Purinergic system: regulations and interactions

Le purine sono molecole segnale coinvolte nella regolazione delle risposte funzionali di molti organi e tessuti, sia in condizioni fisiologiche che patologiche. In condizioni di danno cerebrale larghe quantità di ATP ed adenosina sono rilasciate dai neuroni e dalla glia e svolgono un ruolo importante nel controllo dell’attività cellulare e nella risposta al danno. In questo contesto, studiare il cross-talk funzionale tra nucleotidi e nucleosidi purinergici, nonché tra il sistema purinergico e i mediatori infiammatori, costituisce un target importante per comprendere le risposte funzionali delle cellule del sistema nervoso centrale in seguito al danno cerebrale. In particolare l’obiettivo di questa tesi è stato quello di investigare: - la co-localizzazione, il ruolo dell’eterodimerizzazione e l’interazione funzionale dei recettori A1 dell’adenosina e dei recettori P2Y1 dell’ATP nell’ippocampo e nella regolazione delle risposte di cellule gliali umane; - gli effetti del trattamento con la citochina infiammatoria TNF-α sui recettori A2B dell’adenosina in cellule gliali umane, nonché le vie di segnale intracellulare coinvolte in questo cross-talk

Brain-targeted enzyme loaded nanoparticles: a breach through the blood brain barrier for enzyme replacement therapy in Krabbe disease

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement

The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons

Background: Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway. Methods: Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance. Results: We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber-binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype. Conclusions: We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity

Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evalu- ate the changes in tubulin cytoskeleton organization and cell morphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4- mm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells

Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evaluate the changes in tubulin cytoskeleton organization and cellmorphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4-mm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells

RP-CARS: label-free optical readout of the myelin intrinsic healthiness

Here we present a method based on Rotating-Polarization Coherent Anti-Stokes Raman Scattering (RP-CARS) imaging to assess the myelin health status in mouse sciatic nerves. Differently from the existing techniques, our method is based on the readout of intrinsic molecular architecture rather than on the image analysis, relying on the fact that healthy myelin is characterized by a high degree of molecular order. We exploit RP-CARS imaging to demonstrate that the degree of spatial anisotropy of the CARS signal displays a strong correlation with the g-ratio (a well-known image-based index of myelin damage) in a chemical-damage model and therefore that the former is a good indicator for the local myelin health status