Alice Bertero

Altered functional connectivity networks in acallosal and socially impaired BTBR mice

Abstract Agenesis of the corpus callosum (AgCC) is a congenital condition associated with wide-ranging emotional and social impairments often overlapping with the diagnostic criteria for autism. Mapping functional connectivity in the acallosal brain can help identify neural correlates of the deficits associated with this condition, and elucidate how congenital white matter alterations shape the topology of large-scale functional networks. By using resting-state BOLD functional magnetic resonance imaging (rsfMRI), here we show that acallosal BTBR T+tpr3tf/J (BTBR) mice, an idiopathic model of autism, exhibit impaired intra-hemispheric connectivity in fronto-cortical, but not in posterior sensory cortical areas. We also document profoundly altered subcortical and intra-hemispheric connectivity networks, with evidence of marked fronto-thalamic and striatal disconnectivity, along with aberrant spatial extension and strength of ipsilateral and local connectivity. Importantly, inter-hemispheric tracing of monosynaptic connections in the primary visual cortex using recombinant rabies virus confirmed the absence of direct homotopic pathways between posterior cortical areas of BTBR mice, suggesting a polysynaptic origin for the synchronous rsfMRI signal observed in these regions. Collectively, the observed long-range connectivity impairments recapitulate hallmark neuroimaging findings in autism, and are consistent with the behavioral phenotype of BTBR mice. In contrast to recent rsfMRI studies in high functioning AgCC individuals, the profound fronto-cortical and subcortical disconnectivity mapped suggest that compensatory mechanism may not necessarily restore the full connectional topology of the brain, resulting in residual connectivity alterations that serve as plausible substrates for the cognitive and emotional deficits often associated with AgCC.

Surface functionalisation regulates polyamidoamine dendrimer toxicity on blood–brain barrier cells and the modulation of key inflammatory receptors on microglia

Abstract Dendrimers are branched polymers with spherical morphology. Their tuneable chemistry and surface modification make them valuable nanomaterials for biomedical applications. In view of possible dendrimer uses as brain-aimed nanocarriers, the authors studied amine- and lipid-functionalised (G4) polyamidoamine (PAMAM) biocompatibility with cell population forming the blood–brain barrier (BBB). Both amine-PAMAM and lipid-PAMAM dendrimers were able to enter endothelial and primary neural cells. However, only amine-PAMAM damaged cell membranes in a dose-dependent manner. Transmission electron microscopy evidenced the ability of dendrimers to precipitate salts and serum components present in culture medium that slightly increased toxicity of the amine-PAMAM. Amine- and lipid-PAMAM were both able to cross the BBB and differently induced CD11b and CCR2 overexpression on primary CX3CR1-GFP murine microglia in vitro. These data emphasise the role of dendrimer surface functionalisation in toxicity and neural immune cell activation, raising concerns about possible neuroinflammatory reactions.

A poly(ether-ester) copolymer for the preparation of nanocarriers with improved degradation and drug delivery kinetics

This paper reports the synthesis and the physicochemical, functional and biological characterisations of nanocarriers made of a novel di-block biodegradable poly(ether-ester) copolymer. This material presents tunable, fast biodegradation rates, but its products are less acidic than those of other biosorbable polymers like PLGA, thus presenting a better biocompatibility profile and the possibility to carry pH-sensitive payloads. A method for the production of monodisperse and spherical nanoparticles is proposed; drug delivery kinetics and blood protein adsorption were measured to evaluate the functional properties of these nanoparticles as drug carriers. The copolymer was labelled with a fluorescent dye for internalisation tests, and rhodamine B was used as a model cargo to study transport and release inside cultured cells. Biological tests demonstrated good cytocompatibility, significant cell internalisation and the possibility to vehiculate non-cell penetrating moieties into endothelial cells. Taken together, these results support the potential use of this nanoparticulate system for systemic administration of drugs.

Molecularly Imprinted Biodegradable Nanoparticles

Biodegradable polymer nanoparticles are promising carriers for targeted drug delivery in nanomedicine applications. Molecu- lar imprinting is a potential strategy to target polymer nanoparticles through binding of endogenous ligands that may promote recognition and active transport into specific cells and tissues. However, the lock-and-key mechanism of molecular imprinting requires relatively rigid cross-linked structures, unlike those of many biodegradable polymers. To date, no fully biodegradable molecularly imprinted particles have been reported in the literature. This paper reports the synthesis of a novel molecularly- imprinted nanocarrier, based on poly(lactide-co-glycolide) (PLGA) and acrylic acid, that combines biodegradability and molec- ular recognition properties. A novel three-arm biodegradable cross-linker was synthesized by ring-opening polymerization of glycolide and lactide initiated by glycerol. The resulting macromer was functionalized by introduction of end-functions through reaction with acryloyl chloride. Macromer and acrylic acid were used for the synthesis of narrowly-dispersed nanoparticles by radical polymerization in diluted conditions in the presence of biotin as template molecule. The binding capacity of the imprinted nanoparticles towards biotin and biotinylated bovine serum albumin was twentyfold that of non-imprinted nanoparti- cles. Degradation rates and functional performances were assessed in in vitro tests and cell cultures, demonstrating effective biotin-mediated cell internalization.

