Using integrated meta-omics to appreciate the role of the gut microbiota in epilepsy.

The way the human microbiota may modulate neurological pathologies is a fascinating matter of research. Epilepsy is a common neurological disorder, which has been largely investigated in correlation with microbiota health and function. However, the mechanisms that regulate this apparent connection are scarcely defined, and extensive effort has been conducted to understand the role of microbiota in preventing and reducing epileptic seizures. Intestinal bacteria seem to modulate the seizure frequency mainly by releasing neurotransmitters and inflammatory mediators. In order to elucidate the complex microbial contribution to epilepsy pathophysiology, integrated meta-omics could be pivotal. In fact, the combination of two or more meta-omics approaches allows a multifactorial study of microbial activity within the frame of disease or drug treatments. In this review, we provide information depicting and supporting the use of multi-omics to study the microbiota-epilepsy connection. We described different meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics), focusing on current technical challenges in stool collection procedures, sample extraction methods and data processing. We further discussed the current advantages and limitations of using the integrative approach of multi-omics in epilepsy investigations.

Technological tools and strategies for culturing human gut microbiota in engineered in vitro models.

The gut microbiota directly impacts the pathophysiology of different human body districts. Consequently, microbiota investigation is an hot topic of research and its in vitro culture has gained extreme interest in different fields. However, the high sensitivity of microbiota to external stimuli, such as sampling procedure, and the physicochemical complexity of the gut environment make its in vitro culture a challenging task. New engineered microfluidic gut-on-a-chip devices have the potential to model some important features of the intestinal structure, but they are usually unable to sustain culture of microbiota over an extended period of time. The integration of gut-on-a-chip devices with bioreactors for continuous bacterial culture would lead to fast advances in the study of microbiota-host crosstalk. In this review, we summarize the main technologies for the continuous culture of microbiota as upstream systems to be coupled with microfluidic devices to study bacteria-host cells communication. The engineering of integrated microfluidic platforms, capable of sustaining both anaerobic and aerobic cultures, would be the starting point to unveil complex biological phenomena proper of the microbiota-host crosstalks, paving to way to multiple research and technological applications.

Increased transcription of transglutaminase 1 mediates neuronal death in in vitro models of neuronal stress and Aß1-42-mediated toxicity.

Alzheimer's disease (AD) is the most common cause of dementia. At the pre-symptomatic phase of the disease, the processing of the amyloid precursor protein (APP) produces toxic peptides, called amyloid-ß 1-42 (Aß 1-42). The downstream effects of Aß 1-42 production are not completely uncovered. Here, we report the involvement of transglutaminase 1 (TG1) in in vitro AD models of neuronal toxicity. TG1 was increased at late stages of the disease in the hippocampus of a mouse model of AD and in primary cortical neurons undergoing stress. Silencing of TGM1 gene was sufficient to prevent Aß-mediated neuronal death. Conversely, its overexpression enhanced cell death. TGM1 upregulation was mediated at the transcriptional level by an activator protein 1 (AP1) binding site that when mutated halted TGM1 promoter activation. These results indicate that TG1 acts downstream of Aß-toxicity, and that its stress-dependent increase makes it suitable for pharmacological intervention.

Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture.

Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration.

Unravelling the mechanotransduction pathways in Alzheimer's disease.

Alzheimer's disease (AD) represents one of the most common and debilitating neurodegenerative disorders. By the end of 2040, AD patients might reach 11.2 million in the USA, around 70% higher than 2022, with severe consequences on the society. As now, we still need research to find effective methods to treat AD. Most studies focused on the tau and amyloid hypothesis, but many other factors are likely involved in the pathophysiology of AD. In this review, we summarize scientific evidence dealing with the mechanotransduction players in AD to highlight the most relevant mechano-responsive elements that play a role in AD pathophysiology. We focused on the AD-related role of extracellular matrix (ECM), nuclear lamina, nuclear transport and synaptic activity. The literature supports that ECM alteration causes the lamin A increment in the AD patients, leading to the formation of nuclear blebs and invaginations. Nuclear blebs have consequences on the nuclear pore complexes, impairing nucleo-cytoplasmic transport. This may result in tau hyperphosphorylation and its consequent self-aggregation in tangles, which impairs the neurotransmitters transport. It all exacerbates in synaptic transmission impairment, leading to the characteristic AD patient's memory loss. Here we related for the first time all the evidence associating the mechanotransduction pathway with neurons. In addition, we highlighted the entire pathway influencing neurodegenerative diseases, paving the way for new research perspectives in the context of AD and related pathologies.

