RESUMEN
Diffuse axonal injury is recognized as a progressive and long-term consequence of traumatic brain injury. Axonal injury can have sustained negative consequences on neuronal functions such as anterograde and retrograde transport and cellular processes such as autophagy that depend on cytoarchitecture and axon integrity. These changes can lead to somatic atrophy and an inability to repair and promote plasticity. Obstruction of the autophagic process has been noted after brain injury, and rapamycin, a drug used to stimulate autophagy, has demonstrated positive effects in brain injury models. The optimization of drugs to promote beneficial autophagy without negative side effects could be used to attenuate traumatic brain injury and promote improved outcome. Lanthionine ketimine ethyl ester, a bioavailable derivative of a natural sulfur amino acid metabolite, has demonstrated effects on autophagy both in vitro and in vivo. Thirty minutes after a moderate central fluid percussion injury and throughout the survival period, lanthionine ketimine ethyl ester was administered, and mice were subsequently evaluated for learning and memory impairments and biochemical and histological changes over a 5-week period. Lanthionine ketimine ethyl ester, which we have shown previously to modulate autophagy markers and alleviate pathology and slow cognitive decline in the 3 × TgAD mouse model, spared cognition and pathology after central fluid percussion injury through a mechanism involving autophagy modulation.
Asunto(s)
Aminoácidos Sulfúricos/farmacología , Autofagia/efectos de los fármacos , Lesión Axonal Difusa/tratamiento farmacológico , Aminoácidos Sulfúricos/administración & dosificación , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BLRESUMEN
Cytokines such as TNFα can polarize microglia/macrophages into different neuroinflammatory types. Skewing of the phenotype towards a cytotoxic state is thought to impair phagocytosis and has been described in Alzheimer's Disease (AD). Neuroinflammation can be perpetuated by a cycle of increasing cytokine production and maintenance of a polarized activation state that contributes to AD progression. In this study, 3xTgAD mice, age 6 months, were treated orally with 3 doses of the TNFα modulating compound isoindolin-1,3 dithione (IDT) for 10 months. We demonstrate that IDT is a TNFα modulating compound both in vitro and in vivo. Following long-term IDT administration, mice were assessed for learning & memory and tissue and serum were collected for analysis. Results demonstrate that IDT is safe for long-term treatment and significantly improves learning and memory in the 3xTgAD mouse model. IDT significantly reduced paired helical filament tau and fibrillar amyloid accumulation. Flow cytometry of brain cell populations revealed that IDT increased the infiltrating neutrophil population while reducing TNFα expression in this population. IDT is a safe and effective TNFα and innate immune system modulator. Thus small molecule, orally bioavailable modulators are promising therapeutics for Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Cognición/clasificación , Isoindoles/administración & dosificación , Isoindoles/farmacología , Infiltración Neutrófila/efectos de los fármacos , Tioamidas/administración & dosificación , Tioamidas/farmacología , Tionas/administración & dosificación , Tionas/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas tau/química , Administración Oral , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Disponibilidad Biológica , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Isoindoles/efectos adversos , Isoindoles/uso terapéutico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/metabolismo , Fenotipo , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Seguridad , Solubilidad , Tioamidas/efectos adversos , Tioamidas/uso terapéutico , Tionas/efectos adversos , Tionas/uso terapéutico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Autophagy is a fundamental cellular recycling process vulnerable to compromise in neurodegeneration. We now report that a cell-penetrating neurotrophic and neuroprotective derivative of the central nervous system (CNS) metabolite, lanthionine ketimine (LK), stimulates autophagy in RG2 glioma and SH-SY5Y neuroblastoma cells at concentrations within or below pharmacological levels reported in previous mouse studies. Autophagy stimulation was evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) both in the absence and presence of bafilomycin-A1 which discriminates between effects on autophagic flux versus blockage of autophagy clearance. LKE treatment caused changes in protein level or phosphorylation state of multiple autophagy pathway proteins including mTOR; p70S6 kinase; unc-51-like-kinase-1 (ULK1); beclin-1 and LC3 in a manner essentially identical to effects observed after rapamycin treatment. The LKE site of action was near mTOR because neither LKE nor the mTOR inhibitor rapamycin affected tuberous sclerosis complex (TSC) phosphorylation status upstream from mTOR. Confocal immunofluorescence imaging revealed that LKE specifically decreased mTOR (but not TSC2) colocalization with LAMP2(+) lysosomes in RG2 cells, a necessary event for mTORC1-mediated autophagy suppression, whereas rapamycin had no effect. Suppression of the LK-binding adaptor protein CRMP2 (collapsin response mediator protein-2) by means of shRNA resulted in diminished autophagy flux, suggesting that the LKE action on mTOR localization may occur through a novel mechanism involving CRMP2-mediated intracellular trafficking. These findings clarify the mechanism-of-action for LKE in preclinical models of CNS disease, while suggesting possible roles for natural lanthionine metabolites in regulating CNS autophagy.
