RESUMO
Zellweger syndrome (ZS), a neonatal lethal disorder arising from defective peroxisome biogenesis, features profound neuroanatomical abnormalities and brain dysfunction. Here we used mice with brain-restricted inactivation of the peroxisome biogenesis gene PEX13 to model the pathophysiological features of ZS, and determine the impact of peroxisome dysfunction on neurogenesis and cell maturation in ZS. In the embryonic and postnatal PEX13 mutant brain, we demonstrate key regions with altered brain anatomy, including enlarged lateral ventricles and aberrant cortical, hippocampal and hypothalamic organization. To characterize the underlying mechanisms, we show a significant reduction in proliferation, migration, differentiation, and maturation of neural progenitors in embryonic E12.5 through to P3 animals. An increasing reactive gliosis in the PEX13 mutant brain started at E14.5 in association with the pathology. Together with impaired neurogenesis and associated gliosis, our data demonstrate increased cell death contributing to the hallmark brain anatomy of ZS. We provide unique data where impaired neurogenesis and migration are shown as critical events underlying the neuropathology and altered brain function of mice with peroxisome deficiency.
Assuntos
Gliose/genética , Proteínas de Membrana/deficiência , Mutação/genética , Neurogênese/genética , Síndrome de Zellweger/metabolismo , Animais , Encéfalo/metabolismo , Diferenciação Celular/genética , Modelos Animais de Doenças , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Peroxissomos/genéticaRESUMO
Many neurodegenerative diseases are characterized by the formation of microscopically visible intracellular protein aggregates. α-Synuclein is the key aggregating protein in Parkinson's disease which is characterized by neuronal cytoplasmic Lewy body inclusions. Previous studies have shown relative sparing of neurons in Parkinson's disease and dementia with Lewy bodies that are positive for the vitamin D-dependent calcium-buffering protein, calbindin-D28k, and that α-synuclein aggregates are excluded from calbindin-D28k-positive neurons. Recent cell culture studies have shown that α-synuclein aggregation can be induced by raised intracellular-free Ca(II) and demonstrated that raised intracellular calcium and oxidative stress can act synergistically to promote α-synuclein aggregation. We hypothesized that calcipotriol, a potent vitamin D analogue used pharmaceutically, may be able to suppress calcium-dependent α-synuclein aggregation by inducing calbindin-D28k expression. Immunofluorescence and western blot analysis showed that calcipotriol potently induced calbindin-D28k in a dose-dependent manner in SH-SY5Y human neuroblastoma cells. Calcipotriol significantly decreased the frequency of α-synuclein aggregate positive cells subjected to treatments that cause raised intracellular-free Ca(II) (potassium depolarization, KCl/H2 O2 combined treatment, and rotenone) in a dose-dependent manner and increased viability. Suppression of calbindin-D28k expression in calcipotriol-treated cells using calbindin-D28k-specific siRNA showed significantly higher α-synuclein aggregation levels, indicating that calcipotriol-mediated blocking of calcium-dependent α-synuclein aggregation was dependent on the induction of calbindin-D28k expression. These data indicate that targeting raised intraneuronal-free Ca(II) in the brain by promoting the expression of calbindin-D28k at the transcriptional level using calcipotriol could prevent α-synuclein aggregate formation and ameliorate Parkinson's disease pathogenesis.
Assuntos
Calbindina 1/biossíntese , Calcitriol/análogos & derivados , Neuroblastoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/metabolismo , Antineoplásicos/farmacologia , Calbindina 1/antagonistas & inibidores , Calcitriol/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Agregados Proteicos/fisiologiaRESUMO
α-Synuclein (α-syn) aggregates (Lewy bodies) in Dementia with Lewy Bodies (DLB) may be associated with disturbed calcium homeostasis and oxidative stress. We investigated the interplay between α-syn aggregation, expression of the calbindin-D28k (CB) neuronal calcium-buffering protein and oxidative stress, combining immunofluorescence double labelling and Western analysis, and examining DLB and normal human cases and a unilateral oxidative stress lesion model of α-syn disease (rotenone mouse). DLB cases showed a greater proportion of CB+ cells in affected brain regions compared to normal cases with Lewy bodies largely present in CB- neurons and virtually undetected in CB+ neurons. The unilateral rotenone-lesioned mouse model showed a greater proportion of CB+ cells and α-syn aggregates within the lesioned hemisphere than the control hemisphere, especially proximal to the lesion site, and α-syn inclusions occurred primarily in CB- cells and were almost completely absent in CB+ cells. Consistent with the immunofluorescence data, Western analysis showed the total CB level was 25% higher in lesioned compared to control hemisphere in aged animals that are more sensitive to lesion and 20% higher in aged compared to young mice in lesioned hemisphere, but not significantly different between young and aged in the control hemisphere. Taken together, the findings show α-syn aggregation is excluded from CB+ neurons, although the increased sensitivity of aged animals to lesion was not related to differential CB expression.
