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Microglia derived extracellular vesicles (EVs) in brain aging: a rejuvenation approach
Get PDFBrain aging presents a picture defined "inflammaging", characterized by high oxidative stress, chronic inflammation and elevated production of inflammatory compounds (López-Otín et al. 2023). Microglia, the immunocompetent cells of the central nervous system, shift towards a chronic low-grade inflammatory state and become hyper-responsive. Indeed, these cells undergo the most prominent aging-related changes in both the morphological and functional phenotypes; their progressive loss of neuroprotective functions affects the whole brain homeostasis. This condition could result in cognitive functions deterioration, lack of motor coordination and memory loss. Microglial phenotype is impacted by aging in a region-dependent manner with hippocampus being more susceptible to age-related modifications during late aging (Grabert et al. 2016). In addition, age-related changes occur differently in aged males and females, since sex hormones are involved in microglial activation and inflammatory response.
Extracellular vesicles are key players of the inter-cellular communication between donor and recipient cells, through which brain cells exchange packages of molecular information consisting of lipids, proteins and nucleic acids (Budnik et al. 2016). Microglia-derived EVs transport molecular contents that mirror the inflammatory status of donor cells and modulate the inflammatory phenotype of recipient microglia and other cell types. The principal aim of the project was to investigate whether exogenous administration of EVs deriving from microglial-like BV2 cells could be efficient to slow down neuroinflammation in aged mice. To this aim, BV2-derived EVs were analyzed for their molecular, cellular and behavioral effects when administered to 16- 20 months-old male and female mice. Data obtained demonstrated that the treatment dampened inflammation, restored a juvenile microglia morphology, reduced anxiety-like behavior while increasing spatial learning, with sex-dependent differences. Results of this thesis has been published in 2024 on the journal “Brain, behavior, and immunity” (Rinaldi & Balietti et al., 2024)
Approcci nanotecnologici innovativi per il targeting del glioblastoma
No full textIl glioblastoma (GBM) è la forma più comune di tumore cerebrale maligno. Ad oggi, non esistono terapie curative. L’aggressività del tumore, l’insufficiente accumulo di farmaci al cervello e la resistenza ai trattamenti sono ostacoli maggiori che limitano l’efficacia delle terapie convenzionali. Le nanomedicine (NMeds) rappresentano una strategia promettente per migliorare le attuali terapie per il GBM, incapsulando i farmaci tradizionali per garantirne la protezione, il delivery sito-specifico tramite targeting e il rilascio controllato nel sito target. Nell'ambito del GBM, possono essere sfruttati due principali approcci di delivery di farmaci basati su NMeds: sistemico e locoregionale. Questo lavoro di tesi mira a progettare, caratterizzare e testare nuove NMeds per il delivery sia locoregionale che sistemico di farmaci al GBM.
Nell'approccio sistemico, le NMeds possono essere funzionalizzate in superficie con ligandi specifici per promuovere l’attraversamento della barriera emato-encefalica (BEE) e/o per il targeting alle cellule tumorali. A questo scopo, nella prima parte di questo lavoro, una nanoparticella polimerica è stata decorata in superficie con nuovi ligandi con potenziale abilità di targeting alle cellule di GBM. Diverse NMeds “mirate” sono state formulate, caratterizzate e testate in vitro per valutare la loro specificità verso le cellule di GBM e la capacità di veicolare un agente chemioterapico (paclitaxel). Lo studio ha dimostrato il potenziale di tali ligandi per il targeting al GBM. Tra questi, è stato identificato un anticorpo monoclonale (M08) in grado sia di essere internalizzato selettivamente nelle cellule di GBM rispetto alle cellule sane, sia di causare effetti sinergici con il paclitaxel veicolato. La maggiore specificità verso il GBM costituisce la base per la creazione di NMeds per il delivery sistemico con un maggiore potenziale terapeutico e una minore tossicità sulle cellule sane.
