RESUMEN
Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neoplasias , Inmunidad Adaptativa , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Inmunidad Innata , Ratones , Neoplasias/patologíaRESUMEN
There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos B/inmunología , Neoplasias/inmunología , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/secundario , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos , Sitios de Unión de Anticuerpos/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/secundario , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Masculino , Melanoma/inmunología , Melanoma/secundario , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Plasmáticas/inmunología , Células Precursoras de Linfocitos B , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patologíaRESUMEN
Identifying preventive targets for Alzheimer's disease is a central challenge of modern medicine. Non-steroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzymes COX-1 and COX-2, reduce the risk of developing Alzheimer's disease in normal ageing populations. This preventive effect coincides with an extended preclinical phase that spans years to decades before onset of cognitive decline. In the brain, COX-2 is induced in neurons in response to excitatory synaptic activity and in glial cells in response to inflammation. To identify mechanisms underlying prevention of cognitive decline by anti-inflammatory drugs, we first identified an early object memory deficit in APPSwe-PS1ΔE9 mice that preceded previously identified spatial memory deficits in this model. We modelled prevention of this memory deficit with ibuprofen, and found that ibuprofen prevented memory impairment without producing any measurable changes in amyloid-ß accumulation or glial inflammation. Instead, ibuprofen modulated hippocampal gene expression in pathways involved in neuronal plasticity and increased levels of norepinephrine and dopamine. The gene most highly downregulated by ibuprofen was neuronal tryptophan 2,3-dioxygenase (Tdo2), which encodes an enzyme that metabolizes tryptophan to kynurenine. TDO2 expression was increased by neuronal COX-2 activity, and overexpression of hippocampal TDO2 produced behavioural deficits. Moreover, pharmacological TDO2 inhibition prevented behavioural deficits in APPSwe-PS1ΔE9 mice. Taken together, these data demonstrate broad effects of cyclooxygenase inhibition on multiple neuronal pathways that counteract the neurotoxic effects of early accumulating amyloid-ß oligomers.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Trastornos de la Memoria/prevención & control , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Inhibidores de la Ciclooxigenasa , Modelos Animales de Enfermedad , Regulación hacia Abajo , Electroencefalografía , Ibuprofeno , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Reconocimiento en Psicología/efectos de los fármacos , Triptófano Oxigenasa/efectos de los fármacosRESUMEN
Prostaglandin E2 (PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP(1-4) receptors. Here, we investigated the cell-specific in vivo function of PGE2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2(lox/lox) mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2(lox/lox) and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.
Asunto(s)
Encéfalo/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Encéfalo/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Inflamación/inducido químicamente , Inflamación/genética , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Ratones , Ratones Transgénicos , Microglía/efectos de los fármacos , Subtipo EP2 de Receptores de Prostaglandina E/genéticaRESUMEN
Clinical, epidemiological and experimental studies confirm a connection between the common degenerative movement disorder Parkinson's disease (PD) that affects over 1 million individuals, and Gaucher disease, the most prevalent lysosomal storage disorder. Recently, human imaging studies have implicated impaired striatal dopaminergic neurotransmission in early PD pathogenesis in the context of Gaucher disease mutations, but the underlying mechanisms have yet to be characterized. In this report we describe and characterize two novel long-lived transgenic mouse models of Gba deficiency, along with a subchronic conduritol-ß-epoxide (CBE) exposure paradigm. All three murine models revealed striking glial activation within nigrostriatal pathways, accompanied by abnormal α-synuclein accumulation. Importantly, the CBE-induced, pharmacological Gaucher mouse model replicated this change in dopamine neurotransmission, revealing a markedly reduced evoked striatal dopamine release (approximately 2-fold) that indicates synaptic dysfunction. Other changes in synaptic plasticity markers, including microRNA profile and a 24.9% reduction in post-synaptic density size, were concomitant with diminished evoked dopamine release following CBE exposure. These studies afford new insights into the mechanisms underlying the Parkinson's-Gaucher disease connection, and into the physiological impact of related abnormal α-synuclein accumulation and neuroinflammation on nigrostriatal dopaminergic neurotransmission.
