The main research lines involve (i) the molecular basis of synaptic function and homeostatic plasticity; (ii) the pathogenesis of brain diseases, with particular focus on the role of synaptic genes in neural dysfunctions such as epilepsy, autism and mental retardation; (iii) engineering and testing of novel optogenetic probes able to modulate gene transcription and translation; (iv) neuron-to-chip systems allowing the implementation of new neuron-based biosensors and neuroprosthetic interfaces, including the optimization of a photovoltaic polymer-based artificial retina; (v) biocompatibility and performance studies of graphene-neuron interfaces for implantable devices and regenerative therapies; (vi) study of the Octopus arm as a model of muscle mechanics and sensory-motor systems to be exploited in bio-inspired devices. The NSYN research group has a well-established experience in the fields of neurophysiology, cellular and molecular neurobiology, optogenetics and bioengineering.
The NSYN center occupies about 700 sqm inside the IRCCS San Martino University Hospital in Genova. Space includes offices and electrophysiology, molecular neurobiology and neurotechnology facilities.
Patch-clamp and conventional electrophysiology
Twelve patch-clamp setups for primary neuron cultures and acute brain slices, five MEA setups and three in vivo physiology setups.
Primary and organotypic cultures, stem cells and viral preparations.
Histochemistry and immunocyto/histochemistry; 1 Leica SP8 confocal microscope; 1 Leica TIRF workstation; epifluorescence and chemiluminescence microscopes and Neurolucida system.
Cellular and molecular biology
PCR and real time-PCR machines, phosphoimagers, Biacore SPR, spectrophotometers.
Modeling and Molecular Dynamics simulations of neuronal proteins.
300 sqm laboratory area for general services and in vivo laboratories (surgery and behavior, EEG and mouse genetics).
1. Neural networks and neural interfaces
A. Genetically encoded fluorescence sensor for extracellular pH. We have generated a sensor for extracellular pH to monitor the H+ concentration during neuronal activity and epileptic paroxysms using an engineered variant of GFP (E2GFP) whose fluorescence emission increases when pH drops. We engineered a chimeric probe in which the pH sensor is exposed to the extracellular space and functionally validated it after expression in neurons. Using an in vitro model of epilepsy, we recently demonstrated the magnitude and dynamics of acidic shifts in extracellular pH in neural cultures under epileptic-like conditions. Interestingly, we have shown that such shifts were not uniformly diffused, but rather localized in discrete dots corresponding to active synapses. We plan to investigate the pH dynamics during physiological activity, using electrical and/or optogenetic stimulations and in experimental models of brain pathologies and elucidate the intracellular pathways leading to H+ extrusion in hyperactive neurons.
B. Synaptic Ca2+/exocytosis sensors. A long-standing goal in neuroscience has been to ‘see’ the Ca2+ that triggers synaptic transmission. Intracellular calcium in synapses shapes the dynamics of neurotransmitter release. The lab recently succeeded in imaging Ca2+ signals and vesicle release in the same synapse in response to a single action potential, by targeting the genetically encoded Ca2+ indicator GCaMP6s and the red-shifted pH-sensitive probe mOrange2 to synaptic vesicles, thus allowing simultaneous imaging of the trigger for neurotransmitter release (Ca2+) with the release of neurotransmitter itself. The current spatial resolution for Ca2+ signals is at the level of a single synapse (~1µm). The lab plans to engineer GCaMP6s further to improve spatial resolution down to a single active zone (~200nm), to selectively detect the Ca2+ responsible for vesicle release.
GABAergic neurons, through GABA release, provide a systematic inhibitory tone in the cerebral cortex, and their dysfunction could be involved in a wide range of brain disorders, including epilepsy, autism and neuropsychiatric disturbances. Recently, it has been shown that grafted GABAergic neurons have surprising migratory ability in the adult brain tissue and can alleviate refractory epileptic seizures. Collaborator V. Broccoli’s laboratory at Ospedale S.Raffaele has been able to reprogram fibroblasts from animals and humans into dopaminergic neurons, interneurons or astrocytes. Our electrophysiological analysis suggests that the reprogrammed neurons acquire the typical properties of the mature neurons in terms of excitability, firing and synaptic transmission. The neurons also incorporate into the brain networks upon stereotaxic injection, release neurotransmitter at newly established synaptic connections with the host neurons and receive excitatory and inhibitory synaptic contacts. Implanted, reprogrammed inhibitory GABAergic neurons could provide an optimal treatment for refractory epilepsies, because they can be derived directly from the human patients by reprogramming the patients’ own skin fibroblasts. The lab will further characterize these reprogrammed cells, and will transplant them in pilocarpine-induced epileptic mice or in genetic models of epilepsy to test the remission from spontaneous or sensory-evoked seizures.
