STRUCTURAL EFFECTS OF VERAPAMIL ON CELL MEMBRANES AND MOLECULAR MODELS

ABSTRACT Verapamil is one of the frequently prescribed calcium channel blockers used in the treatment of hypertension and angina pectoris . Results of evaluations of the therapy have led to reports of toxic effects. This study presents several evidences that verapamil affects human cells. Scanning electron microscopy observations of intact human erythrocytes indicated that they underwent morphological alterations as increasing verapamil concentrations starting from 5 μM changed their discoid normal shape, and finally to hemolysis. Fluorescence spectroscopy on isolated unsealed human erythrocyte membranes confirmed these outcomes. In fact, the assays showed that verapamil induced a significant increase of the anisotropy parameters and a moderate one of the generalized polarization, indicative of enhanced order at the acyl chain and polar head regions of the erythrocyte membrane lipid bilayer. X-ray diffraction experiments on dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine bilayers, classes of the major phospholipids present in both outer and inner sides of the erythrocyte membrane, respectively showed that verapamil perturbed the polar head and acyl chain regions of both lipid bilayers. These interactions were found to be stronger with DMPC bilayers. On the other hand, human SH-SY5Y neuroblastoma cells incubated with verapamil suffered a sharp decrease of cell viability

Bone tissue and the nervous system: what do they have in common?

Degenerative diseases affecting bone tissues and the brain represent important problems with high socio-economic impact. Certain bone diseases, such as osteoporosis, are considered risk factors for the progression of neurological disorders. Often, patients with neurodegenerative diseases have bone fractures or reduced mobility linked to osteoarthritis. The bone is a dynamic tissue involved not only in movement but also in the maintenance of mineral metabolism. Bone is also associated with the generation of both hematopoietic stem cells (HSCs), and thus the generation of the immune system, and mesenchymal stem cells (MSCs). Bone marrow is a lymphoid organ and contains MSCs and HSCs, both of which are involved in brain health via the production of cytokines with endocrine functions. Hence, it seems clear that bone is involved in the regulation of the neuronal system and vice versa. This review summarizes the recent knowledge on the interactions between the nervous system and bone and highlights the importance of the interaction between nerve and bone cells. In addition, experimental models that study the interaction between nerve and skeletal cells are discussed, and innovative models are suggested to better evaluate the molecular interactions between these two cell types

Millifluidic culture improves human midbrain organoid vitality and differentiation

Human midbrain-specific organoids (hMOs) serve as an experimental in vitro model for studying the pathogenesis of Parkinson's disease (PD). In hMOs, neuroepithelial stem cells (NESCs) give rise to functional midbrain dopaminergic (mDA) neurons that are selectively degenerating during PD. A limitation of the hMO model is an under-supply of oxygen and nutrients to the densely packed core region, which leads eventually to a "dead core". To reduce this phenomenon, we applied a millifluidic culture system that ensures media supply by continuous laminar flow. We developed a computational model of oxygen transport and consumption in order to predict oxygen levels within the hMOs. The modelling predicts higher oxygen levels in the hMO core region under millifluidic conditions. In agreement with the computational model, a significantly smaller "dead core" was observed in hMOs cultured in a bioreactor system compared to those ones kept under conventional shaking conditions. Comparing the necrotic core regions in the organoids with those obtained from the model allowed an estimation of the critical oxygen concentration necessary for ensuring cell vitality. Besides the reduced "dead core" size, the differentiation efficiency from NESCs to mDA neurons was elevated in hMOs exposed to medium flow. Increased differentiation involved a metabolic maturation process that was further developed in the millifluidic culture. Overall, bioreactor conditions that improve hMO quality are worth considering in the context of advanced PD modelling

Structural Plasticity of Dopaminergic Neurons Requires the Activation of the D3R-nAChR Heteromer and the PI3K-ERK1/2/Akt-Induced Expression of c-Fos and p70S6K Signaling Pathway