Asunto(s)
Cuerpo Estriado/patología , Enfermedad de Gaucher/patología , Glucosilceramidasa , Enfermedad de Parkinson/patología , Sinapsis/patología , alfa-Sinucleína/metabolismo , Animales , Cuerpo Estriado/enzimología , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Potenciales Evocados Motores , Femenino , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/fisiopatología , Humanos , Inflamación , Inositol/administración & dosificación , Inositol/análogos & derivados , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Plasticidad Neuronal , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/fisiopatología , Sinapsis/enzimología , Transmisión Sináptica , alfa-Sinucleína/genéticaRESUMEN
Pathologic accumulation of alpha-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of alpha-synuclein in vivo was indicated by the detection of alpha-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal alpha-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of alpha-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with alpha-synuclein within nigral dopaminergic neurons of paraquat-treated and alpha-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of alpha-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of alpha-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal alpha-synuclein buildup.
Asunto(s)
Lisosomas/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Animales , Autofagia/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Herbicidas/efectos adversos , Herbicidas/farmacología , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/genética , Masculino , Mesencéfalo/patología , Ratones , Ratones Transgénicos , Neuronas/patología , Paraquat/efectos adversos , Paraquat/farmacología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , alfa-Sinucleína/genéticaRESUMEN
Within the past 25 years, discoveries of environmental and monogenetic forms of parkinsonism have shaped the direction of Parkinson's disease (PD) research and development of experimental systems to study PD. In this review, we outline a remarkable array of in vivo models available, with particular emphasis on their benefits and pitfalls and the contribution each has made to enhance our understanding of pathological mechanisms involved in PD. Further, we discuss the increasingly popular approach of "model fusion" to create a new generation of animal systems in which to study gene-environment interactions, and the usefulness of such models in capturing the most common events underlying PD.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Parkinson , Animales , Animales Modificados Genéticamente , Ambiente , Genes/fisiología , Humanos , Neurotoxinas/toxicidad , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/fisiopatologíaRESUMEN
Inflammation is a ubiquitous factor accompanying normal aging and neurodegeneration, and recent studies indicate a major contribution of inducible cyclooxygenase (COX-2) and its downstream prostaglandin signaling pathways in modulating neuroinflammatory responses and neuronal function. We have previously shown that the prostaglandin PGE2 receptor EP4 suppresses innate immune responses in models of systemic inflammation. Here we investigated the role of the EP4 receptor in models of Parkinson's disease (PD). Systemic co-administration of the EP4 agonist ONO-AE1-329 with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prevented loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) without significant changes in glial activation, suggesting a potent neuroprotective effect of EP4 signaling in this acute model of DA neuronal loss. Cell-specific conditional ablation of EP4 in Cd11bCre;EP4lox/lox mice exacerbated MPTP-associated glial activation and T-cell infiltration in SNpc, consistent with anti-inflammatory functions of microglial EP4 signaling. In vitro, in primary microglia stimulated with oligomeric α-synuclein, EP4 receptor activation suppressed generation of pro-inflammatory and oxidative stress factors. Taken together, these findings suggest a dual neuroprotective and anti-inflammatory mechanism of action by the EP4 receptor in models of PD.
Asunto(s)
Antiinflamatorios/uso terapéutico , Dinoprostona/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Trastornos Parkinsonianos/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal/fisiología , Animales , Antiinflamatorios/farmacología , Células Cultivadas , Masculino , Éteres Metílicos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/metabolismo , Fármacos Neuroprotectores/farmacología , Trastornos Parkinsonianos/prevención & control , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
Regenerative medicine for the treatment of motor features in Parkinson's disease (PD) is a promising therapeutic option. Donor cells can simultaneously address multiple pathological mechanisms while responding to the needs of the host tissue. Previous studies have demonstrated that mesenchymal stromal cells (MSCs) promote recovery using various animal models of PD. SanBio Inc. has developed a novel cell type designated SB623, which are adult bone marrow-derived MSCs transfected with Notch intracellular domain. In this preclinical study, SB623 cells protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal injury when transplanted unilaterally into C57BL/6 mouse striatum 3 days prior to toxin exposure. Specifically, mice with the SB623 cell transplants revealed significantly higher levels of striatal dopamine, tyrosine hydroxylase immunoreactivity and stereological nigral cell counts in the ipsilateral hemisphere vs vehicle-treated mice following MPTP administration. Interestingly, improvement in markers of striatal dopaminergic integrity was also noted in the contralateral hemisphere. These data indicate that MSCs transplantation, specifically SB623 cells, may represent a novel therapeutic option to ameliorate damage related to PD, not only at the level of striatal terminals (i.e. the site of implantation) but also at the level of the nigral cell body. Copyright © 2015 John Wiley & Sons, Ltd.
