Lucilla Nobbio

Effect of Recombinant Human Nerve Growth Factor on Cisplatin Neurotoxicity in Rats

In this study we evaluated the effect of recombinant human nerve growth factor (rhNGF) on cisplatin (CDDP)-induced sensory neuronopathy in an experimental paradigm in the rat. Young adult female Wistar rats were treated with CDDP (2 mg/kg ip twice weekly for nine times) alone or in combination with rhNGF (1 mg/kg sc on alternate days). The effect of CDDP +/- NGF treatment was evaluated with behavioral (tail-flick test) and neurophysiological (nerve conduction velocity in the tail) methods immediately after treatment and after a follow-up period of 6 weeks. Pathological and morphometrical examinations of the dorsal root ganglia (DRG) and sciatic and saphenous nerves were also performed. rhNGF treatment induced a significant reduction in the CDDP-induced decrease in nerve conduction velocity (P < 0.05), and this was associated with a significant protection against the decrease in somatic (P < 0.05), nuclear (P < 0.05), and nucleolar size (P < 0.01) caused by CDDP treatment. However, for each of the parameters examined the neuroprotection obtained with rhNGF treatment was not complete. At the follow-up examination no differences between the three groups were observed in tail-flick test and nerve conduction velocity. We conclude that rhNGF, administered according to the schedule used in this experiment, exerts a biologically significant neuroprotective effect against CDDP peripheral neurotoxicity.

Different cellular and molecular mechanisms for early and late-onset myelin protein zero mutations

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

Mycophenolate mofetil in dysimmune neuropathies: A preliminary study

Mycophenolate mofetil (MM) is an immunosuppressant that has been used successfully for preventing the rejection of renal, heart, or liver transplants and for the therapy of immune-mediated diseases.4 It shows modest side effects and has a lower risk for late malignancies compared with other immunosuppressive drugs.1 Recently, MM has been used also for the treatment of myasthenia gravis, polymyositis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and multifocal motor neuropathy (MMN).2,7,10 We treated four patients with possible, probable, or definite MMN11 and two with CIDP8 (Table 1) who were on large doses of intravenous immunoglobulins (IVIg), with the hope of reducing or withdrawing IVIg while maintaining a satisfactory and stable clinical state. Four patients were also receiving other immunomodulating agents. Patients received oral MM in a dosage of 1 g twice daily, for an average of 9 months (range, 6–12 months). Prior to MM treatment, all patients underwent several attempts to reduce IVIg dosage, resulting in clinical worsening, even in subjects receiving other immunomodulating agents. Patients were evaluated at baseline and each month thereafter, using the Medical Research Council (MRC) sumscore6 and the Immune Neuropathy Course and Treatment (INCAT) arm and leg disability scores.5 The values of the INCAT scale at baseline ranged from 0 to 3 (mean, 1.5). Full blood count, renal and liver function, and serum amylase levels were monitored every 2 months. Side effects were recorded and graded according to the Common Terminology Criteria for Adverse Events.9 All the patients provided informed consent to participate in the study. Table 1 summarizes the details of each patient. In five patients we did not observe any worsening in MRC sumscore or INCAT disability scale despite a significant reduction in IVIg dosage. On average, before starting MM treatment patients received 2g/kg every 4 weeks. After 6 months the mean dosage of IVIg was 0.75 g/kg every 4 weeks. In patients 1 and 2, IVIg infusion was reduced by 50% after 2 months and discontinued after 4 months. After 1 year of therapy with MM, IVIg treatment remains discontinued. In patient 4, the addition of MM allowed a reduction in IVIg dose to 50% after 4 months. In patient 3, IVIg dose was reduced by 25% but only for 4 months; symptoms relapse then required a return to the previous IVIg schedule. In patients 5 and 6, who had CIDP, IVIg dosage was reduced by 50%. After follow-up ranging from 6 to 12 months there was no deterioration in the MRC sumscore or in the INCAT disability scale in patients 1, 2, 4, 5, and 6; indeed, in patients 1 and 2 we observed a minimal improvement of overall disability within 2 months of starting MM. Finally, azathioprine (patients 3, 5, and 6) and cyclophosphamide (patient 2) were discontinued after 3 months of combined IVIg and MM treatment. Patients 1 and 4 discontinued MM because of loss of appetite, weight loss, abdominal pain, and increase of amylase levels (grade 1 in both cases). Patient 5 developed mild leukopenia (grade 1). Recently, MM has been introduced for the treatment of immune-mediated neuromuscular disorders,2,3 but the findings in only one patient with MMN and nine patients with CIDP have been published, to our knowledge, with conflicting results.7,10 We found that MM, in patients with MMN and CIDP, is effective in reducing IVIg requirement and in replacing other, more toxic, drugs. Only one patient with MMN relapsed, after 4 months, perhaps because of longer duration of disease, as previously observed in another series of patients.10 In fact, axonal impairment, more than immune-mediated de-

Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22

Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate (PIP2)-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles, and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP2-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane–endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.

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.

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca2+]i) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca2+]i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca2+ into CMT1A SC. Correction of the altered [Ca2+]i in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca2+]i and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

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.

Neuroactive Steroid Levels in a Transgenic Rat Model of CMT1A Neuropathy

Charcot–Marie–Tooth type 1A (CMT1A) represents 80% of all the demyelinating hereditary motor and sensory neuropathies. As recently suggested, neuroactive steroids may have a role in a therapeutic strategy for peripheral neuropathies, including CMT1A. To this aim, an accurate qualitative and quantitative analysis of neuroactive steroid levels in this disease could be extremely important to define effective pharmacological strategies. We here analyzed by liquid chromatography-tandem mass spectrometry the levels of neuroactive steroids present in the sciatic nerve of male and female peripheral myelin protein 22 transgenic rats (PMP22tg rats; i.e., an experimental model of CMT1A) and of the corresponding wild-type littermates. We observed that, both in PMP22tg rats and in the wild types, the levels of neuroactive steroids, such as progesterone, tetrahydroprogesterone (THP), isopregnanolone (3β,5α-THP), testosterone, dihydrotestosterone, and 5α-androstane-3α, 17β-diol (3α-diol) are sexually dimorphic. It is interesting to note that the levels of 3β,5α-THP and of 3α-diol, which are exclusively detectable in sciatic nerve of female and male rats, respectively, are strongly decreased in PMP22tg rats. 3β,5α-THP and 3α-diol are modulators of gamma-amino butyric acid A receptor. Thus, the present findings may be considered an interesting background for experiments aimed to evaluate the possible therapeutic effects of modulators of this neurotransmitter receptor in male and female PMP22tg rats.

Collagen VI regulates peripheral nerve regeneration by modulating macrophage recruitment and polarization.

Macrophages contribute to peripheral nerve regeneration and produce collagen VI, an extracellular matrix protein involved in nerve function. Here, we show that collagen VI is critical for macrophage migration and polarization during peripheral nerve regeneration. Nerve injury induces a robust upregulation of collagen VI, whereas lack of collagen VI in Col6a1−/− mice delays peripheral nerve regeneration. In vitro studies demonstrated that collagen VI promotes macrophage migration and polarization via AKT and PKA pathways. Col6a1−/− macrophages exhibit impaired migration abilities and reduced antiinflammatory (M2) phenotype polarization, but are prone to skewing toward the proinflammatory (M1) phenotype. In vivo, macrophage recruitment and M2 polarization are impaired in Col6a1−/− mice after nerve injury. The delayed nerve regeneration of Col6a1−/− mice is induced by macrophage deficits and rejuvenated by transplantation of wild-type bone marrow cells. These results identify collagen VI as a novel regulator for peripheral nerve regeneration by modulating macrophage function.

Does parkin play a role in the peripheral nervous system? A family report

Two genes were identified for autosomal recessive forms of early onset Parkinsons disease: parkin and DJ‐1. We describe 2 siblings with EOPD due to parkin mutations and peripheral neuropathy, which presented as neuropathy with liability to pressure palsies (HNPP) in the index case. RT‐PCR experiments revealed that the parkin gene is expressed in sural nerves from both controls and patient with parkin‐related disease. Our findings support the view that parkin may play a role in the peripheral nervous system.