RESUMO
Synaptic cell adhesion molecules (SynCAMs) play an important role in the formation and maintenance of synapses and the regulation of synaptic plasticity. SynCAM3 is expressed in the synaptic cleft of the central nervous system (CNS) and is involved in the connection between axons and astrocytes. We hypothesized that SynCAM3 may be related to the astrocytic scar (glial scar, the most important factor of CNS injury treatment) through extracellular matrix (ECM) reconstitution. Thus, we investigated the influence of the selective removal of SynCAM3 on the outcomes of spinal cord injury (SCI). SynCAM3 knock-out (KO) mice were subjected to moderate compression injury of the lower thoracic spinal cord using wild-type (WT) (C57BL/6JJc1) mice as controls. Single-cell RNA sequencing analysis over time, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and immunohistochemistry (IHC) showed reduced scar formation in SynCAM3 KO mice compared to WT mice. SynCAM3 KO mice showed improved functional recovery from SCI by preventing the transformation of reactive astrocytes into scar-forming astrocytes, resulting in improved ECM reconstitution at four weeks after injury. Our findings suggest that SynCAM3 could be a novel therapeutic target for SCI.
Assuntos
Gliose , Traumatismos da Medula Espinal , Animais , Astrócitos/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cicatriz/patologia , Gliose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
Mutations in DJ-1 (PARK7) are a known cause of early-onset autosomal recessive Parkinson's disease (PD). Accumulating evidence indicates that abnormalities of synaptic vesicle trafficking underlie the pathophysiological mechanism of PD. In the present study, we explored whether DJ-1 is involved in CNS synaptic function. DJ-1 deficiency impaired synaptic vesicle endocytosis and reavailability without inducing structural alterations in synapses. Familial mutants of DJ-1 (M26I, E64D, and L166P) were unable to rescue defective endocytosis of synaptic vesicles, whereas WT DJ-1 expression completely restored endocytic function in DJ-1 KO neurons. The defective synaptic endocytosis shown in DJ-1 KO neurons may be attributable to alterations in membrane cholesterol level. Thus, DJ-1 appears essential for synaptic vesicle endocytosis and reavailability, and impairment of this function by familial mutants of DJ-1 may be related to the pathogenesis of PD.
Assuntos
Endocitose/fisiologia , Terminações Nervosas/patologia , Proteína Desglicase DJ-1/fisiologia , Sinapses/patologia , Vesículas Sinápticas/patologia , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Mutação , Terminações Nervosas/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
Intervertebral disc (IVD) degeneration can cause chronic lower back pain (LBP), leading to disability. Despite significant advances in the treatment of discogenic LBP, the limitations of current treatments have sparked interest in biological approaches, including growth factor and stem cell injection, as new treatment options for patients with chronic LBP due to IVD degeneration (IVDD). Gene therapy represents exciting new possibilities for IVDD treatment, but treatment is still in its infancy. Literature searches were conducted using PubMed and Google Scholar to provide an overview of the principles and current state of gene therapy for IVDD. Gene transfer to degenerated disc cells in vitro and in animal models is reviewed. In addition, this review describes the use of gene silencing by RNA interference (RNAi) and gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR) system, as well as the mammalian target of rapamycin (mTOR) signaling in vitro and in animal models. Significant technological advances in recent years have opened the door to a new generation of intradiscal gene therapy for the treatment of chronic discogenic LBP.