Asunto(s)
Cuerpo Estriado , Dopamina/metabolismo , Intoxicación por MPTP , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Adulto , Animales , Células Cultivadas , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Intoxicación por MPTP/terapia , Masculino , Células Madre Mesenquimatosas/patología , RatonesRESUMEN
Alpha-synuclein is likely to play a role in neurodegenerative processes, including the degeneration of nigrostriatal dopaminergic neurons that underlies Parkinson's disease. However, the toxicological properties of alpha-synuclein remain relatively unknown. Here, the relationship between alpha-synuclein expression and neuronal injury was studied in mice exposed to the herbicide paraquat. Paraquat neurotoxicity was compared in control animals versus mice with transgenic expression of human alpha-synuclein driven by the tyrosine hydroxylase (TH) promoter. In control mice, paraquat caused both the formation of alpha-synuclein-containing intraneuronal deposits and the degeneration of nigrostriatal neurons, as demonstrated by silver staining and a reduction of the counts of TH-positive and Nissl-stained cells. Mice overexpressing alpha-synuclein, either the human wild-type or the Ala53Thr mutant form of the protein, displayed paraquat-induced protein aggregates but were completely protected against neurodegeneration. These resistant animals were also characterized by increased levels of HSP70, a chaperone protein that has been shown to counteract paraquat toxicity in other experimental models and could therefore contribute to neuroprotection in alpha-synuclein transgenic mice. The results indicate a dissociation between toxicant-induced alpha-synuclein deposition and neurodegeneration. They support a role of alpha-synuclein against toxic insults and suggest that its involvement in human neurodegenerative processes may arise not only from a gain of toxic function, as previously proposed, but also from a loss of defensive properties.
Asunto(s)
Proteínas del Tejido Nervioso/biosíntesis , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/prevención & control , Paraquat , Animales , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Sustancias Macromoleculares , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Enfermedades Neurodegenerativas/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Trastornos Parkinsonianos/genética , Regiones Promotoras Genéticas , Tinción con Nitrato de Plata , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sinucleínas , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genética , alfa-SinucleínaRESUMEN
Protein deposition diseases involve the aggregation of normally soluble proteins, leading to both fibrillar and amorphous deposits. The aggregation of alpha-synuclein is associated with Parkinson's disease, and the aggregation of the Abeta peptide is associated with Alzheimer's disease. Here we show that L-dopa, dopamine, and other catecholamines dissolve fibrils of alpha-synuclein and Abeta peptide generated in vitro. The catecholamines also inhibited the fibrillation of these proteins. In addition, intraneuronal alpha-synuclein deposits formed in a mouse model were dissolved by incubation of tissue slices with L-dopa. These catecholamines are susceptible to oxidative breakdown, and we show that oxidation products are more effective than the parent compounds in inhibition. The ability to dissolve fibrils provides a new approach for studying mechanisms and consequences (e.g., the relationship between fibril formation and neurodegeneration) of protein aggregation. It is also likely to help in the development of strategies for the prevention and treatment of protein deposition diseases.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloide/química , Amiloide/metabolismo , Dihidroxifenilalanina/farmacología , Dopamina/farmacología , Enfermedad de Parkinson/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cromatografía Líquida de Alta Presión , Dihidroxifenilalanina/química , Dihidroxifenilalanina/metabolismo , Dopamina/química , Dopamina/metabolismo , Epinefrina/metabolismo , Epinefrina/farmacología , Humanos , Ratones , Microscopía de Fuerza Atómica , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/ultraestructura , Oxidación-Reducción , Enfermedad de Parkinson/tratamiento farmacológico , Estructura Cuaternaria de Proteína/efectos de los fármacos , Sinucleínas , alfa-SinucleínaRESUMEN
Evidence discussed in this review article lends strong support in favor of an etiologic role of environmentalfactors in Parkinson's disease. First, thanks to the discovery of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), it is now clear that, by targeting the nigrostriatal system, neurotoxicants can reproduce the neurochemical and pathological features of idiopathic parkinsonism. The sequence of toxic events triggered by MPTP has also provided us with intriguing clues concerning mechanisms of toxicant selectivity and nigrostriatal vulnerability. Relevant examples are (i) the role of the plasma membrane dopamine transporter in facilitating the access of potentially toxic species into dopaminergic neurons; (ii) the vulnerability of the nigrostriatal system to failure of mitochondrial energy metabolism; and (iii) the contribution of inflammatory processes to tissue lesioning. Epidemiological and experimental data suggest the potential involvement of specific agents as neurotoxicants (e.g. pesticides) or neuroprotective compounds (e.g. tobacco products) in the pathogenesis of nigrostriatal degeneration, further supporting a relationship between the environment and Parkinson's disease. A likely scenario that emerges from our current knowledge is that neurodegeneration results from multiple events and interactive mechanisms. These may include (i) the synergistic action of endogenous and exogenous toxins (e.g. the ability of the pesticide diethyldithiocarbamate to promote the toxicity of other compounds); (ii) the interactions of toxic agents with endogenous elements (e.g. the protein alpha-synuclein); (iii) the tissue response to an initial toxic insult; and, last but not least, (iv) the effects of environmental factors on the background of genetic predisposition and aging.
