Mesenchymal stem cells (MSCs) have been used for cell-based therapies in regenerative medicine, with increasing importance in central and peripheral nervous system repair. However, MSCs grafting present disadvantages, such as, a high number of cells required for transplantation and low survival rate when transplanted into the central nervous system (CNS). In line with this, MSCs secretome which present on its composition a wide range of molecules (neurotrophins, cytokines) and microvesicles, can be a solution to surpass these problems. However, the effect of MSCs secretome in axonal elongation is poorly understood. In this study, we demonstrate that application of MSCs secretome to both rat cortical and hippocampal neurons induces an increase in axonal length. In addition, we show that this growth effect is axonal intrinsic with no contribution from the cell body. To further understand which are the molecules required for secretome-induced axonal outgrowth effect, we depleted brain-derived neurotrophic factor (BDNF) from the secretome. Our results show that in the absence of BDNF, secretome-induced axonal elongation effect is lost and that axons present a reduced axonal growth rate. Altogether, our results demonstrate that MSCs secretome is able to promote axonal outgrowth in CNS neurons and this effect is mediated by BDNF.

The intra-axonal events governing formation of presynaptic terminals are still poorly understood. Pinto et al. reveal a mechanism by which a localized decrease in proteasome degradation and resultant accumulation of polyubiquitinated proteins at nascent sites signal assembly of presynaptic terminals.

No abstract

No abstract

SUMMARY Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation.

In recent years, signaling through ubiquitin has been shown to be of great importance for normal brain development. Indeed, fluctuations in ubiquitin levels and spontaneous mutations in (de)ubiquitination enzymes greatly perturb synapse formation and neuronal transmission. In the brain, expression of lysine (K) 48-linked ubiquitin chains is higher at a developmental stage coincident with synaptogenesis. Nevertheless, no studies have so far delved into the involvement of this type of polyubiquitin chains in synapse formation. We have recently proposed a role for polyubiquitinated conjugates as triggering signals for presynaptic assembly. Herein, we aimed at characterizing the axonal distribution of K48 polyubiquitin and its dynamics throughout the course of presynaptic formation. To accomplish so, we used an ubiquitination-induced fluorescence complementation (UiFC) strategy for the visualization of K48 polyubiquitin in live hippocampal neurons. We first validated its use in neurons by analyzing changing levels of polyubiquitin. UiFC signal is diffusely distributed with distinct aggregates in somas, dendrites and axons, which perfectly colocalize with staining for a K48-specific antibody. Axonal UiFC aggregates are relatively stable and new aggregates are formed as an axon grows. Approximately 65% of UiFC aggregates colocalize with synaptic vesicle clusters and they preferentially appear in the axonal domains of axo-somatodendritic synapses when compared to isolated axons. We then evaluated axonal accumulation of K48 ubiquitinated signals in bead-induced synapses. We observed rapid accumulation of UiFC signal and endogenous K48 ubiquitin at the sites of newly formed presynapses. Lastly, we show by means of a microfluidic platform, for the isolation of axons, that presynaptic clustering on beads is dependent on E1-mediated ubiquitination at the axonal level. Altogether, these results indicate that enrichment of K48 polyubiquitin at the site of nascent presynaptic terminals is an important axon-intrinsic event for presynaptic differentiation.

No abstract

No abstract