Proteomic quantitative study of dorsal root ganglia and sciatic nerve in type 2 diabetic mice

Diabetes; Dorsal root ganglia; Sciatic nerveDiabetes; Ganglios de la raíz dorsal; Nervio ciáticoDiabetis; Ganglis de l'arrel dorsal; Nervi ciàticObjective Peripheral neuropathy is the most common and debilitating complication of type 2 diabetes, leading to sensory loss, dysautonomia, hyperalgesia, and spontaneous noxious sensations. Despite the clinical and economic burden of diabetic neuropathy, no effective treatment is available. More preclinical research must be conducted in order to gain further understanding of the aetiology of the disease and elucidate new therapeutic targets. Methods The proteome of lumbar dorsal root ganglia and sciatic nerve of BKS-db/db mice, which contain a mutation of the leptin receptor and are an established type 2 diabetes model, was characterized for the first time by tandem mass tag labelling and mass spectrometry analysis. Results Proteomic analysis showed differentially expressed proteins grouped into functional clusters in db/db peripheral nerves compared to control mice, underlining reduced glycolytic and TCA cycle metabolism, higher lipid catabolism, upregulation of muscle-like proteins in DRG and downregulation in SCN, increased cytoskeleton-related proteins, a mild dysregulation of folding chaperones, activation of acute-phase and inflammatory response, and alterations in glutathione metabolism and oxidative stress related proteins. Conclusions Our data validate previous transcriptomic and metabolomic results and uncover new pathways altered in diabetic neuropathy. Our results point out that energetic deficiency could represent the main mechanism of neurodegeneration observed in diabetic neuropathy. These findings may provide important information to select appropriate targets to develop new therapeutic strategies.This work was supported by the Fundació Marató TV3 (grant 201607.10) and 2017 SGR1468 to M.C. M.L.J, A.O and S.V are recipients of predoctoral fellowships from Generalitat de Catalunya (2019FI_B2 00061; 2020FI_B2 00037 and 2020FI_B1 00054, respectively). The authors declare that there is no conflict of interest

Proteomic quantitative study of dorsal root ganglia and sciatic nerve in type 2 diabetic mice

