Valentina Papa

Satellite cell characterization from aging human muscle

Abstract Objectives: Satellite cells (SCs) are skeletal muscle progenitor cells located between the basal lamina and the sarcolemma of muscle fibers. They are responsible for muscle growth and repair. In humans, aging results in the depletion of the SC population and in its proliferative activity, but not in its function. It has not yet been determined whether under conditions of massive muscle fiber death in vivo, the regenerative potential of SCs is totally or partially compromised in old muscle. No studies have yet tested whether advanced age is a factor that restrains the response of SCs to muscle denervation in humans; this is also due to difficulties in the isolation and in the culture of SCs from a small human surgery fragment. The aim of this study was to study in depth muscle regeneration analysing the SC ability of SCs to proliferate and differentiate in aging human patients. Methods: In order to study in more detail the molecular mechanism, the proliferative and differentiative ability of aging SCs, we isolated SCs from aging human muscle biopsies and analysed their morphology by transmission electron microscopy and immunocytochemical analysis (antibodies against desmin, N-CAM and M-cadherin) and their capacity to grow and to expand in vitro. Moreover, in order to evaluate gene expression of myogenic regulatory factors Myf5, MyoD and myogenin (Myf4), RT-PCR was performed. Results and discussion: SCs isolated from aging human muscle biopsies and plated into favorable proliferation and differentiation conditions were able to proceed through the myogenic program and actively form myotubes, although taking longer than the young control sample. The RT-PCR analysis together with the ultrastructural SC features showed that the myogenic potential seemed to be compromised during the aging human muscle proliferation in vitro.

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

Sleep and cardiovascular phenotype in middle-aged hypocretin-deficient narcoleptic mice

Narcolepsy with cataplexy (NC) is a lifelong disorder caused by loss of hypothalamic hypocretin/orexin (HCRT) neurones, often starting in childhood. NC patients show altered control of heart rate (HR) and a normotensive non‐dipper blood pressure (BP) profile, but the natural history and prognostic significance of these alterations remain unclear. Similar alterations have been observed in HCRT‐ataxin‐3 transgenic (TG) NC mice lacking HCRT neurones, but studies have been limited to young adult individuals <4 months of age. Here we evaluated long‐term effects of NC on derangements in the wake–sleep state and cardiovascular control by studying middle‐aged TG. We chronically instrumented TG and wild‐type mice aged 10–11 months with electrodes for sleep scoring and a telemetric transducer for BP and HR measurements. We then recorded mice in freely behaving conditions. TG showed a NC phenotype including fragmentation of wakefulness, reduced latency to rapid eye movement sleep (REMS) and cataplexy‐like events. TG also showed blunted BP decline on entering non‐rapid eye movement sleep (NREMS), enhanced BP increase on passing to REMS, increased HR, and blunted changes in HR upon arousal and awakening from NREMS. Histological and ultrastructural analysis of cardiovascular and renal tissue did not reveal evidence of subclinical hypertensive organ damage. These data indicate that HCRT neurone loss in TG causes alterations in wake–sleep behaviour and cardiovascular control that are not peculiar to the beginning of the disease but are maintained at least up to middle age. These alterations are similar to those in adult NC patients, but do not produce early subclinical damage to the heart and kidneys.

Validity of internal expression of the major histocompatibility complex class I in the diagnosis of inflammatory myopathies

Objective The inflammatory myopathies (IMs) are a group of disorders characterised by weakness and inflammation of the skeletal muscles. Muscle biopsy is the most crucial test to confirm the clinical diagnosis, but also the most common cause of misdiagnosis. There are currently no markers specific or sensitive enough to distinguish IMs from other diseases with similar clinical and morphological features, and an international multidisciplinary effort is under way to develop new classification criteria for IMs. Methods Standards for Reporting of Diagnostic Accuracy recommendations to validate a diagnostic test based on the quantification of internal major histocompatibility complex class I (MHC-I) positive fibres were adopted. MHC-I immunostained specimens from 64 patients were scored by two independent blinded investigators, and the percentage of positive fibres was determined. Agreement between investigators was evaluated with the k-weighted statistic. The receiver operating characteristic curve, area under the curve, sensitivity, specificity, and positive and negative predictive values of each percentage range of positive fibres versus the diagnosis of IM were calculated. Results The main difference between IM and non-inflammatory samples was the number of internal MHC-I positive fibres. The k-weighted value was 0.89 for a percentage of MHC-I positive fibres above 50%; the positive predictive value was 100%, and the negative predictive value was 94%. Conclusions This is the first study on the validity of a quantitative analysis of internal MHC-I positive fibres for an IM diagnosis performed according to Standards for Reporting of Diagnostic Accuracy recommendations. The interobserver agreement was almost perfect, thus making the method reproducible. Applying an MHC-I cut-off above 50% is an optimal marker for polymyositis (PM) and dermatomyositis (DM) diagnosis.

