PROGETTAZIONE E SINTESI DI PRECURSORI DI 18F-DERIVATI DEGLI ACIDI BILIARI PER LA SPERIMENTAZIONE PET

Gli acidi biliari (BA) per lungo tempo sono stati studiati essenzialmente per la loro capacità di solubilizzare colesterolo, acidi grassi, e vitamine liposolubili, facilitandone la digestione ed il loro trasporto. Recenti studi hanno evidenziato una loro azione più ampia e attualmente sono definiti come molecole di segnalazione in grado di esercitare una vasta gamma di effetti endocrini. Questa nuova definizione della funzione degli acidi biliari è strettamente associata alla scoperta nel 1999 del farnesoid X recettore (FXR), uno specifico recettore nucleare dei BA e nel 2002 del recettore TGR5 (conosciuto anche come M-BAR, GP-BAR1 o BG37) recettore specifico metabotropico dei BA. Attualmente i progressi sullo studio dei percorsi di segnalazione innescati dall’attivazione dei recettori TGR5 e FXR giustificano il riconoscimento di questi recettori come plausibili target per i disordini dell’omeostasi del glucosio e dei lipidi. Gli FXR-agonisti reprimono la sintesi degli acidi biliari endogeni e riducono i livelli plasmatici di trigliceridi, glucosio e colesterolo, mentre i ligandi del recettore TGR5 riducono i livelli di glicemia e aumentano la spesa energetica. Il duplice sviluppo di agonisti per i recettori FXR e TGR5 potrebbe quindi fornire nuove opportunità per il trattamento dei disturbi lipidici e glucidici. Tuttavia, recentemente l’attenzione è stata focalizzata sui percorsi di segnalazione attivati dalla stimolazione dei TGR5 in quanto, tra le numerose azioni biochimiche prodotte, vi è anche l’attivazione del GLP-1 (Glucagon-like peptide 1), non indotta invece dalla stimolazione dei FXR. L’agonismo selettivo sul recettore TGR5 consente inoltre di evitare l’impatto negativo degli agonisti FXR sulla biosintesi dell’HDL. Nei tessuti metabolici (BAT e tessuto muscolare), l’attivazione dei TGR5 è associata all’accumulo intracellulare di cAMP, un importante "secondo messaggero" coinvolto nei meccanismi di trasduzione del segnale, ampiamente espresso in diversi tipi di cellule. Mentre l’attivazione di questo recettore di membrana nei macrofagi riduce la produzione di citochine ad azione pro-infiammatoria, l’attivazione dei TGR5 da parte degli acidi biliari a livello dei miociti e adipociti, induce l’aumento della spesa energetica. Quest’ultimo effetto coinvolge l’induzione cAMP-dipendente dell’enzima D2 (iodotironina deiodinasi di tipo 2) che oltre a modulare il processo di attivazione e inattivazione dell’ormone tiroideo, svolge un ruolo vitale nel controllo dell’energia omeostatica e nel processo di termogenesi adattativa del tessuto adiposo bruno. In linea con il coinvolgimento di TGR5 nell'omeostasi energetica, è stato inoltre osservato un aumento della produzione di GPL1 (Glucagon-like peptide 1), che si verifica attraverso una stimolazione TGR5-dipendente. Sulla base delle osservazioni di cui sopra, il recettore di membrana TGR5 emerge come promettente target nel trattamento dell’obesità, del diabete e dei disordini metabolici. Scopo di questa tesi è stato quello di sintetizzare adatti precursori per ottenere ligandi selettivi del recettore TGR5 a struttura steroidea e caratterizzati dalla presenza un atomo di fluoro. La sostituzione dell’atomo di fluoro di queste molecole con l’isotopo radioattivo 18F, potrebbe consentire di ottenere composti capaci di indagare sulla distribuzione del recettore TGR5 in virtù del segnale radioattivo emesso attraverso la tecnica di imaging PET (Tomografia ad Emissione di Positroni) in vivo. La scelta del Fluoro-18 è legata al suo lungo tempo di dimezzamento (109 min, il più lungo tra i vari radioisotopi con breve emivita) che lo rende il radioisotopo di elezione nella sperimentazione PET. A seguito di una approfondita ricerca bibliografica dei ligandi selettivi verso i recettori TGR5 sono stati selezionati i derivati fluorurati 7 e 8 dell’acido litocolico, ligando fisiologico del recettore TGR5, quali buoni candidati per la progettazione dei corrispondenti radiofarmaci La sintesi dei derivati 7 e 8 e degli opportuni precursori necessari per la radiomarcatura non è riportata in letteratura. E’ stata quindi proposta secondo diversi schemi di sintesi (Schemi 1-3), concepiti per ottenere i composti sia in miscela isomerica che nella forma isomerica pura a partire rispettivamente dall’acido 7-ketolitocolico (7-keto LCA), ursodesossicolico (UDCA)e chenodeossicolico (CDCA)

Integrative Organelle-Based Functional Proteomics: In Silico Prediction of Impaired Functional Annotations in SACS KO Cell Model

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS

Integrative Organelle-Based Functional Proteomics: In Silico Prediction of Impaired Functional Annotations in SACS KO Cell Model

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS

Integrative Organelle-Based Functional Proteomics: In Silico Prediction of Impaired Functional Annotations in SACS KO Cell Model

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS.Peer reviewe

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation

Functional transcriptome analysis in ARSACS KO cell model reveals a role of sacsin in autophagy

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS

Electrophysiological profile remodeling via selective suppression of voltage-gated currents by CLN1/PPT1 overexpression in human neuronal-like cells.

CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.Peer reviewe

Converging Role for REEP1/SPG31 in Oxidative Stress

Mutations in the receptor expression-enhancing protein 1 gene (REEP1) are associated with hereditary spastic paraplegia type 31 (SPG31), a neurological disorder characterized by lengthdependent degeneration of upper motor neuron axons. Mitochondrial dysfunctions have been observed in patients harboring pathogenic variants in REEP1, suggesting a key role of bioenergetics in disease-related manifestations. Nevertheless, the regulation of mitochondrial function in SPG31 remains unclear. To elucidate the pathophysiology underlying REEP1 deficiency, we analyzed in vitro the impact of two different mutations on mitochondrial metabolism. Together with mitochondrial morphology abnormalities, loss-of-REEP1 expression highlighted a reduced ATP production with increased susceptibility to oxidative stress. Furthermore, to translate these findings from in vitro to preclinical models, we knocked down REEP1 in zebrafish. Zebrafish larvae showed a significant defect in motor axon outgrowth leading to motor impairment, mitochondrial dysfunction, and reactive oxygen species accumulation. Protective antioxidant agents such as resveratrol rescued free radical overproduction and ameliorated the SPG31 phenotype both in vitro and in vivo. Together, our findings offer new opportunities to counteract neurodegeneration in SPG31