RESUMEN
Microglia have been found to acquire unique region-dependent deleterious features with age and diseases that contribute to neuronal dysfunction and degeneration in the brain. However, it remains unknown whether microglia exhibit similar phenotypic heterogeneity in the spinal cord. Here, we performed a regional analysis of spinal cord microglia in 3-, 16-, 23-, and 30-month-old mice. Using light and electron microscopy, we discovered that spinal cord microglia acquire an increasingly activated phenotype during the course of aging regardless of regional location. However, aging causes microglia in the ventral but not dorsal horn to lose their spatial organization. Aged ventral horn microglia also aggregate around the somata of motor neurons and increase their contacts with motor synapses, which have been shown to be lost with age. These findings suggest that microglia may affect the ability of motor neurons to receive and relay motor commands during aging. To generate additional insights about aging spinal cord microglia, we performed RNA-sequencing on FACS-isolated microglia from 3-, 18-, 22-, and 29-month-old mice. We found that spinal cord microglia acquire a similar transcriptional identity as those in the brain during aging that includes altered expression of genes with roles in microglia-neuron interactions and inflammation. By 29 months of age, spinal cord microglia exhibit additional and unique transcriptional changes known and predicted to cause senescence and to alter lysosomal and ribosomal regulation. Altogether, this work provides the foundation to target microglia to ameliorate aged-related changes in the spinal cord, and particularly on the motor circuit.
Asunto(s)
Microglía , Neuronas Motoras , Ratones , Animales , Microglía/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Sinapsis/metabolismo , Inflamación/metabolismoRESUMEN
Amyotrophic Lateral Sclerosis (ALS) is a currently incurable disease that causes progressive motor neuron loss, paralysis and death. Skeletal muscle pathology occurs early during the course of ALS. It is characterized by impaired mitochondrial biogenesis, metabolic dysfunction and deterioration of the neuromuscular junction (NMJ), the synapse through which motor neurons communicate with muscles. Therefore, a better understanding of the molecules that underlie this pathology may lead to therapies that slow motor neuron loss and delay ALS progression. Kruppel Like Factor 15 (KLF15) has been identified as a transcription factor that activates alternative metabolic pathways and NMJ maintenance factors, including Fibroblast Growth Factor Binding Protein 1 (FGFBP1), in skeletal myocytes. In this capacity, KLF15 has been shown to play a protective role in Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), however its role in ALS has not been evaluated. Here, we examined whether muscle-specific KLF15 overexpression promotes the health of skeletal muscles and NMJs in the SOD1G93A ALS mouse model. We show that muscle-specific KLF15 overexpression did not elicit a significant beneficial effect on skeletal muscle atrophy, NMJ health, motor function, or survival in SOD1G93A ALS mice. Our findings suggest that, unlike in mouse models of DMD and SMA, KLF15 overexpression has a minimal impact on ALS disease progression in SOD1G93A mice.
Asunto(s)
Esclerosis Amiotrófica Lateral , Factores de Transcripción de Tipo Kruppel , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Longevidad , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Células Musculares/metabolismo , Células Musculares/patología , Músculo Esquelético/patología , Unión Neuromuscular/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
BACKGROUND: Effective treatment of solid tumors requires multi-modality approaches. In many patients with stage IV liver disease, current treatments are not curative. Chimeric antigen receptor T cells (CAR-T) are an intriguing option following success in hematological malignancies, but this has not been translated to solid tumors. Limitations include sub-optimal delivery and elevated interstitial fluid pressures. We developed a murine model to test the impact of high-pressure regional delivery (HPRD) on trafficking to liver metastases (LM) and tumor response. MATERIALS AND METHODS: CAR-T were generated from CD45.1 mice and adoptively transferred into LM-bearing CD45.2 mice via regional or systemic delivery (RD, SD). Trafficking, tumor growth, and toxicity were evaluated with flow cytometry, tumor bioluminescence (TB, photons/sec log2-foldover baseline), and liver function tests (LFTs). RESULTS: RD of CAR-T was more effective at controlling tumor growth versus SD from post-treatment days (PTD) 2-7 (P = 0.002). HPRD resulted in increased CAR-T penetration versus low-pressure RD (LPRD, P = 0.004), suppression of tumor proliferation (P = 0.03), and trended toward improved long-term control at PTD17 (TB=3.7 versus 6.1, P = 0.47). No LFT increase was noted utilizing HPRD versus LPRD (AST/ALT P = 0.65/0.84) while improved LFTs in RD versus SD groups suggested better tumor control (HPRD AST/ALT P = 0.04/0.04, LPRD AST/ALT P = 0.02/0.02). CONCLUSIONS: Cellular immunotherapy is an emerging option for solid tumors. Our model suggests RD and HPRD improved CAR-T penetration into solid tumors with improved short-term tumor control. Barriers associated with SD can be overcome using RD techniques to maximize therapeutic delivery and HPRD may further augment efficacy without increased toxicity.
