In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells.

BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Investigations of cardiac fibrosis rheology by in vitro cardiac tissue modeling with 3D cellular spheroids.

Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ1) followed by a slower one (τ2). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ1 with respect to τ2. Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis.

Defects in AMPAR trafficking and microglia activation underlie socio-cognitive deficits associated to decreased expression of phosphodiesterase 2 a.

Phosphodiesterase 2 A (PDE2A) is an enzyme involved in the homeostasis of cAMP and cGMP and is the most highly expressed PDE in human brain regions critical for socio-cognitive behavior. In cerebral cortex and hippocampus, PDE2A expression level is upregulated in Fmr1-KO mice, a model of the Fragile X Syndrome (FXS), the most common form of inherited intellectual disability (ID) and autism spectrum disorder (ASD). Indeed, PDE2A translation is negatively modulated by FMRP, whose functional absence causes FXS. While the pharmacological inhibition of PDE2A has been associated to its pro-cognitive role in normal animals and in models of ID and ASD, homozygous PDE2A mutations have been identified in patients affected by ID, ASD and epilepsy. To clarify this apparent paradox about the role of PDE2A in brain development, we characterized here Pde2a+/- mice (homozygote animals being not viable) at the behavioral, cellular, molecular and electrophysiological levels. Pde2a+/- females display a milder form of the disorder with reduced cognitive performance in adulthood, conversely males show severe socio-cognitive deficits throughout their life. In males, these phenotypes are associated with microglia activation, elevated glutathione levels and increased externalization of Glutamate receptor (GluR1) in CA1, producing reduced mGluR-dependent Long-term Depression. Overall, our results reveal molecular targets of the PDE2A-dependent pathway underlying socio-cognitive performance. These results clarify the mechanism of action of pro-cognitive drugs based on PDE2A inactivation, which have been shown to be promising therapeutic approaches for Alzheimer's disease, schizophrenia, FXS as well as other forms of ASD.

Nanoscale organization of CaV2.1 splice isoforms at presynaptic terminals: implications for synaptic vesicle release and synaptic facilitation.

The distance between CaV2.1 voltage-gated Ca2+ channels and the Ca2+ sensor responsible for vesicle release at presynaptic terminals is critical for determining synaptic strength. Yet, the molecular mechanisms responsible for a loose coupling configuration of CaV2.1 in certain synapses or developmental periods and a tight one in others remain unknown. Here, we examine the nanoscale organization of two CaV2.1 splice isoforms (CaV2.1[EFa] and CaV2.1[EFb]) at presynaptic terminals by superresolution structured illumination microscopy. We find that CaV2.1[EFa] is more tightly co-localized with presynaptic markers than CaV2.1[EFb], suggesting that alternative splicing plays a crucial role in the synaptic organization of CaV2.1 channels.

The Tumor Suppressor DAB2IP Is Regulated by Cell Contact and Contributes to YAP/TAZ Inhibition in Confluent Cells.

External and internal mechanical forces modulate cell morphology, movement, proliferation and metabolism, and represent crucial inputs for tissue homeostasis. The transcriptional regulators YAP and TAZ are important effectors of mechanical signaling and are frequently activated in solid tumors, correlating with metastasis, chemoresistance, and shorter patient survival. YAP/TAZ activity is controlled by various pathways that sense cell shape, polarity, contacts, and mechanical tension. In tumors, aberrant YAP/TAZ activation may result from cancer-related alterations of such regulatory networks. The tumor suppressor DAB2IP is a Ras-GAP and scaffold protein that negatively modulates multiple oncogenic pathways and is frequently downregulated or inactivated in solid tumors. Here, we provide evidence that DAB2IP expression is sustained by cell confluency. We also find that DAB2IP depletion in confluent cells alters their morphology, reducing cell packing while increasing cell stiffness. Finally, we find that DAB2IP depletion in confluent cells favors YAP/TAZ nuclear localization and transcriptional activity, while its ectopic expression in subconfluent cells increases YAP/TAZ retention in the cytoplasm. Together, these data suggest that DAB2IP may function as a sensor of cell interactions, contributing to dampening cellular responses to oncogenic inputs in confluent cells and that DAB2IP loss-of-function would facilitate YAP/TAZ activation in intact epithelia, accelerating oncogenic transformation.