Development of an Induced Pluripotent Stem Cell-Based Liver-on-a-Chip Assessed with an Alzheimer's Disease Drug.

Liver-related drug metabolism is a key aspect of pharmacokinetics and possible toxicity. From this perspective, the availability of advanced in vitro models for drug testing is still an open need, also to the end of reducing the burden of in vivo experiments. In this scenario, organ-on-a-chip is gaining attention as it couples a state-of-the art in vitro approach to the recapitulation of key in vivo physiological features such as fluidodynamics and a tri-dimensional cytoarchitecture. We implemented a novel liver-on-a-chip (LoC) device based on an innovative dynamic device (MINERVA 2.0) where functional hepatocytes (iHep) have been encapsulated into a 3D hydrogel matrix interfaced through a porous membrane with endothelial cells (iEndo)]. Both lines were derived from human-induced pluripotent stem cells (iPSCs), and the LoC was functionally assessed with donepezil, a drug approved for Alzheimer's disease therapy. The presence of iEndo and a 3D microenvironment enhanced the expression of liver-specific physiologic functions as in iHep, after 7 day perfusion, we noticed an increase of albumin, urea production, and cytochrome CYP3A4 expression compared to the iHep static culture. In particular, for donepezil kinetics, a computational fluid dynamic study conducted to assess the amount of donepezil diffused into the LoC indicated that the molecule should be able to pass through the iEndo and reach the target iHep construct. Then, we performed experiments of donepezil kinetics that confirmed the numerical simulations. Overall, our iPSC-based LoC reproduced the in vivo physiological microenvironment of the liver and was suitable for potential hepatotoxic screening studies.

Human gut epithelium features recapitulated in MINERVA 2.0 millifluidic organ-on-a-chip device.

We developed an innovative millifluidic organ-on-a-chip device, named MINERVA 2.0, that is optically accessible and suitable to serial connection. In the present work, we evaluated MINERVA 2.0 as millifluidic gut epithelium-on-a-chip by using computational modeling and biological assessment. We also tested MINERVA 2.0 in a serially connected configuration prodromal to address the complexity of multiorgan interaction. Once cultured under perfusion in our device, human gut immortalized Caco-2 epithelial cells were able to survive at least up to 7 days and form a three-dimensional layer with detectable tight junctions (occludin and zonulin-1 positive). Functional layer development was supported by measurable trans-epithelial resistance and FITC-dextran permeability regulation, together with mucin-2 expression. The dynamic culturing led to a specific transcriptomic profile, assessed by RNASeq, with a total of 524 dysregulated transcripts (191 upregulated and 333 downregulated) between static and dynamic condition. Overall, the collected results suggest that our gut-on-a-chip millifluidic model displays key gut epithelium features and, thanks to its modular design, may be the basis to build a customizable multiorgan-on-a-chip platform.

3D photopolymerized microstructured scaffolds influence nuclear deformation, nucleo/cytoskeletal protein organization, and gene regulation in mesenchymal stem cells.

Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications.

Microbiota-Host Immunity Communication in Neurodegenerative Disorders: Bioengineering Challenges for In Vitro Modeling.

Human microbiota communicates with its host by secreting signaling metabolites, enzymes, or structural components. Its homeostasis strongly influences the modulation of human tissue barriers and immune system. Dysbiosis-induced peripheral immunity response can propagate bacterial and pro-inflammatory signals to the whole body, including the brain. This immune-mediated communication may contribute to several neurodegenerative disorders, as Alzheimer's disease. In fact, neurodegeneration is associated with dysbiosis and neuroinflammation. The interplay between the microbial communities and the brain is complex and bidirectional, and a great deal of interest is emerging to define the exact mechanisms. This review focuses on microbiota-immunity-central nervous system (CNS) communication and shows how gut and oral microbiota populations trigger immune cells, propagating inflammation from the periphery to the cerebral parenchyma, thus contributing to the onset and progression of neurodegeneration. Moreover, an overview of the technological challenges with in vitro modeling of the microbiota-immunity-CNS axis, offering interesting technological hints about the most advanced solutions and current technologies is provided.

The nuclear import of the transcription factor MyoD is reduced in mesenchymal stem cells grown in a 3D micro-engineered niche.

Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.

