PNN. We found that Nptx2 binds to the glycosaminoglycans hyaluronan and chondroitin sulphate E in the PNN. Furthermore, in primary cortical neuron cultures, the addition of NPTX2 to the culture medium enhances PNN formation during PNN development. These findings suggest Nptx2 as a novel PNN binding protein with a role in the mechanism of PNN formation.


Assuntos
Proteína C-Reativa/metabolismo , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Satélites Perineuronais/metabolismo , Córtex Visual/metabolismo , Animais , Células Cultivadas , Feminino , Rede Nervosa/química , Rede Nervosa/citologia , Plasticidade Neuronal/fisiologia , Neurônios/química , Neurônios/metabolismo , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Células Satélites Perineuronais/química , Córtex Visual/química , Córtex Visual/citologia
11.
Int J Mol Sci ; 19(4)2018 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-29649136

RESUMO

Perineuronal nets (PNNs) are extracellular matrix structures surrounding neuronal sub-populations throughout the central nervous system, regulating plasticity. Enzymatically removing PNNs successfully enhances plasticity and thus functional recovery, particularly in spinal cord injury models. While PNNs within various brain regions are well studied, much of the composition and associated populations in the spinal cord is yet unknown. We aim to investigate the populations of PNN neurones involved in this functional motor recovery. Immunohistochemistry for choline acetyltransferase (labelling motoneurones), PNNs using Wisteria floribunda agglutinin (WFA) and chondroitin sulphate proteoglycans (CSPGs), including aggrecan, was performed to characterise the molecular heterogeneity of PNNs in rat spinal motoneurones (Mns). CSPG-positive PNNs surrounded ~70-80% of Mns. Using WFA, only ~60% of the CSPG-positive PNNs co-localised with WFA in the spinal Mns, while ~15-30% of Mns showed CSPG-positive but WFA-negative PNNs. Selective labelling revealed that aggrecan encircled ~90% of alpha Mns. The results indicate that (1) aggrecan labels spinal PNNs better than WFA, and (2) there are differences in PNN composition and their associated neuronal populations between the spinal cord and cortex. Insights into the role of PNNs and their molecular heterogeneity in the spinal motor pools could aid in designing targeted strategies to enhance functional recovery post-injury.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Neurônios Motores/citologia , Medula Espinal/citologia , Animais , Colina O-Acetiltransferase/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Neurônios Motores/metabolismo , Plasticidade Neuronal , Ratos , Medula Espinal/metabolismo
12.
J Neurosci ; 36(45): 11459-11468, 2016 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-27911749

RESUMO

Perineuronal nets (PNNs) are unique extracellular matrix structures that wrap around certain neurons in the CNS during development and control plasticity in the adult CNS. They appear to contribute to a wide range of diseases/disorders of the brain, are involved in recovery from spinal cord injury, and are altered during aging, learning and memory, and after exposure to drugs of abuse. Here the focus is on how a major component of PNNs, chondroitin sulfate proteoglycans, control plasticity, and on the role of PNNs in memory in normal aging, in a tauopathy model of Alzheimer's disease, and in drug addiction. Also discussed is how altered extracellular matrix/PNN formation during development may produce synaptic pathology associated with schizophrenia, bipolar disorder, major depression, and autism spectrum disorders. Understanding the molecular underpinnings of how PNNs are altered in normal physiology and disease will offer insights into new treatment approaches for these diseases.


Assuntos
Encéfalo/fisiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Rede Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Animais , Humanos , Modelos Neurológicos
13.
Mol Cell Neurosci ; 68: 1-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25771432

RESUMO

Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two 'activated' forms of talin produced by point mutation (on laminin and aggrecan-laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract.


