RESUMEN
Efficient in vivo delivery of anti-inflammatory proteins to modulate the microenvironment of an injured spinal cord and promote neuroprotection and functional recovery is a great challenge. Nucleoside-modified messenger RNA (mRNA) has become a promising new modality that can be utilized for the safe and efficient delivery of therapeutic proteins. Here, we used lipid nanoparticle (LNP)-encapsulated human interleukin-10 (hIL-10)-encoding nucleoside-modified mRNA to induce neuroprotection and functional recovery following rat spinal cord contusion injury. Intralesional administration of hIL-10 mRNA-LNP to rats led to a remarkable reduction of the microglia/macrophage reaction in the injured spinal segment and induced significant functional recovery compared to controls. Furthermore, hIL-10 mRNA treatment induced increased expression in tissue inhibitor of matrix metalloproteinase 1 and ciliary neurotrophic factor levels in the affected spinal segment indicating a time-delayed secondary effect of IL-10 5 d after injection. Our results suggest that treatment with nucleoside-modified mRNAs encoding neuroprotective factors is an effective strategy for spinal cord injury repair.
RESUMEN
BACKGROUND: Spinal cord injuries induce a critical loss of motoneurons followed by irreversible locomotor function impairment. Surgical approaches combined with neuroprotective agents effectively rescue the damaged motoneurons and improve locomotor function. Our aim was to develop a reliable method which is able to provide quantifiable and in-depth data on the locomotor recovery during skeletal muscle reinnervation. NEW METHOD: Sprague-Dawley rats underwent lumbar 4 ventral root avulsion and reimplantation followed by riluzole treatment in order to rescue the injured motoneurons of the damaged pool. Control animals were operated, but received no riluzole treatment. The locomotor pattern of the hind limb was recorded biweekly on a special runway equipped with high resolution and high speed digital cameras producing both lateral and rear views simultaneously. All together 12 parameters of the hind limb movement pattern were evaluated by measuring specific joint angles, footprints and gait parameters in single video frames. Four months after the operation Fast Blue, a fluorescent retrograde tracer was applied to the L4 spinal nerve in order to label the reinnervating motoneurons. RESULTS: Our results confirmed the sensitivity of our arrangement and established strong relationship between the functional improvement and the morphological reinnervation. Moreover, we developed a correction method to make the system tolerant to the differences in the weight, step duration and step length. COMPARISON WITH EXISTING METHODS: There are no commercially available cheap, multi-parametric analysing equipment to characterise the gait in its complexity. CONCLUSIONS: Our system offers a modular, adaptable and expandable analysis on the reinnervation of the limb musculature in rodents.
Asunto(s)
Neuronas Motoras , Regeneración Nerviosa , Animales , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Regeneración Nerviosa/fisiología , Ratas , Ratas Sprague-Dawley , Raíces Nerviosas Espinales/fisiologíaRESUMEN
Spinal cord injury results in irreversible tissue damage followed by a very limited recovery of function. In this study we investigated whether transplantation of undifferentiated human induced pluripotent stem cells (hiPSCs) into the injured rat spinal cord is able to induce morphological and functional improvement. hiPSCs were grafted intraspinally or intravenously one week after a thoracic (T11) spinal cord contusion injury performed in Fischer 344 rats. Grafted animals showed significantly better functional recovery than the control rats which received only contusion injury. Morphologically, the contusion cavity was significantly smaller, and the amount of spared tissue was significantly greater in grafted animals than in controls. Retrograde tracing studies showed a statistically significant increase in the number of FB-labeled neurons in different segments of the spinal cord, the brainstem and the sensorimotor cortex. The extent of functional improvement was inversely related to the amount of chondroitin-sulphate around the cavity and the astrocytic and microglial reactions in the injured segment. The grafts produced GDNF, IL-10 and MIP1-alpha for at least one week. These data suggest that grafted undifferentiated hiPSCs are able to induce morphological and functional recovery after spinal cord contusion injury.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Traumatismos de la Médula Espinal , Nicho de Células Madre , Trasplante de Células Madre , Animales , Quimiocina CCL3/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/trasplante , Interleucina-10/metabolismo , Ratas , Ratas Endogámicas F344 , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapiaRESUMEN
Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration.SIGNIFICANCE STATEMENT Although most of the cells in our body have a very short lifespan, postmitotic neurons must survive for many decades. Longevity of a cell within the organism depends on its ability to properly regulate signaling pathways that counteract perturbations, such as DNA damage, oxidative stress, or protein misfolding. Here, we provide evidence that tissue-specific regulators of stress tolerance exist in postmitotic neurons. Specifically, we identify protein arginine methyltransferase 8 (PRMT8) as a cell-type-restricted arginine methyltransferase in spinal cord motoneurons (MNs). PRMT8-dependent arginine methylation is required for neuroprotection against age-related increased of cellular stress. Tissue-restricted expression and the enzymatic activity of PRMT8 make it an attractive target for drug development to delay the onset of neurodegenerative disorders.
Asunto(s)
Daño del ADN/fisiología , Neuronas Motoras/enzimología , Proteína-Arginina N-Metiltransferasas/fisiología , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Contracción Isométrica , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Musculares/enzimología , Células Musculares/fisiología , Unión Neuromuscular/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/deficiencia , Proteína-Arginina N-Metiltransferasas/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Reflejo Anormal , Prueba de Desempeño de Rotación con Aceleración Constante , Médula Espinal/citología , Médula Espinal/crecimiento & desarrolloRESUMEN
Besides their deleterious action on cardiac muscle, anthracycline-type cytostatic agents exert significant neurotoxic effects on primary sensory neurons. Since cardiac sensory nerves confer protective effects on heart muscle and share common traits with cutaneous chemosensitive nerves, this study examined the effects of cardiotoxic doses of adriamycin on the function and morphology of epidermal nerves. Sensory neurogenic vasodilatation, plasma extravasation, and the neural CGRP release evoked by TRPV1 and TRPA1 agonists in vitro were examined by using laser Doppler flowmetry, the Evans blue technique, and ELISA, respectively. Carrageenan-induced hyperalgesia was assessed with the Hargreaves method. Immunohistochemistry was utilized to study cutaneous innervation. Adriamycin treatment resulted in profound reductions in the cutaneous neurogenic sensory vasodilatation and plasma extravasation evoked by the TRPV1 and TRPA1 agonists capsaicin and mustard oil, respectively. The in vitro capsaicin-, but not high potassium-evoked neural release of the major sensory neuropeptide, CGRP, was markedly attenuated after adriamycin treatment. Carrageenan-induced inflammatory hyperalgesia was largely abolished following the administration of adriamycin. Immunohistochemistry revealed a substantial loss of epidermal TRPV1-expressing nociceptive nerves and a marked thinning of the epidermis. These findings indicate impairments in the functions of TRPV1 and TRPA1 receptors expressed on cutaneous chemosensitive nociceptive nerves and the loss of epidermal axons following the administration of cardiotoxic doses of adriamycin. Monitoring of the cutaneous nociceptor function in the course of adriamycin therapy may well be of predictive value for early detection of the deterioration of cardiac nerves which confer protection against the deleterious effects of the drug.