Davide Visigalli

Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells

BackgroundWe investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU) in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells.MethodsWe analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC), bonesialoprotein (BSP), membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146).ResultsWe report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow.ConclusionsHU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for homeostasis related genes and modulation of mRNAs and proteins for ECM deposition, maturation and remodeling which may be key factors for bone maintenance.

Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot‐Marie‐Tooth type 1A (CMT1A) rats. The latter used as a model of dys‐demyelination. O2 consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O2 consumption by IMV from Wt and CMT1A 1‐month‐old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys‐demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys‐demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.

PMP22 messenger RNA levels in skin biopsies: testing the effectiveness of a Charcot–Marie–Tooth 1A biomarker

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with increased gene dosage for PMP22. Therapeutic approaches are currently aiming at correcting PMP22 over-expression. It is unknown whether PMP22 can be used as a biological marker of disease progression and therapy efficacy. We performed quantitative real-time polymerase chain reaction on skin biopsies of 45 patients with CMT1A, obtained at study entry and after 24-months of treatment either with ascorbic acid or placebo. Data of a subgroup of patients were also compared with matched healthy subjects. Finally, we analysed PMP22 messenger RNA levels in sural nerve biopsies. We did not find significant differences in the levels of any known PMP22 transcripts in treated or untreated patients with CMT1A, thus confirming that ascorbic acid does not impact on the molecular features of CMT1A. Most importantly, we did not observe any correlation between PMP22 messenger RNA levels and the different clinical and electrophysiological outcome measures, underscoring the weakness of PMP22 to mirror the phenotypic variability of patients with CMT1A. We did not find increased PMP22 messenger RNA levels in skin and sural nerve biopsies of patients with CMT1A compared with relative controls. In conclusion, this study shows that ascorbic acid does not impact on PMP22 transcriptional regulation and PMP22 is not a suitable biomarker for CMT1A.

The carboxyl terminal trimer of procollagen I induces pro-metastatic changes and vascularization in breast cancer cells xenografts

BackgroundThe COOH terminal peptide of Pro-collagen type I (PICP, also called C3) is chemotactic for endothelial melanoma and breast cancer cells. PICP induces the expression of Metalloproteinases-2 and -9, of Vascular endothelial growth factor and of the chemokine CXCL-12 receptor CXCR4 in MDA MB231 breast carcinoma cells in vitro.MethodsWe used a model of xenografts in BalbC/nude mice obtaining tumors by implanting in contro-lateral subcutaneous positions MDA MB231 cells added or not with purified PICP and studied the earlier phases of tumor development, up to 48 days from implant, by histology, immunostain and in situ hybridization.ResultsAddition of PICP promotes rapid vascularization of the tumors while does not affect mitotic and apoptotic indexes and overall tumor growth. PICP-treated, relative to control tumors, show up-modulation of Vascular endothelial factor, Metalloproteinase-9 and CXCR4, all tumor prognostic genes; they also show down-modulation of the endogenous Metalloproteinase inhibitor, reversion-inducing-cysteine-rich protein with kazal motifs, and a different pattern of modulation of Tissue Inhibitor of Metalloproteinase-2. These changes occur in absence of detectable expression of CXCL-12, up to 38 days, in control and treated tumors.ConclusionPICP has an early promoting effect in the acquisition by the tumors of prometastatic phenotype. PICP may be play a relevant role in the productive interactions between stroma and tumor cells by predisposing the tumor cells to respond to the proliferation stimuli ensuing the activation of signaling by engagement of CXCR4 by cytokines and by fostering their extravasion, due to the induction of increased vascular development.

The Diadenosine Homodinucleotide P18 Improves In Vitro Myelination in Experimental Charcot‐Marie‐Tooth Type 1A

Charcot‐Marie‐Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca2+ concentration ([Ca2+]i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca2+ levels through down‐regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5′,5′′′‐P1, P2‐diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11‐overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild‐type (wt) cells. P18 increased [cAMP]i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non‐phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]i‐increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype. J. Cell. Biochem. 115: 161–167, 2014.

Tolerability and efficacy study of P2X7 inhibition in experimental Charcot-Marie-Tooth type 1A (CMT1A) neuropathy.

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy for which pharmacological treatments are not yet available. An abnormally high intracellular Ca(2+) concentration was observed in Schwann cells (SC) from CMT1A rats, caused by the PMP22-mediated overexpression of the P2X7 purinoceptor. The purpose of this study was to investigate the tolerability and therapeutic potential of a pharmacological antagonist of the P2X7 receptor (A438079) in CMT1A. A438079 ameliorated in vitro myelination of organotypic DRG cultures from CMT1A rats. Furthermore, we performed an experimental therapeutic trial in PMP22 transgenic and in wild-type rats. A preliminary dose-escalation trial showed that 3mg/kg A438079 administered via intraperitoneal injection every 24h for four weeks was well tolerated by wild type and CMT1A rats. Affected rats treated with 3mg/kg A438079 revealed a significant improvement of the muscle strength, when compared to placebo controls. Importantly, histologic analysis revealed a significant increase of the total number of myelinated axons in tibial nerves. Moreover, a significant decrease of the hypermyelination of small caliber axons and a significant increase of the frequency and diameter of large caliber myelinated axons was highlighted. An improved distal motor latencies was recorded, whereas compound muscle action potentials (CMAP) remained unaltered. A438079 reduced the SC differentiation defect in CMT1A rats. These results show that pharmacological inhibition of the P2X7 receptor is well tolerated in CMT1A rats and represents a proof-of-principle that antagonizing this pathway may correct the molecular derangements and improve the clinical phenotype in the CMT1A neuropathy.

