Tiziana Vigo

Predictors of response to rituximab in patients with neuropathy and anti–myelin associated glycoprotein immunoglobulin M

Abstract  We evaluated the efficacy and safety of rituximab in an open‐label, uncontrolled study of 13 patients with polyneuropathy associated with antibodies to myelin‐associated glycoprotein (MAG) and correlated the response to therapy with clinical and laboratory features. One year after rituximab therapy, anti‐MAG immunoglobulin M (IgM) titers were significantly reduced. At that time, eight patients (62%) had improved in both the inflammatory neuropathy cause and treatment (INCAT) sensory sumscore and the Medical Research Council sumscore for muscle strength and seven of them also in the INCAT disability score. The improvement in the mean INCAT sensory sumscore was significant at 12 months and correlated with lower anti‐MAG antibody at entry and at follow‐up. This study suggests that rituximab may be efficacious in patients with anti–MAG associated neuropathy and particularly on sensory impairment and in those with moderately elevated antibody titers. These findings suggest that antibody reduction below a critical level may be necessary to achieve clinical improvement.

Experimental Charcot-Marie-Tooth type 1A: A cDNA microarrays analysis

To reveal the spectrum of genes that are modulated in Charcot-Marie-Tooth neuropathy type 1A (CMT1A), which is due to overexpression of the gene coding for the peripheral myelin protein 22 (pmp22), we performed a cDNA microarray experiment with cDNA from sciatic nerves of a rat model of the disease. In homozygous pmp22 overexpressing animals, we found a significant down-regulation of 86 genes, while only 23 known genes were up-regulated, suggesting that the increased dosage of pmp22 induces a general down-regulation of gene expression in peripheral nerve tissue. Classification of the modulated genes into functional categories leads to the identification of some pathways altered by overexpression of pmp22. In particular, a selective down-regulation of the ciliary neurotrophic factor transcript and of genes coding for proteins involved in cell cycle regulation, for cytoskeletal components and for proteins of the extracellular matrix, was observed. Cntf expression was further studied by real-time PCR and ELISA technique in pmp22 transgenic sciatic nerves, human CMT1A sural nerve biopsies, and primary cultures of transgenic Schwann cells. According to the results of cDNA microarray analysis, a down-regulation of cntf, both at the mRNA and protein level, was found in all the conditions tested. These results are relevant to reveal the molecular function of PMP22 and the pathogenic mechanism of CMT1A. In particular, finding a specific reduction of cntf expression in CMT1A Schwann cells suggests that overexpression of pmp22 significantly affects the ability of Schwann cells to offer a trophic support to the axon, which could be a factor, among other, responsible for the development of axonal atrophy in human and experimental CMT1A.

Impairment of PMP22 transgenic Schwann cells differentiation in culture: implications for Charcot-Marie-Tooth type 1A disease

Charcot-Marie-Tooth type 1A (CMT1A) is a hereditary demyelinating neuropathy due to an increased genetic dosage of the peripheral myelin protein 22 (PMP22). The mechanisms leading from PMP22 overexpression to impairment of myelination are still unclear. We evaluated expression and processing of PMP22, viability, proliferation, migration, motility and shaping properties, and ability of forming myelin of PMP22 transgenic (PMP22(tg)) Schwann cells in culture. In basal conditions, PMP22(tg) Schwann cells, although expressing higher PMP22 levels than control ones, show normal motility, migration and shaping properties. Addition of forskolin to the media induces an additional stimulation of PMP22 expression and results in an impairment of cells migration and motility, and a reduction of cell area and perimeter. Similarly, co-culturing transgenic Schwann cells with neurons causes an altered cells differentiation and an impairment of myelin formation. In conclusion, exposure of PMP22(tg) Schwann to the axon or to axonal-mimicking stimuli significantly affects the transition of transgenic Schwann cells to the myelinating phenotype.

Axonal damage and demyelination in long-term dorsal root ganglia cultures from a rat model of Charcot-Marie-Tooth type 1A disease.

