Extracellular Vesicles as Promising Carriers in Drug Delivery: Considerations from a Cell Biologist's Perspective.

The use of extracellular vesicles as cell-free therapy is a promising approach currently investigated in several disease models. The intrinsic capacity of extracellular vesicles to encapsulate macromolecules within their lipid bilayer membrane-bound lumen is a characteristic exploited in drug delivery to transport active pharmaceutical ingredients. Besides their role as biological nanocarriers, extracellular vesicles have a specific tropism towards target cells, which is a key aspect in precision medicine. However, the little knowledge of the mechanisms governing the release of a cargo macromolecule in recipient cells and the Good Manufacturing Practice (GMP) grade scale-up manufacturing of extracellular vesicles are currently slowing their application as drug delivery nanocarriers. In this review, we summarize, from a cell biologist's perspective, the main evidence supporting the role of extracellular vesicles as promising carriers in drug delivery, and we report five key considerations that merit further investigation before translating Extracellular Vesicles (EVs) to clinical applications.

Tau Seeds in Extracellular Vesicles Induce Tau Accumulation in Degradative Organelles of Cells.

Clinical progression of tauopathies may result from transcellular propagation of pathogenic Tau seeds with the possible involvement of extracellular vesicles (EVs) as transport vectors. We established a cell model for investigating EV delivery of proteins, since the mechanism regulating EV cargo delivery to recipient cells is poorly understood. In our cell model, EVs are readily internalized and accumulate in degradative organelles (DOs). We then show for the first time that in this acidic compartment, profibrillogenic Tau delivered by EVs interacts with Tau expressed by the recipient cells and cause its accumulation by a process that involves the participation of autophagy. Thus, the degradative compartment of cells may represent the subcellular site initiating a cascade of events resulting in early hallmarks of tauopathies. These are characterized by seeded Tau accumulation, pathology-associated epitopes, DO stress, and cytotoxicity. The involvement of autophagy to this process and the relative accessibility of the degradative pathway for extracellular agents, support possible modes of intervention to slow down the progression of neurodegeneration.

Hijacking Endocytosis and Autophagy in Extracellular Vesicle Communication: Where the Inside Meets the Outside.

Extracellular vesicles, phospholipid bilayer-membrane vesicles of cellular origin, are emerging as nanocarriers of biological information between cells. Extracellular vesicles transport virtually all biologically active macromolecules (e.g., nucleotides, lipids, and proteins), thus eliciting phenotypic changes in recipient cells. However, we only partially understand the cellular mechanisms driving the encounter of a soluble ligand transported in the lumen of extracellular vesicles with its cytosolic receptor: a step required to evoke a biologically relevant response. In this context, we review herein current evidence supporting the role of two well-described cellular transport pathways: the endocytic pathway as the main entry route for extracellular vesicles and the autophagic pathway driving lysosomal degradation of cytosolic proteins. The interplay between these pathways may result in the target engagement between an extracellular vesicle cargo protein and its cytosolic target within the acidic compartments of the cell. This mechanism of cell-to-cell communication may well own possible implications in the pathogenesis of neurodegenerative disorders.

Chloroquine, the Coronavirus Crisis, and Neurodegeneration: A Perspective.

On the verge of the ongoing coronavirus pandemic, in vitro data suggested that chloroquine, and its analog hydroxychloroquine, may be useful in controlling SARS-CoV-2 infection. Efforts are ongoing in order to test this hypothesis in clinical trials. Some studies demonstrated no evidence of efficacy, whereas in some cases results were retracted after reporting. Despite the lack of scientific validation, support for the use of these compounds continues from various influencers. At the cellular level, the lysosomotropic drug chloroquine accumulates in acidic organelles where it acts as an alkalizing agent with possible downstream effects on several cellular pathways. In this perspective, we discuss a possible modulatory role of these drugs in two shared features of neurodegenerative diseases, the cellular accumulation of aberrantly folded proteins and the contribution of neuroinflammation in this pathogenic process. Certainly, the decision on the use of chloroquine must be determined by its efficacy in the specific clinical situation. However, at an unprecedented time of a potential widespread use of chloroquine, we seek to raise awareness of its potential impact in ongoing clinical trials evaluating disease-modifying therapies in neurodegeneration.

Tau affects P53 function and cell fate during the DNA damage response.

Cells are constantly exposed to DNA damaging insults. To protect the organism, cells developed a complex molecular response coordinated by P53, the master regulator of DNA repair, cell division and cell fate. DNA damage accumulation and abnormal cell fate decision may represent a pathomechanism shared by aging-associated disorders such as cancer and neurodegeneration. Here, we examined this hypothesis in the context of tauopathies, a neurodegenerative disorder group characterized by Tau protein deposition. For this, the response to an acute DNA damage was studied in neuroblastoma cells with depleted Tau, as a model of loss-of-function. Under these conditions, altered P53 stability and activity result in reduced cell death and increased cell senescence. This newly discovered function of Tau involves abnormal modification of P53 and its E3 ubiquitin ligase MDM2. Considering the medical need with vast social implications caused by neurodegeneration and cancer, our study may reform our approach to disease-modifying therapies.

Split GFP technologies to structurally characterize and quantify functional biomolecular interactions of FTD-related proteins.

Protein multimerization in physiological and pathological conditions constitutes an intrinsic trait of proteins related to neurodegeneration. Recent evidence shows that TDP-43, a RNA-binding protein associated with frontotemporal dementia and amyotrophic lateral sclerosis, exists in a physiological and functional nuclear oligomeric form, whose destabilization may represent a prerequisite for misfolding, toxicity and subsequent pathological deposition. Here we show the parallel implementation of two split GFP technologies, the GFP bimolecular and trimolecular fluorescence complementation (biFC and triFC) in the context of TDP-43 self-assembly. These techniques coupled to a variety of assays based on orthogonal readouts allowed us to define the structural determinants of TDP-43 oligomerization in a qualitative and quantitative manner. We highlight the versatility of the GFP biFC and triFC technologies for studying the localization and mechanisms of protein multimerization in the context of neurodegeneration.