Graphene related materials and the nervous system
Due to their peculiar physico-chemical properties, bidimensional nanomaterials, such as graphene related materials (GRMs) and transition metal dichalcogenides, have attracted attention for the development of new technologies for the treatment of neurodisorders. In the framework of the Graphene Flagship project (the largest-ever research initiative in the EU), we have characterized the use of graphene and its derivatives for neuro–applications. We are using graphene and other 2D materials, to investigate in vitro and in vivo how the nanomaterials interface to nervous system cells and impact on their functionality, for the development of stimulating/recording devices and of tridimensional scaffolds to support neuronal growth and reconnection. To this aim, we combine electrophysiology, calcium imaging, behavioral studies and structural characterization of the tissue. Another line of research regards the use of soluble GRMs as platform for targeted drug delivery systems. We found that some soluble GRMs, when unconjugated, interact per se with nervous cells, modifying the activity of both neurons and glial cells. This ability can be exploited for therapeutic intervention and, in this perspective, we have tested GRMs, free or in complexation to bioactive compounds, in models of anxiety, ischemia and neuroinflammation. On the other hand, safety assessment of graphene and other 2D materials is a key goal of the project. We are contributing to this aspect by using the zebrafish model, in which we combine high throughput approaches for the screening of behavioral alterations with the monitoring of single cell function through live imaging in whole organism upon exposure to nanomaterials.
Nanoscale optical interfaces for single-cell stimulation
Silicon-based nanoscale photodiodes (Si-nPDs) represent an emerging nano-technology, characterized by a great spatial-temporal resolution and specificity and here employed to apply single neuron stimulation. Si-nPDs are biocompatibility and represent a light-sensitive semiconductor which responds to light stimulation with the generation of spatially confined electrical current. We are employing Si-nPDs to exert single-cell stimulation over visually identified neuron in organotypic spinal cord culture. Neuronal and astrocytic activity is evaluated taking advantage of genetically encoded calcium indicators. The outcome of the stimulation is reflected onto the whole network activity, and it differs basing on the identity and localization of the stimulated neuron. Si-nPDs are used as well to study dorsal-ventral microcircuits cross-talk, with the two separate networks displaying stereotyped responses which are coherent with each circuit core activity features. In addition, astrocytic network response is evaluated, by stimulating a single neuron an observing how astrocytes parallel neuronal synchronization. Finally, nPDs specificity can be furtherly increased, by applying chemical functionalisation on their external shell, as linking to it GluA2 antibody which will direct the material specifically onto excitatory synapses, or increasing cellular adhesive properties with the usage of TAT peptide.
Neuroinflammation and nanovesicles: unraveling neuron-astrocyte interplay in mouse spinal circuits
Small extracellular vesicles (sEVs) have emerged as potent nano-scale mediators orchestrating intercellular physio-pathological signal propagation. In the CNS, sEVs trafficking can facilitate the spread of neuroinflammation, triggering astrocytic reactivity, microglial activation and neuronal dysfunction. To dissect sEVs dynamics and their impact on neuron-glia interactions, we employ an ex-vivo organotypic spinal model immuno-challenged by a cytokine-based neuroinflammation trigger, recapitulating in vivo neuroinflammation observed in Multiple Sclerosis. Thus, tissue cultures that preserve the spinal cyto-architecture offer a relevant paradigm to investigate the diverse cellular components (neurons and resident glial cells) in disease-relevant contexts. Neuronal and astrocytic activity – recorded both individually and simultaneously - is monitored in real-time through whole-cell patch-clamp electrophysiology and viral transduction of genetically encoded calcium indicators, respectively. In parallel, nanovesicle characterization and functional assessments are deepened by a broad array of molecular biology approaches. These range from SDS-PAGE electrophoresis and Western blotting for vesicular protein composition analysis to the design and production of adeno-associated viral (AAV) vectors to enable vesicular cargo manipulation. This exploratory study aims to elucidate sEVs-mediated signaling, contributing to the growing body of evidence highlighting these nanovesicles as both disease-defining vectors, informative in ethio-pathological contexts, and physiological messengers with untapped therapeutic potential.
The effects of pro-inflammatory cytokines on oligodendrocyte function and myelination.
By using the ex vivo spinal organotypic cultures, we are investigating also the impact of proinflammatory cytokines on oligodendrocytes. Oligodendrocytes are the myelin-forming cells of CNS, essential for insulating axons and facilitating efficient, rapid synaptic transmission. Dysfunction of oligodendrocytes is linked to a range of neurological disorders, most notably multiple sclerosis. Despite their critical role, our understanding of the behavior and function of oligodendrocytes and their precursor cells remains limited. To advance this understanding, we employ advanced imaging techniques and cell-specific genetic manipulations. Using live imaging and genetically encoded calcium indicators, we track and analyze dynamic changes in oligodendrocyte function within the organotypic spinal cultures, providing deeper insight into their roles in health and disease.
Zebrafish as translational model towards healthy aging strategies
Zebrafish exhibit age-related changes in synaptic integrity which have been correlated, as in mammals, to cognitive functions decline, making them precious model organism for studying neurobiological and behavioral changes due to aging, with the potential to identify pro-healthy aging interventions. We are investigating the effects of aging on the CNS evaluating synaptic integrity and integrated physiological features using behavioral analysis, live imaging and confocal microscopy. In particular, we focus on age-related declines in cognitive functions correlated to altered synaptic integrity combining aging conditions to stress factors, such as anxiety induced by social isolation.