Clinical progression in hereditary and acquired demyelinating disorders of both the central and peripheral nervous system is mainly due to a time‐dependent axonal impairment. We established 90‐day dorsal root ganglia (DRG) cultures from a rat model of Charcot–Marie–Tooth type 1A (CMT1A) neuropathy to evaluate the structure of myelin and axons, and the expression of myelin‐related proteins and cytoskeletal components, by morphological and molecular techniques. Both wild‐type and CMT1A cultures were rich in myelinated fibres. Affected cultures showed dysmyelinated internodes and focal myelin swellings. Furthermore, uncompacted myelin and smaller axons with increased neurofilament (NF) density were found by electron microscopy, and Western blots showed higher levels of nonphosphorylated NF. Confocal microscopy demonstrated an abnormal distribution of the myelin‐associated glycoprotein which, instead of being expressed at the noncompact myelin level, showed focal accumulation along the internodes while other myelin proteins were normally distributed. These findings suggest that CMT1A DRG cultures, similarly to the animal model and human disease, undergo axonal atrophy over a period of time. This model may be utilized to study the molecular changes underlying demyelination and secondary axonal impairment. As axonal damage may occur after just 3 months and tissue cultures represent a strictly controlled environment, this model may be ideal for testing neuroprotective therapies.

IFN-γ orchestrates mesenchymal stem cell plasticity through the signal transducer and activator of transcription 1 and 3 and mammalian target of rapamycin pathways.

Background: Mesenchymal stem cells (MSCs) display a therapeutic plasticity because of their ability to modulate immunity, foster tissue repair, and differentiate into mesodermal cells. IFN‐&ggr; has been described to differently affect human mesenchymal stem cell (hMSC) and mouse mesenchymal stem cell (mMSC) immunomodulation and differentiation, depending on the inflammatory milieu. Objective: We aimed at dissecting the relevant intracellular pathways through which IFN‐&ggr; affects MSC plasticity and the consequence of their manipulation on MSC functions. Methods: Modification of relevant IFN‐&ggr;–dependent pathways in mMSCs was carried out in vitro through gene silencing or chemical inhibition of key components. Functional outcomes were assessed by means of Western blotting, real‐time PCR, differentiation, and proliferation assays on MSCs. The effect on T cells was addressed by T‐cell proliferation assays; the effect of mammalian target of rapamycin (mTOR) manipulation in MSCs was studied in vivo in a mouse model of delayed‐type hypersensitivity assay. To address whether similar mechanisms are involved also in hMSCs on IFN‐&ggr; stimulation, the effect of chemical inhibition on the same intracellular pathways was assessed by means of Western blotting, and the final outcome on immunomodulatory properties was evaluated based on real‐time PCR and T‐cell proliferation. Results: We revealed that in mMSCs IFN‐&ggr;–induced immunoregulation is mediated by early phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3, which is significantly enhanced by an extracellular signal‐regulated kinase 1/2–dependent mTOR inhibition, thereby promoting pSTAT1 nuclear translocation. Accordingly, after intracellular mTOR inhibition, MSCs augmented their ability to inhibit T‐cell proliferation and control delayed‐type hypersensitivity in vivo. Similarly, on mTOR blockade, hMSCs also enhanced their immunoregulatory features. A sustained exposure to IFN‐&ggr; led to inhibition of STAT3 activity, which in mMSCs resulted in an impaired proliferation and differentiation. Conclusion: These results provide new insights about MSC intracellular pathways affected by IFN‐&ggr;, demonstrating that pharmacologic or genetic manipulation of MSCs can enhance their immunomodulatory functions, which could be translated into novel therapeutic approaches.

Expression of ciliary neurotrophic factor (CNTF) in charcot‐marie‐tooth type 1A (CMT1A) disease

cDNA microarray experiments on sciatic nerves from 30‐day‐old PMP22 transgenic (PMP22tg) rats were performed to detect genes modulated in CMT1A. Among the downregulated genes, CNTF mRNA was significantly reduced, whereas other neurotrophic factors (BDNF, NT3, NGF) and cognate receptors (TRKA, TRKB, TRKC) were unchanged. We further studied CNTF expression in transgenic nerves using an ELISA technique. We observed significantly (p < 0.001) lower concentrations of the protein in sciatic nerves from homozygous (59 ± 7.8 pg/mg of proteins) and heterozygous (140.8 ± 17.56 pg/mg of proteins) PMP22tg nerves compared to normal controls (713.8 ± 168.7 pg/mg of proteins). Moreover, using real time PCR, we studied CNTF expression in human sural nerves from 2 CMT1A patients and in 3 control nerves. According to the animal results, CNTF mRNA expression was absent in CMT1A patients whereas it was detectable in control nerves (1.99 ± 0.9). Finally, we studied CNTF mRNA expression in aging PMP22tg rats; preliminary results show a further decrease of CNTF mRNA in heterozygous transgenic rats compared to normal age‐matched littermates. Our results suggest that reduced CNTF expression may account for the development of axonal atrophy in CMT1A.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 33