Controlled Quenching of Agarose Defines Hydrogels with Tunable Structural, Bulk Mechanical, Surface Nanomechanical, and Cell Response in 2D Cultures.

The scaffolding of agarose hydrogel networks depends critically on the rate of cooling (quenching) after heating. Efforts are made to understand the kinetics and evolution of biopolymer self-assembly upon cooling, but information is lacking on whether quenching might affect the final hydrogel structure and performance. Here, a material strategy for the fine modulation of quenching that involves temperature-curing steps of agarose is reported. Combining microscopy techniques, standard and advanced macro/nanomechanical tools, it is revealed that agarose accumulates on the surface when the curing temperature is set at 121 °C. The inhomogeneity can be mostly recovered when it is reduced to 42 °C. This has a drastic effect on the stiffness of the surface, but not on the viscoelasticity, roughness, and wettability. When hydrogels are strained at small/large deformations, the curing temperature has no effect on the viscoelastic response of the hydrogel bulk but does play a role in the onset of the non-linear region. Cells cultured on these hydrogels exhibit surface stiffness-sensing that affects cell adhesion, spreading, F-actin fiber tension, and assembly of vinculin-rich focal adhesions. Collectively, the results indicate that the temperature curing of agarose is an efficient strategy to produce networks with tunable mechanics and is suitable for mechanobiology studies.

Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice.

Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.

Advanced photodynamic therapy with an engineered M13 phage targeting EGFR: Mitochondrial localization and autophagy induction in ovarian cancer cell lines.

Photodynamic therapy (PDT) is a potential synergistic approach to chemotherapy for treating ovarian cancer, the most lethal gynecologic malignancy. Here we used M13 bacteriophage as a targeted vector for the efficient photodynamic killing of SKOV3 and COV362 cells. The M13 phage was refactored (M13r) to display an EGFR binding peptide in its tip that is frequently overexpressed in ovarian cancer. The refactored phage was conjugated with chlorin e6 (Ce6), one of the most widely used photosensitizers (M13r-Ce6). The new platform, upon irradiation, generated ROS by type I mechanism and showed activity in killing SKOV3 and COV362 cells even at concentrations in which Ce6 alone was ineffective. A microscopy analysis demonstrated an enhanced cellular uptake of M13r-Ce6 compared to free Ce6 and its mitochondrial localization. Western blot analysis revealed significant downregulation in the expression of EGFR in cells exposed to M13r-Ce6 after PDT. Following PDT treatment, autophagy induction was supported by an increased expression of LC3II, along with a raised autophagic fluorescent signal, as observed by fluorescence microscopy analysis for autophagosome visualization. As a conclusion we have herein proposed a bacteriophage-based receptor targeted photodynamic therapy for EGFR-positive ovarian cancer.

Long-Term Effects of Biliverdin Reductase a Deficiency in Ugt1-/- Mice: Impact on Redox Status and Metabolism.

Accumulation of neurotoxic bilirubin due to a transient neonatal or persistent inherited deficiency of bilirubin glucuronidation activity can cause irreversible brain damage and death. Strategies to inhibit bilirubin production and prevent neurotoxicity in neonatal and adult settings seem promising. We evaluated the impact of Bvra deficiency in neonatal and aged mice, in a background of unconjugated hyperbilirubinemia, by abolishing bilirubin production. We also investigated the disposal of biliverdin during fetal development. In Ugt1-/- mice, Bvra deficiency appeared sufficient to prevent lethality and to normalize bilirubin level in adults. Although biliverdin accumulated in Bvra-deficient fetuses, both Bvra-/- and Bvra-/-Ugt1-/- pups were healthy and reached adulthood having normal liver, brain, and spleen histology, albeit with increased iron levels in the latter. During aging, both Bvra-/- and Bvra-/-Ugt1-/- mice presented normal levels of relevant hematological and metabolic parameters. Interestingly, the oxidative status in erythrocytes from 9-months-old Bvra-/- and Bvra-/-Ugt1-/- mice was significantly reduced. In addition, triglycerides levels in these 9-months-old Bvra-/- mice were significantly higher than WT controls, while Bvra-/-Ugt1-/- tested normal. The normal parameters observed in Bvra-/-Ugt1-/- mice fed chow diet indicate that Bvra inhibition to treat unconjugated hyperbilirubinemia seems safe and effective.