Asunto(s)
Aminoácidos Sulfúricos/farmacología , Autofagia/efectos de los fármacos , Complejos Multiproteicos/metabolismo , Fármacos Neuroprotectores/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Aminoácidos Sulfúricos/química , Animales , Autofagia/fisiología , Línea Celular Tumoral , Humanos , Inmunosupresores/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Ratas , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Lanthionine ketimine ([LK] 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylic acid) is the archetype for a family of naturally occurring brain sulfur amino acid metabolites, the physiologic function of which is unknown. Lanthionine ketimine and its synthetic derivatives have recently demonstrated neurotrophic, neuroprotective, and antineuroinflammatory properties in vitro through a proposed mechanism involving the microtubule-associated protein collapsin response mediator protein 2. Therefore, studies were undertaken to test the effects of a bioavailable LK ester in the 3 × Tg-AD mouse model of Alzheimer disease. Lanthionine ketimine ester treatment substantially diminished cognitive decline and brain amyloid-ß (Aß) peptide deposition and phospho-Tau accumulation in 3 × Tg-AD mice and also reduced the density of Iba1-positive microglia. Furthermore, LK ester treatment altered collapsin response mediator protein 2 phosphorylation. These findings suggest that LK may not be a metabolic waste but rather a purposeful neurochemical, the synthetic derivatives of which constitute a new class of experimental therapeutics for Alzheimer disease and related entities.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Aminoácidos Sulfúricos/uso terapéutico , Encéfalo/efectos de los fármacos , Cognición/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Aminoácidos Sulfúricos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Comportamiento de Nidificación/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Fosforilación/efectos de los fármacosRESUMEN
BACKGROUND: Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer. PRINCIPAL FINDINGS: Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls. SIGNIFICANCE: Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/inmunología , Células Mieloides/patología , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Modelos Animales de Enfermedad , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Metástasis de la Neoplasia , Ovalbúmina/inmunología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Resultado del Tratamiento , Carga Tumoral , VacunaciónRESUMEN
BACKGROUND: Chronic neuroinflammation is an important component of Alzheimer's disease and could contribute to neuronal dysfunction, injury and loss that lead to disease progression. Multiple clinical studies implicate tumor necrosis factor-α as an inflammatory mediator of neurodegeneration in patients with Alzheimer's because of elevated levels of this cytokine in the cerebrospinal fluid, hippocampus and cortex. Current Alzheimer's disease interventions are symptomatic treatments with limited efficacy that do not address etiology. Thus, a critical need exists for novel treatments directed towards modifying the pathophysiology and progression. METHODS: To investigate the effect of early immune modulation on neuroinflammation and cognitive outcome, we treated triple transgenic Alzheimer's disease mice (harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes) with the small molecule tumor necrosis factor-α inhibitors, 3,6'-dithiothalidomide and thalidomide, beginning at four months of age. At this young age, mice do not exhibit plaque or tau pathology but do show mild intraneuronal amyloid beta protein staining and a robust increase in tumor necrosis factor-α. After 10 weeks of treatment, cognitive performance was assessed using radial arm maze and neuroinflammation was assessed using biochemical, stereological and flow cytometric endpoints. RESULTS: 3,6'-dithiothalidomide reduced tumor necrosis factor-α mRNA and protein levels in the brain and improved working memory performance and the ratio of resting to reactive microglia in the hippocampus of triple transgenic mice. In comparison to non-transgenic controls, triple transgenic Alzheimer's disease mice had increased total numbers of infiltrating peripheral monomyelocytic/granulocytic leukocytes with enhanced intracytoplasmic tumor necrosis factor-α, which was reduced after treatment with 3,6'-dithiothalidomide. CONCLUSIONS: These results suggest that modulation of tumor necrosis factor-α with small molecule inhibitors is safe and effective with potential for the long-term prevention and treatment of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Trastornos del Conocimiento/prevención & control , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/uso terapéutico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Células Cultivadas , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fármacos Neuroprotectores/farmacología , Talidomida/análogos & derivados , Talidomida/farmacología , Talidomida/uso terapéutico , Factores de Tiempo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Numerous studies suggest a central role for the low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V in Alzheimer's Disease. We continue our investigation of a ligand for this receptor, transforming growth factor beta2, which is also implicated in Alzheimer Disease pathogenesis, but whose mechanism(s) remain elusive. Confocal imaging reveals that transforming growth factor beta2 rapidly targets amyloid beta peptide to the lysosomal compartment in cortical neurons and induces cell death. Low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V is known as an endocytic receptor, delivering proteins to the lysosomal compartment for degradation. Transforming growth factor beta2 may alter this pathway resulting in increased uptake, intracellular accumulation and toxicity of amyloid beta peptide. RT-PCR and Western blot analysis of transforming growth factor beta2-treated cells demonstrate that transforming growth factor beta2 modestly increases the mRNA and protein levels of low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V as well as increases the uptake activity. Furthermore, transforming growth factor beta2 alters the morphology and numbers of lysosomes in neurons. Lucifer Yellow and lysosomal hydrolase analysis show that transforming growth factor beta2 makes lysosomal membranes unstable and leaky and this effect is exacerbated with the addition of amyloid beta protein. Our data support a key role for low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V in mediating transforming growth factor beta2 enhancement of amyloid beta peptide uptake and neurotoxicity.