A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha- synuclein to lysosomes

The nervous system spread of alpha-synuclein fibrils leads to Parkinson′s disease (PD) and other synucleinopathies, yet the mechanisms underlying internalization and cell-to-cell transfer are enigmatic. Here we use confocal and superresolution microscopy, subcellular fractionation and electron microscopy of immunogold labelled alpha-synuclein pre-formed fibrils (PFF) to demonstrate that this toxic protein species enters cells using a novel form of ultra-rapid macropinocytosis with transfer to lysosomes in as little as 2 minutes, an unprecedented cell biological kinetic for lysosomal targeting. PFF uptake circumvents classical endosomal pathways and is independent of clathrin. Immunogold-labelled PFF are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, and in lysosomes. While many of the fibrils remain in lysosomes that continue to take up PFF for hours, a portion are transferred to neighboring naive cells on the external face of vesicles, likely exosomes. These data indicate that PFF uses a novel internalization mechanism as a component of cell-to-cell propagation.

Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy

<p>Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown in co-culture; an activator strain expressing the nitroreductase E. coli nfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. In contrast with observations in human cell multilayers, the PR-104A and SN27686 metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase variant library and negatively impact the recovery of more active variants. Significantly fewer nfsA-expressing cells were recovered from FACS when using CB1954 and nitro-CBI-DEI, when the bystander effect was given time to occur, as compared to controls in which the bystander effect was given no time to occur. In contrast, at the preferred challenge concentrations the mustard prodrugs PR-104A and SN27686 did not yield significantly lower proportions of nfsA-expressing cells under bystander condition. A subsequent investigation compared the evolutionary outcomes arising from screening a nitroreductase variant library using FACS, in which the bystander effect can occur, in parallel to a manual pre-selection method of individual clones for detoxification of structurally divergent nitroaromatic antibiotics. Overall the results of this investigation were inconclusive after just a single round of selection, but there is some evidence that the FACS strategy was more effective than niclosamide/chloramphenicol pre-selection in enriching for superior CB1954-reducing variants. Finally, a panel of nitroreductase candidates was evaluated with the next generation prodrugs PR-104A and SN36506 for possible Clostridia-DEPT development. It was found that the Vibrio vulnificus NfsB F70A/F108Y variant displayed the highest catalytic efficiency with PR-104A reported thus far compared to any other nitroreductase, and was the only NfsB family nitroreductase to exhibit any activity with SN36506 at the purified protein level. At the time this research was performed only NfsB family nitroreductases had been successfully expressed in C. sporogenes by our collaborators, hence the V. vulnificus NfsB F70A/F108Y variant was selected as a promising lead enzyme for further development.</p>

Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy

<p>Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown in co-culture; an activator strain expressing the nitroreductase E. coli nfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. In contrast with observations in human cell multilayers, the PR-104A and SN27686 metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase variant library and negatively impact the recovery of more active variants. Significantly fewer nfsA-expressing cells were recovered from FACS when using CB1954 and nitro-CBI-DEI, when the bystander effect was given time to occur, as compared to controls in which the bystander effect was given no time to occur. In contrast, at the preferred challenge concentrations the mustard prodrugs PR-104A and SN27686 did not yield significantly lower proportions of nfsA-expressing cells under bystander condition. A subsequent investigation compared the evolutionary outcomes arising from screening a nitroreductase variant library using FACS, in which the bystander effect can occur, in parallel to a manual pre-selection method of individual clones for detoxification of structurally divergent nitroaromatic antibiotics. Overall the results of this investigation were inconclusive after just a single round of selection, but there is some evidence that the FACS strategy was more effective than niclosamide/chloramphenicol pre-selection in enriching for superior CB1954-reducing variants. Finally, a panel of nitroreductase candidates was evaluated with the next generation prodrugs PR-104A and SN36506 for possible Clostridia-DEPT development. It was found that the Vibrio vulnificus NfsB F70A/F108Y variant displayed the highest catalytic efficiency with PR-104A reported thus far compared to any other nitroreductase, and was the only NfsB family nitroreductase to exhibit any activity with SN36506 at the purified protein level. At the time this research was performed only NfsB family nitroreductases had been successfully expressed in C. sporogenes by our collaborators, hence the V. vulnificus NfsB F70A/F108Y variant was selected as a promising lead enzyme for further development.</p>

Impact of Sterilization Methods on the Seeding Ability of Human Tau Proteopathic Seeds

