Induced pluripotent stem cell-based organ-on-a-chip as personalized drug screening tools: A focus on neurodegenerative disorders

The Organ-on-a-Chip (OoC) technology shows great potential to revolutionize the drugs development pipeline by mimicking the physiological environment and functions of human organs. The translational value of OoC is further enhanced when combined with patient-specific induced pluripotent stem cells (iPSCs) to develop more realistic disease models, paving the way for the development of a new generation of patient-on-a-chip devices. iPSCs differentiation capacity leads to invaluable improvements in personalized medicine. Moreover, the connection of single-OoC into multi-OoC or body-on-a-chip allows to investigate drug pharmacodynamic and pharmacokinetics through the study of multi-organs cross-talks. The need of a breakthrough thanks to this technology is particularly relevant within the field of neurodegenerative diseases, where the number of patients is increasing and the successful rate in drug discovery is worryingly low. In this review we discuss current iPSC-based OoC as drug screening models and their implication in development of new therapies for neurodegenerative disorders

The Parkinson's disease-related protein DJ-1 protects dopaminergic neurons in vivo and cultured cells from alpha-synuclein and 6-hydroxydopamine toxicity

BACKGROUND Dopaminergic degeneration is a major finding in brains of patients with Parkinson's disease (PD), together with Lewy bodies, intraneuronal inclusions mainly composed of the fibrillogenic protein \textgreeka-synuclein (\textgreeka-syn). The familial-PD-related protein DJ-1 was reported to reduce dopaminergic degeneration triggered by \textgreeka-syn or by the dopaminergic-selective neurotoxin 6-hydroxydopamine (6-OHDA). OBJECTIVE The aim was to further investigate the role of DJ-1 in dopaminergic degeneration and to see whether a cell-permeable recombinant form of DJ-1 (TAT-DJ-1) could restore dopamine depletion in vivo, thus representing an innovative therapeutic approach. METHODS We developed in vitro (PC12/TetOn cells and mouse primary mesencephalic neurons) and in vivo models including DJ-1 knockout (-/-) mice to investigate DJ-1 in dopaminergic degeneration. RESULTS We found that in PC12/TetOn cells overexpressing \textgreek{a}-syn with the familial-PD linked mutation A30P, DJ-1 silencing increased \textgreek{a}-syn (A30P) toxicity. Primary mesencephalic neurons from DJ-1 (-/-) mice were more vulnerable to a cell-permeable form of \textgreek{a}-syn (TAT-\textgreek{a}-syn) and to 6-OHDA. Intrastriatally administered TAT-DJ-1 reduced 6-OHDA toxicity in vivo in C57BL/6 mice. Finally, when we injected TAT-\textgreek{a}-syn (A30P) in the striatum of DJ-1 (-/-) animals, dopamine was depleted more than in the control strain. CONCLUSION DJ-1 appears to have a protective role against dopaminergic degeneration triggered by \textgreek{a}-syn or 6-OHDA, reinforcing the possible therapeutic importance of this protein in PD

Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases

COVID-19 MORTALITY IN LOMBARDY: THE VULNERABILITY OF THE OLDEST OLD AND THE RESILIENCE OF MALE CENTENARIANS

Italy was the first European nation to be affected by COVID-19. The biggest cluster of cases occurred in Lombardy, the most populous Italian region, and elderly men were the population hit in the hardest way. Besides its high infectivity, COVID-19 causes a severe cytokine storm and old people, especially those with comorbidities, appear to be the most vulnerable, presumably in connection to inflammaging. In centenarians inflammaging is much lower than predicted by their chronological age and females, presenting survival advantage in almost all centenarian populations, outnumber males, a phenomenon particularly evident in Northern Italy. Within this scenario, we wondered if: a) the COVID-19 mortality in centenarians was lower than that in people aged between 50 and 80 and b) the mortality from COVID-19 in nonagenarians and centenarians highlighted gender differences. We checked COVID-19-related vulnerability/mortality at the peak of infection (March 2020), using data on total deaths (i.e. not only confirmed COVID-19 cases). Our conclusion is that excess mortality increases steadily up to very old ages and at the same time men older than 90 years become relatively more resilient than age-matched females