Assuntos
Edição de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Degeneração do Disco Intervertebral/terapia , Animais , Humanos , Degeneração do Disco Intervertebral/genéticaRESUMO
Due to the safety issues and poor engraftment of mesenchymal stem cell (MSC) implantation, MSC-derived exosomes have been spotlighted as an alternative therapy for spinal cord injury (SCI). However, insufficient productivity of exosomes limits their therapeutic potential for clinical application. Moreover, low targeting ability of unmodified exosomes is a critical obstacle for their further applications as a therapeutic agent. In the present study, we fabricated macrophage membrane-fused exosome-mimetic nanovesicles (MF-NVs) from macrophage membrane-fused umbilical cord blood-derived MSCs (MF-MSCs) and confirmed their therapeutic potential in a clinically relevant mouse SCI model (controlled mechanical compression injury model). MF-NVs contained larger quantity of ischemic region-targeting molecules compared to normal MSC-derived nanovesicles (N-NVs). The targeting molecules in MF-NVs, which were derived from macrophage membranes, increased the accumulation of MF-NVs in the injured spinal cord after the in vivo systemic injection. Increased accumulation of MF-NVs attenuated apoptosis and inflammation, prevented axonal loss, enhanced blood vessel formation, decreased fibrosis, and consequently, improved spinal cord function. Synthetically, we developed targeting efficiency-potentiated exosome-mimetic nanovesicles and present their possibility of clinical application for SCI.
Assuntos
Exossomos , Células-Tronco Mesenquimais/citologia , Traumatismos da Medula Espinal/terapia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose , Feminino , Sangue Fetal/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos/citologia , Fusão de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas , Neovascularização Fisiológica , Fármacos Neuroprotetores/farmacologia , Células PC12 , Células RAW 264.7 , Ratos , Medula Espinal/irrigação sanguínea , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologiaRESUMO
Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.
Assuntos
Ácido Hialurônico , Hidrogéis/administração & dosagem , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Metilcelulose , Geleia de Wharton/citologia , Animais , Biomarcadores , Técnicas de Cultura de Células , Sobrevivência Celular , Modelos Animais de Doenças , Matriz Extracelular , Regulação Enzimológica da Expressão Gênica , Hidrogéis/química , Imuno-Histoquímica , Degeneração do Disco Intervertebral/etiologia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/terapia , RatosRESUMO
Tauroursodeoxycholic acid (TUDCA) is a US FDA-approved hydrophilic bile acid for the treatment of chronic cholestatic liver disease. In the present study, we investigate the effects of TUDCA on the proliferation and differentiation of osteoblasts and its therapeutic effect on a mice model of osteoporosis. Following treatment with different concentrations of TUDCA, cell viability, differentiation, and mineralization were measured. Three-month-old female C57BL/6 mice were randomly divided into three groups (n = 8 mice per group): (i) normal mice as the control group, (ii) ovariectomy (OVX) group (receiving phosphate-buffered saline (PBS) treatment every other day for 4 weeks), and (iii) OVX group with TUDCA (receiving TUDCA treatment every other day for 4 weeks starting 6 weeks after OVX). At 11 weeks post-surgery, serum levels of procollagen type I N-terminal propeptides (PINP) and type I collagen crosslinked C-telopeptides (CTX) were measured, and all mice were sacrificed to examine the distal femur by micro-computed tomography (CT) scans and histology. TUDCA (100 nM, 1 µM) significantly increased the proliferation and viability of osteoblasts and osteoblast differentiation and mineralization when used in vitro. Furthermore, TUDCA neutralized the detrimental effects of methylprednisolone (methylprednisolone-induced osteoblast apoptosis). In the TUDCA treatment group the PINP level was higher and the CTX level was lower, but these levels were not significantly different compared to the PBS treatment group. Micro-CT and histology showed that the TUDCA treatment group preserved more trabecular structures in the distal femur compared to the PBS treatment group. In addition, the TUDCA treatment group increased the percentage bone volume with respect to the total bone volume, bone mineral density, and mice distal femur trabeculae compared with the PBS treatment group. Taken together, our findings suggest that TUDCA may provide a favorable effect on bones and could be used for the prevention and treatment of osteoporosis.
Assuntos
Osteoporose/tratamento farmacológico , Ovariectomia/efeitos adversos , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Ácido Tauroquenodesoxicólico/administração & dosagem , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metilprednisolona/efeitos adversos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoporose/etiologia , Osteoporose/metabolismo , Distribuição Aleatória , Ácido Tauroquenodesoxicólico/farmacologia , Resultado do TratamentoRESUMO
Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.