We have previously shown that the heteromer composed by the dopamine D3 receptor (D3R) and the nicotinic acetylcholine receptor (nAChR) (D3R-nAChR heteromer) is expressed in dopaminergic neurons, activated by nicotine and represents the molecular unit that, in these neurons, contributes to the modulation of critical events such as structural plasticity and neuroprotection. We now extended this study by investigating the D3R-nAChR heteromer properties using various cell models such as transfected HEK293 cells, primary cultures of mouse dopaminergic neurons and human dopaminergic neurons derived from induced pluripotent stem cells. We found that the D3R-nAChR heteromer is the molecular effector that transduces the remodeling properties not only associated with nicotine but also with D3R agonist stimulation: neither nAChR nor D3R, in fact, when express as monomers, are able to elicit these effects. Moreover, strong and sustained activation of the PI3K-ERK1/2/Akt pathways is coupled with D3R-nAChR heteromer stimulation, leading to the expression of the immediate-early gene c-Fos and to sustained phosphorylation of cytosolic p70 ribosomal S6 kinase (p70S6K), critical for dendritic remodeling. By contrast, while D3R stimulation results in rapid and transient activation of both Erk1/2 and Akt, that is PI3K-dependent, stimulation of nAChR is associated with persistent activation of Erk1/2 and Akt, in a PI3K-independent way. Thus, the D3R-nAChR heteromer and its ability to trigger the PI3K-ERK1/2/Akt signaling pathways may represent a novel target for preserving dopaminergic neurons healthy and for conferring neuronal protection against injuries

Targeting Human Endothelial Cells with Glutathione and Alanine Increases the Crossing of a Polypeptide Nanocarrier through a Blood–Brain Barrier Model and Entry to Human Brain Organoids

Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood–brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson’s disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain

A Triple Combination of Targeting Ligands Increases the Penetration of Nanoparticles across a Blood-Brain Barrier Culture Model

Nanosized drug delivery systems targeting transporters of the blood-brain barrier (BBB) are promising carriers to enhance the penetration of therapeutics into the brain. The expression of solute carriers (SLC) is high and shows a specific pattern at the BBB. Here we show that targeting ligands ascorbic acid, leucine and glutathione on nanoparticles elevated the uptake of albumin cargo in cultured primary rat brain endothelial cells. Moreover, we demonstrated the ability of the triple-targeted nanovesicles to deliver their cargo into midbrain organoids after crossing the BBB model. The cellular uptake was temperature- and energy-dependent based on metabolic inhibition. The process was decreased by filipin and cytochalasin D, indicating that the cellular uptake of nanoparticles was partially mediated by endocytosis. The uptake of the cargo encapsulated in triple-targeted nanoparticles increased after modification of the negative zeta potential of endothelial cells by treatment with a cationic lipid or after cleaving the glycocalyx with an enzyme. We revealed that targeted nanoparticles elevated plasma membrane fluidity, indicating the fusion of nanovesicles with endothelial cell membranes. Our data indicate that labeling nanoparticles with three different ligands of multiple transporters of brain endothelial cells can promote the transfer and delivery of molecules across the BBB

Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation

peer reviewedMidbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson’s disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity

Impaired serine metabolism complements LRRK2-G2019S pathogenicity in PD patients

Parkinson's disease (PD) is a multifactorial disorder with complex etiology. The most prevalent PD associated mutation, LRRK2-G2019S is linked to familial and sporadic cases. Based on the multitude of genetic predispositions in PD and the incomplete penetrance of LRRK2-G2019S, we hypothesize that modifiers in the patients' genetic background act as susceptibility factors for developing PD. To assess LRRK2-G2019S modifiers, we used human induced pluripotent stem cell-derived neuroepithelial stem cells (NESCs). Isogenic controls distinguish between LRRK2-G2019S dependent and independent cellular phenotypes. LRRK2-G2019S patient and healthy mutagenized lines showed altered NESC self-renewal and viability, as well as impaired serine metabolism. In patient cells, phenotypes were only partly LRRK2-G2019S dependent, suggesting a significant contribution of the genetic background. In this context we identified the gene serine racemase (SRR) as a novel patient-specific, developmental, genetic modifier contributing to the aberrant phenotypes. Its enzymatic product, n-serine, rescued altered cellular phenotypes. Susceptibility factors in the genetic background, such as SRR, could be new targets for early PD diagnosis and treatment.Analytical BioScience

Experimental approaches in vitro and in vivo for the pathogenic understanding of Alzheimer's Disease: potential role of metal ions

