Prion protein involvement in Reelin/Dab1 signalling: physiological and pathological aspects

Cellular prion protein (PrPC) has been widely investigated since its misfolded isoform, scrapie prion protein (PrPSc), which form aggregates that accumulate in the brain, causes prion diseases. The pathogenesis of prion diseases is currently under debate as they could be gain of toxic function consequences of the formation of PrPSc, or could be due to the loss of the normal physiological function of PrPC. For this reason, a better definition of the exact physiological function of PrPC represents a critical challenge since Prnp knockout mice lack striking phenotype. PrPC is highly abundant in the nervous system where it plays an important role in modulating cell signalling, acting as a dynamic scaffold for the assembly of many different signalling molecules at the neuronal surface. Importantly for the present study, it has been reported that PrPC is able to modulate Fyn, PI3K, AKT/PKB and GSK3\u3b2 activity. All these intracellular kinases could be also regulated by yet another protein, Reelin, an extracellular matrix glycoprotein that activates a linear downstream signalling pathway. Interestingly, it has been reported that Reelin-signalling pathway is involved in both Alzheimer\u2019s and prion diseases. Indeed, the intracellular adaptor protein Dab1 is able to affect APP processing and intracellular trafficking and to influence amyloid beta deposition in sporadic Creutzfeldt\u2013Jakob disease. In the present study, taking advantage of different mouse models, we analysed the functional interaction between the prion protein and Dab1 signalling cascade from physiological and pathological point of view. In the first part of the project, using a Prnp knockout mouse model, we addressed the question whether PrPC could modulate the Reelin-signalling cascade. If this is the case, we could expect impairments in Reelin downstream signalling when PrPC expression is ablated. Indeed, the expression level and the activation state of Dab1 were evaluated in wild-type (Prnp+/+) and PrPC-null (Prnp0/0) mice brain and primary neuronal cultures. Reelin-conditioned medium, obtained from stably transfected HEK293T cells, was used to stimulate Dab1 activation in vitro and analyse a putative differential activation between the two genotypes. Moreover, recombinant PrP and fusion PrP-hFc proteins were applied in the same system to evaluate the ability of PrPC to stimulate directly the signalling cascade. Dab1 protein resulted upregulated in Prnp0/0 brain in comparison to wild-type animals, with no changes in mdab1 gene transcription levels. Moreover, Prnp0/0 neurons appeared less responsive to Reelin stimulation than controls, leading to reduced phosphorylation of Dab1. Unfortunately, in our conditions, neither recombinant PrP nor PrP-hFc fusion protein were able to induce Dab1 phosphorylation, suggesting a modulating role, instead of a direct effect, of PrPC on the pathway. Expression analysis of different component of the pathway revealed no statistical differences between the two genotypes, with the exception of Fyn kinase, which resulted less phosphorylated in Prnp0/0 mice compared to wild-type controls. Immunoprecipitation experiments failed to unravel an interaction between PrPC and Reelin or its receptor ApoER2, suggesting that impairments in Dab1 expression and phosphorylation are not due to a direct coupling between these proteins at the cell surface. Interestingly, in Prnp0/0 neurons, reduced levels of NCAM protein, a known PrPC interactor, were detected. In the second part of the project, taking advantage of different mouse models of Reelin pathway disruption, we addressed the opposite question to understand whether alteration of the Reelin-signalling pathway could affect the expression of PrPC. However, PrPC expression levels did not resulted significantly different in comparison to their respective controls in none of the genotypes analysed. These results suggest that, while PrPC is able to modulate Reelin-signalling cascade and in particular Dab1 activation state, alterations of this pathway do not affect PrPC expression. Finally, terminally sick mice intracerebrally inoculated with RML prion strain were used as prion pathology model. Interestingly, in terminal stages of prion infection, Dab1 and Reelin receptors signals are no longer detectable. NCAM expression level seems not affected by prion infection, while Fyn kinase and AKT levels are reduced by 30% and 50% respectively. Total Reelin amount is not modified by prion infection, while full-length protein is reduced by almost 50% in RML-infected samples compared to controls with a concomitant 2-fold increase of both cleavage products. Taken together, these findings suggest the existence of a functional interplay between prion protein and Dab1 signalling. Results obtained using PrPC-null mice as model of loss-of-function indicate a role for PrPC in promoting Dab1 signalling. However, as Reelin and its receptors are not affected by Prnp ablation, more likely PrPC indirectly modulate Dab1 signalling through NCAM and Fyn pathway. Interestingly, Dab1 signalling resulted completely abolished in RML-inoculated terminally sick mice, used as prion disease model, together with strong alteration in Reelin processing, already reported in Alzheimer\u2019s disease patients. Further experiments are in progress in order to clarify the mechanisms by which PrPC and PrPSc modulates Dab1 signalling pathway