In vitro modeling of dendritic atrophy in Rett syndrome: determinants for phenotypic drug screening in neurodevelopmental disorders.

Dendritic atrophy, defined as the reduction in complexity of the neuronal arborization, is a hallmark of several neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, affecting 1:10,000 girls worldwide, is mainly caused by mutations in the MECP2 gene and has no cure. We describe here an in vitro model of dendritic atrophy in Mecp2-/y mouse hippocampal primary cultures, suitable for phenotypic drug-screening. Using High-Content Imaging techniques, we systematically investigated the impact of culturing determinants on several parameters such as neuronal survival, total dendritic length, dendritic endpoints, soma size, cell clusterization, spontaneous activity. Determinants included cell-seeding density, glass or polystyrene substrates, coating with poly-Ornithine with/without Matrigel and miniaturization from 24 to 96-half surface multiwell plates. We show that in all plate-sizes at densities below 320 cells/mm2, morphological parameters remained constant while spontaneous network activity decreased according to the cell-density. Mecp2-/y neurons cultured at 160 cells/mm2 density in 96 multiwell plates, displayed significant dendritic atrophy and showed a marked increase in dendritic length following treatment with Brain-derived neurotrophic factor (BDNF) or Mirtazapine. In conclusion, we have established a phenotypic assay suitable for fast screening of hundreds of compounds, which may be extended to other neurodevelopmental diseases with dendritic atrophy.

BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo.

Bdnf exon-IV and exon-VI transcripts are driven by neuronal activity and are involved in pathologies related to sleep, fear or memory disorders. However, how their differential transcription translates activity changes into long-lasting network changes is elusive. Aiming to trace specifically the network controlled by exon-IV and -VI derived BDNF during activity-dependent plasticity changes, we generated a transgenic reporter mouse for B DNF- l ive- e xon- v isualization (BLEV), in which expression of Bdnf exon-IV and -VI can be visualized by co-expression of CFP and YFP. CFP and YFP expression was differentially activated and targeted in cell lines, primary cultures and BLEV reporter mice without interfering with BDNF protein synthesis. CFP and YFP expression, moreover, overlapped with BDNF protein expression in defined hippocampal neuronal, glial and vascular locations in vivo. So far, activity-dependent BDNF cannot be explicitly monitored independent of basal BDNF levels. The BLEV reporter mouse therefore provides a new model, which can be used to test whether stimulus-induced activity-dependent changes in BDNF expression are instrumental for long-lasting plasticity modifications.

Novel NOD2 Mutation in Early-Onset Inflammatory Bowel Phenotype.

Background: Nucleotide-binding oligomerization domain 2 (NOD2) is a key intracellular protein of the innate immune system. NOD2 variants are associated with inflammatory bowel disease (IBD) and other inflammatory phenotypes. We described the case of a baby with a very early-onset IBD who is characterized by a rare homozygous variant in NOD2, found through whole-exome sequencing, Its pathogenic effect was investigated through bioinformatics and functional studies. Methods: The microbicide activity of the patient's phagocytes was analyzed using Escherichia coli. HEK293 and Caco2 cell lines were transfected with wild-type and mutated NOD2 cDNA to evaluate the NF-kB activity and the protein distribution. The functionality of the NOD2 pathway was assessed through analysis of the expression of tumor nectrosis factor alpha (TNFα) on monocytes. The levels of various cytokines were quantified in the patient plasma by a multiplex suspension array. Results: A missense NOD2 mutation, c.G1277A; p.R426H in homozygosis, was found. The patient's microbicide activity was comparable to that observed in controls. HEK293 cells transfected with the mutated cDNA showed a 20-fold increase of NF-kB activation in basal condition. Moreover, Caco2 immunostaining revealed a different cytoplasmic distribution of the mutated protein compared with wild-type. A higher production of TNFα by monocytes and elevated levels of plasmatic cytokines and chemokines were evidenced in the patient. Conclusions: This homozygous mutation is functionally relevant and shows a different NOD2 involvement in the IBD phenotype. In our patient, this mutation caused a gain of function typical of the Blau syndrome phenotype, manifesting, however, an IBD-like phenotype.

3D Carbon-Nanotube-Based Composites for Cardiac Tissue Engineering.