Assuntos
Integrinas/metabolismo , Neurônios/efeitos dos fármacos , Talina/metabolismo , Agrecanas/metabolismo , Animais , Axônios/fisiologia , Células Cultivadas , Gânglios Espinais/citologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrinas/genética , Laminina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Talina/genética , Transfecção
14.
Molecules ; 20(3): 3527-48, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25706756

RESUMO

With recent advances in the construction of synthetic glycans, selective targeting of the extracellular matrix (ECM) as a potential treatment for a wide range of diseases has become increasingly popular. The use of compounds that mimic the structure or bioactive function of carbohydrate structures has been termed glycomimetics. These compounds are mostly synthetic glycans or glycan-binding constructs which manipulate cellular interactions. Glycosaminoglycans (GAGs) are major components of the ECM and exist as a diverse array of differentially sulphated disaccharide units. In the central nervous system (CNS), they are expressed by both neurons and glia and are crucial for brain development and brain homeostasis. The inherent diversity of GAGs make them an essential biological tool for regulating a complex range of cellular processes such as plasticity, cell interactions and inflammation. They are also involved in the pathologies of various neurological disorders, such as glial scar formation and psychiatric illnesses. It is this diversity of functions and potential for selective interventions which makes GAGs a tempting target. In this review, we shall describe the molecular make-up of GAGs and their incorporation into the ECM of the CNS. We shall highlight the different glycomimetic strategies that are currently being used in the nervous system. Finally, we shall discuss some possible targets in neurological disorders that may be addressed using glycomimetics.


Assuntos
Biomimética , Sistema Nervoso Central/metabolismo , Matriz Extracelular/metabolismo , Glicoconjugados/metabolismo , Glicosaminoglicanos/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Animais , Humanos
15.
J Neurosci ; 33(1): 234-43, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23283337

RESUMO

Monocular deprivation (MD) during a critical period of postnatal development produces significant changes in the anatomy and physiology of the visual cortex, and the deprived eye becomes amblyopic. Extracellular matrix molecules have a major role in restricting plasticity such that the ability to recover from MD decreases with age. Chondroitin sulfate proteoglycans (CSPGs) act as barriers to cell migration and axon growth. Previous studies showing that degradation of CSPGs by the bacterial enzyme chondroitinase can restore plasticity in the adult rat visual cortex suggest a potential treatment for amblyopia. Here MD was imposed in cats from the start of the critical period until 3.5 months of age. The deprived eye was reopened, the functional architecture of the visual cortex was assessed by optical imaging of intrinsic signals, and chondroitinase was injected into one hemisphere. Imaging was repeated 1 and 2 weeks postinjection, and visually evoked potentials (VEPs) and single-cell activity were recorded. Immunohistochemistry showed that digestion of CSPGs had been successful. After 2 weeks of binocular exposure, some recovery of deprived-eye responses occurred when chondroitinase had been injected into the hemisphere contralateral to that eye; when injected into the ipsilateral hemisphere, no recovery was seen. Deprived-eye VEPs were no larger in the injected hemisphere than in the opposite hemisphere. The small number of neurons dominated by the deprived eye exhibited poor tuning characteristics. These results suggest that despite structural effects of chondroitinase in adult cat V1, plasticity was not sufficiently restored to enable significant functional recovery of the deprived eye.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Condroitinases e Condroitina Liases/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Privação Sensorial/fisiologia , Córtex Visual/efeitos dos fármacos , Ambliopia/metabolismo , Ambliopia/fisiopatologia , Animais , Mapeamento Encefálico , Gatos , Potenciais Evocados Visuais/fisiologia , Feminino , Masculino , Plasticidade Neuronal/fisiologia , Córtex Visual/metabolismo , Córtex Visual/fisiopatologia
16.
J Biol Chem ; 288(38): 27384-27395, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23940048

RESUMO

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186-200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.


Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Semaforina-3A/metabolismo , Motivos de Aminoácidos , Animais , Química Encefálica/fisiologia , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Matriz Extracelular/química , Matriz Extracelular/genética , Células HEK293 , Humanos , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Ratos , Semaforina-3A/química , Semaforina-3A/genética
17.
Mol Cell Neurosci ; 56: 186-200, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23665579

RESUMO

In the adult rodent brain, subsets of neurons are surrounded by densely organised extracellular matrix called perineuronal nets (PNNs). PNNs consist of hyaluronan, tenascin-R, chondroitin sulphate proteoglycans (CSPGs), and the link proteins Crtl1 and Bral2. PNNs restrict plasticity at the end of critical periods and can be visualised with Wisteria floribunda agglutinin (WFA). Using a number of antibodies raised against the different regions of semaphorin3A (Sema3A) we demonstrate that this secreted chemorepulsive axon guidance protein is localised to WFA-positive PNNs around inhibitory interneurons in the cortex and several other PNN-bearing neurons throughout the brain and co-localises with aggrecan, versican, phosphacan and tenascin-R. Chondroitinase ABC (ChABC) was injected in the cortex to degrade glycosaminoglycans (GAGs) from the CSPGs, abolishing WFA staining of PNNs around the injection site. Sema3A-positive nets were no longer observed in the area devoid of WFA staining. In mice lacking the link protein Crtl1 in the CNS only vestigial PNNs are present, and in these mice there were no Sema3A-positive PNN structures. A biochemical analysis shows that Sema3A protein binds with high-affinity to CS-GAGs and aggrecan and versican extracted from PNNs in the adult rat brain, and a significant proportion of Sema3A is retrieved in brain extracts that are enriched in PNN-associated GAGs. The Sema3A receptor components PlexinA1 and A4 are selectively expressed by inhibitory interneurons in the cortex that are surrounded by Sema3A positive PNNs. We conclude that the chemorepulsive axon guidance molecule Sema3A is present in PNNs of the adult rodent brain, bound to the GAGs of the CSPGs. These observations suggest a novel concept namely that chemorepulsive axon guidance molecules like Sema3A may be important functional attributes of PNNs in the adult brain.


Assuntos
Córtex Cerebral/metabolismo , Matriz Extracelular/metabolismo , Semaforina-3A/metabolismo , Agrecanas/metabolismo , Animais , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Células HEK293 , Humanos , Interneurônios/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteoglicanas/genética , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Semaforina-3A/genética , Versicanas/metabolismo
18.
Brain Commun ; 6(4): fcae244, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39144751

RESUMO

Pleckstrin homology-like domain family A-member 3 (PHLDA3) has recently been identified as a player in adaptive and maladaptive cellular stress pathways. The outcome of pleckstrin homology-like domain family A-member 3 signalling was shown to vary across different cell types and states. It emerges that its expression and protein level are highly increased in amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Whether it orchestrates a supportive or detrimental function remains unexplored in the context of neurodegenerative pathologies. To directly address the role of pleckstrin homology-like domain family A-member 3 in healthy and ALS astrocytes, we used overexpression and knockdown strategies. We generated cultures of primary mouse astrocytes and also human astrocytes from control and ALS patient-derived induced pluripotent stem cells harbouring the superoxide dismutase 1 mutation. Then, we assessed astrocyte viability and the impact of their secretome on oxidative stress responses in human stem cell-derived cortical and spinal neuronal cultures. Here, we show that PHLDA3 overexpression or knockdown in control astrocytes does not significantly affect astrocyte viability or reactive oxygen species production. However, PHLDA3 knockdown in ALS astrocytes diminishes reactive oxygen species concentrations in their supernatants, indicating that pleckstrin homology-like domain family A-member 3 can facilitate stress responses in cells with altered homeostasis. In support, supernatants of PHLDA3-silenced ALS and even control spinal astrocytes with a lower pleckstrin homology-like domain family A-member 3 protein content could prevent sodium arsenite-induced stress granule formation in spinal neurons. Our findings provide evidence that reducing pleckstrin homology-like domain family A-member 3 levels may transform astrocytes into a more neurosupportive state relevant to targeting non-cell autonomous ALS pathology.

