Roberto Cotrufo

Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product.

To clarify the role and function of the N‐myc product in cell differentiation and apoptosis, we used the antisense oligonucleotide technique to inhibit N‐myc gene expression in neuroblastoma cells with different phenotypes: intermediate (I) and neuronal (N), or Schwann‐glia (S), respectively. The results suggest that N‐myc operates along different pathways. Inhibiting N‐myc gene expression either results in suppression of cell proliferation or in induction of differentiation and/or apoptosis. J. Cell. Biochem. 73:97–105, 1999.

A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Accumulating evidence indicates that various miRNAs, expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, misregulation of microRNAs contributes to some mental disorders and neurodegeneration diseases. Here, we analyzed the expression profiles of 911 human miRNAs using microarray technology in leukocytes, the most readily available human tissue cells, obtained from 8 patients affected by sporadic amyotrophic lateral sclerosis (sALS) and 12 healthy controls. An independent group of 14 sALS patients and 14 controls was used for validation by TaqMan real-time polymerase chain reaction assay. We identified 8 miRNAs that were significantly up- or downregulated in sALS patients as compared to healthy controls. The significant variations in miRNAs profiles detected in leukocytes have been related to miRNAs predominantly expressed in the nervous system. One of these miRNAs, miR-338-3p, has previously been shown to be de-regulated in ALS brains. This study, for the first time, detected specific microRNAs disease-related changes at an earlier stage of sALS. We suggest that miRNAs profiles found in the peripheral blood leukocytes from sALS patients can be relevant to understand the pathogenesis of sALS and/or used as biomarkers of the disease.

EGF-responsive rat neural stem cells: Molecular follow-up of neuron and astrocyte differentiation in vitro

Neural stem cells (NSCs) could be very useful for the “cell therapy” treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF‐responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF‐containing medium, cells were plated on poly‐L‐ornithine‐coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the “p53 network,” was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.

Azathioprine and Interferon β1a in Relapsing-Remitting Multiple Sclerosis Patients: Increasing Efficacy of Combined Treatment

Current treatments of relapsing-remitting multiple sclerosis (RRMS) with immunosuppressive or immunomodulatory drugs have been shown to modify the course of the disease in a significative number of patients. However, in many cases, the response to either interferon β (IFN-β) or azathioprine (AZA) treatments was not satisfactory and new therapeutic approaches are needed. We studied clinical and MRI efficacy, safety and tolerance of AZA and IFN-β1a combined therapy in 23 patients with clinically definite RRMS, who had not previously been responsive to either monotherapies. Our cases were divided into three subgroups: 8 previously untreated patients (subgroup A) with at least 2 years of natural course of the disease, 8 patients (subgroup B) previously treated with AZA for 2 years and 7 patients (subgroup C) previously treated with IFN-β1a for 2 years. The baseline Expanded Disability Status Scale (EDSS) ranged from 2 to 4 in all subgroups. All patients completed 2 years of combined treatment with a dose of AZA adjusted to reduce lymphocyte count down to 1,000 ± 100/µl in association with IFN-β1a at a dose of 6 MIU every other day. The mean number of relapses during the combined treatment period was significantly lower than that observed before combined therapy in all the three subgroups. Also, the mean ΔEDSS score was significantly lower during combined treatment than in monotherapy in subgroups B and C. Moreover, after 2 years of combined treatment, the number of new T1 hypointense lesions, the number and volume of proton density/T2 hyperintense lesions and the gadolinium enhancement of T1 hypointense lesions were significantly lower than before combined treatment. After 2 years of treatment, this combination therapy appears to be safe and well tolerated and no serious side effects were reported. Despite some limitations of our study design, the information regarding efficacy, safety and tolerance of the association of AZA and IFN-β is most encouraging.

Increased expression of IGF-binding protein-5 in Duchenne Muscular Dystrophy (DMD) fibroblasts correlates with the fibroblast-induced downregulation of DMD myoblast growth: An in vitro analysis

In DMD the progressive loss of muscle ability and concomitant increasing fibrosis might originate from, besides other causes, the fibroblast paracrine inhibition of satellite cell “growth.” In this study we report that in myoblast/fibroblast coculture experiments, the presence of DMD fibroblasts negatively interfered with DMD myoblast growth to an extent directly proportional to the percentage of DMD fibroblasts present in the mixed‐cell cultures. Moreover, the observation that media conditioned with proliferating DMD fibroblasts inhibited the growth of DMD myoblasts more seriously than did control fibroblast‐conditioned media suggested a paracrine effect by diffusible factors. IGF‐binding proteins could act as such diffusible factors; in fact, IGFBP‐5 transcript increased threefold in DMD fibroblasts proliferating in DMD muscle extracts, whereas IGFBP‐3 mRNA decreased. In addition, high levels of IGFBP‐5 protein were detected in DMD fibroblast‐conditioned media. In neutralizing IGFBP‐5 in DMD fibroblast‐conditioned media by means of specific antibodies, or inhibiting IGFBP‐5 gene expression in DMD fibroblasts by means of oligo antisense, the fibroblast‐conditioned media lost inhibitory power over DMD myoblast proliferation. J. Cell. Physiol. 185:143–153, 2000.