The goal of this project, in collaboration with IIT-CNST, is to use organic semiconducting polymers as organic prostheses to stimulate neurons, such as in an artificial retina for treating genetic retinal degenerative diseases. The lab has already shown that primary neurons can be successfully grown onto a photovoltaic organic polymer and electrically stimulated by light, and that blind retinas in contact with the polymer recover basic light-sensitivity. Recently, the lab has improved the efficiency of neural stimulation by testing various types of polymers, by varying thickness and patterns of deposition, and by developing a biocompatible, flexible and possibly porous substrate. The lab currently implants a silk-based prosthesis subretinally in the eye of genetically blind Royal College of Surgeons rats to evaluate the efficacy of the implant in restoring light sensitivity in vivo. Preliminary results indicate an improvement in the papillary light reflex, the recovery of the visually evoked field potentials and a behavior in the Dark/Light test comparable with normal animals. The analysis of the implanted rat will be extended to the recovery of visual acuity and sensitivity using advanced visual-behavioral tests. The interface will be optimized for larger eyes to implant the artificial retina in pigs with chemically induced photoreceptor degeneration to analyze its physiological performance and long-term tolerability as a final assessment before human experimentation. Main goals are to: (i) improve the device for surgical procedures in the human eye; (ii) test its structure and the implantation procedure; and (iii) verify the recovery of light sensitivity and visual acuity in the photoreceptor-depleted pig and/or monkeys as final stage preceding the clinical application. The lab will also explore the possibility of implanting infrared-sensitive polymers in animals without photoreceptor degeneration. The lab also plans to exploit new polymeric interfaces, based on novel materials and geometries, to develop more advanced retinal prostheses and new opto-neural devices.
A. Biocompatibility and performances of graphene-neuron interfaces. In view of its exceptional properties including stability, conducibility and flexibility, graphene is the future material of choice for constructing environmentally friendly devices, particularly neuroprosthesis and neurointerfaces. We plan to evaluate the biocompatibility of this material with the survival and growth of primary neurons and astrocytes in vitro. Cell viability, growth and proliferation will be tested on graphene substrates, in comparison to standard culture conditions. The studies on the biocompatibility will successively be extended to living rodents implanted with the graphene-based devices. After the assessment of biocompatibility, we will proceed to the functional characterization of graphene-neuronal hybrid interfaces to be employed in biological applications.
B. Innovative scaffolds for neural growth and regeneration. Scaffolds made of different materials have been constructed for the repair of different tissues. However, attempts to construct scaffolds for the repair of the central nervous system have had limited success for the functional recovery of the damaged nervous system, because of the intrinsic poor regenerative properties of this tissue. For an effective repair of nervous system injuries, it is necessary to control the growth and functional connectivity of 3D neuronal networks. 3D scaffolds for neuronal network must have a hierarchical level of complexity in which the macroscopic structure is determined by imaging at a millimeter and micrometer resolution, while the scaffold substrate is patterned and decorated with guidance molecules at a nanometer resolution. To this aim, we plan to use multichannel conduits for peripheral nervous system regeneration and super-hydrophobic (SH) scaffolds to create three-dimensional conditions of neuron development and regrowth. As neurons cultured under these 3D conditions display a lower mortality rate, this 3D culture system represents a valuable tool to support the long-term growth and maintenance of neuronal cells. We plan to develop these scaffolds using flexible, biocompatible and reabsorbable materials, such as polycaprolactone (PCL) that are very promising as new materials for implantable biomedical microdevices. We will use several biological assays to evaluate functional properties of neurons cultivated in these scaffolds, and study expression of physiological markers of neuronal maturation as well as the correct localization and distribution of neuron-specific proteins required for proper neuronal excitability and synaptic transmission. Moreover, the scaffold absorption and slow release of agents stimulating neuron growth and survival, such as neurotrophins, will be exploited to boost the regenerative properties of the nervous tissue.
The aim of the research is to investigate the properties of a hyper-redundant arm from molecular motors to the embodied limb properties. The main interest is to explore the muscle mechanics and control principles of the Octopus vulgaris arm by employing techniques of electrophysiology, histochemistry and biophysics. We will investigate: (i) the arm muscle mechanics and sensory-motor systems to determine the properties of the various muscle types and their role in movement generation; (ii) the sucker adhesive mechanism and sensory-motor system to find solutions for bio-robotic applications. To characterize the muscular hydrostatic properties of the octopus arm and of the properties of its neuromuscular junction, we investigate on the biophysical properties of entire arm buds as well as of single muscle type. Single muscle type relaxation/contraction properties are investigated. We are particularly interested in deciphering the occurrence and functional role of stiffening in various muscle behaviors. We are also interested in investigating the adhesive mechanism of the octopus sucker to find solutions to be implemented for bio-robotic applications in collaboration with B. Mazzolai at the CMBR@IIT. In particular, we are investigating the morphology and physiological properties of sucker sensory and motor system. The physiological properties of the sucker receptors will be dissected, by monitoring the responses of an isolated sucker under various environmental stimulations and their characteristics will be used as bio-inspiration for artificial sensors.