Nella seconda parte di questo lavoro, sono state progettate nuove NMed per il delivery di molecole innovative con potenziale terapeutico per il GBM: i siRNA (small interfering RNA). Sono state sviluppate due nuove NMeds ibride polimero-lipide, destinate a complessare un siRNA modello in superficie e a veicolarlo sotto forma di complessi NMed/siRNA. Le NMed e i complessi sono stati caratterizzati per proprietà fisico-chimiche, capacità di complessare il siRNA e protezione del siRNA dalla degradazione enzimatica. L’uptake, l'efficacia e la biocompatibilità sono state valutate in vitro su linee cellulari di GBM. Entrambi i complessi hanno mostrato un'elevata capacità di legare a proteggere il siRNA. Uno di essi ha inoltre mostrato un'elevata attività di silenziamento genico e una buona citocompatibilità in vitro. Date le proprietà promettenti, i complessi NMed/siRNA sono stati sfruttati per una strategia di delivery locoregionale al GBM. In particolare, l'uso di scaffold biopolimerici per la somministrazione locale di siRNA nella cavità di resezione tumorale rappresenta una strategia utile per bypassare la BEE, aumentare la concentrazione di siRNA nel sito tumorale e prolungarne il rilascio. Pertanto, la formulazione di NMed/siRNA più promettente, in termini di efficacia e citocompatibilità in vitro, è stata incorporata in un idrogel iniettabile e termosensibile, destinato ad essere iniettato nella cavità di resezione e agire come sistema di depot per rallentare il rilascio di siRNA in-situ. I nanocompositi NMed-idrogel sono stati caratterizzati per proprietà fisico-chimiche e reologiche e cinetica di rilascio del siRNA. Il sistema sviluppato, che gelifica alla temperatura corporea, ha dimostrato inoltre un'elevata citocompatibilità in vitro. Studi di efficacia su modelli di GBM in vitro sono in corso.Glioblastoma (GBM) is the most common malignant tumor of the brain, for which there are no curative therapies. The tumor aggressiveness, the insufficient drug accumulation in the brain, and resistance to treatments, are major obstacles limiting the effectiveness of conventional therapies. Nanomedicine (NMed) delivery systems are a promising strategy to improve current therapies for GBM, by encapsulating traditional drugs to ensure their protection, site-specific delivery via targeting, enhanced accumulation in the brain, as well as controlled release at the target site. In the context of GBM, two main NMed-based drug delivery approaches can be exploited: systemic or locoregional ones. The aim of this project was to design, characterize and test novel NMeds for both locoregional and systemic delivery of drugs to GBM.
In the systemic approach, NMed can be targeted with specific ligands on their surface to deliver the cargo across the blood brain barrier (BBB) and/or target GBM cells. To that aim, in the first part of this work, an injectable polymeric NMed was decorated onto the surface with novel ligands selected for their GBM-targeting potential. Different targeted NMeds were therefore produced, characterized, and tested in vitro to evaluate their GBM cell specificity and ability to deliver an anticancer drug (paclitaxel) to GBM cells. By optimizing surface modifications, this study demonstrated the potential of this ligands for BBB and/or GBM targeting. In addition, a monoclonal antibody (M08) that can specifically enter GBM cells over healthy cells and cause synergistic effects when delivering paclitaxel was identified. The improved GBM specificity sets the basis to create NMeds for systemic administration with higher therapeutic potential and lower off-target toxicity to healthy cells.
In the second part of this work, novel NMeds were designed to deliver cutting-edge molecules with a therapeutic potential for GBM. In particular, small interfering RNA (siRNA) was selected as a promising drug candidate. Two novel polymer-lipid hybrid NMed platforms were developed, intended to complex a model siRNA onto their surface and deliver it in the form of NMed/siRNA complexes. NMed and their complexes were characterized for physicochemical properties, siRNA binding ability, and siRNA protection from enzymatic degradation. Cell uptake in GBM cells, efficacy and biocompatibility were assessed in vitro. Both complexes showed high siRNA binding, enhanced siRNA protection, and high cell uptake in a GBM cell model in vitro. One of them also displayed superior gene silencing activity and good cytocompatibility in vitro.
Given the promising properties, we decided to explore the potential of the developed siRNA/NMed formulation for a locoregional drug delivery strategy for GBM. For such approach, the use of biocompatible scaffolds for the local delivery of siRNA into the tumor resection cavity is a promising means to bypass the blood–brain barrier, increase siRNA concentration at the tumor site, and prolong its local release. Therefore, the most promising siRNA/NMed formulation, in terms of cytocompatibility and efficacy in vitro, was incorporated into an injectable and thermo-sensitive hydrogel, intended to be injected into the resection cavity and act as a depot for the NMeds to slow down siRNA release in situ. The NMed-hydrogel nanocomposites were characterized for physicochemical and rheological properties, and siRNA release kinetics. The optimized system gelified at the body temperature and possessed high cytocompatibility in vitro. In vitro studies are ongoing to assess whether the nanocomposite can ensure prolonged siRNA gene silencing activity in vitro on GBM model cell lines
Spatial memory, plasticity and nucleus accumbens