Assuntos
Exocitose/efeitos dos fármacos , Fosfatos de Inositol/farmacologia , Sinaptotagmina I/fisiologia , Animais , Hipocampo/citologia , Neurônios/fisiologia , Células PC12 , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimer's disease (AD). However, it remains to be determined whether inhibition of excessive mitochondrial fission is beneficial in mammal models of AD. To determine whether dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fragmentation, can be a disease-modifying therapeutic target for AD, we examined the effects of Drp1 inhibitor on mitochondrial and synaptic dysfunctions induced by oligomeric amyloid-ß (Aß) in neurons and neuropathology and cognitive functions in Aß precursor protein/presenilin 1 double-transgenic AD mice. Inhibition of Drp1 alleviates mitochondrial fragmentation, loss of mitochondrial membrane potential, reactive oxygen species production, ATP reduction, and synaptic depression in Aß-treated neurons. Furthermore, Drp1 inhibition significantly improves learning and memory and prevents mitochondrial fragmentation, lipid peroxidation, BACE1 expression, and Aß deposition in the brain in the AD model. These results provide evidence that Drp1 plays an important role in Aß-mediated and AD-related neuropathology and in cognitive decline in an AD animal model. Therefore, inhibiting excessive Drp1-mediated mitochondrial fission may be an efficient therapeutic avenue for AD.SIGNIFICANCE STATEMENT Mitochondrial fission relies on the evolutionary conserved dynamin-related protein 1 (Drp1). Drp1 activity and mitochondria fragmentation are significantly elevated in the brains of sporadic Alzheimer's disease (AD) cases. In the present study, we first demonstrated that the inhibition of Drp1 restored amyloid-ß (Aß)-mediated mitochondrial dysfunctions and synaptic depression in neurons and significantly reduced lipid peroxidation, BACE1 expression, and Aß deposition in the brain of AD mice. As a result, memory deficits in AD mice were rescued by Drp1 inhibition. These results suggest that neuropathology and combined cognitive decline can be attributed to hyperactivation of Drp1 in the pathogenesis of AD. Therefore, inhibitors of excessive mitochondrial fission, such as Drp1 inhibitors, may be a new strategy for AD.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Transtornos Cognitivos/fisiopatologia , Dinaminas/metabolismo , Depressão Sináptica de Longo Prazo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/complicações , Animais , Encéfalo/fisiopatologia , Transtornos Cognitivos/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição NeuralRESUMO
Variation in PPP3CC, the gene that encodes the γ isoform of the calcineurin catalytic subunit, has been reported to be associated with schizophrenia. Because of its low expression level in most tissues, there has been little research devoted to the specific function of the calcineurin Aγ (CNAγ) versus the calcineurin Aα (CNAα) and calcineurin Aß (CNAß) catalytic isoforms. Consequently, we have a limited understanding of the role of altered CNAγ function in psychiatric disease. In this study, we demonstrate that CNAγ is present in the rodent and human brain and dephosphorylates a presynaptic substrate of calcineurin. Through a combination of immunocytochemistry and immuno-EM, we further show that CNAγ is localized to presynaptic terminals in hippocampal neurons. Critically, we demonstrate that RNAi-mediated knockdown of CNAγ leads to a disruption of synaptic vesicle cycling in cultured rat hippocampal neurons. These data indicate that CNAγ regulates a critical aspect of synaptic vesicle cycling and suggest that variation in PPP3CC may contribute to psychiatric disease by altering presynaptic function.