Defective growth in vitro of Duchenne Muscular Dystrophy myoblasts: the molecular and biochemical basis.

As the molecular basis of Duchenne Muscular Dystrophy (DMD) was being discovered, increasing focus was placed on the mechanisms of progressive failure of myoregeneration. In this study, we propose a pathogenesis model for DMD, where an autocrine growth factor release of TGF‐β1—from necrotic myofibers—could contribute to the increasing loss of muscle regeneration. In fact, we report evidence that DMD myoblasts reduce their proliferation rate, in time and later cultures; in connection with this, we observed TGF‐β1 increase in conditioned media of DMD myoblasts, able to control the myoblast growth by reducing fusion and differentiation of DMD satellite cells. J. Cell. Biochem. 76:118–132, 1999.

miR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Ninety percent of ALS patients are sporadic cases (sALS) with no clear genetic linkage. Accumulating evidence indicates that various microRNAs (miRNAs), expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, microRNA dysregulation contributes to some mental disorders and neurodegeneration diseases. In our research, the expression of one selected miRNA, miR-338-3p, which previously we have found over-expressed in blood leukocytes, was studied in several different tissues from sALS patients. For the first time, we detected a specific microRNA disease-related upregulation, miR-338-3p, in blood leukocytes as well in cerebrospinal fluid, serum, and spinal cord from sALS patients. Besides, staining of in situ hybridization showed that the signals of miR-338-3p were localized in the grey matter of spinal cord tissues from sALS autopsied patients. We propose that miRNA profiles found in tissue samples from sALS patients can be relevant to understand sALS pathogenesis and lead to set up effective biomarkers for sALS early diagnosis.

Sporadic Creutzfeldt-Jakob disease: the extent of microglia activation is dependent on the biochemical type of PrPSc.

In prion-related encephalopathies, microglial activation occurs early and is dependent on accumulation of disease-specific forms of the prion protein (PrPSc) and may play a role in nerve cell death. Previously, we found that different types of PrPSc (i.e. type 1 and type 2) coexisted in approximately 25% of patients with sporadic Creutzfeldt-Jakob disease (CJD); and a close relationship was detected between PrPSc type, the pattern of PrP immunoreactivity, and extent of spongiform degeneration. To investigate whether microglial reaction is related to the biochemical type and deposition pattern of PrPSc, we carried out a neuropathologic and biochemical study on 26 patients with sporadic CJD, including all possible genotypes at codon 129 of the prion protein gene. By quantitative analysis, we demonstrated that strong microglial activation was associated with type 1 PrPSc and diffuse PrP immunoreactivity, whereas type 2 PrPSc and focal PrP deposits were accompanied by mild microglia reaction. These findings support the view that the phenotypic heterogeneity of sporadic CJD is largely determined by the physicochemical properties of distinct PrPSc conformers.

Charcot–Marie–Tooth disease with giant axons A clinicopathological and genetic entity

The authors report an Italian family with autosomal-dominant Charcot–Marie–Tooth disease (CMT) in which there were giant axons in the sural nerve biopsy. Linkage to the known CMT2 loci (CMT2A, CMT2B, CMT2D, CMT2F) and mutations in the known CMT2 genes (Cx32, MPZ, NEFL), GAN, NEFM, and CMT1A duplication/HNPP deletion were excluded. This family with CMT and giant axons has a pathologic and genetic entity distinct from classic CMT.

Experimental hyperthyroidism fails to expedite reinnervation of muscles denervated by crushing sciatic nerves in rabbits.

Abstract The time for initial reinnervation of tibialis anterior muscle after sciatic nerve crushing was measured in rabbits treated with intraperitoneal injections of 3,3′,5-triiodothyronine, 1 μg/kg body weight/day. The sciatic nerve was crushed in the thigh, just above its division into lateral and medial popliteal nerves. The method used to determine the beginning of reinnervation consisted of EMG recordings of (a) muscular potentials evoked by electrical stimulation of the sciatic nerve, below the crushed point, and (b) spontaneous action potentials during activity; the muscle tested was the tibialis anterior. The time for initial reinnervation averaged 43.6 ± 1.15 days in treated rabbits, and 43.2 ± 1.78 days in vehicle-injected animals. The distance from the site of crushing to the point where the anterior tibial nerve enters the tibialis anterior muscle was measured in each animal and the regeneration rate was calculated; this parameter averaged 1.48 ± 0.05 mm/day in treated rabbits, and 1.50 ± 0.04 mm/day in control animals. Therefore, no effect of 3,3′,5-triiodothyronine at the dosage used on the reinnervation process of muscles denervated by a crushed sciatic nerve could be demonstrated.