The Parkinson-related E193K LRRK2 variant impacts neuronal vesicles dynamics through perturbed protein interactions

The Leucine-Rich Repeat Kinase 2 (LRRK2) is a complex protein, expressed in neurons and implicated in Parkinson disease (PD). LRRK2 contains a dual enzymatic activity and several structural domains that constitute a versatile platform for multiple protein interactions at the synapses. In this study, we characterize the functional role of the N-terminal Armadillo repeats domain of LRRK2 and the impact on synaptic vesicle (SV) dynamics of a novel variant, E193K, harboured within this domain and identified in an Italian family affected by PD. Using a genetically encoded sensor of recycling, synaptopHluorine, and total internal reflection fluorescence microscopy, we visualized SV trafficking in the N2A neuroblastoma cells expressing the wild type LRRK2 protein, a mutant lacking the Armadillo domain (\u394N LRRK2) or the E193K variant. We found that expression of the \u394N construct increased the frequency and the amplitude of spontaneous synaptic events. A similar phenotype was detected in the presence of the E193K variant, suggesting that this mutation behaves as a loss-of-function mutation. A domain-based pulldown approach demonstrated that the LRRK2 N-terminus binds to cytoskeletal (\u3b2-actin and \u3b1-tubulin) and SV (synapsin I) proteins and the E193K substitution alters strength and quality of LRRK2 interactions. The results support a role of the Armadillo domain in interaction with synaptic proteins and suggest that the E193K mutation affects LRRK2 function via perturbation of its physiological network of interactors, resulting in impaired vesicular trafficking. These findings may have important implications for understanding the role of LRRK2 at the synapses and the pathophysiological mechanism for LRRK2-linked disease

Mechanotransduction in human and mouse beta cell lines: reliable models to characterize novel signaling pathways controlling beta cell fate

Background and aims: Attempts to influence \u3b2-cell differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/transduction complexity remain elusive. We recently demonstrated that human islets of Langerhans sense the ECM nanotopography and activate a mechanotransductive pathway, which is essential for preserving long-term \u3b2-cell differentiation and function in vitro. However, human islets of Langerhans are extremely heterogeneous and their availability for research purpose is limited. Therefore, aim of the proposed research was to investigate whether mouse and human \u3b2-cell lines might sense changes innthe ECM topography and might be used as a simplified model to dissect the molecular pathways involved in mechanotransduction. Materials and methods: We used supersonic cluster beam deposition to fabricate nanostructured substrates characterized by a quantitatively controllable ECM-like nanoroughness. Mouse \u3b2TC3 and human 1.1B4 cells were seeded on these substrates and after five days in culture, the activation of the mechanotransductive pathway was verified by means of morphological (super-resolution fluorescence microscopy), functional and proteomic techniques. Results: Quantitative immunofluorescence studies demonstrated that the cell-nanotopography interaction affects the focal adhesion structures (smaller vinculin clusters), the organization of the actin cytoskeleton (shorter actin fiber) and the nuclear architecture. Functional studies revealed that nanostructured surfaces improve the \u3b2-cell mitochondrial activity and increase the glucose-stimulated Ca2+currents and insulin release. Label-free shotgun proteomics broadly confirmed the morphological and functional studies and showed the upregulation of a number of mechanosensors and transcription factors involved in \u3b2-cell differentiation in cells grown on nanostructured substrates compared to those grown on flat standard control surfaces. Conclusion: Our data reveal that mouse and human \u3b2-cell lines sense changes in extracellular mechanical forces and activate a mechanotransductive pathway. The findings from this study will be useful to clarify the link between mechanotransduction and cell fate and to successfully engineer scaffolds in order to have functional beta cells

Proteomic Analysis Reveals a Mitochondrial Remodeling of βTC3 Cells in Response to Nanotopography

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that \u3b2 cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing \u3b2-cell differentiation and function in vitro in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of \u3b2TC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in \u3b2TC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different \u3b2-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs

Malattie virali dell’apparato respiratorio

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Eastern European parents' experiences of parenting a child with SEN in England