Assuntos
Calcineurina/metabolismo , Endocitose , Vesículas Sinápticas/enzimologia , Animais , Calcineurina/genética , Células Cultivadas , Hipocampo/citologia , Hipocampo/enzimologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/enzimologia , Ratos , Vesículas Sinápticas/genéticaRESUMO
Osteoporotic vertebral compression fractures (OVCFs) are serious health problems. We conducted a randomized, open-label, phase I/IIa study to determine the feasibility, safety, and effectiveness of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) and teriparatide (parathyroid hormone 1-34) in OVCFs. Twenty subjects with recent OVCFs were randomized to teriparatide (20 µg/day, daily subcutaneous injection for 6 months) treatment alone or combined treatment of WJ-MSCs (intramedullary [4 × 107 cells] injection and intravenous [2 × 108 cells] injection after 1 week) and teriparatide (20 µg/day, daily subcutaneous injection for 6 months). Fourteen subjects (teriparatide alone, n = 7; combined treatment, n = 7) completed follow-up assessment (visual analog scale [VAS], Oswestry Disability Index [ODI], Short Form-36 [SF-36], bone mineral density [BMD], bone turnover measured by osteocalcin and C-terminal telopeptide of type 1 collagen, dual-energy x-ray absorptiometry [DXA], computed tomography [CT]). Our results show that (a) combined treatment with WJ-MSCs and teriparatide is feasible and tolerable for the patients with OVCFs; (b) the mean VAS, ODI, and SF-36 scores significantly improved in the combined treatment group; (c) the level of bone turnover markers were not significantly different between the two groups; (d) BMD T-scores of spine and hip by DXA increased in both control and experimental groups without a statistical difference; and (e) baseline spine CT images and follow-up CT images at 6 and 12 months showed better microarchitecture in the combined treatment group. Our results indicate that combined treatment of WJ-MSCs and teriparatide is feasible and tolerable and has a clinical benefit for fracture healing by promoting bone architecture. Clinical trial registration: https://nedrug.mfds.go.kr/, MFDS: 201600282-30937.
Assuntos
Conservadores da Densidade Óssea , Fraturas por Compressão , Transplante de Células-Tronco Mesenquimais , Fraturas por Osteoporose/terapia , Fraturas da Coluna Vertebral , Teriparatida , Geleia de Wharton , Densidade Óssea , Conservadores da Densidade Óssea/uso terapêutico , Fraturas por Compressão/terapia , Humanos , Fraturas da Coluna Vertebral/terapia , Teriparatida/uso terapêutico , Geleia de Wharton/citologiaRESUMO
Resolvins, a new family from the endogenous specialized pro-resolving mediators (SPMs), promote the resolution of the inflammatory response. Resolvin D3 (RvD3), a docosahexaenoic acid (DHA) product, has been known to suppress the inflammatory response. However, the anti-inflammatory and neuroprotective effects of RvD3 are not known in a model of spinal cord injury (SCI). Here, we investigated the anti-inflammatory and neuroprotective effect of RvD3 in a mouse model of SCI. Processes associated with anti-inflammation and angiogenesis were studied in RAW 264.7 cells and the human brain endothelial cell line hCMEC/D3, respectively. Additionally, female C57BL/6 mice were subjected to moderate compression SCI (20-g weight compression for 1 min) followed by intrathecal injection of vehicle or RvD3 (1 µg/20 µL) at 1 h post-SCI. RvD3 decreased the lipopolysaccharide (LPS)-induced production of inflammatory mediators and nitric oxide (NO) in RAW 264.7 cells and promoted an angiogenic effect in the hCMEC/D3 cell line. Treatment with RvD3 improved locomotor recovery and reduced thermal hyperalgesia in SCI mice compared with vehicle treatment at 14 days post-SCI. Remarkably, RvD3-treated mice exhibited reduced expression of inflammatory cytokines (TNF-α, IL6, IL1ß) and chemokines (CCL2, CCL3). Also, RvD3-treated mice exhibited increased expression of tight junction proteins such as zonula occludens (ZO)-1 and occludin. Furthermore, immunohistochemistry showed a decreased level of gliosis (GFAP, Iba-1) and neuroinflammation (CD68, TGF-ß) and enhanced neuroprotection. These data provide evidence that intrathecal injection of RvD3 represents a promising therapeutic strategy to promote inflammatory resolution, neuroprotection, and neurological functional recovery following SCI.