Liver transplantation for mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal, recessive disease caused by mutations in the gene encoding thymidine phosphorylase, leading to reduced enzymatic activity, toxic nucleoside accumulation, and secondary mitochondrial DNA damage. Thymidine phosphorylase replacement has been achieved by allogeneic hematopoietic stem cell transplantation, a procedure hampered by high mortality. Based on high thymidine phosphorylase expression in the liver, a 25‐year‐old severely affected patient underwent liver transplantation. Serum levels of toxic nucleosides rapidly normalized. At 400 days of follow‐up, the patients clinical conditions are stable. We propose liver transplantation as a new therapy for MNGIE. Ann Neurol 2016;80:448–455

Homozygous NOTCH3 null mutation and impaired NOTCH3 signaling in recessive early‐onset arteriopathy and cavitating leukoencephalopathy

Notch signaling is essential for vascular physiology. Neomorphic heterozygous mutations in NOTCH3, one of the four human NOTCH receptors, cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Hypomorphic heterozygous alleles have been occasionally described in association with a spectrum of cerebrovascular phenotypes overlapping CADASIL, but their pathogenic potential is unclear. We describe a patient with childhood‐onset arteriopathy, cavitating leukoencephalopathy with cerebral white matter abnormalities presented as diffuse cavitations, multiple lacunar infarctions and disseminated microbleeds. We identified a novel homozygous c.C2898A (p.C966*) null mutation in NOTCH3 abolishing NOTCH3 expression and causing NOTCH3 signaling impairment. NOTCH3 targets acting in the regulation of arterial tone (KCNA5) or expressed in the vasculature (CDH6) were downregulated. Patients vessels were characterized by smooth muscle degeneration as in CADASIL, but without deposition of granular osmiophilic material (GOM), the CADASIL hallmark. The heterozygous parents displayed similar but less dramatic trends in decrease in the expression of NOTCH3 and its targets, as well as in vessel degeneration. This study suggests a functional link between NOTCH3 deficiency and pathogenesis of vascular leukoencephalopathies.

Skeletal Muscle Satellite Cells in Amyotrophic Lateral Sclerosis

Abstract Objectives: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving progressive muscular paralysis reflecting degeneration of motor neurons. Skeletal muscle tissue seems to play a significant role in ALS pathogenesis. Here, the role of satellite cells (SCs) in ALS muscle atrophy is investigated. Methods: We isolated SCs from ALS human muscle biopsies and we analyzed their ability to grow and expand in vitro. Ultrastructural and immunophenotypical features were analyzed. Quantitative real-time RT-QPCR and western blot (WB) analyses were performed to evaluate MRFs and MyH1 expression. Results: ALS SCs showed a high proliferative potential, but their capacity to proceed through the myogenic program and form myotubes seems altered compared to controls (Ctrls). We observed that differentiating ALS SCs showed some specific features, but they displayed an altered morphology, with a large number of vacuoles. RT-QPCR and WB showed lower Myf-4 and MyH1 compared to Ctrls. Conclusions: Our data suggest that the capacity of ALS SCs to proceed through the myogenic program seems to be altered: SCs seem to lose their ability to regenerate and restore mature myofibers.

Proteomic analysis of extracellular vesicles from medullospheres reveals a role for iron in the cancer progression of medulloblastoma

BackgroundMedulloblastoma (MB) is the most common malignant childhood brain tumor with the propensity to disseminate at an early stage, and is associated with high morbidity. New treatment strategies are needed to improve cure rates and to reduce life-long cognitive and functional deficits associated with current therapies. Extracellular Vesicles (EVs) are important players in cell-to-cell communication in health and diseases. A clearer understanding of cell-to-cell communication in tumors can be achieved by studying EV secretion in medullospheres. This can reveal subtle modifications induced by the passage from adherent to non-adherent growth, as spheres may account for the adaptation of tumor cells to the mutated environment.MethodsFormation of medullospheres from MB cell lines stabilized in adherent conditions was obtained through culture conditioning based on low attachment flasks and specialized medium. EVs collected by ultracentrifugation, in adherent conditions and as spheres, were subjected to electron microscopy, NanoSight measurements and proteomics.ResultsInterestingly, iron carrier proteins were only found in EVs shed by CSC-enriched tumor cell population of spheres. We used iron chelators when culturing MB cell lines as spheres. Iron chelators induced a decrease in number/size of spheres and in stem cell populations able to initiate in vitro spheres formation.ConclusionsThis work suggests a not yet identified role of iron metabolism in MB progression and invasion and opens the possibility to use chelators as adjuvants in anti-tumoral chemotherapy.

Ultrastructural changes in LGMD1F.