19.
J Neurosci ; 32(21): 7325-35, 2012 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-22623678

RESUMO

Growing and regenerating axons need to interact with the molecules in the extracellular matrix as they traverse through their environment. An important group of receptors that serve this function is the integrin superfamily of cell surface receptors, which are evolutionarily conserved αß heterodimeric transmembrane proteins. The function of integrins is controlled by regulating the affinity for ligands (also called "integrin activation"). Previous results have shown that CNS inhibitory molecules inactivate axonal integrins, while enhancing integrin activation can promote axon growth from neurons cultured on inhibitory substrates. We tested two related molecules, kindlin-1 and kindlin-2 (Fermitin family members 1 and 2), that can activate ß1, ß2, and ß3 integrins, for their effects on integrin signaling and integrin-mediated axon growth in rat sensory neurons. We determined that kindlin-2, but not kindlin-1, is endogenously expressed in the nervous system. Knocking down kindlin-2 levels in cultured sensory neurons impaired their ability to extend axons, but this was partially rescued by kindlin-1 expression. Overexpression of kindlin-1, but not kindlin-2, in cultured neurons increased axon growth on an inhibitory aggrecan substrate. This was found to be associated with enhanced integrin activation and signaling within the axons. Additionally, in an in vivo rat dorsal root injury model, transduction of dorsal root ganglion neurons to express kindlin-1 promoted axon regeneration across the dorsal root entry zone and into the spinal cord. These animals demonstrated improved recovery of thermal sensation following injury. Our results therefore suggest that kindlin-1 is a potential tool for improving axon regeneration after nervous system lesions.


Assuntos
Agrecanas/farmacologia , Axônios/fisiologia , Gânglios Espinais/fisiologia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células Ganglionares da Retina/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiologia , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/lesões , Gânglios Espinais/metabolismo , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Integrinas/metabolismo , Laminina/farmacologia , Regeneração Nervosa/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , Cultura Primária de Células , Células de Purkinje/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
20.
Front Neuroanat ; 17: 1152131, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37025098

RESUMO

Microvascular integrity is disrupted following spinal cord injury (SCI) by both primary and secondary insults. Changes to neuronal structures are well documented, but little is known about how the capillaries change and recover following injury. Spatiotemporal morphological information is required to explore potential treatments targeting the microvasculature post-SCI to improve functional recovery. Sprague-Dawley rats were given a T10 moderate/severe (200 kDyn) contusion injury and were perfuse-fixed at days 2, 5, 15, and 45 post-injury. Unbiased stereology following immunohistochemistry in four areas (ventral and dorsal grey and white matter) across seven spinal segments (n = 4 for each group) was used to calculate microvessel density, surface area, and areal density. In intact sham spinal cords, average microvessel density across the thoracic spinal cord was: ventral grey matter: 571 ± 45 mm-2, dorsal grey matter: 484 ± 33 mm-2, ventral white matter: 90 ± 8 mm-2, dorsal white matter: 88 ± 7 mm-2. Post-SCI, acute microvascular disruption was evident, particularly at the injury epicentre, and spreading three spinal segments rostrally and caudally. Damage was most severe in grey matter at the injury epicentre (T10) and T11. Reductions in all morphological parameters (95-99% at day 2 post-SCI) implied vessel regression and/or collapse acutely. Transmission electron microscopy (TEM) revealed disturbed aspects of neurovascular unit fine structure at day 2 post-SCI (n = 2 per group) at T10 and T11. TEM demonstrated a more diffuse and disrupted basement membrane and wider intercellular clefts at day 2, suggesting a more permeable blood spinal cord barrier and microvessel remodelling. Some evidence of angiogenesis was seen during recovery from days 2 to 45, indicated by increased vessel density, surface area, and areal density at day 45. These novel results show that the spinal cord microvasculature is highly adaptive following SCI, even at chronic stages and up to three spinal segments from the injury epicentre. Multiple measures of gross and fine capillary structure from acute to chronic time points provide insight into microvascular remodelling post-SCI. We have identified key vascular treatment targets, namely stabilising damaged capillaries and replacing destroyed vessels, which may be used to improve functional outcomes following SCI in the future.

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