Assuntos
Calbindina 1/metabolismo , Doença por Corpos de Lewy/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Calbindina 1/genética , Humanos , Doença por Corpos de Lewy/etiologia , Doença por Corpos de Lewy/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Estresse Oxidativo , Agregados Proteicos , Rotenona/toxicidade , alfa-Sinucleína/genéticaRESUMO
Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.
Assuntos
DNA/biossíntese , Corantes Fluorescentes/química , Timidina/análogos & derivados , Timidina/química , Animais , Bromodesoxiuridina/química , Proliferação de Células , Células Cultivadas , Química Click , DNA/química , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Humanos , Coloração e Rotulagem , Nicho de Células-TroncoRESUMO
Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro. We present here a novel thymidine analogue, ethynyl deoxyuridine (EdU) for labelling dividing cells, detected with a fluorescent azide which forms a covalent bond via the "click" chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). Unlike the commonly used BrdU, EdU detection requires no heat or acid treatment. It is quick and easy and compatible with multiple probes for fluorescence immunochemistry, facilitating the characterisation of proliferating cells at high resolution.
Assuntos
Sistema Nervoso/citologia , Neurogênese/fisiologia , Neurônios/fisiologia , Compostos de Fenilureia/metabolismo , Actinas/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Indóis , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Gravidez , Timidina/metabolismoRESUMO
Aggregation of the pre-synaptic protein, α-synuclein (α-syn), is the key etiological factor in Parkinson's disease (PD) and other alpha-synucleinopathies, such as multiple system atrophy (MSA) and Dementia with Lewy bodies (DLB). Various triggers for pathological α-syn aggregation have been elucidated, including post-translational modifications, oxidative stress, and binding of metal ions, such as calcium. Raised neuronal calcium levels in PD may occur due to mitochondrial dysfunction and/or may relate to calcium channel dysregulation or the reduced expression of the neuronal calcium buffering protein, calbindin-D28k. Recent results on human tissue and a mouse oxidative stress model show that neuronal calbindin-D28k expression excludes α-syn inclusion bodies. Previously, cell culture model studies have shown that transient increases of intracellular free Ca(II), such as by opening of the voltage-gated plasma calcium channels, could induce cytoplasmic aggregates of α-syn. Raised intracellular free calcium and oxidative stress also act cooperatively to promote α-syn aggregation. The association between raised neuronal calcium, α-syn aggregation, oxidative stress, and neurotoxicity is reviewed in the context of neurodegenerative α-syn disease and potential mechanism-based therapies.
RESUMO
Intratumor heterogeneity is a primary feature of high-grade gliomas, complicating their therapy. As accumulating evidence suggests that intratumor heterogeneity is a consequence of cellular subsets with different cycling frequencies, we developed a method for transcriptional profiling of gliomas, using a novel technique to dissect the tumors into two fundamental cellular subsets, namely, the proliferating and non-proliferating cell fractions. The tumor fractions were sorted whilst maintaining their molecular integrity, by incorporating the thymidine analog 5-ethynyl-2'-deoxyuridine into actively dividing cells. We sorted the actively dividing versus non-dividing cells from cultured glioma cells, and parental and clonally derived orthotopic tumors, and analyzed them for a number of transcripts. While there was no significant difference in the transcriptional profiles between the two cellular subsets in cultured glioma cells, we demonstrate â¼2-6 fold increase in transcripts of cancer and neuronal stem cell and tumor cell migration/invasion markers, and â¼2-fold decrease in transcripts of markers of hypoxia and their target genes, in the dividing tumor cells of the orthotopic glioma when compared to their non-proliferative counterparts. This suggests the influence of the brain microenvironment in transcriptional regulation and, thereby, the physiology of glioma cells in vivo. When clonal glioma cells were derived from a parental glioma and the resultant orthotopic tumors were compared, their transcriptional profiles were closely correlated to tumor aggression and consequently, survival of the experimental animals. This study demonstrates the resolution of intratumor heterogeneity for profiling studies based on cell proliferation, a defining feature of cancers, with implications for treatment design.