Autophagy and Inflammasome Activation in Dilated Cardiomyopathy

Background: The clinical outcome of patients affected by dilated cardiomyopathy (DCM) is heterogeneous, since its pathophysiology is only partially understood. Interleukin 1 beta levels could predict the mortality and necessity of cardiac transplantation of DCM patients. Objective: To investigate mechanisms triggering sterile inflammation in dilated cardiomyopathy (DCM). Methods: Hearts explanted from 62 DCM patients were compared with 30 controls, employing immunohistochemistry, cellular and molecular biology, as well as metabolomics studies. Results: Although misfolded protein accumulation and aggresome formation characterize DCM hearts, aggresomes failed to trigger the autophagy lysosomal pathway (ALP), with consequent accumulation of both p62(SQSTM1) and dysfunctional mitochondria. In line, DCM hearts are characterized by accumulation of lipoperoxidation products and activation of both redox responsive pathways and inflammasome. Consistently with the fact that mTOR signaling may impair ALP, we observed, an increase in DCM activation, together with a reduction in the nuclear localization of Transcription Factor EB -TFEB- (a master regulator of lysosomal biogenesis). These alterations were coupled with metabolomic alterations, including accumulation of branched chain amino acids (BCAAs), known mTOR activators. Consistently, reduced levels of PP2Cm, a phosphatase that regulates the key catabolic step of BCAAs, coupled with increased levels of miR-22, a regulator of PP2Cm levels that triggers senescence, characterize DCM hearts. The same molecular defects were present in clinically relevant cells isolated from DCM hearts, but they could be reverted by downregulating miR-22. Conclusion: We identified, in human DCM, a complex series of events whose key players are miR-22, PP2Cm, BCAA, mTOR, and ALP, linking loss of proteostasis with inflammasome activation. These potential therapeutic targets deserve to be further investigated

The longevity-associated BPIFB4 gene supports cardiac function and vascularization in ageing cardiomyopathy

Aims The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene–environment interaction. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector improves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV-BPIFB4 gene could address the unmet therapeutic need to delay the heart’s spontaneous ageing. Methods and results Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supplementation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. Conclusions We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart’s ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people

Cell Senescence in Cardiac Repair and Failure

Although the lack of a robust cardiomyocyte proliferative response has been considered to be a crucial determinant of cardiac pathology and Heart Failure in adult mammalians, the emerging picture is that myocardial regeneration is a complex phenotype involving many actors, including acute cellular senescence and inflammation. However, three major and interconnected events occur in response to tissue injury: loss of protein homeostasis, accumulation of dysfunctional mitochondria and chronic inflammation. These events blunt the reparative response of the heart, are associated with the accumulation of chronically senescent cells and progressively lead to cardiac dysfunction. Therefore, it is crucial to understand which are the pivotal players of this process, in order to devise strategies aimed at reducing the occurrence of chronic cell senescence in the heart in vivo

The Longevity-Associated Variant of BPIFB4 Reduces Senescence in Glioma Cells and in Patients’ Lymphocytes Favoring Chemotherapy Efficacy

Glioblastoma (GBM) is the most common primary brain cancer with the median age at diagnosis around 64 years, thus pointing to aging as an important risk factor. Indeed, aging, by increasing the senescence burden, is configured as a negative prognostic factor for GBM stage. Furthermore, several anti-GBM therapies exist, such as temozolomide (TMZ) and etoposide (ETP), that unfortunately trigger senescence and the secretion of proinflammatory senescence-associated secretory phenotype (SASP) factors that are responsible for the improper burst of (i) tumorigenesis, (ii) cancer metastasis, (iii) immunosuppression, and (iv) tissue dysfunction. Thus, adjuvant therapies that limit senescence are urgently needed. The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) gene previously demonstrated a modulatory activity in restoring age-related immune dysfunction and in balancing the low-grade inflammatory status of elderly people. Based on the above findings, we tested LAV-BPIFB4 senotherapeutic effects on senescent glioblastoma U87-MG cells and on T cells from GBM patients. We interrogated SA-β-gal and HLA-E senescence markers, SASP factors, and proliferation and apoptosis assays. The results highlighted a LAV-BPIFB4 remodeling of the senescent phenotype of GBM cells, enhancement of their sensitivity to temozolomide and a selective reduction of the T cells’ senescence from GBM patients. Overall, these findings candidate LAV-BPIFB4 as an adjuvant therapy for GBM

Data set related to the article: "The longevity-associated BPIFB4 gene supports cardiac function and vascularization in ageing cardiomyopathy"

Aims The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene–environment inter- action. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector im- proves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV- BPIFB4 gene could address the unmet therapeutic need to delay the heart’s spontaneous ageing. Methods and results Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supple- mentation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. Conclusions We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart’s ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people

Data set related to the article: "The longevity-associated BPIFB4 gene supports cardiac function and vascularization in ageing cardiomyopathy"

Aims The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene–environment inter- action. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector im- proves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV- BPIFB4 gene could address the unmet therapeutic need to delay the heart’s spontaneous ageing. Methods and results Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supple- mentation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. Conclusions We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart’s ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people

Onasemnogene abeparvovec in spinal muscular atrophy: predictors of efficacy and safety in naïve patients with spinal muscular atrophy and following switch from other therapiesResearch in context

Summary: Background: Efficacy and safety of onasemnogene abeparvovec (OA) for Spinal Muscular Atrophy infants under 7 months and <8.5 kg has been reported in clinical trials. This study examines efficacy and safety predictors in a wide age (22 days–72 months) and weight (3.2–17 kg) range, also including patients previously treated with other drugs. Methods: 46 patients were treated for 12 months between January 2020 and March 2022. Safety profile was also available for another 21 patients with at least 6 month follow-up after OA infusion. 19/67 were treatment naïve when treated with OA. Motor function was measured with the CHOP-INTEND. Findings: CHOP-INTEND changes varied among age groups. Baseline score and age at OA treatment best predicted changes. A mixed model post-hoc analysis showed that in patients treated before the age of 24 months the CHOP-INTEND changes were already significant 3 months after OA while in those treated after the age of 24 months the difference was only significant 12 months after OA. Adverse events occurred in 51/67. The risk for elevated transaminases serum levels was higher in older patients. This was also true for weight and for pre-treatment with nusinersen when analysed individually. A binomial negative regression analysis showed that only age at OA treatment had a significant effect on the risk of elevated transaminases. Interpretation: Our paper describes OA 12-month follow-up showing efficacy across various age and weight groups not targeted by clinical trials. The study identifies prognostic factors for safety and efficacy in treatment selection. Funding: None