Parenting a child with Special Educational Needs (SEN) presents numerous challenges for families. For immigrant parents, these challenges can be particularly difficult to overcome when faced with structural, cultural and linguistic barriers. This qualitative study explored the lived experiences of 8 Eastern European immigrants parenting a child with SEN in England. Semi-structured interviews were conducted, and a data-driven thematic analysis of a series of interviews was carried out. The study identified two key themes: (a) embarking on an unpredicted journey and (b) navigating through challenges. The analyses highlight discrepancies in partnership working between parents and educators and shortcomings in advice that professionals provided to these parents, potentially placing pupils and their families at a disadvantage. The implications for educational psychologists (EP) and other professionals working with Eastern European parents raising a child with SEN are also discussed

Eastern European parents' experiences of parenting a child with SEN in England

Parenting a child with Special Educational Needs (SEN) presents numerous challenges for families. For immigrant parents, these challenges can be particularly difficult to overcome when faced with structural, cultural and linguistic barriers. This qualitative study explored the lived experiences of 8 Eastern European immigrants parenting a child with SEN in England. Semi-structured interviews were conducted, and a data-driven thematic analysis of a series of interviews was carried out. The study identified two key themes: (a) embarking on an unpredicted journey and (b) navigating through challenges. The analyses highlight discrepancies in partnership working between parents and educators and shortcomings in advice that professionals provided to these parents, potentially placing pupils and their families at a disadvantage. The implications for educational psychologists (EP) and other professionals working with Eastern European parents raising a child with SEN are also discussed

Interactions of airway pathogens and inflammatory processes

The clinical history of chronic obstructive pulmonary disease (COPD) is punctuated by recurrent episodes of increases in dyspnea, cough, or sputum production named exacer- bations. In addition to increasing COPD-associated morbidity and mortality, exacerbations contribute to loss of lung function and impaired health status in COPD patients (1). Although it is often assumed that exacerbations are associated with increased airway inflammation, there is little information on the nature of the acute-on-chronic inflammation that characterizes these episodes. Most of the data currently available refer to soluble indirect markers of airway inflammation rather than inflammatory cell infiltration per se (2). Infections of the tracheobronchial tree, together with air pollution, are considered the most common causes of COPD exacerbations (1). Whether different patterns of airway inflammation correspond to different etiologies is largely unknown. Better understanding of these relationships and of the underlying pathophysiological mechanisms would give the opportunity to identify relevant targets (pathogens and inflammation) for the treatment and prevention of COPD exacerbations. Many exacerbations are associated with symptoms of infection of the tracheobron- chial tree, and bacteria have been considered the main infective cause of exacerbations (1). Determining the contribution of bacteria to exacerbations is difficult, as COPD patients are often colonized with bacteria even when clinically stable (3). The proportion of patients with positive bacterial cultures and a high bacterial load increases during exacerbations in most, although not in all, studies (4–6). Newer molecular techniques have recently shown that colonization is not a static condition and there is a frequent turnover of different strains of bacteria evoking specific host responses (7). Thus, it is likely that a change in the strain but not the organism may be responsible for the exacerbations. Therefore, previous studies lacking in the molecular characterization of bacterial strains may have missed evidence of a new infection. Indeed, it has been documented that the acquisition of a new strain of colonizing bacteria increases the risk of an exacerbation (8). In the last few decades, the use of highly sensitive diagnostic methods, such as polymerase chain reaction (PCR), to evaluate the association between respiratory virus infections and COPD exacerbations has shown that viruses are responsible for a much higher proportion of exacerbations than was previously realized. In a study of the East London COPD cohort, respiratory viruses were detected in 39% of exacerbations, the most common being rhinoviruses that accounted for 58% of viruses (9). A respiratory virus was detected in around 50% of patients with severe COPD exacerbation admitted to hospitals in Germany and Italy, with rhinovirus again being the most common (5,10). In patients with very severe COPD exacerbations requiring intubation and mechanical ventilation, viruses were identified in 47% of patients (11). At variance with bacterial infections, the respiratory viruses more commonly found at exacerbations were virtually absent in stable state (5,12), suggesting that they play a relevant role in the etiology of the acute episodes