The protein tau, when misfolded in neurodegenerative diseases, has several prion-like properties including being able to spread by cell-to-cell transfer, induce templated seeding, and exist in distinct conformational strains. These properties of transmission may present health hazards when lesion-containing biospecimens are used in research and neuropathology laboratories. We evaluated the impact standard sterilization and cleaning methods have on the capacity of tau seeds to induce aggregation. We employed a previously developed, highly sensitive FRET-based biosensor assay to assess remnant tau seeding after exposure to these procedures. For tau species derived from human Alzheimer disease tissue (brain homogenate and sarkosyl-insoluble fibrils), both autoclaving and incubation in 90.6% formic acid were sufficient to reduce tau bioactivity. By contrast, boiling was not always effective in completely blocking bioactivity in the seeding assay. Notably, only formic acid incubation was able to produce a similar reduction in tissue from a P301L mutant tau mouse model of tauopathy. Our study highlights nuances in methods for inactivation of tau seeding which may support adapted tissue processing procedures, especially in research settings. These findings also highlight the importance of universal precautions when handling human neuropathological and research laboratory materials.

Mitotherapy: Unraveling a Promising Treatment for Disorders of the Central Nervous System and Other Systemic Conditions

Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer’s disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.

Rapid and Efficient Cell-to-Cell Transmission of Avian Influenza H5N1 Virus in MDCK Cells Is Achieved by Trogocytosis

Viruses have developed direct cell-to-cell transfer strategies to enter target cells without being released to escape host immune responses and antiviral treatments. These strategies are more rapid and efficient than transmission through indirect mechanisms of viral infection between cells. Here, we demonstrate that an H5N1 influenza virus can spread via direct cell-to-cell transfer in Madin-Darby canine kidney (MDCK) cells. We compared cell-to-cell transmission of the H5N1 virus to that of a human influenza H1N1 virus. The H5N1 virus has been found to spread to recipient cells faster than the human influenza H1N1 virus. Additionally, we showed that plasma membrane exchange (trogocytosis) occurs between co-cultured infected donor cells and uninfected recipient cells early point, allowing the intercellular transfer of viral material to recipient cells. Notably, the H5N1 virus induced higher trogocytosis levels than the H1N1 virus, which could explain the faster cell-to-cell transmission rate of H5N1. Importantly, this phenomenon was also observed in A549 human lung epithelial cells, which are representative cells in the natural infection site. Altogether, our results provide evidence demonstrating that trogocytosis could be the additional mechanism utilized by the H5N1 virus for rapid and efficient cell-to-cell transmission.

Recent Progress on Exosomes in RNA Virus Infection

Recent research indicates that most tissue and cell types can secrete and release membrane-enclosed small vesicles, known as exosomes, whose content reflects the physiological/pathological state of the cells from which they originate. These exosomes participate in the communication and cell-to-cell transfer of biologically active proteins, lipids, and nucleic acids. Studies of RNA viruses have demonstrated that exosomes release regulatory factors from infected cells and deliver other functional host genetic elements to neighboring cells, and these functions are involved in the infection process and modulate the cellular responses. This review provides an overview of the biogenesis, composition, and some of the most striking functions of exosome secretion and identifies physiological/pathological areas in need of further research. While initial indications suggest that exosome-mediated pathways operate in vivo, the exosome mechanisms involved in the related effects still need to be clarified. The current review focuses on the role of exosomes in RNA virus infections, with an emphasis on the potential contributions of exosomes to pathogenesis.

Dangerous Liaisons: Interactions of Cryptococcus neoformans with Host Phagocytes

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of death in immunocompromised individuals. The interactions of this yeast with host phagocytes are critical to disease outcome, and C. neoformans is equipped with an array of factors to modulate these processes. Cryptococcal infection begins with the deposition of infectious particles into the lungs, where the fungal cells deploy various antiphagocytic factors to resist internalization by host cells. If the cryptococci are still engulfed, they can survive and proliferate within host cells by modulating the phagolysosome environment in which they reside. Lastly, cryptococcal cells may escape from phagocytes by host cell lysis, nonlytic exocytosis, or lateral cell-to-cell transfer. The interactions between C. neoformans and host phagocytes also influence the dissemination of this pathogen to the brain, where it may cross the blood-brain barrier and cause an often-fatal meningoencephalitis. In this review, we highlight key cryptococcal factors involved in various stages of cryptococcal-host interaction and pathogenesis.

Bacterial nanotubes as a manifestation of cell death

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells’ genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.