Assuntos
Neoplasias Encefálicas/patologia , Proliferação de Células , Perfilação da Expressão Gênica , Glioma/patologia , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Zellweger syndrome (ZS) is a peroxisome biogenesis disorder that involves significant neuropathology, the molecular basis of which is still poorly understood. Using a mouse model of ZS with brain-restricted deficiency of the peroxisome biogenesis protein PEX13, we demonstrated an expanded and morphologically modified brain mitochondrial population. Cultured fibroblasts from PEX13-deficient mouse embryo displayed similar changes, as well as increased levels of mitochondrial superoxide and membrane depolarization; this phenotype was rescued by antioxidant treatment. Significant oxidative damage to neurons in brain was indicated by products of lipid and DNA oxidation. Similar overall changes were observed for glial cells. In toto, these findings suggest that mitochondrial oxidative stress and aberrant mitochondrial dynamics are associated with the neuropathology arising from PEX13 deficiency.
Assuntos
Encéfalo/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Síndrome de Zellweger/metabolismo , Animais , Western Blotting , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Superóxido Dismutase/metabolismo , Triptofano Hidroxilase/metabolismo , Síndrome de Zellweger/patologiaRESUMO
BACKGROUND: The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. NEW METHOD: We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. RESULTS: 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. COMPARISON WITH EXISTING METHOD: BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. CONCLUSIONS: The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation.
Assuntos
Neoplasias Encefálicas , Encéfalo , Perfilação da Expressão Gênica/métodos , Neurogênese , Neurônios , Análise de Célula Única/métodos , Animais , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Neoplasias Encefálicas/fisiopatologia , Células Cultivadas , Química Click , Desoxiuridina/análogos & derivados , Células-Tronco Embrionárias/fisiologia , Feminino , Glioma/fisiopatologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Mucosa Olfatória/fisiologia , RNA/metabolismoRESUMO
The motor innervation of the duodenum and distal colon remains uncharacterized within the same species. Our aim was to compare the projections and neurochemical properties of duodenal and distal colon circular muscle motor neurons. Circular muscle motor neurons were retrogradely traced by using a neural tracer in vitro, processed for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity and then visualized by using indirect immunofluorescence. A mean of 372 +/- 64 and 156 +/- 23 neurons (mean +/- SEM) were tracer-labeled within the duodenum and colon, respectively. The ChAT+/NOS- neurons comprised 57.6 +/- 6.6% and 39.6 +/- 4.4% of all labeled cells in the duodenum and colon, respectively, and projected mainly in the oral direction. Of all labeled cells, the ChAT-/NOS+ neurons comprised 8.5 +/- 2.3% in the duodenum and 46.6 +/- 5.0% in the distal colon and projected mainly in the anal direction. Of the remainder, 20.6 +/- 5.0% and 8.2 +/- 2.4% were ChAT+/NOS+ and 13.2 +/- 0.9% and 5.6 +/- 1.4% were ChAT-/NOS- in the duodenum and distal colon, respectively. Within both regions, the distribution of the ChAT+/NOS- and ChAT-/NOS+ neurons are consistent with the ascending excitatory and descending inhibitory reflexes. The proportion of ChAT-/NOS+ neurons is greater within the colon in comparison with the duodenum. A considerable proportion of duodenal motor neurons were ChAT+/NOS+ and ChAT-/NOS-. These two classes may underlie nonperistaltic motor patterns, which predominate within the duodenum. These findings demonstrate regional differences in the innervation of intestinal circular muscle.
Assuntos
Colina O-Acetiltransferase/metabolismo , Colo/inervação , Duodeno/inervação , Neurônios Motores/citologia , Músculo Liso/inervação , Plexo Mientérico/citologia , Óxido Nítrico Sintase/metabolismo , Gambás/anatomia & histologia , Acetilcolina/metabolismo , Animais , Carbocianinas , Tamanho Celular/fisiologia , Células Cultivadas , Colo/metabolismo , Duodeno/metabolismo , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Masculino , Microscopia Confocal , Neurônios Motores/metabolismo , Músculo Liso/metabolismo , Plexo Mientérico/metabolismo , Óxido Nítrico/metabolismo , Gambás/metabolismo , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestruturaRESUMO
ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity.