On-going frontal alpha rhythms are dominant in passive state and desynchronize in active state in adult gray mouse lemurs

The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8-12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7-9 Hz) during passive state. During active state, there was a reduction in alpha power density (8-12 Hz) and an increase of power density at slow frequencies (1-4 Hz). Relative EMG activity was related to EEG power density at 2-4 Hz (positive correlation) and at 8-12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology

A Novel Study and Meta-Analysis of the Genetic Variation of the Serotonin Transporter Promoter in the Italian Population Do Not Support a Large Effect on Alzheimer's Disease Risk

Alzheimer's disease (AD) is a neurodegenerative disorder whose clinical onset is mainly characterized by memory loss. During AD progression, behavioral and psychological symptoms of dementia (BPSD) frequently occur. In this paper we evaluated the association between AD and the short/long (S/L) functional polymorphism of the promoter region of the 5-hydroxytryptamine (5-HT) transporter gene (SLC6A4). The S-allele shows a 2-fold reduced transcriptional rate, causing an imbalance in 5-HT intracellular availability that might in turn trigger behavioral and cognitive alterations. We also genotyped the SLC6A4 promoter functional variant rs25531 (A → G). By comparing the genotypic and allelic frequencies in an Italian population of 235 AD and 207 controls, we found an association between 5-HTTLPR and AD (odds ratio for the L-allele versus the S-allele: 0.74, associated P value = .03), while no difference was found for the rs25531. A meta-analysis of studies in Italy assessing 5-HTTLPR and AD risk gave an estimation of odds ratio for the L-allele versus the S-allele of 0.85 (associated P value = .08). Overall, our findings are not supportive of a large genetic effect of the explored polymorphisms on AD risk

Chemical exchange saturation transfer MRI shows low cerebral 2-deoxy-D-glucose uptake in a model of Alzheimer's Disease

Glucose is the central nervous system's only energy source. Imaging techniques capable to detect pathological alterations of the brain metabolism are useful in different diagnostic processes. Such techniques are also beneficial for assessing the evaluation efficacy of therapies in pre-clinical and clinical stages of diseases. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a possible alternative to positron emission tomography (PET) imaging that has been widely explored in cancer research in humans and animal models. We propose that pathological alterations in brain 2-deoxy-D-glucose (2DG) uptake, typical of neurodegenerative diseases, can be detected with CEST MRI. Transgenic mice overexpressing a mutated form of amyloid precusrsor protein (APP23), a model of Alzheimer's disease, analyzed with CEST MRI showed a clear reduction of 2DG uptake in different brain regions. This was reminiscent of the cerebral condition observed in Alzheimer's patients. The results indicate the feasibility of CEST for analyzing the brain metabolic state, with better image resolution than PET in experimental models

Synthetic miniprion PrP106.

Elucidation of structure and biological properties of the prion protein scrapie (PrP(Sc)) is fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease-specific isoforms and the pathogenesis of prion diseases. Unfortunately, the insolubility and heterogeneity of PrP(Sc) have limited these studies. The observation that a construct of 106 amino acids (termed PrP106 or miniprion), derived from mouse PrP and containing two deletions (Delta 23-88, Delta 141-176), becomes protease-resistant when expressed in scrapie-infected neuroblastoma cells and sustains prion replication when expressed in PrP(0/0) mice prompted us to generate a corresponding synthetic peptide (sPrP106) to be used for biochemical and cell culture studies. sPrP106 was obtained successfully with a straightforward procedure, which combines classical stepwise solid phase synthesis with a purification strategy based on transient labeling with a lipophilic chromatographic probe. sPrP106 readily adopted a beta-sheet structure, aggregated into branched filamentous structures without ultrastructural and tinctorial properties of amyloid, exhibited a proteinase K-resistant domain spanning residues 134-217, was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. These features are central properties of PrP(Sc) and make sPrP106 an excellent tool for investigating the molecular basis of the conformational conversion of PrP(C) into PrP(Sc) and prion disease pathogenesis