Asunto(s)
Ambiente , Enfermedad de Parkinson/etiología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Dopaminérgicos , Humanos , Degeneración Nerviosa/patología , Fármacos Neuroprotectores/uso terapéutico , Neurotoxinas/toxicidad , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Factores de RiesgoRESUMEN
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Asunto(s)
Lipooxigenasa/metabolismo , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Interacción Gen-Ambiente , Predisposición Genética a la Enfermedad , Isoenzimas , Ratones , Sustancia Negra/efectos de los fármacos , Sustancia Negra/enzimología , Sustancia Negra/patologíaRESUMEN
Thiamine (vitamin B1) deficiency, associated with a variety of conditions, including chronic alcoholism and bariatric surgery for morbid obesity, can result in the neurological disorder Wernicke's encephalopathy (WE). Recent work building upon early observations in animal models of thiamine deficiency has demonstrated an inflammatory component to the neuropathology observed in thiamine deficiency. The present, multilevel study including in vivo magnetic resonance imaging (MRI) and spectroscopy (MRS) and postmortem quantification of chemokine and cytokine proteins sought to determine whether a combination of these in vivo neuroimaging tools could be used to characterize an in vivo MR signature for neuroinflammation. Thiamine deficiency for 12days was used to model neuroinflammation; glucose loading in thiamine deficiency was used to accelerate neurodegeneration. Among 38 animals with regional brain tissue assayed postmortem for cytokine/chemokine protein levels, three groups of rats (controls+glucose, n=6; pyrithiamine+saline, n=5; pyrithiamine+glucose, n=13) underwent MRI/MRS at baseline (time 1), after 12days of treatment (time 2), and 3h after challenge (glucose or saline, time 3). In the thalamus of glucose-challenged, thiamine deficient animals, correlations between in vivo measures of pathology (lower levels of N-acetyle aspartate and higher levels of lactate) and postmortem levels of monocyte chemotactic protein-1 (MCP-1, also known as chemokine ligand 2, CCL2) support a role for this chemokine in thiamine deficiency-related neurodegeneration, but do not provide a unique in vivo signature for neuroinflammation.
Asunto(s)
Encéfalo/metabolismo , Citocinas/metabolismo , Imagen por Resonancia Magnética , Encefalopatía de Wernicke/metabolismo , Encefalopatía de Wernicke/patología , Análisis de Varianza , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Creatina/metabolismo , Modelos Animales de Enfermedad , Hígado/patología , Espectroscopía de Resonancia Magnética , Masculino , Examen Neurológico , Ratas , Ratas Wistar , Tiamina/metabolismo , Factores de TiempoRESUMEN
Loss of DJ-1 function contributes to pathogenesis in Parkinson's disease. Here, we investigate the impact of aging and DJ-1 deficiency in transgenic mice. Ventral midbrain from young DJ-1-deficient mice revealed no change in 4-hydroxy-2-nonenal (4-HNE), but HSP60, HSP40 and striatal dopamine turnover were significantly elevated compared to wildtype. In aged mice, the chaperone response observed in wildtype animals was absent from DJ-1-deficient transgenics, and nigral 4-HNE immunoreactivity was enhanced. These changes were concomitant with increased striatal dopamine levels and uptake. Thus, increased oxidants and diminished protein quality control may contribute to nigral oxidative damage with aging in the model.