Assuntos
Encéfalo/microbiologia , Burkholderia pseudomallei/fisiologia , Interações Hospedeiro-Patógeno , Melioidose/microbiologia , Nervo Olfatório/microbiologia , Nervo Trigêmeo/microbiologia , Animais , Encéfalo/patologia , Feminino , Imuno-Histoquímica , Melioidose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Cavidade Nasal/microbiologia , Nervo Olfatório/patologia , Fatores de Tempo , Nervo Trigêmeo/patologiaRESUMO
Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs, with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms, depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans, represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES), suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II, involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Mitocôndrias/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , alfa-Tocoferol/farmacologia , Linhagem Celular Tumoral , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Feminino , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Triptofano/metabolismoRESUMO
Oligodendroglial inclusion bodies characterize a subset of neurodegenerative diseases. Multiple system atrophy (MSA) is characterized by α-synuclein glial cytoplasmic inclusions and progressive supranuclear palsy (PSP) is associated with glial tau inclusions. The ubiquitin homologue, SUMO-1, has been identified in inclusion bodies in MSA, located in discrete sub-domains in α-synuclein-positive inclusions. We investigated SUMO-1 associated with oligodendroglial inclusion bodies in brain tissue from MSA and PSP and in glial cell models. We examined MSA and PSP cases and compared to age-matched normal controls. Fluorescence immunohistochemistry revealed frequent SUMO-1 sub-domains within and surrounding inclusions bodies in both diseases and showed punctate co-localization of SUMO-1 and the lysosomal marker, cathepsin D, in affected brain regions. Cell counting data revealed that 70-75 % of lysosomes in inclusion body-positive oligodendrocytes were SUMO-1-positive consistently across MSA and PSP cases, compared to 20 % in neighbouring inclusion body negative oligodendrocytes and 10 % in normal brain tissue. Hsp90 co-localized with some SUMO-1 puncta. We examined the SUMO-1 status of lysosomes in 1321N1 human glioma cells over-expressing α-synuclein and in immortalized rat oligodendrocyte cells over-expressing the four repeat form of tau following treatment with the proteasome inhibitor, MG132. We also transfected 1321N1 cells with the inherently aggregation-prone huntingtin exon 1 mutant, HttQ74-GFP. Each cell model showed the association of SUMO-1-positive lysosomes around focal cytoplasmic accumulations of α-synuclein, tau or HttQ74-GFP, respectively. Association of SUMO-1 with lysosomes was also detected in glial cells bearing α-synuclein aggregates in a rotenone-lesioned rat model. SUMO-1 labelling of lysosomes showed a major increase between 24 and 48 h post-incubation of 1321N1 cells with MG132 resulting in an increase in a 90 kDa SUMO-1-positive band that was immunopositive for Hsp90 and immunoprecipitated with an anti-SUMO-1 antibody. That SUMO-1 co-localizes with a subset of lysosomes in neurodegenerative diseases with glial protein aggregates and in glial cell culture models of protein aggregation suggests a role for SUMO-1 in lysosome function.
Assuntos
Lisossomos/metabolismo , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Proteína SUMO-1/metabolismo , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Lisossomos/patologia , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Neuroglia/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Trauma to the spinal cord creates an initial physical injury damaging neurons, glia, and blood vessels, which then induces a prolonged inflammatory response, leading to secondary degeneration of spinal cord tissue, and further loss of neurons and glia surrounding the initial site of injury. Angiogenesis is a critical step in tissue repair, but in the injured spinal cord angiogenesis fails; blood vessels formed initially later regress. Stabilizing the angiogenic response is therefore a potential target to improve recovery after spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) can initiate angiogenesis, but cannot sustain blood vessel maturation. Platelet-derived growth factor (PDGF) can promote blood vessel stability and maturation. We therefore investigated a combined application of VEGF and PDGF as treatment for traumatic spinal cord injury, with the aim to reduce secondary degeneration by promotion of angiogenesis. Immediately after hemisection of the spinal cord in the rat we delivered VEGF and PDGF and to the injury site. One and 3 months later the size of the lesion was significantly smaller in the treated group compared to controls, and there was significantly reduced gliosis surrounding the lesion. There was no significant effect of the treatment on blood vessel density, although there was a significant reduction in the numbers of macrophages/microglia surrounding the lesion, and a shift in the distribution of morphological and immunological phenotypes of these inflammatory cells. VEGF and PDGF delivered singly exacerbated secondary degeneration, increasing the size of the lesion cavity. These results demonstrate a novel therapeutic intervention for SCI, and reveal an unanticipated synergy for these growth factors whereby they modulated inflammatory processes and created a microenvironment conducive to axon preservation/sprouting.