Advantages and limitations of a supernegative GFP in facilitating MyoD intracellular tracking.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (-30)GFP, to a myogenic transcription factor, MyoD. The (-30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (-30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (-30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

Mechanical regulation of nucleocytoplasmic translocation in mesenchymal stem cells: characterization and methods for investigation.

Mesenchymal stem cells (MSCs) have immune-modulatory and tissue-regenerative properties that make them a suitable and promising tool for cell-based therapy application. Since the bio-chemo-mechanical environment influences MSC fate and behavior, the understanding of the mechanosensors involved in the transduction of mechanical inputs into chemical signals could be pivotal. In this context, the nuclear pore complex is a molecular machinery that is believed to have a key role in force transmission and in nucleocytoplasmic shuttling regulation. To fully understand the nuclear pore complex role and the nucleocytoplasmic transport dynamics, recent advancements in fluorescence microscopy provided the possibility to study passive and facilitated nuclear transports also in mechanically stimulated cell culture conditions. Here, we review the current available methods for the investigation of nucleocytoplasmic shuttling, including photo-perturbation-based approaches, fluorescence correlation spectroscopy, and single-particle tracking techniques. For each method, we analyze the advantages, disadvantages, and technical limitations. Finally, we summarize the recent knowledge on mechanical regulation of nucleocytoplasmic translocation in MSC, the relevant progresses made so far, and the future perspectives in the field.

Advanced Organ-on-a-Chip Devices to Investigate Liver Multi-Organ Communication: Focus on Gut, Microbiota and Brain.

The liver is a key organ that can communicate with many other districts of the human body. In the last few decades, much interest has focused on the interaction between the liver and the gut microbiota, with their reciprocal influence on biosynthesis pathways and the integrity the intestinal epithelial barrier. Dysbiosis or liver disorders lead to0 epithelial barrier dysfunction, altering membrane permeability to toxins. Clinical and experimental evidence shows that the permeability hence the delivery of neurotoxins such as LPS, ammonia and salsolinol contribute to neurological disorders. These findings suggested multi-organ communication between the gut microbiota, the liver and the brain. With a view to in vitro modeling this liver-based multi-organ communication, we describe the latest advanced liver-on-a-chip devices and discuss the need for new organ-on-a-chip platforms for in vitro modeling the in vivo multi-organ connection pathways in physiological and pathological situations.

Plasma Aß42 as a Biomarker of Prodromal Alzheimer's Disease Progression in Patients with Amnestic Mild Cognitive Impairment: Evidence from the PharmaCog/E-ADNI Study.

It is an open issue whether blood biomarkers serve to diagnose Alzheimer's disease (AD) or monitor its progression over time from prodromal stages. Here, we addressed this question starting from data of the European FP7 IMI-PharmaCog/E-ADNI longitudinal study in amnesic mild cognitive impairment (aMCI) patients including biological, clinical, neuropsychological (e.g., ADAS-Cog13), neuroimaging, and electroencephalographic measures. PharmaCog/E-ADNI patients were classified as "positive" (i.e., "prodromal AD" n = 76) or "negative" (n = 52) based on a diagnostic cut-off of Aß42/P-tau in cerebrospinal fluid as well as APOE ε 4 genotype. Blood was sampled at baseline and at two follow-ups (12 and 18 months), when plasma amyloid peptide 42 and 40 (Aß42, Aß40) and apolipoprotein J (clusterin, CLU) were assessed. Linear Mixed Models found no significant differences in plasma molecules between the "positive" (i.e., prodromal AD) and "negative" groups at baseline. In contrast, plasma Aß42 showed a greater reduction over time in the prodromal AD than the "negative" aMCI group (p = 0.048), while CLU and Aß40 increased, but similarly in the two groups. Furthermore, plasma Aß42 correlated with the ADAS-Cog13 score both in aMCI patients as a whole and the prodromal AD group alone. Finally, CLU correlated with the ADAS-Cog13 only in the whole aMCI group, and no association with ADAS-Cog13 was found for Aß40. In conclusion, plasma Aß42 showed disease progression-related features in aMCI patients with prodromal AD.

Exome sequencing in an Italian family with Alzheimer's disease points to a role for seizure-related gene 6 (SEZ6) rare variant R615H.