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Neoplasias Colorrectales , Neoplasias Hepáticas , Neoplasias , Receptores Quiméricos de Antígenos , Animales , Neoplasias Colorrectales/terapia , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias Hepáticas/patología , Ratones , Neoplasias/terapia , Linfocitos TRESUMEN
Spinal motor neurons have been implicated in the loss of motor function that occurs with advancing age. However, the cellular and molecular mechanisms that impair the function of these neurons during aging remain unknown. Here, we show that motor neurons do not die in old female and male mice, rhesus monkeys, and humans. Instead, these neurons selectively and progressively shed excitatory synaptic inputs throughout the soma and dendritic arbor during aging. Thus, aged motor neurons contain a motor circuitry with a reduced ratio of excitatory to inhibitory synapses that may be responsible for the diminished ability to activate motor neurons to commence movements. An examination of the motor neuron translatome (ribosomal transcripts) in male and female mice reveals genes and molecular pathways with roles in glia-mediated synaptic pruning, inflammation, axonal regeneration, and oxidative stress that are upregulated in aged motor neurons. Some of these genes and pathways are also found altered in motor neurons affected with amyotrophic lateral sclerosis (ALS) and responding to axotomy, demonstrating that aged motor neurons are under significant stress. Our findings show mechanisms altered in aged motor neurons that could serve as therapeutic targets to preserve motor function during aging.
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Esclerosis Amiotrófica Lateral , Neuronas Motoras , Humanos , Ratones , Masculino , Femenino , Animales , Anciano , Neuronas Motoras/metabolismo , Envejecimiento/metabolismo , Esclerosis Amiotrófica Lateral/genética , Sinapsis/metabolismo , PrimatesRESUMEN
Lynx1 is a glycosylphosphatidylinositol (GPI)-linked protein shown to affect synaptic plasticity through modulation of nicotinic acetylcholine receptor (nAChR) subtypes in the brain. Because of this function and structural similarity to α-bungarotoxin, which binds muscle-specific nAChRs with high affinity, Lynx1 is a promising candidate for modulating nAChRs in skeletal muscles. However, little is known about the expression and roles of Lynx1 in skeletal muscles and neuromuscular junctions (NMJs). Here, we show that Lynx1 is expressed in skeletal muscles, increases during development, and concentrates at NMJs. We also demonstrate that Lynx1 interacts with muscle-specific nAChR subunits. Additionally, we present data indicating that Lynx1 deletion alters the response of skeletal muscles to cholinergic transmission and their contractile properties. Based on these findings, we asked if Lynx1 deletion affects developing and adult NMJs. Loss of Lynx1 had no effect on NMJs at postnatal day 9 (P9) and moderately increased their size at P21. Thus, Lynx1 plays a minor role in the structural development of NMJs. In 7- and 12-month-old mice lacking Lynx1, there is a marked increase in the incidence of NMJs with age- and disease-associated morphological alterations. The loss of Lynx1 also reduced the size of adult muscle fibers. Despite these effects, Lynx1 deletion did not alter the rate of NMJ reinnervation and stability following motor axon injury. These findings suggest that Lynx1 is not required during fast remodeling of the NMJ, as is the case during reformation following crushing of motor axons and development. Instead, these data indicate that the primary role of Lynx1 may be to maintain the structure and function of adult and aging NMJs.
RESUMEN
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that proliferate in the setting of cancer and have potent immunosuppressive functions hindering anti-tumor immunity. Here we establish that the immunologic landscape and tumor microenvironments (TME) vary between different organs which discretely shape MDSC repertoires. We found that pSTAT3 signaling exerts a dominant effect on MDSC programming in liver metastasis (LM). In contrast, in lung metastasis (LuM), MDSC programming is driven mainly by pSTAT5. Adoptive transfer of LM-MDSC into LuM resulted in a shift from pSTAT3 signaling to pSTAT5, in association with an overall shift toward lung MDSC programming. A shift from more immunosuppressive M-MDSC to G-MDSC, along with enhanced differentiation of MDSCs into pro-inflammatory M1 macrophages in LuM, indicated that MDSC plasticity and differentiation patterns are environmentally dependent. Using mass spectroscopy, we confirmed that LM-MDSCs showed enhanced expression of key proliferation pathway markers. This confirmed that liver-specific MDSC programing was comprehensive but reversible, implying that therapeutic targeting of LM-MDSC could prime the TME in a favorable manner. Our data suggest that MDSC programming in response to malignancy is highly dependent on organ-specific conditions and is modifiable.
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Granulocitos/metabolismo , Neoplasias Pulmonares/metabolismo , Monocitos/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Microambiente Tumoral , Animales , Línea Celular Tumoral , Granulocitos/patología , Neoplasias Pulmonares/patología , Masculino , Ratones , Monocitos/patología , Células Supresoras de Origen Mieloide/patología , Especificidad de ÓrganosRESUMEN
Metastatic liver tumors have presented challenges with the use of checkpoint inhibitors (CPIs), with only limited success. We hypothesize that regional delivery (RD) of CPIs can improve activity in the liver and minimize systemic exposure, thereby reducing immune-related adverse events (irAE). Using a murine model of colorectal cancer liver metastases (LM), we confirmed high levels of PD-L1 expression on the tumor cells and liver myeloid-derived suppressor cells (L-MDSC). In vivo, we detected improved LM response at 3 mg/kg on PTD7 via portal vein (PV) regional delivery as compared to 3 mg/kg via tail vein (TV) systemic delivery (p = 0.04). The minimal effective dose at PTD7 was 5 mg/kg (p = 0.01) via TV and 0.3 mg/kg (p = 0.02) via PV. We detected 6.7-fold lower circulating CPI antibody levels in the serum using the 0.3 mg/kg PV treatment compared to the 5 mg/kg TV cohort (p < 0.001) without increased liver toxicity. Additionally, 3 mg/kg PV treatment resulted in increased tumor cell apoptotic signaling compared to 5 mg/kg TV (p < 0.05). Therefore, RD of an anti-PD-1 CPI therapy for CRCLM may improve the therapeutic index by reducing the total dose required and limiting the systemic exposure. These advantages could expand CPI indications for liver tumors.