Heart failure is a disease of epidemic proportion and a leading cause of mortality in the world. Because cardiac myocytes are terminally differentiated cells with minimal intrinsic ability to self-regenerate, cardiac tissue engineering has emerged as one of the most realistic therapeutic strategies for cardiac repair. We have previously proven the ability of carbon nanotube scaffolds to promote cardiomyocyte proliferation, maturation, and long-term survival. Here, we tested if three-dimensional scaffolds of carbon nanotube-based composites can also promote cardiomyocyte growth, electrophysiological maturation, and formation of functional syncytia. To this purpose, we developed an elastomeric scaffold that consists of a microporous and self-standing material made of polydimethylsiloxane (PDMS) containing micrometric cavities, and integrated multiwall carbon nanotubes (MWCNTs) into the scaffold. We combined microscopy, cell biology, and calcium imaging to investigate whether neonatal rat ventricular myocytes (NRVMs) cultured on the 3D-PDMS+MWCNT acquire a more viable and mature phenotype compared to control. We found that when cultured in the 3D-PDMS+MWCNTs, NRVMs showed improved viability (p < 0.005 at day 3) and more defined and mature sarcomeric phenotype compared to 3D-PDMS control. These modifications were associated with an increase of connexin-43 gene expression, gap junction areas (p < 0.005 at day 3), and a more mature electrophysiological phenotype of syncytia and calcium transients. Finally, 3D-PDMS+MWCNT boosted NRVMs proliferation (p < 0.005 at day 3) while hindering cardiac fibroblasts proliferation compared to control PDMS. Thus, 3D-PDMS+MWCNT has the ability to promote viability, proliferation and functional maturation of cardiac myocytes. These properties are essential in cardiac tissue engineering and offer novel perspectives in the development of innovative therapies for cardiac repair.

Targeted Delivery of Neutralizing Anti-C5 Antibody to Renal Endothelium Prevents Complement-Dependent Tissue Damage.

Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney.

Signaling pathways controlling activity-dependent local translation of BDNF and their localization in dendritic arbors.

Brain-derived neurotrophic factor (BDNF) is encoded by multiple mRNA variants whose differential subcellular distribution constitutes a 'spatial code' for local translation of BDNF and selective morphological remodeling of dendrites. Here, we investigated where BDNF translation takes place and what are the signaling pathways involved. Cultured hippocampal neurons treated with KCl showed increased BDNF in the soma, proximal and distal dendrites, even in quaternary branches. This activity-dependent increase of BDNF was abolished by cycloheximide, suggesting local translation, and required activation of glutamate and Trk receptors. Our data showed that BDNF translation was regulated by multiple signaling cascades including RAS-Erk and mTOR pathways, and CaMKII-CPEB1, Aurora-A-CPEB1 and Src-ZBP1 pathways. Aurora-A, CPEB1, ZBP1 (also known as IGF2BP1), eiF4E, S6 (also known as rpS6) were present throughout the dendritic arbor. Neuronal activity increased the levels of Aurora-A, CPEB1 and ZBP1 in distal dendrites whereas those of eiF4E and S6 were unaffected. BDNF-6, the main dendritic BDNF transcript, was translated in the same subcellular domains and in response to the same pathways as total BDNF. In conclusion, we identified the signaling cascades controlling BDNF translation and we describe how the translational machinery localization is modulated in response to electrical activity.

Combined cisplatin and aurora inhibitor treatment increase neuroblastoma cell death but surviving cells overproduce BDNF.

Drug-resistance to chemotherapics in aggressive neuroblastoma (NB) is characterized by enhanced cell survival mediated by TrkB and its ligand, brain-derived neurotrophic factor (BDNF); thus reduction in BDNF levels represent a promising strategy to overcome drug-resistance, but how chemotherapics regulate BDNF is unknown. Here, cisplatin treatment in SK-N-BE neuroblastoma upregulated multiple BDNF transcripts, except exons 5 and 8 variants. Cisplatin increased BDNF mRNA and protein, and enhanced translation of a firefly reporter gene flanked by BDNF 5'UTR exons 1, 2c, 4 or 6 and 3'UTR-long. To block BDNF translation we focused on aurora kinases inhibitors which are proposed as new chemotherapeutics. NB cell survival after 24 h treatment was 43% with cisplatin, and 22% by cisplatin+aurora kinase inhibitor PHA-680632, while the aurora kinases inhibitor alone was less effective; however the combined treatment induced a paradoxical increase of BDNF in surviving cells with strong translational activation of exon6-3'UTR-long transcript, while translation of BDNF transcripts 1, 2C and 4 was suppressed. In conclusion, combined cisplatin and aurora kinase inhibitor treatment increases cell death, but induces BDNF overproduction in surviving cells through an aurora kinase-independent mechanism.