A large Italo‐Spanish kindred with autosomal‐dominant inheritance has been reported with proximal limb and axial muscle weakness. Clinical, histological and genetic features have been described. A limb girdle muscular dystrophy 1F (LGMD1F) disease locus at chromosome 7q32.1–32.2 has been previously identified. We report a muscle pathological study of two patients (mother and daughter) from this family. Muscle morphologic findings showed increased fiber size variability, fiber atrophy, and acid‐phosphatase‐positive vacuoles. Immunofluorescence against desmin, myotilin, p62 and LC3 showed accumulation of myofibrils, ubiquitin binding protein aggregates and autophagosomes. The ultrastructural study confirmed autophagosomal vacuoles. Many alterations of myofibrillar component were detected, such as prominent disarray, rod‐like structures with granular aspect, and occasionally, cytoplasmic bodies. Our ultrastructural data and muscle pathological features are peculiar to LGMD1F and support the hypothesis that the genetic defect leads to a myopathy phenotype associated with disarrangement of the cytoskeletal network.

Familial polyglucosan body myopathy with unusual phenotype

Polyglucosan (PG) is an abnormal polysaccharide that, compared with glycogen, has fewer branched points and excessively long peripheral chains that structurally resemble the plant polysaccharide ‘amylopectin’. Under electron microscopy, PG bodies are round, nonmembrane-bound cytoplasmic particles with irregular branched filaments, which often displace myofibrils, leading to Z disk streaming. PG bodies react strongly to PAS stain and are partially resistant to diastase digestion. PG accumulation constitutes the histopathological manifestation of several clinically different conditions. It occurs mainly in the brain (Lafora disease, double athetosis) and in skeletal muscle (glycogenosis type IV, glycogenosis type VII, other polysaccharide storage myopathies [1–6]). Recently, recessive mutations in the RBCK1 gene, which encodes for an E3 ubiquitin ligase, have been recognized as causing two different disorders with PG accumulation in skeletal and cardiac muscle: a myopathy with cardiomyopathy [7,8], and a fatal immunodeficiency with myopathy and cardiomyopathy [9]. To date, only 10 families have been reported in which PG body myopathy is caused by RBCK1 gene mutations. Here we describe a new family (including one pair of siblings, Patient 1 and Patient 2, and their paternal female cousin, Patient 3), presenting as an autosomal recessive form of late-onset myopathy with limb girdle muscular dystrophy phenotype and PG accumulation in muscle. Patient 1. This 66-year-old man had complained for many years of back pain, progressive difficulty walking and climbing stairs and weakness in muscles of both girdles. He presented with a waddling gait, weakness in ileopsoas, leg and arm flexor muscles, calf hypertrophy, pes cavus and moderately increased creatine kinase level. At age 74 years he was unable to rise from sitting (Figure 1) and was able to walk only with the aid of a cane. Cardiac investigations revealed a bundle branch block and an ischemic cardiomyopathy. He also presented with skin lesions characteristic of generalized vitiligo (Figure 1). Patient 2. At age 52 years she complained of progressive difficulty in climbing stairs and rising from a chair, and noticed asthenia in the lower limbs. Creatine kinase level was mildly increased. At age 57 years she was diagnosed with familial spastic paraparesis with axonal motor polyneuropathy, and with chronic inflammatory demyelinating polyneuropathy (CIDP). She presented with a waddling gait, inability to rise from the floor without support, weakness in proximal limb muscles, pes cavus, calf hypertrophy and hand muscle atrophy. No cardiac symptoms were reported. At age 60 years she became unable to walk unsupported. Patient 3. Since she was 50 years she had complained of limb myalgia. After an ischemic stroke at age 52, causing sensory-motor hemi-syndrome, she noticed progressive weakness in proximal limb girdle muscles. At age 63 years, when the first muscle biopsy was done, she presented with a waddling gait, Gowers sign, normal EMG and CK levels. At age 70 years, when a second muscle biopsy was performed, she was unable to raise her arms over her head, had scapular winging, and quadriceps muscle wasting. No cardiac symptoms were reported. Two out of the four muscle biopsies available for this study (Patient 1, first biopsy Patient 3) showed myopathic features but neither vacuoles nor accumulation of PASpositive material. Conversely, the other two muscles (Patient 2, second biopsy Patient 3) were characterized by violet or hyaline PG inclusions in about 10–20% of fibres (Figure 2). The vacuoles were filled with strongly PASpositive reacting material, whereas the cytoplasmic areas surrounding the inclusions were depleted in glycogen. The vacuoles were outlined by a rim of intense oxidative enzyme reaction, but their membranes were not immunolabelled by caveolin-3. The accumulated material showed absent staining with Lugol’s iodine and acid phosphatase, negative immunolabelling for desmin and myotilin; it was resistant to pre-digestion with both diastase/amylase and proteinase-k, and was strongly immunolabelled for ubiquitin and for p62/SQSTM1 (protein aggregates) (Figure 2). Scattered LC3-positive reaction was present in most fibres. Immunoblot analysis