Soluble Neuregulin1 is strongly up-regulated in the rat model of Charcot-Marie-Tooth 1A disease:

Neuregulin1 (NRG1) is a growth factor playing a pivotal role in peripheral nerve development through the activation of the transmembrane co-receptors ErbB2–ErbB3. Soluble NRG1 isoforms, mainly secreted by Schwann cells, are strongly and transiently up-regulated after acute peripheral nerve injury, thus suggesting that they play a crucial role also in the response to nerve damage. Here we show that in the rat experimental model of the peripheral demyelinating neuropathy Charcot-Marie-Tooth 1A (CMT1A) the expression of the different NRG1 isoforms (soluble, type α and β, type a and b) is strongly up-regulated, as well as the expression of NRG1 co-receptors ErbB2–ErbB3, thus showing that CMT1A nerves have a gene expression pattern highly reminiscent of injured nerves. Because it has been shown that high concentrations of soluble NRG1 negatively affect myelination, we suggest that soluble NRG1 over-expression might play a negative role in the pathogenesis of CMT1A disease, and that a therapeutic approach, aimed to interfere with NRG1 activity, might be beneficial for CMT1A patients. Further studies will be necessary to test this hypothesis in animal models and to evaluate NRG1 expression in human patients. Impact statement Charcot-Marie-Tooth1A (CMT1A) is one of the most frequent inherited neurological diseases, characterized by chronic demyelination of peripheral nerves, for which effective therapies are not yet available. It has been recently proposed that the treatment with soluble Neuregulin1 (NRG1), a growth factor released by Schwann cells immediately after acute nerve injury, might be effective in CMT1A treatment. However, the expression of the different isoforms of endogenous NRG1 in CMT1A nerves has not been yet investigated. In this preliminary study, we demonstrate that different isoforms of soluble NRG1 are strongly over-expressed in CMT1A nerves, thus suggesting that a therapeutic approach based on NRG1 treatment should be carefully reconsidered. If soluble NRG1 is over-expressed also in human CMT1A nerves, a therapeutic approach aimed to inhibit (instead of stimulate) the signal transduction pathways driven by NRG1 might be fruitfully developed. Further studies will be necessary to test these hypotheses.

Sphingomyelin as a myelin biomarker in CSF of acquired demyelinating neuropathies

Fast, accurate and reliable methods to quantify the amount of myelin still lack, both in humans and experimental models. The overall objective of the present study was to demonstrate that sphingomyelin (SM) in the cerebrospinal fluid (CSF) of patients affected by demyelinating neuropathies is a myelin biomarker. We found that SM levels mirror both peripheral myelination during development and small myelin rearrangements in experimental models. As in acquired demyelinating peripheral neuropathies myelin breakdown occurs, SM amount in the CSF of these patients might detect the myelin loss. Indeed, quantification of SM in 262 neurological patients showed a significant increase in patients with peripheral demyelination (p = 3.81 * 10 − 8) compared to subjects affected by non-demyelinating disorders. Interestingly, SM alone was able to distinguish demyelinating from axonal neuropathies and differs from the principal CSF indexes, confirming the novelty of this potential CSF index. In conclusion, SM is a specific and sensitive biomarker to monitor myelin pathology in the CSF of peripheral neuropathies. Most importantly, SM assay is simple, fast, inexpensive, and promising to be used in clinical practice and drug development.

Alternative Splicing in the Human PMP22 Gene: Implications in CMT1A Neuropathy

CMT1A patients commonly share PMP22 genetic overloading but they show phenotypic heterogeneity and variability in PMP22 mRNA and protein expression. Moreover, PMP22 mRNA levels do not correlate with clinical outcome measures in these patients, suggesting their uselessness as a disease biomarker. Thus, in‐depth analysis of PMP22 transcription and translation might help to define its pathogenic role in CMT1A. We focused on the alternative splicing of PMP22 gene to verify whether mRNA processing is altered in CMT1A. We identified three new PMP22 transcripts enriched in human sural nerve biopsies. One of them was an untranslated variant, whereas the other two originated from a PMP22 undescribed exon and encoded for a new putative protein localized in the endoplasmic reticulum. As splicing events in the PMP22 gene are differently regulated in tissues and during development, we analyzed the levels of PMP22 transcripts and their splicing pattern in human and experimental CMT1A. We found an altered PMP22 splicing ratio in the CMT1A rat. In addition, we showed a remarkable derangement in rat QKI expression, which is a critical regulator of splicing during myelination. Overall, our data suggest that an alteration of mRNA processing could be a pathogenic mechanism in CMT1A.