Asunto(s)
Envejecimiento/fisiología , Proteínas Oncogénicas/genética , Estrés Oxidativo/genética , Factores de Edad , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Química Encefálica/genética , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/fisiología , Femenino , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/fisiología , Ratones , Ratones Transgénicos , Neostriado/metabolismo , Peroxirredoxinas , Proteína Desglicasa DJ-1 , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
Oxidative damage of membrane polyunsaturated fatty acids (PUFA) is thought to play a major role in mitochondrial dysfunction related to Parkinson's disease (PD). The toxic products formed by PUFA oxidation inflict further damage on cellular components and contribute to neuronal degeneration. Here, we tested the hypothesis that isotopic reinforcement, by deuteration of the bisallylic sites most susceptible to oxidation in PUFA may provide at least partial protection against nigrostriatal injury in a mouse model of oxidative stress and cell death, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model. Mice were fed a fat-free diet supplemented with saturated acids, oleic acid and essential PUFA: either normal, hydrogenated linoleic (LA, 18:2n-6) and α-linolenic (ALA, 18:3n-3) or deuterated 11,11-D2-LA and 11,11,14,14-D4-ALA in a ratio of 1:1 (to a total of 10% mass fat) for 6 days; each group was divided into two cohorts receiving either MPTP or saline and then continued on respective diets for 6 days. Brain homogenates from mice receiving deuterated PUFA (D-PUFA) vs. hydrogenated PUFA (H-PUFA) demonstrated a significant incorporation of deuterium as measured by isotope ratio mass-spectrometry. Following MPTP exposure, mice fed H-PUFA revealed 78.7% striatal dopamine (DA) depletion compared to a 46.8% reduction in the D-PUFA cohort (as compared to their respective saline-treated controls), indicating a significant improvement in DA concentration with D-PUFA. Similarly, higher levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were detected in MPTP-exposure mice administered D-PUFA; however, saline-treated mice revealed no change in DA or DOPAC levels. Western blot analyses of tyrosine hydroxylase (TH) confirmed neuroprotection with D-PUFA, as striatal homogenates showed higher levels of TH immunoreactivity in D-PUFA (88.5% control) vs. H-PUFA (50.4% control) in the MPTP-treated cohorts. In the substantia nigra, a significant improvement was noted in the number of nigral dopaminergic neurons following MPTP exposure in the D-PUFA (79.5% control) vs. H-PUFA (58.8% control) mice using unbiased stereological cell counting. Taken together, these findings indicate that dietary isotopic reinforcement with D-PUFA partially protects against nigrostriatal damage from oxidative injury elicited by MPTP in mice.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Ácidos Grasos Insaturados/farmacología , Enfermedad de Parkinson/prevención & control , Trastornos Parkinsonianos/prevención & control , Sustancia Negra/efectos de los fármacos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Deuterio , Modelos Animales de Enfermedad , Ácido Linoleico/farmacología , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Ácido Oléico/farmacología , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Trastornos Parkinsonianos/inducido químicamente , Ácido alfa-Linolénico/farmacologíaRESUMEN
BACKGROUND: Parkinson's disease is a neurodegenerative disorder characterized pathologically by the loss of nigrostriatal dopamine neurons that project from the substantia nigra in the midbrain to the putamen and caudate nuclei, leading to the clinical features of bradykinesia, rigidity, and rest tremor. Oxidative stress from oxidized dopamine and related compounds may contribute to the degeneration characteristic of this disease. RESULTS: To investigate a possible role of the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4) in protection from oxidative stress, we investigated GPX4 expression in postmortem human brain tissue from individuals with and without Parkinson's disease. In both control and Parkinson's samples, GPX4 was found in dopaminergic nigral neurons colocalized with neuromelanin. Overall GPX4 was significantly reduced in substantia nigra in Parkinson's vs. control subjects, but was increased relative to the cell density of surviving nigral cells. In putamen, GPX4 was concentrated within dystrophic dopaminergic axons in Parkinson's subjects, although overall levels of GPX4 were not significantly different compared to control putamen. CONCLUSIONS: This study demonstrates an up-regulation of GPX4 in neurons of substantia nigra and association of this protein with dystrophic axons in striatum of Parkinson's brain, indicating a possible neuroprotective role. Additionally, our findings suggest this enzyme may contribute to the production of neuromelanin.
