Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagic as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a CTSD-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance, but also restored endo-lysosome and autophagy function in human and murine neurons as well as tissue. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.authorsversionepub_ahead_of_prin

Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

El estrés oxidativo (OS) daña las membranas celulares. Los lisosomas son particularmente sensibles a la permeabilización de la membrana ya que sus funciones requieren gradientes de protones estables dependientes de la membrana. La lipocalina Apolipoproteiń a D (ApoD) es una proteiń a de unión a liṕ idos dotada de capacidad antioxidante. Los astrocitos secretan ApoD para ayudar a las neuronas a afrontar el desafió . En esta tesis, realizamos un análisis exhaustivo del tráfico intracelular de ApoD y demostramos su papel en la homeostasis del pH lisosomal sobre el OS inducido por paraquat. Demostramos que ApoD se endocita y se dirige a un subconjunto de lisosomas vulnerables de una manera dependiente del estrés, es funcionalmente estable en los lisosomas, y su presencia es suficiente y necesaria para que se recuperen de la alcalinización lisosómica inducida por OS, tanto en astrocitos como neuronas. También demostramos que existe transferncia de vesiculas extracelulares (EV) con ApoD entre diferentes células de cultivos in vitro. Esta tesis caracteriza diferentes EVs ApoD positivos implicados en la comunicación glia-neurona. Además, la función ApoD garantiza procesos tan diversos como la supervivencia celular sobre el estrés oxidativo, la compactación adecuada de la mielina (controlando los procesos de reciclado de glucoliṕ idos) o una actividad fagocit́ ica adecuada. Describiremos su papel en estos procesos. El papel crucial de ApoD dentro del lisosoma nos permite estudiar los efectos potenciales de ApoD en enfermedades neurodegenerativas de diferentes oriǵ enes: como la neurodegeneración proteinopática en la Ataxia Espinocerebelosa Tipo I (SCA1) y una enfermedad de almacenamiento Lysosomal (LSD) particularmente devastadora, la enfermedad Niemann Pick tipo A (NPA). Mientras que el estrés oxidativo induce una entrada acelerada de ApoD en el compartimiento lisosomal en las células sanas, tal objetivo se pierde en las células enfermas, lo que contribuye a la vulnerabilidad de los lisosomas. Demostramos que la ApoD agregada exógenamente es capaz de reducir significativamente la permeabilización y la alcalinización lisosomal promovida por NPA, dando como resultado un aumento significativo en la supervivencia celular. Nuestros resultados descubren propiedades biológicas (ubicación y transporte) y funciones de ApoD previamente desconocidas, lo que nos permite comprender mejor los procesos biológicos en los que está involucrada y abrir oportunidades terapéuticas para reparar los lisosomas en situaciones patológicas.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Kõrgrõhu-spektroskoopiline uurimus fotosünteetiliste bakterite valgust koguvate valkude kromofooride vesiniksidemetest

