No abstract

Spine pathology has been implicated in the early onset of Alzheimer’s disease (AD), where Aβ-Oligomers (AβOs) cause synaptic dysfunction and loss. Previously, we described that pharmacological inhibition of c-Abl prevents AβOs-induced synaptic alterations. Hence, this kinase seems to be a key element in AD progression. Here, we studied the role of c-Abl on dendritic spine morphological changes induced by AβOs using c-Abl null neurons (c-Abl-KO). First, we characterized the effect of c-Abl deficiency on dendritic spine density and found that its absence increases dendritic spine density. While AβOs-treatment reduces the spine number in both wild-type (WT) and c-Abl-KO neurons, AβOs-driven spine density loss was not affected by c-Abl. We then characterized AβOs-induced morphological changes in dendritic spines of c-Abl-KO neurons. AβOs induced a decrease in the number of mushroom spines in c-Abl-KO neurons while preserving the populations of immature stubby, thin, and filopodia spines. Furthermore, synaptic contacts evaluated by PSD95/Piccolo clustering and cell viability were preserved in AβOs-exposed c-Abl-KO neurons. In conclusion, our results indicate that in the presence of AβOs c-Abl participates in synaptic contact removal, increasing susceptibility to AβOs damage. Its deficiency increases the immature spine population reducing AβOs-induced synapse elimination. Therefore, c-Abl signaling could be a relevant actor in the early stages of AD.

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV’s secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV’s production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV’s characteristics and functionality and the parental cell’s phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method’s interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.