Development of Serotonergic Fibers in the Post-Natal Mouse Brain

Serotonin (5-HT)-synthetizing neurons, which are confined in the raphe nuclei of the rhombencephalon, provide a pervasive innervation of the central nervous system (CNS) and are involved in the modulation of a plethora of functions in both developing and adult brain. Classical studies have described the post-natal development of serotonergic axons as a linear process of terminal field innervation. However, technical limitations have hampered a fine morphological characterization. With the advent of genetic mouse models, the possibility to label specific neuronal populations allowed the rigorous measurement of their axonal morphological features as well as their developmental dynamics. Here, we used the Tph2GFP knock-in mouse line, in which GFP expression allows punctual identification of serotonergic neurons and axons, for confocal microscope imaging and we performed 3-dimensional reconstruction in order to morphologically characterize the development of serotonergic fibers in specified brain targets from birth to adulthood. Our analysis highlighted region-specific developmental patterns of serotonergic fiber density ranging from a linear and progressive colonization of the target (Caudate/Putamen, Basolateral Amygdala, Geniculate Nucleus and Substantia Nigra) to a transient increase in fiber density (medial Prefrontal Cortex, Globus Pallidus, Somatosensory Cortex and Hippocampus) occurring with a region-specific timing. Despite a common pattern of early post-natal morphological maturation in which a progressive rearrangement from a dot-shaped to a regular and smooth fiber morphology was observed, starting from post-natal day 28 serotonergic fibers acquire the region specific morphological features present in the adult. In conclusion, we provided novel, target-specific insights on the morphology and temporal dynamics of the developing serotonergic fibers.

eIF4B phosphorylation at Ser504 links synaptic activity with protein translation in physiology and pathology

Neuronal physiology requires activity-driven protein translation, a process in which translation initiation factors are key players. We focus on eukaryotic initiation factor 4B (eIF4B), a regulator of protein translation, whose function in neurons is undetermined. We show that neuronal activity affects eIF4B phosphorylation and identify Ser504 as a phosphorylation site regulated by casein kinases and sensitive to the activation of metabotropic glutamate receptors. Ser504 phosphorylation increases eIF4B recruitment to the pre-initiation complex and influences eIF4B localization at synapses. Moreover, Ser504 phosphorylation modulates the translation of protein kinase Mζ. Therefore, by sensing synaptic activity, eIF4B could adjust translation to neuronal needs, promoting adaptive changes in synaptic plasticity. We also show that Ser504 phosphorylation is increased in vivo in a rat model of epilepsy during epileptogenesis i.e. when translation drives maladaptive synaptic changes. We propose eIF4B as a mediator between neuronal activity and translation, with relevance in the control of synaptic plasticity.

Serotonergic Signaling Controls Input-Specific Synaptic Plasticity at Striatal Circuits

Monoaminergic modulation of cortical and thalamic inputs to the dorsal striatum (DS) is crucial for reward-based learning and action control. While dopamine has been extensively investigated in this context, the synaptic effects of serotonin (5-HT) have been largely unexplored. Here, we investigated how serotonergic signaling affects associative plasticity at glutamatergic synapses on the striatal projection neurons of the direct pathway (dSPNs). Combining chemogenetic and optogenetic approaches reveals that impeding serotonergic signaling preferentially gates spike-timing-dependent long-term depression (t-LTD) at thalamostriatal synapses. This t-LTD requires dampened activity of the 5-HT4 receptor subtype, which we demonstrate controls dendritic Ca2+ signals by regulating BK channel activity, and which preferentially localizes at the dendritic shaft. The synaptic effects of 5-HT signaling at thalamostriatal inputs provide insights into how changes in serotonergic levels associated with behavioral states or pathology affect striatal-dependent processes.

Autism-associated 16p11.2 microdeletion impairs prefrontal functional connectivity in mouse and human

Human genetic studies are rapidly identifying variants that increase risk for neurodevelopmental disorders. However, it remains unclear how specific mutations impact brain function and contribute to neuropsychiatric risk. Chromosome 16p11.2 deletion is one of the most common copy number variations in autism and related neurodevelopmental disorders. Using resting state functional MRI data from the Simons Variation in Individuals Project (VIP) database, we show that 16p11.2 deletion carriers exhibit impaired prefrontal connectivity, resulting in weaker long-range functional coupling with temporal-parietal regions. These functional changes are associated with socio-cognitive impairments. We also document that a mouse with the same genetic deficiency exhibits similarly diminished prefrontal connectivity, together with thalamo-prefrontal miswiring and reduced long-range functional synchronization. These results reveal a mechanistic link between specific genetic risk for neurodevelopmental disorders and long-range functional coupling, and suggest that deletion in 16p11.2 may lead to impaired socio-cognitive function via dysregulation of prefrontal connectivity.

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Deletion of autism risk gene Shank3 disrupts prefrontal connectivity

Mutations in the synaptic scaffolding protein Shank3 are a major cause of autism, and are associated with prominent intellectual and language deficits. However, the neural mechanisms whereby SHANK3 deficiency affects higher order socio-communicative functions remain unclear. Using high-resolution functional and structural MRI in mice, here we show that loss of Shank3 (Shank3B-/-) results in disrupted local and long-range prefrontal functional connectivity, as well as fronto-striatal decoupling. We document that prefrontal hypo-connectivity is associated with reduced short-range cortical projections density, and reduced gray matter volume. Finally, we show that prefrontal disconnectivity is predictive of social communication deficits, as assessed with ultrasound vocalization recordings. Collectively, our results reveal a critical role of SHANK3 in the development of prefrontal anatomy and function, and suggest that SHANK3 deficiency may predispose to intellectual disability and socio-communicative impairments via dysregulation of higher-order cortical connectivity.