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Fotosünteesivate purpurbakterite valgust koguvad antennid (LH1 ja LH2) on sobivad mudelsüsteemid membraansete valkude ehitusliku stabiilsuse uurimiseks. Käesolevas töös on uuritud kõrge rõhu all vesiniksidemete (H-side) tugevust, mis seovad uuritud valkudes bakterklorofülle (BChl). BChl-d uuritud valkudes (LH1-s 32-56 ja LH2-s 27) moodustavad ringe ja ahelaid, ja nende ülesandeks on koguda valguse energia ning suunata see bakteri elutegevuseks. H-sidemed, mis seovad BChl, moodustuvad valgu aminohappe külgahela positiivse laenguga vesiniku ja negatiivse laenguga BChl hapniku vahel, on suhteliselt nõrgad sidemed, kuid neil on väga suur roll elufunktsioone hoidvate valkude funktsioonide ja struktuuri stabiilsuse säilitamisel. Käesolevas töös on kasutatud BChl-le, kui sensoreid, mis kajastavad enda seisundit valgu sisemuses H-sidemete kaudu ning selle jälgimiseks on rakendatud nende valgust neelavate omaduste muutusi. Uurimises on leitud, et kõrge hüdrostaatilise rõhu all oma loomulikus membraanses ümbruses paiknevad antennid on äärmiselt stabiilsed (taluvad kuni 30000 atm rõhku). Kuid detergenti viiduna (mis on kunstlik membraan) ilmutavad need antennid oma neeldumisspektri tavapärases punanihkumises rõhu kasvamise käigus nihkumist spektri sinisesse ossa, mis toimub LH1 puhul 10000 atm juures ja LH2 puhul 4000 atm juures, ja sellega kaasneb ka spektririba laienemine. Toodud efektid viitavad BChl liikumisvabaduse kasvule valgus, mis viitab BChl ja valgu vaheliste H-sidemete katkemisele. Rõhu vähendamisel sininihe ja laienemine on pöörduvad. Katsetulemuste alusel hinnatud BChl-e paigal hoidvate H-sidemete katkemise energia ületab 10-20 korda neid tavapäraselt ümbritsevast termilisest energiast. See tagab nende valkude märkimisväärse stabiilsuse nende elukeskkonnas. Lisaks on tuvastatud võimalik H-sidemete kooperatiivne katkemine ning valgus sisalduva karotenoidi ja valgu kontsentratsiooni ning glütserooli stabiliseeriv mõju valgu ehituse stabiliseerimisel.The light-harvesting antenna complexes from purple photosynthetic bacteria are convenient model systems to examine the poorly understood role of hydrogen bonds as stabilizing factors in membrane protein complexes. The non-covalently bound arrays of bacteriochlorophyll chromophores within native and genetically modified variants of light-harvesting complexes were used to monitor local changes in the chromophore binding sites induced by externally applied hydrostatic pressure. A unique combination of optical spectroscopy with genetic and noninvasive physical (high-pressure) engineering applied in this work provides the first demonstration and quantification of the rupture of multiple hydrogen bonds in the bacteriochlorophyll binding pockets of the LH1 and LH2 membrane chromoproteins. While the membrane-bound complexes demonstrate very high resilience to pressures reaching 3 GPa, the detergent-isolated complexes reveal characteristic discontinuities of the absorption band shifts and broadenings around 1.1 GPa and 0.5 GPa in the case of the wild type LH1 and LH2 complexes, respectively, which evidence reversible and cooperative breakage of H-bonds. Genetic manipulations leading to exchange of native carotenoids, partial loss of chromophores, and/or H-bonds that bind the chromophores to the surrounding protein scaffold were found to significantly destabilize the membrane chromoproteins under high pressure. Co-solvents such as glycerol as well as high protein concentration, on the other hand, were able to stabilize not only detergent-isolated, which was known previously, but also the membrane-embedded chromoproteins. The energy required to break the H-bonds in wild type LH1 and LH2 complexes is 10–20 times greater than the average thermal energy at physiological temperatures, which secures their great stability under functional conditions. This study thereby provides important insights into design principles of natural photosynthetic complexes

Articular cartilage regenerative strategies based on platelet derivatives with perspective view in 3D-bioprinting technology