2. Molecular networks brain function and diseases
Mutations in some presynaptic proteins are associated with epilepsy, autism spectrum disorder (ASD) and cognitive impairment. The lab will study the effects of down-regulation or over-expression of these pathogenic candidates both in vitro and in vivo to uncover their physiological role in synaptic transmission, excitation/inhibition balance and network activity. The lab currently generates iPS cells derived from fibroblasts of patients with mutations in synaptic proteins that have been implicated in the ASD or epilepsy. The iPS cells will be differentiated into excitatory and inhibitory neurons for in vitro modeling of brain diseases, in a patient-specific manner. Using computational modeling and Molecular Dynamics simulations we will investigate the fine detailed structural mechanisms underlying presynaptic proteins function and their modulation of synaptic activity. Among others, we will study how ATP and Ca2+ influence functional conformational changes in synapsin I/II and other Ca2+-binding proteins such as synaptotagmin or rabphilin. We will examine how synapsin oligomerization, governing synaptic vesicle trafficking, depends on ligand binding, and how point mutations at the binding site affect the process. We will also explore the structural basis of different Ca2+ regulation of ATP binding observed in synapsin isoforms and their mutants and extend this analysis to other Ca2+-binding proteins of the synapse.
In this context, we will investigate the molecular mechanisms of homeostatic plasticity and its involvement in the pathogenesis of cortical circuit dysfunctions. Our working hypothesis is that homeostatic plasticity and defects thereof represent a common basis for epilepsy, autism and mental retardation. First, to unravel the fundamental mechanisms that fine tune the level of neuronal excitability in response to average changes in network activity, we will investigate how transcription factors and specifically the transcriptional repressor REST are involved in scaling excitability and synaptic transmission following sustained hyperexcitation or silencing of neuronal activity. Second, to understand the role of homeostatic synaptic plasticity in brain disorders, we will investigate how deficits in proteins that mediate cell-cell recognition and interactions, such as the integrins, prevent neuronal networks from homeostatically readjusting the strength and specificity of their connections in response to deafferentation or sensory deprivation.
Perturbations of transcriptional activity or epigenetic modifications can dramatically influence neuronal phenotype. The lab will engineer light-sensitive probes to modulate gene transcription with high spatial and temporal precision. By employing the light-sensitive LOV2 domain of Avena sativa, the lab has recently created recombinant proteins that modulate the activity of the RE1-silencing transcription factor (REST), a master regulator of nervous system development and lineage specification. REST represses the transcription of a large panel of specific genes necessary for the acquisition of the unique phenotype of neural cells. Accordingly, REST is downregulated during neuronal differentiation and in mature neurons and is dysregulated, most often strongly upregulated, in many brain diseases, including epilepsy, ischemia, Huntington disease, Down Syndrome and brain tumors. Thus, the reversible modulation of REST activity by optogenetics could have an impact not only in orchestrating the expression of neural genes during development and in mature neurons, but also in treating brain pathologies linked to REST dysregulation. Chimeric proteins with a LOV2 domain and a REST-interacting domain were simulated, to direct and optimize chimeric strategies and linker optimization. Engineered LOV2-REST inhibitors were effective in regulating neural gene expression in neuroblastoma cell lines. The lab will express the optogenetic probe in mouse primary neurons under normal conditions and epileptogenic-like conditions to study the role of REST on the basic physiological properties of neurons. The lab will also use the probes in other models of REST over-expression/over-activity, such as murine models of Huntington’s disease, epilepsy or brain ischemia, as well as in neuron- or astrocyte-derived tumors, which display high REST expression. New LOV-operated probes will also be designed to boost REST action or to regulate the activity of the associated factor MeCP2, which is involved in Rett syndrome.
Neurotrophins represent a promising target for the development of novel therapeutic approaches aimed at curing a number of pathologies of the central and peripheral nervous system, such as Alzheimer’s disease and neuropathic pain. This project aims at gaining novel insights about the neurotrophin-dependent mechanisms that modulate the physiology of neuronal circuits in the adult brain, by studying the excitability and plasticity properties of mature networks. To this purpose, we will employ a number of transgenic mouse lines, which will be crossed with Cre-expressing lines driving the postnatal- and nervous system-specific ablation of the proteins of interest. We will focus on membrane proteins and protein adaptors that are target of trophic stimuli, and participate in the modulation of the intracellular signaling pathways initiated by such cues. Moreover, we will address the mechanisms by which neurotrophin signaling impacts on neural excitability, by studying neurotrophin-dependent processes that modulate the function and localization of a number of neuron-specific, voltage-gated sodium channel isoforms.