Aptamer targeting of the elongation factor 1A impairs hepatocarcinoma cells viability and potentiates bortezomib and idarubicin effects.

The high morbidity and mortality of hepatocellular carcinoma (HCC) is mostly due to the limited efficacy of the available therapeutic approaches. Here we explore the anti-HCC potential of an aptamer targeting the elongation factor 1A (eEF1A), a protein implicated in the promotion of HCC. As delivery methods, we have compared the effectiveness of cationic liposome and cholesterol-mediated approaches. A75 nucleotide long aptamer containing GT repetition (GT75) was tested in three HCC cell lines, HepG2, HuH7 and JHH6. When delivered by liposomes, GT75 was able to effectively reducing HCC cells viability in a dose and time dependent fashion. Particular sensitive were JHH6 where increased apoptosis with no effects on cell cycle were observed. GT75 effect was likely due to the interference with eEF1A activity as neither the mRNA nor the protein levels were significantly affected. Notably, cholesterol-mediated delivery of GT75 abrogated its efficacy due to cellular mis-localization as proven by fluorescence and confocal microscopic analysis. Finally, liposome-mediated delivery of GT75 improved the therapeutic index of the anticancer drugs bortezomib and idarubicin. In conclusion, liposome but not cholesterol-mediated delivery of GT75 resulted in an effective delivery of GT75, causing the impairment of the vitality of a panel of HCC derived cells.

The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells.

Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells.

Geranylgeraniol and Neurological Impairment: Involvement of Apoptosis and Mitochondrial Morphology.

Deregulation of the cholesterol pathway is an anomaly observed in human diseases, many of which have in common neurological involvement and unknown pathogenesis. In this study we have used Mevalonate Kinase Deficiency (MKD) as a disease-model in order to investigate the link between the deregulation of the mevalonate pathway and the consequent neurodegeneration. The blocking of the mevalonate pathway in a neuronal cell line (Daoy), using statins or mevalonate, induced an increase in the expression of the inflammasome gene (NLRP3) and programmed cell death related to mitochondrial dysfunction. The morphology of the mitochondria changed, clearly showing the damage induced by oxidative stress and the decreased membrane potential associated with the alterations of the mitochondrial function. The co-administration of geranylgeraniol (GGOH) reduced the inflammatory marker and the damage of the mitochondria, maintaining its shape and components. Our data allow us to speculate about the mechanism by which isoprenoids are able to rescue the inflammatory marker in neuronal cells, independently from the block of the mevalonate pathway, and about the fact that cell death is mitochondria-related.

Pharmacological treatment with mirtazapine rescues cortical atrophy and respiratory deficits in MeCP2 null mice.

Loss of MeCP2 (Methyl CpG binding protein 2) in Rett syndrome (RTT) causes brain weight decrease, shrinkage of the cortex with reduced dendritic arborization, behavioral abnormalities, seizures and cardio-respiratory complications. The observed monoamine neurotransmitters reduction in RTT suggested antidepressants as a possible therapy. We treated MeCP2-null mice from postnatal-day 28 for two weeks with desipramine, already tested in RTT, or mirtazapine, an antidepressant with limited side-effects, known to promote GABA release. Mirtazapine was more effective than desipramine in restoring somatosensory cortex thickness by fully rescuing pyramidal neurons dendritic arborization and spine density. Functionally, mirtazapine treatment normalized heart rate, breath rate, anxiety levels, and eliminated the hopping behavior observed in MeCP2-null mice, leading to improved phenotypic score. These morphological and functional effects of mirtazapine were accompanied by reestablishment of the GABAergic and glutamatergic receptor activity recorded in cortex and brainstem tissues. Thus, mirtazapine can represent a new potential pharmacological treatment for the Rett syndrome.