Articular cartilage is an unique and highly specialized connective tissue adapted to bear compressive loads and shear forces in synovial joints, which is a crucial function during body\u2019s motion. In adult joints, articular cartilage is composed by chondrocytes immersed in an intricate extracellular matrix, displaying a complex multi-zonal organization. Despite a relatively low metabolic activity within a harsh physical environment, chondrocytes are active in maintaining the matrix, thus allowing healthy tissue to sustain itself and carry out its functions. Damage to tissue high level of organization and molecular architecture is a major source of morbidity for articular cartilage, thus it is usually susceptible to malfunction following acute injury or chronic diseases such as osteoarthritis. Cartilage has poor intrinsic repair and regenerative capacity, although it has been demonstrated to contain a subset of progenitors even in adults. These cells have shown to react to injury, but do not seem to be able to mount an effective tissue reparative or regenerative action when needed. Hence, much effort has been devoted to finding ways by which articular cartilage repair could be induced and enhanced. Surgical techniques and bioengineered treatment options developed over recent decades have led to several clinical treatment modalities for acutely injured or osteoarthritic joints. To prevent progressive cartilage degeneration or to replace damaged tissue, the surgical treatment is often the only option, but it does not ensure full tissue function recovery. Therefore, regenerative medicine has emerged as an important field of research in the treatment of cartilage disorders. In this context, the medical community have shown great interest in therapeutic strategies based on the use of platelet-derived products, such as platelet rich plasma (PRP) and platelet lysate (PL). Since these derivatives are a mix of growth factors, cytokines and chemokines normally involved in tissue healing, the rationale behind their application is the re-activation of latent endogenous regenerative mechanisms. PRP therapeutic use in musculoskeletal disorders have led to promising outcomes, although mechanism of action and efficacy of platelet products in orthopaedics still need to be elucidate. The main objective of this PhD thesis is to study the effects of platelet derivatives on cartilage cell behaviour, including chondrocytes and chondro-progenitors, in order to highlight potentialities and even identify limits concerning their use, both useful in better direct biomedical applications in the field of articular cartilage therapy. Indeed, a better understanding of the events that could induce cartilage repair by PRP or PL may allow to clarify current experimental outcomes and offer the opportunity to conceive innovative strategies or tools in cell therapy approach and in the latest tissue engineering technologies. In this regard, it will be beneficial to find alternative options to cell transplantation (based on mesenchymal stem cells or chondrocytes) aimed in achieving cartilage regeneration, planning interventions focused on in situ stimulation of resident cell population or local progenitor niche in the joint, that are developmentally endowed with greater chondrogenic potential than traditional cell sources. In parallel, the research in the field of tissue engineering continues to be active and innovative strategies for the fabrication of enhanced bioengineered grafts are recently emerging by the spreading of 3D-printing technology. 3D-bioprinting especially represents a developmental biology inspired alternative to classic scaffold-based approaches in the field, since it shows the ability to assemble biological components replicating complex native-like tissue architecture more faithfully than traditional methods of assembly as well as patient customization. In this context, bioprinted constructs may provide a solution for cartilage injuries and defects, despite 3D-bioprinting is still a technology in progress and consequently some challenges have to be overcome before its translation to clinical applications. The research tasks of this work and the main obtained results are: 1) the in vitro characterization of human articular chondrocyte responses at PL treatment with focus on the recruitment/re-activation of a chondro-progenitor cell population from PL-treated cartilage explant cultures. Stem cell-based therapies to achieve articular cartilage regeneration attract great interest. Stem cell niches are located within the joint, where they could participate directly in tissue homeostasis and repair processes. It is reasonable that exploiting local chondro-progenitors for cartilage repair may be a better and more efficient cell-based therapeutic strategy compared to the use of mesenchymal stem cell from different sources, given that they are developmentally primed for differentiation into chondrocytes. Furthermore, in the field of regenerative medicine therapeutic strategies targeting stem cells in situ could be more attractive and more advantageous than stem cell transplantation. According to this approach, endogenous stem cells could be recruited to the injury site by administration of bioactive factors. Thus, in the last decades, among a wide range of products, PRP has spread as a clinical treatment tool for musculoskeletal diseases. Several studies have investigated PRP or PL roles both in vitro and in vivo, highlighting their capacity to exert anti-inflammatory and proliferating effects on cells, as well as to stimulate resident progenitors or to recruit circulating ones. Primary cultures of human articular chondrocytes and cartilage chips were set up from donor biopsies and were treated in vitro with PL. Proliferation, clonogenic potential and phenotype of chondrocytes and chondro-progenitor cells derived from explant cultures in PL were characterized. Tri-lineage differentiation potential were tested in vitro and scaffold-assisted chondrogenesis of these cells were studied in nude mice. Moreover, secretory profile of chondro-progenitors were analysed together with their migratory capabilities by mimic osteoarthritis in vitro. Finally, it was reported that ex vivo treatment of human articular cartilage with PL induced activation and outgrowth of cells showing features of stemness, such as clonogenicity and expression of nestin. The stimulation of nestin-positive progenitor cells induced by PL in articular cartilage is of particular interest for the future development of therapeutic strategies given the involvement of these cells in tissue regenerative processes. In addition, their high proliferation capacity with concurrent chondrogenic potential maintenance further sustain the potential of PL-mobilized chondro-progenitor cells as promising tool in the field of cartilage tissue engineering. Moreover, PL-induced effects on phenotype of mature articular chondrocytes were further characterized, showing that they can revert to an earlier stage similar to chondro-progenitor one. 2) Embedding and re-differentiation of primary human articular chondrocytes in PRP-based hydrogel suitable for 3D-bioprinting. Although the implantation of cultured chondrocytes intended for injured cartilage therapy is performed worldwide, there are still unresolved challenges associated with the maintenance of their chondrogenic phenotype. The expansion of chondrocytes in vitro is associated with de-differentiation, which is a reduction in the expression of cartilage-specific markers. Accordingly, such cells often produce fibrocartilage rather than native hyaline cartilage when used in clinical procedures. Several strategies to counteract this phenomenon have been adopted, such as 3D-culture of chondrocytes encapsulated in biomaterials. Adoption of hydrogels attracts particular interest in cartilage regeneration since they provide a highly hydrated environment similar to that of native tissue. In this context, progresses are expected thanks to the application of the 3D-bioprinting. However, the field of biofabrication often strongly focuses on the biomaterial rheology to allow controlled production, taking less care of its inherent impact on cellular phenotype. The combination of both aspects can be achieved by incorporation of biological components in printable hydrogels that often lack of bioactivity. Primary human articular chondrocytes derived from previous monolayer culture expansion were bioprinted by embedding them in a commercial available alginate-based ink and their ability to regain the chondrogenic potential both in vitro and in vivo was evaluated. Interestingly, an improved ability to sustain cell viability, proliferation and a certain degree of chondrogenic phenotype rescue were found inside the bioprointed constructs by adding PRP as a source of biological agents in the ink formulation