La neuropatia perifèrica és la complicació més freqüent i debilitant de la diabetis tipus 2 (DMT2) donant lloc a pèrdua sensorial, disfunció del sistema nerviós autònom, hiperalgèsia i sensació espontània de dolor. Malgrat l'elevat impacte clínic i econòmic, actualment no hi ha cap tractament efectiu per la neuropatia diabètica (ND). En l'última dècada, l'estudi del transcriptoma i, més recentment, del metaboloma ha permès conèixer el paisatge molecular del sistema nerviós perifèric (SNP) en models murins i pacients humans de diabetis. Tot i això, no hi ha estudis explorant l'efecte de la DMT2 en el proteoma del SNP. Donada la baixa correlació entre les dades transcriptòmiques i proteòmiques, una aproximació multiestratègica que inclogui l'anàlisi del proteoma seria essencial per caracteritzar completament la ND. Per aquesta raó, l'objectiu principal d'aquesta tesi és caracteritzar el proteoma del SNP sota la síndrome metabòlica amb la finalitat de comprendre amb major profunditat els mecanismes patològics de la malaltia i desenvolupar noves aproximacions terapèutiques. En aquest treball, vam escollir el ratolí obès i hiperglucèmic BKS-Leprdb/db per l'anàlisi proteòmic donat que és el model de ND més estudiat en el camp de la DMT2. En primer lloc, vam avaluar el desenvolupament de neuropatia per mitjà d'electrofisiologia, test de sudoració i anàlisi morfomètric del nervi tibial. A continuació, vam caracteritzar el proteoma dels ganglis de l'arrel dorsal, així com del nervi ciàtic utilitzant marcatge TMT™ (Tandem Mass Tag) i espectrometria de masses. L'anàlisi proteòmic va mostrar un llistat de proteïnes diferencialment expressades, agrupades en grups funcionals per mitjà d'eines bioinformàtiques, destacant una alteració en el metabolisme de la glucosa i lipídic, anomalies de proteïnes estructurals, incloses proteïnes musculars i del citoesquelet, desregulació en xaperones, augment de la resposta de fase aguda i inflamatòria, i alteracions en proteïnes relacionades amb estrès oxidatiu. A més, vam estudiar amb vectors virals adenoassociats la funció de MUP en el SNP, una de les proteïnes més disminuïdes en el nostre estudi, així com el possible paper terapèutic de KLOTHO, una hormona pleiotròpica involucrada en neuroprotecció per mitjà de la modulació de la neuroinflamació, l'estrès oxidatiu i la mielinització, pel tractament de la ND. En conclusió, les nostres dades han permès trobar noves vies afectades en ND, així com validar resultats previs obtinguts per transcriptòmica i metabolòmica, confirmant que el SNP presenta alteracions moleculars, fins i tot abans que s'observin canvis morfomètrics. Els nostres resultats, en conjunt amb altres publicacions, indiquen que la disfunció metabòlica observada i, la conseqüent reducció en la producció d'ATP, podrien ser el principal mecanisme neurodegeneratiu en la ND. Aquests resultats proporcionarien noves dianes per el desenvolupament d'estratègies terapèutiques efectives pel tractament de la neuropatia en diabetis.La neuropatía periférica es la complicación más frecuente y debilitante de la diabetes tipo 2 (DMT2) dando lugar a pérdida sensorial, disfunción del sistema nervioso autónomo, hiperalgesia y sensación espontanea de dolor. A pesar del elevado impacto clínico y económico, actualmente no hay ningún tratamiento efectivo para la neuropatía diabética (ND). En la última década, el estudio del transcriptoma y, más recientemente, del metaboloma ha permitido conocer el paisaje molecular del sistema nervioso periférico (SNP) en modelos murinos y pacientes humanos de diabetes. Aun así, no hay estudios que exploren el efecto de la DMT2 en el proteoma del SNP. Dada la baja correlación entre los datos transcriptómicos y proteómicos, una aproximación multiestratégica que incluya el análisis del proteoma sería esencial para caracterizar completamente la ND. Por esta razón, el objetivo principal de esta tesis es caracterizar el proteoma del SNP bajo el síndrome metabólico con la finalidad de comprender con mayor profundidad los mecanismos patológicos de la enfermedad y desarrollar nuevas aproximaciones terapéuticas. En este trabajo, escogimos el ratón obeso e hiperglucémico BKS-Leprdb/db para su análisis proteómico dado que es el modelo de ND más estudiado en el campo de la DMT2. En primer lugar, evaluamos el desarrollo de neuropatía por medio de electrofisiología, test de sudoración y análisis morfométrico del nervio tibial. A continuación, caracterizamos el proteoma de los ganglios de la raíz dorsal, así como del nervio ciático utilizando marcaje TMT™ (Tandem Mass Tag) y espectrometría de masas. El análisis proteómico mostró un listado de proteínas diferencialmente expresadas, agrupadas en grupos funcionales por medio de herramientas bioinformáticas, destacando una alteración en el metabolismo de la glucosa y lipídico, anomalías de proteínas estructurales, incluyendo proteínas musculares y del citoesqueleto, desregulación en chaperonas, aumento de la respuesta de fase aguda e inflamatoria, y alteraciones en proteínas relacionadas con estrés oxidativo. Además, estudiamos con vectores virales adeno-asociados la función de MUP en el SNP, una de las proteínas más disminuidas en nuestro estudio, así como el posible papel terapéutico de KLOTHO, una hormona pleiotrópica involucrada en neuroprotección por medio de la modulación de la neuroinflamación, el estrés oxidativo y la mielinización, para el tratamiento de la ND. En conclusión, nuestros datos han permitido encontrar nuevas vías afectadas en ND, así como validar resultados previos obtenidos por transcriptómica y metabolómica, confirmando que el SNP presenta alteraciones moleculares, incluso antes que se observen cambios morfométricos. Nuestros resultados, en conjunto con otras publicaciones, indican que la disfunción metabólica observada y, la consecuente reducción en la producción de ATP, podrían ser el mecanismo principal neurodegenerativo en la ND. Estos resultados proporcionarían nuevas dianas para el desarrollo de estrategias terapéuticas efectivas para el tratamiento de la neuropatía en diabetes.Peripheral neuropathy is the most common and debilitating complication of type 2 diabetes (T2DM) leading to sensory loss, dysautonomia, hyperalgesia and spontaneous noxious sensations. Despite the huge clinical and economic burden of diabetic neuropathy (DN), no effective treatment is currently available. Over the past decade, high-throughput gene expression profiling technologies and, more recently, metabolomics have enabled to study the molecular landscape of the peripheral nervous system (PNS) of diabetic murine models and diabetic patients. However, there are no studies exploring the effect of T2DM on the PNS proteome. Since poor correlation between transcriptomics and proteomics has been demonstrated, a multi-strategy approach that includes the analysis of the proteome would be essential to fully characterise DN. For that reason, the main purpose of this work is to characterise the PNS proteome under metabolic syndrome aiming to gain further understanding of the pathogenic mechanisms of the disease and elucidate new therapeutic approaches. We chose the obese and hyperglycaemic BKS-Leprdb/db mouse for proteomic analysis since is the most studied model of DN in T2DM research. After neuropathy evaluation by electrophysiological and sweating tests, as well as, tibial nerve morphometry, the proteome of lumbar dorsal root ganglia and sciatic nerve was characterized by TMT™ (Tandem Mass Tag) labelling and mass spectrometry analysis. Proteomic analysis showed differentially expressed proteins, grouped into functional clusters by bioinformatic tools, underlining glucose and lipid metabolism dysregulation, abnormalities of structural proteins, including muscle-like proteins and cytoskeleton, dysregulation of folding chaperones, upregulation of acute-phase response and inflammatory proteins, and alterations in oxidative stress related proteins. Moreover, using adeno-associated viral vectors, we studied the unknown function of MUP protein in the PNS, one of the most downregulated proteins found in our data set, and analysed KLOTHO, a pleiotropic hormone involved in neuroprotection by modulating neuroinflammation, oxidative stress and myelination, as a therapeutic approach for DN. In conclusion, our data uncover new pathways altered in DN and validate previous transcriptomic and metabolomic results confirming that PNS exhibits molecular alterations, even before morphometric changes developed. Our results, in conjunction with other publications, point out that low ATP bioavailability due to metabolic impairment could represent the main mechanism of neurodegeneration observed in DN. These findings may provide important information to select appropriate targets to develop new therapeutic strategies for this severe complication of diabetes