The etiopathogenesis of Alzheimer’s disease (AD) is far from being clearly understood. However, the involvement of metal ions as a potential key factor to-wards conformational modifications and aggregation of beta-amyloid peptide is widely recognized. The aim of this thesis was to investigate the potential and differ-ential role of metal ions (aluminum, copper, iron and zinc) in affecting the fibril-logenesis of beta-amyloid. Data herein reported demonstrated that the aggregational profile of the beta-amyloid peptide was greatly different according to the metal bound. Particularly, aluminum was the most effective, among the tested metals, in promoting a conforma-tional modification of beta-amyloid which resulted in an increased aggregation rate and ex-pousure of hydrophobic clusters. This modification stabilized the peptide in an oli-gomeric state which is higly toxic to neurons. Furthemore, the peculiar hydrophobic conformation of beta-amyloid-aluminum complex was markedly enhanced in the presence of the beta-amyloid 17-28 fragment with a consequent increase of deleterious effects on cell culture. Moreover, the geno-toxic role of beta-amyloid and beta-amyloid-metal complexes was investigated on neuroblastoma cell culture as a model for the understanding of AD ethiopatho-genesis. In collaboration with the "G. Rossi" hospital (VR), several genetic alterations determined by the beta-amyloid-metal complexes have been correlated with the genetic expression pattern of pheripheral blood samples from AD patients, with the aim of identifying potential diagnostic biomarkers. The most promising data was obtained for the gene which encodes for glutaminyl cyclase. According to the recent literature, this enzyme could be involved in the pathology, even if the mechanism is still elusive, and a significant overexpression of this gene was indeed found in AD patients compared to age-match controls. Additionally, the effect of a 3 month copper-deficient diet on the distribution of several metal ions (aluminum, calcium, copper, iron and zinc) in organs and tissues of adult mice was uncovered. The study highlighted that the copper-deficient diet, besides being effectively in determining copper decrease especially in the frontal area and liver, was also able to determine a decrease in the concentration of the other tested metals, creating a sort of domino effect which detrimentally altered the general metal homeostasis. This aspect has to be taken into account especially when the use of chelating compounds, aiming at restoring the correct metal homeostasis in the brain, is proposed as therapeutic approach for AD.Il mio lavoro di tesi ha riguardato lo studio dell’implicazione di alcuni metal-loioni come promotori del misfolding del peptide amiloidogenico beta-amiloide, che sembra essere coinvolto nella patogenesi del morbo di Alzheimer (AD). I risultati ottenuti dimostrano come la conformazione del peptide beta-amiloide cambi in funzione del metallo ad esso legato (alluminio, rame, zinco e ferro). In particolare, lo studio ha chiaramente messo in luce come tra i vari ioni metallici testati, l’alluminio sia il catione più efficiente nel promuovere l’aumento sia dell’idrofobicità superficiale che dell’aggregazione del peptide in vitro. Queste modificazioni si riflettono in un notevole aumento della neurotossicità di tale complesso sia rispetto al solo peptide che agli altri complessi metallici. La conformazione ed i diversi effetti biologici sono quindi da attribuirsi unicamente al diverso tipo di aggregato formato in seguito al le-game del peptide con i singoli metalli. Si è inoltre confrontata la propensione a for-mare aggregati dei complessi di beta-amiloide-metalli in presenza ed in assenza del frammento beta-amiloide 17-28. I dati ottenuti indicano una maggiore idrofobicità e capacità di aggregazione del complesso beta-amiloide-alluminio in presenza del frammento beta-amiloide 17-28, caratteristiche che determinano una maggiore tossicità rispetto a tutti gli altri complessi testati. Si è approfondito inoltre il ruolo geno-tossico dei complessi beta-amiloide-ioni metallici su colture cellulari di neuroblastoma umano, come modelli per la comprensione dell’eziopatogenesi molecolare di AD. Le alterazioni genetiche prodotte da tali com-plessi sono state correlate con il pattern di espressione genica di campioni di sangue periferico di pazienti AD selezionati dall’ospedale “G. Rossi” di Verona al fine di identificare test potenzialmente prognostici da sperimentare sulla definizione clinica della patologia. Il dato ottenuto più interessante è relativo alla glutaminil ciclasi, en-zima che potrebbe essere coinvolto nella patologia ed il cui gene è stato visto essere sovraespresso nel sangue periferico di soggetti malati di AD. Complessivamente, i dati sperimentali finora ottenuti fanno quindi supporre un possibile coinvolgimento dell’alluminio, complessato con beta-amiloide, nel processo eziopatogenico dell’AD. È stato infine condotto uno studio sull’effetto della carenza di ra-me su topi adulti che ha messo in evidenza come la rimozione di un metallo essenziale dalla dieta per un periodo di tre mesi determini, a livello cerebrale, una variazione significativa nella concentrazione non solo del rame ma anche di altri metalli essen-ziali quali ferro, zinco e non-fisiologici come l’alluminio. Questo a sottolineare come esista una stretta correlazione tra i diversi sistemi di regolazione nell’omeostasi degli ioni metallici. Tale aspetto è di fondamentale importanza per la progettazione di te-rapie chelanti che mirino a rimuovere depositi anomali di ioni metallici che potrebbero stimolare la patologica aggregazione del peptide beta-amiloide