Osteocyte regulation of bone quality in homeostasis and in skeletal disease

Osteocytes are the most numerous cells in bone, working from within the mineralized matrix to exert control over their local environment and cells on the bone surface. Healthy osteocytes are key to bone homeostasis, but the full functional importance of their local remodeling activity, called perilacunar/canalicular remodeling (PLR), is only recently being discovered. This work uses multiple mouse models to investigate the molecular mechanisms controlling osteocyte PLR and demonstrates that repression of PLR causes severe loss of bone material quality. Additionally, osteocyte dysfunction is shown to disrupt bone homeostasis during skeletal disease and to promote degradation of overlying cartilage in the joint. Finally, we begin to determine the extent to which PLR is mechanosensitive, revealing distinct site- and magnitude-specific modulation of osteocyte behavior with applied load. Together these findings reveal new essential roles for osteocytes and PLR within bone and joints during homeostasis and disease

Visualising the subcellular distribution of antibiotics against tuberculosis

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), remains the world’s deadliest infectious disease. Although treatable, effective chemotherapy requires at least six months of treatment with a minimum of four antibiotics. Novel antibiotics are needed to quell the pandemic. However, we do not fully understand why current treatments take so long to work in patients. Mtb has a dynamic intracellular lifestyle, and this thesis tests the hypothesis that not all antibiotics penetrate into, or are effective within, all compartments containing Mtb during infection. Our understanding of the intracellular pharmacokinetics of drugs against TB has been limited by a lack of technologies for studying the subcellular distribution of antibiotics. This work developed a correlative imaging workflow incorporating fluorescence, electron and nanoscale ion microscopy (CLEIM) to map the subcellular distribution of two antibiotics, bedaquiline (BDQ) and pyrazinamide (PZA), at sub-micrometre resolution in Mtb-infected human macrophages. This workflow was complemented with orthogonal methods, including high-content live-cell imaging, to study the dynamic processes that contribute to antibiotic activity. BDQ accumulated primarily in host cell lipid droplets (LD), but heterogeneously in Mtb within a variety of intracellular compartments. Surprisingly, LD did not sequester the antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Lipid binding therefore facilitated drug trafficking by host organelles to an intracellular target. PZA is a pro-drug, and the accumulation of its active metabolite pyrazinoic acid has been hypothesised to depend on the bacteria being in an acidic environment. Direct analysis of antibiotic accumulation by ion microscopy, combined with live-cell imaging at the single cell level, revealed that, whilst acidic intracellular environments support PZA activity, they are not necessary for antibiotic efficacy. Many intracellular pathogens interact with LD or reside in partially acidified vacuoles, and these results therefore have broad implications for our understanding of antibiotic activity

Performance of nasal chondrocyte-based engineered tissues in osteoarthritis simulating environments