BACKGROUND: The typical familial form of Alzheimer's disease (FAD) accounts for about 5% of total Alzheimer's disease (AD) cases. Presenilins (PSEN1 and PSEN2) and amyloid-ß (A4) precursor protein (APP) genes carry all reported FAD-linked mutations. However, other genetic loci may be involved in AD. For instance, seizure-related gene 6 (SEZ6) has been reported in brain development and psychiatric disorders and is differentially expressed in the cerebrospinal fluid of AD cases. METHODS: We describe a targeted exome sequencing analysis of a large Italian kindred with AD, negative for PSEN and APP variants, that indicated the SEZ6 heterozygous mutation R615H is associated with the pathology. RESULTS: We overexpressed R615H mutation in H4-SW cells, finding a reduction of amyloid peptide Aß(1-42). Sez6 expression decreased with age in a mouse model of AD (3xTG-AD), but independently from transgene expression. CONCLUSIONS: These results support a role of exome sequencing for disease-associated variant discovery and reinforce available data on SEZ6 in AD models.

Secretome released from hydrogel-embedded adipose mesenchymal stem cells protects against the Parkinson's disease related toxin 6-hydroxydopamine.

Neurodegenerative diseases, as Parkinson's disease (PD), involve irreversible neural cell damage and impairment. In PD, there is a selective degeneration of the dopaminergic neurons leading to motor symptoms. A common finding in PD neurodegeneration is the increase of reactive oxygen species (ROS), leading to oxidative stress. To date there are only interventions to relieve PD symptoms, however progress has been made in the development of therapies that target the immune system or use its components as therapeutic agents; among these, mesenchymal stem cells (MSCs), which are able to express neuroprotective factors as cytokines, chemokines and angiogenic molecules, collectively named secretome, that accumulate in MSC culture medium. However, lasting cell-free administration of secretome in vitro or in vivo is challenging. We used the conditioned media from rat adipose tissue-derived MSCs (RAA-MSCs) to check for neuroprotective activity towards pro-oxidizing agents such as hydrogen peroxide (H2O2) or the dopaminergic selective toxin 6-hydroxydopamine (6-OHDA) that is commonly used to model PD neurodegeneration. When neuroblastoma SH-SY5Y cells were pre-conditioned with 100% RAA-MSC media, then treated with H2O2 and 6-OHDA, mortality and ROS generation were reduced. We implemented the controlled release of RAA-MSC secretome from injectable biodegradable hydrogels that offer a possible in situ implant with mini-invasive techniques. The hydrogels were composed of type I bovine collagen (COLL) and low-molecular-weight hyaluronic acid (LMWHA) or COLL and polyethylene glycol (PEG). Hydrogels were suitable for RAA-MSC embedding up to 48h and secretome from these RAA-MSCs was active and counteracted 6-OHDA toxicity, with upregulation of the antioxidant enzyme sirtuin 3 (SIRT3). These results support a biomaterials-based approach for controlled delivery of MSC-produced neuroprotective factors in a PD-relevant experimental context.

The SIRT1 promoter polymorphic site rs12778366 increases IL-6 related human mortality in the prospective study "Treviso Longeva (TRELONG)".

Studies on sirtuins (SIRT), a family of proteins with deacetylase activity, have provided convergent evidence of the key role of these enzymes in aging-linked physiological functions. The link between SIRT1 and longevity has emerged in model organism but few data are available in humans, in particular relying on longitudinal studies. Here, we assessed whether a genetic variant within SIRT1 gene promoter (rs12778366) was associated to human longevity. We analyzed 586 genomic DNA (gDNA) collected in the study "Treviso Longeva" (TRELONG), including elderly over 70 years of age from the municipality of Treviso, a town in the Northeast of Italy, with a 11-year follow-up. We genotyped SIRT1 rs12778366 by real-time polymerase chain reaction (RT-PCR) allelic discrimination assay. A cross-sectional analysis performed by comparing people over and under 85 years of age did not evidence association between rs12778366 and longevity. When we performed a longitudinal analysis considering mortality as dependent variable, we did not observe an association of rs12778366 with longevity in the whole population (corrected P-value = 0.33). However, when we stratified the TRELONG subjects according to circulating level of interleukin-6 (IL-6), a predictor of disability and mortality, we found that rs12778366 (TC+CC) carriers were at increased risk of mortality in comparison to the TT reference group (corrected P-value = 0.03, HR 1.47). Our data do not support a major role of rs12778366 in human longevity, but the stratified analysis on IL-6 suggests that this variant may be involved in the detrimental effect of high circulating IL-6 in the elderly.