The lack of techniques to trace brain cell behavior in vivo hampers the ability to monitor status of cells in a living brain. Extracellular vesicles (EVs), nanosized membrane-surrounded vesicles, released by virtually all brain cells might be able to report their status in easily accessible biofluids, such as blood. EVs communicate among tissues using lipids, saccharides, proteins, and nucleic acid cargo that reflect the state and composition of their source cells. Currently, identifying the origin of brain-derived EVs has been challenging, as they consist of a rare population diluted in an overwhelming number of blood and peripheral tissue-derived EVs. Here, we developed a sensitive platform to select out pre-labelled brain-derived EVs in blood as a platform to study the molecular fingerprints of brain cells. This proof-of-principle study used a transducible construct tagging tetraspanin (TSN) CD63, a membrane-spanning hallmark of EVs equipped with affinity, bioluminescent, and fluorescent tags to increase detection sensitivity and robustness in capture of EVs secreted from pre-labelled cells into biofluids. Our platform enables unprecedented efficient isolation of neural EVs from the blood. These EVs derived from pre-labelled mouse brain cells or engrafted human neuronal progenitor cells (hNPCs) were submitted to multiplex analyses, including transcript and protein levels, in compliance with the multibiomolecule EV carriers. Overall, our novel strategy to track brain-derived EVs in a complex biofluid opens up new avenues to study EVs released from pre-labelled cells in near and distal compartments into the biofluid source.

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the “click”-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether “click DNA” (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

The central nervous system (CNS) consists of a heterogeneous population of cells with highly specialized functions. For optimal functioning of the CNS, in disease and in health, intricate communication between these cells is vital. One important mechanism of cellular communication is the release and uptake of extracellular vesicles (EVs). EVs are membrane enclosed particles actively released by cells, containing a wide array of proteins, lipids, RNA, and DNA. These EVs can be taken up by neighboring or distant cells, and influence a wide range of processes. Due to the complexity and relative inaccessibility of the CNS, our current understanding of the role of EVs is mainly derived in vitro work. However, recently new methods and techniques have opened the ability to study the role of EVs in the CNS in vivo. In this review, we discuss the current developments in our understanding of the role of EVs in the CNS in vivo.

Polyglutamine (polyQ) diseases are a group of inherited neurodegenerative disorders caused by the expansion of the cytosine-adenine-guanine (CAG) repeat. This mutation encodes extended glutamine (Q) tract in the disease protein, resulting in the alteration of its conformation/physiological role and in the formation of toxic fragments/aggregates of the protein. This group of heterogeneous disorders shares common molecular mechanisms, which opens the possibility to develop a pan therapeutic approach. Vast efforts have been made to develop strategies to alleviate disease symptoms. Nonetheless, there is still no therapy that can cure or effectively delay disease progression of any of these disorders. Mesenchymal stromal cells (MSC) are promising tools for the treatment of polyQ disorders, promoting protection, tissue regeneration, and/or modulation of the immune system in animal models. Accordingly, data collected from clinical trials have so far demonstrated that transplantation of MSC is safe and delays the progression of some polyQ disorders for some time. However, to achieve sustained phenotypic amelioration in clinics, several treatments may be necessary. Therefore, efforts to develop new strategies to improve MSC’s therapeutic outcomes have been emerging. In this review article, we discuss the current treatments and strategies used to reduce polyQ symptoms and major pre-clinical and clinical achievements obtained with MSC transplantation as well as remaining flaws that need to be overcome. The requirement to cross the blood-brain-barrier (BBB), together with a short rate of cell engraftment in the lesioned area and low survival of MSC in a pathophysiological context upon transplantation may contribute to the transient therapeutic effects. We also review methods like pre-conditioning or genetic engineering of MSC that can be used to increase MSC survival in vivo, cellular-free approaches—i.e., MSC-conditioned medium (CM) or MSC-derived extracellular vesicles (EVs) as a way of possibly replacing the use of MSC and methods required to standardize the potential of MSC/MSC-derived products. These are fundamental questions that need to be addressed to obtain maximum MSC performance in polyQ diseases and therefore increase clinical benefits.