Assuntos
Ácidos Graxos Insaturados/uso terapêutico , Inflamação/tratamento farmacológico , Neuroproteção , Fármacos Neuroprotetores/uso terapêutico , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Movimento Celular/efeitos dos fármacos , Cicatriz/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Ácidos Graxos Insaturados/farmacologia , Feminino , Fibrose , Locomoção/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Neuroglia/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/biossíntese , Dor/complicações , Dor/fisiopatologia , Fenótipo , Células RAW 264.7 , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/complicações , Proteínas de Junções Íntimas/metabolismoRESUMO
Spinal cord injury (SCI) is a devastating condition of the central nervous system that can lead to permanent motor and sensory deficits. Carbon monoxide-releasing molecule-2 (CORM-2) has been shown to have anti-inflammatory, anti-apoptotic, and angiogenic properties that may be useful for the treatment of SCI. However, it has a short carbon monoxide (CO) release half-life (approximately 1 min). To address this challenge, we developed a CORM-2-incorporated solid lipid nanoparticle (CORM-2-SLN) and evaluated its ameliorating effects for preventing blood-spinal cord barrier (BSCB) disruption and endothelial cell death following SCI. After a moderate compression injury of the spinal cord (compression with a 35-g impounder for 5 min), groups of rats were treated with a CORM-2-solution and CORM-2-SLNs at an equal dose of 10 mg/kg each via an intraperitoneal injection for 8 consecutive days. Behavior analysis was performed and animals were later sacrificed at different time points and evaluated for whether the CORM-2-SLNs prevented BSCB disruption and rescued endothelial cell damage following SCI. The CORM-2-SLN-treated group showed significantly diminished extravasation of Evans Blue dye with enhanced expression of tight junction proteins following SCI. Likewise, significantly diminished endothelial cell markers after SCI were optimally stabilized at 21 days. Additionally, lipopolysaccharide (LPS)-induced loss of tight junction integrity was significantly preserved after CORM-2-SLN treatment in human cerebral microvascular endothelial cell line (hCMEC/D3). Clinically, CORM-2-SLNs were associated with a significantly improved functional recovery, as compared with the CORM-2-solution. CORM-2-SLNs may help potentially to maintain BSCB integrity following SCI.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Nanopartículas/administração & dosagem , Compostos Organometálicos/administração & dosagem , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Nanopartículas/uso terapêutico , Compostos Organometálicos/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
The original version of this article, the name of author was incorrectely presented. That is Kyungjae Won (K. Won) should be presented as Jae Won Kyung (J.W. Kyung).
RESUMO
Neuropathic pain is a devastating chronic condition and effective treatments are still lacking. Carbon monoxide-releasing molecule-2 (CORM-2) as a carbon monoxide (CO) carrier, exerts potent anti-neuropathic pain effects; however, its poor water solubility and short half-life hinder its clinical utility. Therefore, the aim of this study was to investigate whether CORM-2-loaded solid lipid nanoparticles (CORM-2-SLNs) enhance the anti-allodynic and anti-hyperalgesic effects of CORM-2 in a rat chronic constriction injury (CCI) model. CORM-2-SLNs were prepared using a nanotemplate engineering technique with slight modifications. The physiochemical properties of CORM-2-SLNs were characterized and CO release from CORM-2-SLNs was assessed using a myoglobin assay. CO was slowly released from CORM-2-SLNs, was observed, and the half-life of CO release was 50 times longer than that of CORM-2. In vivo results demonstrate that intraperitoneal administration of CORM-2-SLNs (5 and 10 mg/kg/day, ip) once daily for seven consecutive days significantly reduced the mechanical allodynia and mechanical hyperalgesia compared with CORM-2 (10 mg/kg/day, ip). RT-PCR and Western blot analyses on days 7 and 14, revealed that treatment with CORM-2-SLNs resulted in greater reductions in the CCI-elevated levels of heme-oxygenase-2 (HO-2); inducible nitric oxide synthase (iNOS); neuronal NOS (nNOS); and inflammatory mediators (TNF-α, IBA-1, and GFAP) in the spinal cord and dorsal root ganglions compared with treatment with CORM-2. In contrast, HO-1 and IL-10 were significantly increased in the CORM-2-SLN-treated group compared with the group treated with CORM-2. These data indicate that CORM-2-SLNs are superior to CORM-2-S in alleviating mechanical allodynia and mechanical hyperalgesia.