RESUMEN
A growing body of experimental and clinical literature indicates an association between Gaucher disease and parkinsonism, raising the possibility that convergent mechanisms may contribute to neurodegeneration in these disorders. The aim of this study was to determine whether there is a relationship between alpha-synuclein (alpha-syn), a key protein in Parkinson's disease pathogenesis, and abnormalities in glucocerebroside (GC) catabolism that lead to the development of Gaucher disease. We inhibited glucocerebrosidase (GCase) with conduritol B epoxide (CBE) in neuroblastoma cells and mice to test whether a biological link exists between GCase activity and alpha-syn. After CBE exposure, enhanced alpha-syn protein was detected in differentiated cells challenged with CBE as compared to vehicle, with no change in alpha-syn mRNA. In the mouse model, after one injection of CBE, elevated nigral alpha-syn levels were also detected. Analyses by Western blot and confocal microscopy revealed that normal alpha-syn distribution was perturbed after CBE exposure with its accumulation apparent within nigral cell bodies as well as astroglia. These findings raise the possibility that alpha-syn may contribute to the cascade of events that promote neuronal dysfunction in Gaucher disease and are the first to implicate this protein as a plausible biological intersection between Gaucher disease and parkinsonism using a pharmacological model.
Asunto(s)
Enfermedad de Gaucher/metabolismo , alfa-Sinucleína/metabolismo , Análisis de Varianza , Animales , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Enfermedad de Gaucher/inducido químicamente , Enfermedad de Gaucher/patología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inositol/análogos & derivados , Inositol/toxicidad , Ratones , Ratones Endogámicos C57BL , Neuroblastoma/patología , ARN Mensajero/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , alfa-Sinucleína/genéticaRESUMEN
Mutations in the gene for DJ-1 have been associated with early-onset autosomal recessive parkinsonism. Previous studies of null DJ-1 mice have shown alterations in striatal dopamine (DA) transmission with no DAergic cell loss. Here we characterize a new line of DJ-1-deficient mice. A subtle locomotor deficit was present in the absence of a change in striatal DA levels. However, increased [(3)H]-DA synaptosomal uptake and [(125)I]-RTI-121 binding were measured in null DJ-1 vs. wild-type mice. Western analyses of synaptosomes revealed significantly higher dopamine transporter (DAT) levels in pre-synaptic membrane fractions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure exacerbated striatal DA depletion in null DJ-1 mice with no difference in DAergic nigral cell loss. Furthermore, increased 1-methyl-4-phenylpyridinium (MPP(+)) synaptosomal uptake and enhanced MPP(+) accumulation were measured in DJ-1-deficient vs. control striatum. Thus, under null DJ-1 conditions, DAT changes likely contribute to altered DA neurotransmission and enhanced sensitivity to toxins that utilize DAT for nigrostriatal entry.
Asunto(s)
Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Ratones Transgénicos , Proteínas Oncogénicas/deficiencia , Terminales Presinápticos/metabolismo , Sustancia Negra/metabolismo , Animales , Western Blotting , Cuerpo Estriado/patología , Dopamina/metabolismo , Inmunohistoquímica , Intoxicación por MPTP , Ratones , Actividad Motora/fisiología , Proteínas Oncogénicas/genética , Peroxirredoxinas , Terminales Presinápticos/patología , Proteína Desglicasa DJ-1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sustancia Negra/patología , Sinaptosomas/metabolismoRESUMEN
Systemic administration of ubiquitin-proteasome system inhibitors to rodents has been reported to induce certain behavioral and neuropathological features of Parkinson's disease. The goal of this study was to replicate these observations by administering a proteasome inhibitor (PSI) to rats using McNaught and colleagues' protocol. No alterations in locomotor activity or striatal dopamine and its metabolites were observed. Differences in nigral dopaminergic cell number between proteasome inhibitor- and vehicle-treated rats and inclusion bodies were not found. Extending the time of survival after administration and using different solvents failed to alter results, indicating this proteasome inhibitor does not consistently produce the selective toxicity and pathological hallmarks characterizing Parkinson's disease.