No full textResearch on the function of the nucleus accumbens, the most ventral component of the striatal complex, has traditionally focused on locomotor activity, reward, motivation and addiction. However, based on the existence of projections to the nucleus accumbens from the allocortical regions involved in spatial navigation, it has been suggested that this structure plays a role in spatial learning and memory. Lesion and neuropharmacological studies confirm this view, also revealing the complex dynamics of the receptors involved in these processes. Moreover, the effects of post-training intra-nucleus accumbens drug administrations demonstrate the necessity of off-line neural activity within this structure in order to consolidate spatial memory. Blockade of molecular processes implicated in synaptic plasticity, such as cAMP response element-binding protein (CREB)-induced transcription or extracellular matrix remodeling, provides further experimental support to this hypothesis. These observations imply that experience-dependent synaptic plasticity responsible for long-term stabilization of spatial information might occur within the nucleus accumbens, similarly to what has been observed in the hippocampus. This suggests that a comprehensive understanding of spatial memory processing should be viewed in the context of a wider neural circuit
Active integration of glutamatergic input to the inferior olive generates bi-directional postsynaptic potentials
Get PDFThe inferior olive plays a critical role in motor coordination and learning by integrating diverse afferent signals to generate climbing fibre inputs to the cerebellar cortex. While it is well established that climbing fibre signals are important for motor coordination, the mechanisms by which neurones in the inferior olive integrate synaptic inputs and the roles of particular ion channels are unclear. Here, we test the hypothesis that neurones in the inferior olive actively integrate glutamatergic synaptic inputs. We demonstrate that optogenetically activated long-range synaptic inputs to the inferior olive, including projections from the motor cortex, generate rapid excitatory potentials followed by slower inhibitory potentials. Synaptic projections from the motor cortex preferentially target the principal olivary nucleus. We show that inhibitory and excitatory components of the bidirectional synaptic potentials are dependent upon GluA receptors, are GABAA -independent, and originate from the same presynaptic axons. Consistent with models that predict active integration of synaptic inputs by inferior olive neurones, we find that the inhibitory component is reduced by blocking large conductance calcium-activated potassium channels with iberiotoxin, and is abolished by blocking small conductance calcium-activated potassium channels with apamin. Summation of excitatory components of synaptic responses to inputs at intervals ≤ 20 ms is increased by apamin, suggesting a role for the inhibitory component of glutamatergic responses in temporal integration. Our results indicate that neurones in the inferior olive implement novel rules for synaptic integration and suggest new principles for the contribution of inferior olive neurones to coordinated motor behaviours
REPEATED ADMINISTRATION OF PHENCYCLIDINE, AMPHETAMINE, AND MK-801 SELECTIVELY IMPAIRS SPATIAL LEARNING IN MICE: A POSSIBLE MODEL OF PSYCHOTOMIMETIC DRUG-INDUCED COGNITIVE DEFICITS
No full textDendritic excitation-inhibition balance shapes cerebellar output during motor behaviour
Get PDFFeedforward excitatory and inhibitory circuits regulate cerebellar output, but how these circuits interact to shape the somatodendritic excitability of Purkinje cells during motor behaviour remains unresolved. Here we perform dendritic and somatic patch-clamp recordings in vivo combined with optogenetic silencing of interneurons to investigate how dendritic excitation and inhibition generates bidirectional (that is, increased or decreased) Purkinje cell output during self-paced locomotion. We find that granule cells generate a sustained depolarization of Purkinje cell dendrites during movement, which is counterbalanced by variable levels of feedforward inhibition from local interneurons. Subtle differences in the dendritic excitation–inhibition balance generate robust, bidirectional changes in simple spike (SSp) output. Disrupting this balance by selectively silencing molecular layer interneurons results in unidirectional firing rate changes, increased SSp regularity and disrupted locomotor behaviour. Our findings provide a mechanistic understanding of how feedforward excitatory and inhibitory circuits shape Purkinje cell output during motor behaviour
D1 and D2 receptors antagonist injections in the prefrontal cortex selectively impair spatial learning in mice
No full texton line publication 9.8.200
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The neural substrate of spatial memory stabilization depends on the distribution of the training sessions
Get PDFDistributed training is known to lead to more robust memory formation as compared to training experiences with short intervals. Although this phenomenon, termed distributed practice effect, ubiquitous over a wide variety of tasks and organisms, has long been known by psychologists, its neurobiological underpinning is still poorly understood. Using the striatum as a model system here we tested the hypothesis that the ability of distributed training to optimize memory might depend upon the recruitment of different neural substrates compared to those engaged by massed training. First, by contrasting the medial and the lateral domains of the dorsal striatum after massed and distributed training we demonstrated that neuronal activity, as assessed using c-Fos expression, is differentially affected by the training protocol in the two striatal subregions. Next, by blocking the AMPA receptors before recall we provide evidence to support a selective role of the medial and the lateral striatum in the storage of information acquired respectively by massed and distributed training. Finally, we found that optogenetic stimulation of the dorsolateral striatum during massed training enables the formation of an enduring memory similarly to what is observed with distributed learning. Overall, these findings identify a possible mechanism for the distributed practice effect, a still poorly understood aspect of learning
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