CMT1A is a hereditary demyelinating neuropathy due to increased genetic dosage of peripheral myelin protein 22 (PMP22). The functions of PMP22 as a structural protein of peripheral myelin and a regulator of the proliferation, differentiation, shaping and apoptosis of Schwann cells (SCs) are well known. However, the effects of its overexpression on the molecular phenotype of SCs are still unclear. In order to detect genes that are modulated by PMP22 overexpression, we performed a cDNA microarrays experiment on PMP22 overexpressing sciatic nerves from a transgenic model of CMT1A. Differential expression of 117 known genes that are sensitive to PMP22 dosage was observed. The number of up- and down-regulated genes in the transgenic nerves was 99 and 144 respectively, of 22.000 spotted genes. In the up-regulated group, the number of known genes was 30 and the number of EST was 69. In the down-regulated group, the number of known genes was 87 and that of EST 57. We classified the differentially expressed genes into functional categories. Among these, we focused our attention on groups of down-regulated genes coding for cytoskeletal and transmembrane proteins and for extracellular matrix components, like collagens. Moreover we identified a group of genes with chromosomal localization in candidate regions for demyelinating neuropathies. The differential expression of several sequences was confirmed by RT-PCR, performed both on normal and transgenic rat sciatic nerves, and on primary SCs cultures obtained from normal and CMT1A rats. Therefore, we point out the importance of the interaction between SCs and extracellular matrix and the involvement of SCs cytoskeletal organization in the pathogenesis of CMT1A. Moreover, this study suggests us new candidate genes that could be submitted to sequence analysis in demyelinating CMT.

PHENOTYPE OF PMP22 TRANSGENIC SCHWANN CELLS IN CULTURE

Charcot-Marie-Tooth type 1A (CMT1A) neuropathy is due to a duplication of the peripheral myelin protein 22 (PMP22) gene. In the sciatic nerves of PMP22 transgenic (PMP22tg) animals, Schwann cells (SC) switch on the myelinating program by producing detectable levels of myelin related proteins (PMP22, P0, MBP), but express also early maturational markers and antigens typical of non-myelinating SC (p75NGFR). To further investigate PMP22tg SC behaviour, we established short-term (five days) SC cultures from P30 normal and PMP22tg rats. Semiquantitative RT-PCR was used to study PMP22, P0, MBP and p75NGFR mRNA expression, both in basal conditions and after forskolin (FSK) addition to mimic the axonal contact. SC proliferation rate was studied using a colorimetric assay (MTT based). Moreover, a gold particle motility assay was used to evaluate cell motility and shaping. Finally, preliminary experiments on SC migration in presence of IGFI were performed using a Boyden chamber. Homozygous and heterozygous PMP22tg SC, compared to normal ones, showed higher basal levels of PMP22 (0.82 vs 0.6 vs 0.09), P0 (2.24 vs 1.92 vs 0.53) and p75NGFR (1.33 vs 1.38 vs 0.34) mRNAs. FSK treatment (24 hrs) further increased PMP22 levels in homozygous (1.57 vs 0.82), heterozygous (0.93 vs 0.6) and normal (0.194 vs 0.09) cultures. PMP22tg SC showed a lower proliferation rate than normal ones. In basal conditions no differences were found between PMP22tg and normal SC in terms of cell area and motility, whereas FSK treatment determined, in PMP22tg SC, a significant decrease in cell area and motility. Finally, cell migration in the presence of a chemoattractant molecule did not differ between PMP22tg and normal SC. Cultured PMP22tg SC, besides maintaining the ability to overexpress PMP22, further increase its expression after FSK treatment. As PMP22tg SC in the presence of FSK also show a decreased motility and area, but migration seems to be identical between normal and transgenic cells, we conclude that axonal contact may increase PMP22 expression at a level that affects SC ability of forming myelin.