Proteomic quantitative study of dorsal root ganglia and sciatic nerve in type 2 diabetic mice

Peripheral neuropathy is the most common and debilitating complication of type 2 diabetes, leading to sensory loss, dysautonomia, hyperalgesia, and spontaneous noxious sensations. Despite the clinical and economic burden of diabetic neuropathy, no effective treatment is available. More preclinical research must be conducted in order to gain further understanding of the aetiology of the disease and elucidate new therapeutic targets. The proteome of lumbar dorsal root ganglia and sciatic nerve of BKS- db/db mice, which contain a mutation of the leptin receptor and are an established type 2 diabetes model, was characterized for the first time by tandem mass tag labelling and mass spectrometry analysis. Proteomic analysis showed differentially expressed proteins grouped into functional clusters in db/db peripheral nerves compared to control mice, underlining reduced glycolytic and TCA cycle metabolism, higher lipid catabolism, upregulation of muscle-like proteins in DRG and downregulation in SCN, increased cytoskeleton-related proteins, a mild dysregulation of folding chaperones, activation of acute-phase and inflammatory response, and alterations in glutathione metabolism and oxidative stress related proteins. Our data validate previous transcriptomic and metabolomic results and uncover new pathways altered in diabetic neuropathy. Our results point out that energetic deficiency could represent the main mechanism of neurodegeneration observed in diabetic neuropathy. These findings may provide important information to select appropriate targets to develop new therapeutic strategie

Safety and immunogenicity of a recombinant protein RBD fusion heterodimer vaccine against SARS-CoV-2

Abstract In response to COVID-19 pandemic, we have launched a vaccine development program against SARS-CoV-2. Here we report the safety, tolerability, and immunogenicity of a recombinant protein RBD fusion heterodimeric vaccine against SARS-CoV-2 (PHH-1V) evaluated in a phase 1-2a dose-escalation, randomized clinical trial conducted in Catalonia, Spain. 30 young healthy adults were enrolled and received two intramuscular doses, 21 days apart of PHH-1V vaccine formulations [10 µg (n = 5), 20 µg (n = 10), 40 µg (n = 10)] or control [BNT162b2 (n = 5)]. Each PHH-1V group had one safety sentinel and the remaining participants were randomly assigned. The primary endpoint was solicited events within 7 days and unsolicited events within 28 days after each vaccination. Secondary endpoints were humoral and cellular immunogenicity against the variants of concern (VOCs) alpha, beta, delta and gamma. All formulations were safe and well tolerated, with tenderness and pain at the site of injection being the most frequently reported solicited events. Throughout the study, all participants reported having at least one mild to moderate unsolicited event. Two unrelated severe adverse events (AE) were reported and fully resolved. No AE of special interest was reported. Fourteen days after the second vaccine dose, all participants had a >4-fold change in total binding antibodies from baseline. PHH-1V induced robust humoral responses with neutralizing activities against all VOCs assessed (geometric mean fold rise at 35 days p < 0.0001). The specific T-cell response assessed by ELISpot was moderate. This initial evaluation has contributed significantly to the further development of PHH-1V, which is now included in the European vaccine portfolio. ClinicalTrials.gov Identifier NCT05007509 EudraCT No. 2021-001411-8