Assuntos
Degeneração Neural/prevenção & controle , Fator de Crescimento Derivado de Plaquetas/farmacologia , Traumatismos da Medula Espinal/patologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Imunofluorescência , Masculino , Degeneração Neural/etiologia , Ratos , Ratos WistarRESUMO
Bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promise in in vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease. However, the effects of intracerebral transplantation are not well defined, and studies do not agreed on the optimal neuronal differentiation method. Here, we investigated three growth factor-based neuronal differentiation procedures (using FGF-2/EGF/PDGF/SHH/FGF-8/GDNF), and found all to be capable of eliciting an immature neural phenotype, in terms of cell morphology and gene/protein expression. The neuronal-priming (FGF-2/EGF) method induced neurosphere-like formation and the highest NES and NR4A2 expression by hMSCs. Transplantation of undifferentiated and neuronal-primed hMSCs into the striatum and substantia nigra of 6-OHDA-lesioned hemiparkinsonian rats revealed transient graft survival of 7 days, despite the reported immunosuppressive properties of MSCs and cyclosporine-immunosuppression of rats. Neither differentiation of hMSCs nor induction of host neurogenesis was observed at injection sites, and hMSCs continued producing mesodermal fibronectin. Strategies for improving engraftment and differentiation post-transplantation, such as prior in vitro neuronal-priming, nigral and striatal grafting, and co-transplantation of olfactory ensheathing cells that promote neural regeneration, were unable to provide advantages. Innate inflammatory responses (Iba-1-positive microglia/macrophage and GFAP-positive astrocyte activation and accumulation) were detected around grafts within 7 days. Our findings indicate that growth factor-based methods allow hMSC differentiation toward immature neuronal-like cells, and contrary to previous reports, only transient survival and engraftment of hMSCs occurs following transplantation in immunosuppressed hemiparkinsonian rats. In addition, suppression of host innate inflammatory responses may be a key factor for improving hMSC survival and engraftment.
Assuntos
Medula Óssea/metabolismo , Modelos Animais de Doenças , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Doença de Parkinson/terapia , Animais , Western Blotting , Medula Óssea/crescimento & desenvolvimento , Diferenciação Celular , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Imunofluorescência , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Neurônios/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Rotenone, a mitochondrial complex 1 inhibitor, causes oxidative damage via production of reactive oxygen species. We examined the pathophysiology of neuronal and glial cells of the nigrostriatal pathway following unilateral infusion of varying doses of rotenone into the substantia nigra or medial forebrain bundle of adult male Sprague-Dawley rats, sacrificed 14 and 60 days after infusion. Immunofluorescence techniques were used to qualitatively and quantitatively assay dopaminergic neurons, their projections, glial cells, synapses, and oxidative stress. Rotenone infusion into the substantia nigra at all concentrations caused extensive damage and tissue necrosis, therefore of limited relevance for producing a Parkinson disease model. Infusion of 0.5µg of rotenone targeting the medial forebrain bundle induced oxidative stress in dopaminergic neurons causing ongoing cell stress as defined by an elevation of stress granule and oxidative stress markers. This treatment resulted in the loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra (p≤0.01) and loss of tyrosine hydroxylase immunoreactive nerve fibres and synaptic specialisations in the striatum (p≤0.01). The infusion of 0.5µg of rotenone also caused an increase in astrocytes and microglial cells in the substantia nigra in comparison to control (p≤0.01). We examined the time-dependent reduction of tyrosine hydroxylase-positive nerve fibres and cell bodies in the striatum and substantia nigra respectively, with a progressive reduction evident 60days after infusion (p≤0.01, p≤0.05). Dopaminergic axons exposed to low-dose rotenone undergo oxidative stress, with a resultant ongoing loss of dopaminergic neurons, providing an animal model relevant to Parkinson disease.
Assuntos
Dopamina/fisiologia , Feixe Prosencefálico Mediano/fisiologia , Neurônios/efeitos dos fármacos , Rotenona/farmacologia , Desacopladores/farmacologia , Animais , Apoptose/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/metabolismo , Contagem de Células , Densitometria , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Proteínas dos Microfilamentos , Microglia/metabolismo , Neurônios/fisiologia , Estresse Oxidativo/fisiologia , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Ratos , Ratos Sprague-Dawley , Rotenona/administração & dosagem , Substância Negra/enzimologia , Substância Negra/patologia , Superóxido Dismutase/metabolismo , Sinapses/efeitos dos fármacos , Sinaptofisina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Desacopladores/administração & dosagem , alfa-Sinucleína/metabolismoRESUMO
The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.