Osteoarthritis (OA) is the most prevalent musculoskeletal disease in humans, characterized by a progressive degeneration of the articulation that causes pain and disability in a large percentage of the population. In this pathology, the joint environment becomes predominantly catabolic and concentrations of circulating pro-inflammatory factors significantly increase as compared to nonpathological conditions. Up to date, existing treatments can be effective in reduction of pain and improvement of mobility, but none of the available therapies is able to stop the progression of the disease. Non-degenerative cartilaginous lesions can be currently treated with cell-based approaches, consisting of the implantation of autologous chondrocytes into the defect site, those cells being isolated from presumable non-affected areas of articular cartilage. Differentially, OA is considered a contraindication for such treatments and, in the scarce cases they have been used for patients with degenerative traces, failure is reported as the more common long-term outcome. Possible causes of these results are the inferior chondrogenic capacity and phenotype stability demonstrated for articular chondrocytes (AC) harvested from affected joints, but also, the detrimental conditions of the OA environment, potentially compromising the performance of any implanted cell-based product. Nasal chondrocytes (NC) represent an alternative source for cell and tissue engineering approaches, since they can be obtained from a compartment that is not affected (i.e., the nasal septum), and show more reproducible capacity to generate functional cartilaginous tissues as well as similar responses to mechanical and inflammatory stimuli than AC. In fact, tissue engineered cartilage derived from nasal chondrocytes (N-TEC) have been already used in the clinic for the treatment of post-traumatic cartilage lesions, but not results are generated regarding their potential to additionally treat OA defects. In order to assess such potential, it is necessary to evaluate if N-TEC can survive and maintain their tissue-like properties in the pro-inflammatory and catabolic OA environment, to which cells from the different joint tissues (cartilage, synovial membrane and subchondral bone) contribute. The pillar of this thesis, my PhD dissertation, consists on the exploration of the suitability of NTEC for the treatment of OA lesions. Therefore, this manuscript summarizes methods and outcomes resulting from investigating the interactions between nasal chondrocytes and cells/tissues from OA-joints, as an approach to establish the possible compatibility of N-TEC within an OA cartilage defect. Results showed that N-TEC could maintain their cartilaginous properties, when exposed in vitro to inflammatory stimuli as those found in OA joints, and positively influence the inflammatory profile of cells from OA joints through secreted factors. Moreover, N-TEC were able to survive and engraft into OA compartments simulated in vivo, while preserving cartilaginous matrix properties and dampening inflammation, as observed in vitro. Acknowledging the positive and wide compatibility of N-TEC within OA environments that I demonstrated, the clinical application of autologous N-TEC was tested in two patients with advanced OA, who would have been otherwise considered for partial knee – prosthetic - replacement. After 14 months of implantation, patients have reported reduced pain as well as improved joint function and life quality; all findings indicating that N-TEC can be envisioned as a therapeutic approach for the repair of osteoarthritic knee cartilage defects. To assess efficacy of this procedure, a phase II trial would be required in a larger cohort of patients

The spatiotemporal dynamics of autophagy during Mycobacterium tuberculosis infection of human induced pluripotent stem cell derived macrophages

The interaction of macrophages with the intracellular pathogen Mycobacterium tuberculosis (Mtb) is critical in determining disease outcomes. Presently, we lack human macrophage models that are genetically tractable, karyotypically normal, and available in large cell numbers. Thus, new human macrophage models are required to facilitate the study of host-pathogen interactions, especially where genetic manipulation is required. In this thesis I set up and characterise a stem cell derived model of human macrophages (iPSDM). iPSDM recapitulate many known interactions between Mtb and human macrophages. An RNA-seq comparison of the transcriptional response to infection between Mtb WT and the attenuated strain Mtb ΔRD1, which lacks the ESX-1 secretion system, revealed commonalities with previous studies along with a novel, RD1 dependent response at 48 h post infection. In macrophages, the autophagy pathway plays diverse roles in infection with intracellular pathogens, including capturing cytosol invading pathogens and targeting them for lysosomal killing. The role of the autophagy pathway during Mtb infection is still unclear. Live cell imaging in iPSDM revealed, following membrane damage by Mtb, the induction of LC3 positive tubulovesicular structures, which fail to capture the bacteria. Correlative 3D focussed ion beam scanning electron microscopy showed the bacteria in the macrophage cytosol following successful dissociation from autophagic structures. Genetic disruption of the autophagy pathway through the knockout of Atg7 failed to alter intracellular Mtb replication or rescue replication of the attenuated mutants Mtb ΔRD1 and Mtb ΔCpsA, which has previously been shown to be more susceptible to restriction by non-canonical autophagy. On the other hand, knockout of Atg14 resulted in enhanced Mtb replication, likely through an autophagy-independent mechanism. This work validates iPSDM to study hitherto unexplored aspects of human macrophage-Mtb interactions. Moreover, these data revealed novel autophagy dynamics in space and time and evasion mechanisms employed by Mtb to subvert this host defence pathway

Exploring the regeneration potential of salivary glands using organoids as a model

Radiotherapy is a potential life-saving treatment for head and neck cancer patients. However, despite improvements in precision of radiation dose delivered, the unavoidable co-irradiation of salivary gland still leads to irreversible diminishing of saliva secretion affecting among others the ability to speak, eat and sleep, drastically decreasing the quality of life of the patients. Existing treatments for this dry mouth syndrome, only provide short-term relief. Therefore, the development of new therapies, such as stem cell therapy, is crucial to alleviate this side effect caused by irradiation. Combining mouse injury models with 3D organoid cultures of mouse and human salivary gland derived cells, it is attempted to identify the optimal cell source for, as well as the regulatory mechanisms involved in, the salivary gland regeneration process. Thereby this work focused on the development and optimization of a potentially clinically relevant regenerative therapy approach to functionally restore the irradiated salivary gland tissue