Francisca C. Bronfman

Age-related decline of the neuromuscular junction (NMJ), the peripheral synapse that controls muscle contraction, contributes to muscle weakness and impaired motor function in aging. The NMJ comprises a motor axon terminal, a skeletal muscle fibre, and terminal Schwann cells (tSC). Neurotrophin signalling is essential for mature NMJ organisation, with the p75 receptor acting as a key regulator of its morphology and function. However, the potential contribution of p75 to age-related NMJ decline remains unexplored. In this study, we used germline p75 knockout (p75⁻/⁻) mice to examine how the lifelong absence of p75 impacts NMJ stability and muscle function during aging through quantitative morphometric analyses, postsynaptic receptor dynamics, muscle histology, and functional strength testing. Although NMJ morphology was preserved, aged p75⁻/⁻ mice exhibited pronounced denervation and reduced tSC coverage, hallmarks of age-associated NMJ degeneration. Moreover, postsynaptic domains of aged p75⁻/⁻ mice displayed reduced stability of membrane-bound acetylcholine receptors. Glycolytic muscle fibres also showed signs of atrophy. Notably, aged p75⁻/⁻ mice exhibited a significant reduction in muscle strength compared with age-matched controls. Together, our findings are consistent with cumulative effects of persistent p75 deficiency on NMJ integrity and function during aging, supporting its potential relevance for interventions aimed at preventing age-associated neuromuscular decline.

Brain-derived neurotrophic factor (BDNF) promotes neuronal plasticity through retrograde signaling from axon terminals to the nucleus, activating CREB-dependent transcription. While this relies on signaling endosomes, the molecular mechanisms enabling their axonal transport remain poorly understood. Using compartmentalized cultures of mouse cortical neurons, we show that axonal BDNF activates mTOR signaling and stimulates local protein synthesis. Translation inhibitors blocked both BDNF-enhanced retrograde transport and nuclear CREB phosphorylation, indicating that axonal protein synthesis is required for long-distance signaling. Among locally synthesized proteins, we identified Rab5, a master regulator of endosomal trafficking. BDNF increased axonal Rab5 levels through TrkB- and mTOR-dependent mechanisms, confirmed by puromycin-PLA for Rab5 and soma-free axon preparations. Axon-specific Rab5 knockdown abolished BDNF-induced retrograde transport and CREB activation, demonstrating that local axonal translation of Rab5 mRNA is essential for neurotrophin signaling propagation. Remarkably, even basal retrograde transport depended on ongoing axonal Rab5 synthesis, revealing a constitutive role for local translation in maintaining axonal trafficking capacity. These findings establish that local axonal translation of trafficking regulators is a prerequisite for axon-to-nucleus neurotrophin signaling, positioning on-demand protein synthesis as a central node in long-distance neuronal communication.

Prostate cancer (PCa) is the second leading cause of cancer-related death among men in most Western countries. Current therapies for PCa are limited, often ineffective, and associated with significant side effects. As a result, there is a growing interest in exploring new therapeutic agents, particularly from the polyphyletic group of algae, which offers a promising source of compounds with anticancer properties. Our research group has focused on investigating the effects of a novel oleoresin from Gracilaria chilensis, known as Gracilex^®, as a potential therapeutic agent against PCa using both in vitro and in vivo models. Our findings indicate that Gracilex^® exhibits a time- and dose-dependent inhibitory effect on cell survival in LNCaP and PC-3 PCa, reducing viability by over 50% and inducing apoptosis, as evidenced by a significant increase in activated caspase-3 expression in both cell lines. Moreover, Gracilex^® significantly reduces the proliferation rate of both LNCaP and PC-3 prostate cancer cell lines, as evidenced by a marked decrease in the growth curve slope (p = 0.0034 for LNCaP; p < 0.0001 for PC-3) and a 40–50% reduction in the proportion of Ki-67-positive PCa cells. In addition, Gracilex^® significantly reduces in vitro cell migration and invasion in LNCaP and PC-3 cell lines. Lastly, Gracilex^® inhibits tumor growth in an in vivo xenograft model, an effect that correlates with the reduced PCa cell proliferation observed in tumor tissue sections. Collectively, our data strongly support the broad antitumoral effects of Gracilex^® on PCa cells in vitro and in vivo. These findings advance our understanding of its potential therapeutic role in PCa and highlight the relevance of further investigating algae-derived compounds for cancer treatment.

Endogenous viral elements (EVEs) are genomic sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express co-opted proteins in their host. Furthermore, some EVEs that are expressed as proteins have become part of cellular genes that are fusions of hosts and EVE sequences. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and some of them contain ORFs and can be expressed as proteins. We have shown that an EPV containing an ORF is part of the guinea pig gene enRep-M9l. This gene is broadly transcribed in vivo, indicating that it can be translated into a protein. By generating antibodies against the enRep coding sequence of the enRep-M9l ORF, we showed that the protein enRep-M9l is expressed in vivo and in the guinea pig-derived cell line JH4. By immunofluorescence and in situ proximity ligation assays, we observed that enRep-M9l protein has a cytoplasmic localization near microtubules. The results of this study suggest that the guinea pig EPV-derived protein enRep-M9l is a microtubule-associated protein. To our knowledge, this is the second demonstration that an EPV-derived protein is expressed in vivo.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca²⁺ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca²⁺ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca²⁺ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.

Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.

Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-γ), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors’ signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.

Seaweed aquaculture is affected by natural and anthropogenic stressors, which put the biomass productivity of the cultures at risk. Seaweed biomass for commercial purposes, principally in pharmaceutical and/or nutraceutical applications, needs to be free of pollutants; therefore, controlled cultures have relevance in regulating the quality of biomass. The aim of this work was to demonstrate the successful utilization of controlled outdoor cultures to remove excess heavy metal accumulation in Gracilaria chilensis, an important commercial seaweed farming model. Specifically, we designed a simple and operational heavy metal depuration protocol, utilizing seawater and tap water removal, which permitted the concentration reduction of 10 heavy metals, including As, Cu, and Cd but not Zn, from the biomass at 7 days of culture. The percentage of depuration of the heavy metals ranged from 32 to 92% at 7 days, which was maintained throughout 21 days of culture. During the culture period, the monitored physicochemical parameters (temperature, salinity, and dissolved oxygen, among others) remained stable, with an increase in the daily growth rate (DGR% d⁻¹) of the biomass recorded after 14 days of culture. Consequently, the experimental setup was successful for heavy metal depuration, which highlights the importance of controlled outdoor cultures as important tools of sustainability.