Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative condition characterized by the loss of motor neurons in a progressive manner. The severity of the disorder, as well as substantial genetic and phenotypic variability among patients, further complicates the picture, making it difficult to translate findings from genetically similar animal models into effective human clinical trials. Even though motor neurons are the primary cell type affected in ALS, there is increasing evidence that neuroinflammatory responses play a role in the disease and influence neurodegeneration. Recent evidences, including unpublished observations from our group, highlighted that Glycoprotein nonmetastatic melanoma protein B (GPNMB) could play a critical role in the modulation of neuroinflammatory responses, mediating neuroprotective and neuroimmunomodulatory responses. Considering these evidences, in this study we investigated GPNMB localization and distribution in the spinal cord and bloodstream of SOD1.G93A transgenic rat model of ALS. GPNMB has never been investigated in this model. We applied a combination of: immunohistochemistry analyses on both intracellular and extracellular GPNMB domains; in situ hybridization for GPNMB mRNA and ELISA to detect GPNMB as potential biomarker in the serum. Our analyses highlighted that GPNMB is mainly expressed in motor neurons in healthy conditions. However, both mRNA and protein levels progressively decrease in this cell type in ALS rats as the disease progresses. Interestingly, we found an upregulation of GPNMB mRNA and protein in non-neuronal cells, identified as astrocytes and microglia by immunohistochemistry. Our data support the hypothesis that reactive astrocytes could be a major source of GPNMB in ALS, even though we cannot exclude that also some microglia subpopulations could participate in the GPNMB signal. We clearly highlighted that GPNMB mRNA decrease in motor neurons as the disease progresses whereas the GPNMB extracellular domain is still detected in few surviving motor neurons in the relatively spared spinal cord districts in TG animals. We were able to spot a significant difference in GPNMB serum concentration between non transgenic animals and sick SOD1.G93A animals at the end stage of the disease. Overall, this supports the hypothesis that early upregulation of GPNMB ectodomain could be a therapeutic strategy to enhance motor neuron survival and, at the same time, contributing to modulation of neuroinflammatory responses potentially enhancing therapeutic benefit for ALS patients.
La sclerosi laterale amiotrofica (SLA) è una patologia neurodegenerativa letale caratterizzata dalla perdita progressiva dei motoneuroni. La gravità del disturbo, così come la sostanziale variabilità genetica e fenotipica tra i pazienti, complica ulteriormente il quadro, rendendo difficile tradurre i risultati di modelli animali geneticamente simili in sperimentazioni cliniche umane efficaci. Anche se i motoneuroni sono la tipologia cellulare maggiormente colpita nella SLA, emergono sempre maggiori evidenze sul ruolo delle risposte neuroinfiammatorie che giocano un ruolo nella malattia e influenzano i processi neurodegenerativi. Recenti evidenze, comprese osservazioni non ancora pubblicate dal nostro gruppo di studio, hanno evidenziato che la Glicoproteina B del melanoma non metastatico (GPNMB) potrebbe svolgere un ruolo critico nella modulazione delle risposte neuroinfiammatorie, mediando le risposte neuroprotettive e neuroimmunomodulatorie. Alla luce di queste evidenze, in questo studio abbiamo studiato la localizzazione e la distribuzione di GPNMB nel midollo spinale e nel flusso sanguigno del modello di ratto transgenico SOD1G93A di SLA. GPNMB non è mai stato studiato in questo modello. Abbiamo applicato una combinazione di: analisi immunoistochimiche sui domini intracellulari ed extracellulari di GPNMB; ibridazione in situ per la localizzazione dell’mRNA di GPNMB ed ELISA per rilevare GPNMB come potenziale biomarcatore nel siero. Le nostre analisi hanno evidenziato che GPNMB è espresso principalmente nei motoneuroni in animali sani. Tuttavia, sia i livelli di mRNA che della proteina diminuiscono progressivamente in questo tipo di cellule nei ratti SLA man mano che la malattia progredisce. È interessante notare che abbiamo trovato una sovra regolazione dell'mRNA e della proteina GPNMB in cellule non neuronali, identificate come astrociti e microglia mediante immunoistochimica. I nostri dati supportano l'ipotesi che gli astrociti reattivi potrebbero essere una delle principali fonti di GPNMB nella SLA, anche se non possiamo escludere che anche alcune sottopopolazioni di microglia possano partecipare al segnale GPNMB. Abbiamo chiaramente evidenziato la diminuzione dell'mRNA di GPNMB nei motoneuroni con il progredire della malattia, mentre il dominio extracellulare di GPNMB permane in pochi motoneuroni sopravvissuti nei distretti del midollo spinale di animali TG. Siamo stati in grado di individuare una differenza significativa nella concentrazione sierica di GPNMB tra animali non transgenici e animali SOD1.G93A malati allo stadio terminale della malattia. Nel complesso, questo supporta l'ipotesi che la sovra regolazione precoce dell'ectodominio di GPNMB potrebbe essere una strategia terapeutica per migliorare la sopravvivenza dei motoneuroni e, allo stesso tempo, contribuire alla modulazione delle risposte neuroinfiammatorie, aumentando potenzialmente il beneficio terapeutico per i pazienti affetti da SLA.
Indagine sul ruolo della Glicoproteina B del melanoma non metastatico (GPNMB) in un modello di ratto di sclerosi laterale amiotrofica
SPATAFORA, MAURO GIUSEPPE
2019/2020
Abstract
Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative condition characterized by the loss of motor neurons in a progressive manner. The severity of the disorder, as well as substantial genetic and phenotypic variability among patients, further complicates the picture, making it difficult to translate findings from genetically similar animal models into effective human clinical trials. Even though motor neurons are the primary cell type affected in ALS, there is increasing evidence that neuroinflammatory responses play a role in the disease and influence neurodegeneration. Recent evidences, including unpublished observations from our group, highlighted that Glycoprotein nonmetastatic melanoma protein B (GPNMB) could play a critical role in the modulation of neuroinflammatory responses, mediating neuroprotective and neuroimmunomodulatory responses. Considering these evidences, in this study we investigated GPNMB localization and distribution in the spinal cord and bloodstream of SOD1.G93A transgenic rat model of ALS. GPNMB has never been investigated in this model. We applied a combination of: immunohistochemistry analyses on both intracellular and extracellular GPNMB domains; in situ hybridization for GPNMB mRNA and ELISA to detect GPNMB as potential biomarker in the serum. Our analyses highlighted that GPNMB is mainly expressed in motor neurons in healthy conditions. However, both mRNA and protein levels progressively decrease in this cell type in ALS rats as the disease progresses. Interestingly, we found an upregulation of GPNMB mRNA and protein in non-neuronal cells, identified as astrocytes and microglia by immunohistochemistry. Our data support the hypothesis that reactive astrocytes could be a major source of GPNMB in ALS, even though we cannot exclude that also some microglia subpopulations could participate in the GPNMB signal. We clearly highlighted that GPNMB mRNA decrease in motor neurons as the disease progresses whereas the GPNMB extracellular domain is still detected in few surviving motor neurons in the relatively spared spinal cord districts in TG animals. We were able to spot a significant difference in GPNMB serum concentration between non transgenic animals and sick SOD1.G93A animals at the end stage of the disease. Overall, this supports the hypothesis that early upregulation of GPNMB ectodomain could be a therapeutic strategy to enhance motor neuron survival and, at the same time, contributing to modulation of neuroinflammatory responses potentially enhancing therapeutic benefit for ALS patients.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12967