Assuntos
Portadores de Fármacos/química , Hiperalgesia/tratamento farmacológico , Nanopartículas/química , Compostos Organometálicos/uso terapêutico , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Monóxido de Carbono/metabolismo , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hiperalgesia/patologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipídeos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Neuralgia/complicações , Neuralgia/patologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Compostos Organometálicos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Medula Espinal/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment.
Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Axônios/metabolismo , Células Piramidais/citologia , Células Piramidais/fisiologia , Potenciais de Ação , Animais , Biomarcadores , Cálcio/metabolismo , Sinalização do Cálcio , Expressão Gênica , Técnicas de Silenciamento de Genes , Modelos Biológicos , Ratos , Sinapses/genética , Sinapses/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
Synaptic vesicle retrieval is an essential process for continuous maintenance of neural information flow after synaptic transmission. Epsin1, originally identified as an EPS15-interacting protein, is a major component of clathrin-mediated endocytosis. However, the role of Epsin1 in synaptic vesicle endocytosis at CNS synapses remains elusive. Here, we showed significantly altered synaptic vesicle endocytosis in neurons transfected with shRNA targeting Epsin1 during/after neural activity. Endocytosis was effectively restored by introducing shRNA-insensitive Epsin1 into Epsin1-depleted neurons. Domain studies performed on neurons in which domain deletion mutants of Epsin1 were introduced after Epsin1 knockdown revealed that ENTH, CLAP, and NPFs are essential for synaptic vesicle endocytosis, whereas UIMs are not. Strikingly, the efficacy of the rate of synaptic vesicle retrieval (the "endocytic capacity") was significantly decreased in the absence of Epsin1. Thus, Epsin1 is required for proper synaptic vesicle retrieval and modulates the endocytic capacity of synaptic vesicles.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células Cultivadas , Endocitose , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Domínios Proteicos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Our previous studies demonstrated that transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) into the hippocampus of a transgenic mouse model of Alzheimer's disease (AD) reduced amyloid-ß (Aß) plaques and enhanced cognitive function through paracrine action. Due to the limited life span of hUCB-MSCs after their transplantation, the extension of hUCB-MSC efficacy was essential for AD treatment. In this study, we show that repeated cisterna magna injections of hUCB-MSCs activated endogenous hippocampal neurogenesis and significantly reduced Aß42 levels. To identify the paracrine factors released from the hUCB-MSCs that stimulated endogenous hippocampal neurogenesis in the dentate gyrus, we cocultured adult mouse neural stem cells (NSCs) with hUCB-MSCs and analyzed the cocultured media with cytokine arrays. Growth differentiation factor-15 (GDF-15) levels were significantly increased in the media. GDF-15 suppression in hUCB-MSCs with GDF-15 small interfering RNA reduced the proliferation of NSCs in cocultures. Conversely, recombinant GDF-15 treatment in both in vitro and in vivo enhanced hippocampal NSC proliferation and neuronal differentiation. Repeated administration of hUBC-MSCs markedly promoted the expression of synaptic vesicle markers, including synaptophysin, which are downregulated in patients with AD. In addition, in vitro synaptic activity through GDF-15 was promoted. Taken together, these results indicated that repeated cisterna magna administration of hUCB-MSCs enhanced endogenous adult hippocampal neurogenesis and synaptic activity through a paracrine factor of GDF-15, suggesting a possible role of hUCB-MSCs in future treatment strategies for AD.