Elena Stoppani

Optimal Plasma Progranulin Cutoff Value for Predicting Null Progranulin Mutations in Neurodegenerative Diseases: A Multicenter Italian Study

Background: Recently, attention was drawn to a role for progranulin in the central nervous system with the identification of mutations in the progranulin gene (GRN) as an important cause of frontotemporal lobar degeneration. GRN mutations are associated with a strong reduction of circulating progranulin and widely variable clinical phenotypes: thus, the dosage of plasma progranulin is a useful tool for a quick and inexpensive large-scale screening of carriers of GRN mutations. Objective: To establish the best cutoff threshold for normal versus abnormal levels of plasma progranulin. Methods: 309 cognitively healthy controls (25–87 years of age), 72 affected and unaffected GRN+ null mutation carriers (24–86 years of age), 3 affected GRN missense mutation carriers, 342 patients with neurodegenerative diseases and 293 subjects with mild cognitive impairment were enrolled at the Memory Clinic, IRCCS S. Giovanni di Dio-Fatebenefratelli, Brescia, Italy, and at the Alzheimer Unit, Ospedale Maggiore Policlinico and IRCCS Istituto Neurologico C. Besta, Milan, Italy. Plasma progranulin levels were measured using an ELISA kit (AdipoGen Inc., Seoul, Korea). Results: Plasma progranulin did not correlate with age, gender or body mass index. We established a new plasma progranulin protein cutoff level of 61.55 ng/ml that identifies, with a specificity of 99.6% and a sensitivity of 95.8%, null mutation carriers among subjects attending to a memory clinic. Affected and unaffected GRN null mutation carriers did not differ in terms of circulating progranulin protein (p = 0.686). A significant disease anticipation was observed in GRN+ subjects with the lowest progranulin levels. Conclusion: We propose a new plasma progranulin protein cutoff level useful for clinical practice.

Point mutated Caveolin-3 form (P104L) impairs myoblast differentiation via Akt and p38 signalling reduction, leading to an immature cell signature

Unbalanced levels of caveolin-3 (Cav3) are involved in muscular disorders. In the present study we show that differentiation of immortalized myoblasts is affected by either lack or overexpression of Cav3. Nevertheless, depletion of Cav3 induced by delivery of the dominant-negative Cav3 (P104L) form elicited a more severe phenotype, characterized by the simultaneous attenuation of the Akt and p38 signalling networks, leading to an immature cell and molecular signature. Accordingly, differentiation of myoblasts harbouring Cav3 (P104L) was improved by countering the reduced Akt and p38 signalling network via administration of IGF-1 or trichostatin A. Furthermore, loss of Cav3 correlated with a deregulation of the TGF-β-induced Smad2 and Erk1/2 pathways, confirming that Cav3 controls TGF-β signalling at the plasma membrane. Overall, these data suggest that loss of Cav3, primarily causing attenuation of both Akt and p38 pathways, contributes to impair myoblast fusion.

Differentiation of human rhabdomyosarcoma RD cells is regulated by reciprocal, functional interactions between myostatin, p38 and extracellular regulated kinase signalling pathways

Rhabdomyosarcoma (RMS) includes heterogeneous tumours of mesenchymal derivation which are genetically committed to the myogenic lineage, but fail to complete terminal differentiation. Previous works have reported on deregulated myostatin, p38 and extracellular regulated kinase (ERK) signalling in RMS cell lines; however, the functional link between these pathways and their relative contribution to RMS pathogenesis and/or maintenance of the transformed phenotype in vitro are unclear. Herein we show that the constitutive expression of a dominant-negative form of activin receptor type IIb (dnACTRIIb), which inhibits myostatin signalling, decreased proliferation and promoted differentiation of the human RMS RD cell line. DnACTRIIb-dependent differentiation of RD cells correlated with a reduced SMAD2/3 (small mother against decapentaplegic) and ERK signalling and the activation of p38 pathway. Conversely, the expression of a constitutively activated ALK5 (activin receptor-like kinase) (caALK5) form, activating SMAD3 and ERK pathways, led to further impairment of RD differentiation. Pharmacological blockade of ERK pathway in RD cells was sufficient to replicate the biological phenotype observed in dnACTRIIb-expressing RD cells, and also recovered the differentiation of caALK5-expressing RD cells. Conversely, deliberate activation of p38 signalling mimics the effect of dnActRIIb and overcame the differentiation block in RD cells. These data indicate the existence of a network formed by myostatin/SMAD2/3, ERK and p38 pathways that, when deregulated, might contribute to the pathogenesis of RMS. The components of this network might, therefore, be a valuable target for interventions towards correcting the malignant phenotype of RMS.

Phenotypic behavior of C2C12 myoblasts upon expression of the dystrophy-related caveolin-3 P104L and TFT mutants

Caveolin‐3 (Cav‐3) is the main scaffolding protein present in myofiber caveolae. We transfected C2C12 myoblasts with dominant negative forms of Cav‐3, P104L or ΔTFT, respectively, which cause the limb‐girdle muscular dystrophy 1‐C. Both these forms triggered Cav‐3 loss during C2C12 cell differentiation. The P104L mutation reduced myofiber formation by impaired AKT signalling, accompanied by dramatic expression of the E3 ubiquitin ligase Atrogin. On the other hand, the ΔTFT mutation triggered hypertrophic myotubes sustained by prolonged AKT activation, but independent of increased levels of follistatin and interleukin 4 expression. These data suggest that separated mutations within the same dystrophy‐related gene may cause muscle degeneration through different mechanisms.

The cytosolic sialidase Neu2 is degraded by autophagy during myoblast atrophy

BACKGROUND The sialidase Neu2 is a cytosolic enzyme which is fully expressed in mature muscle myofibers. METHODS To investigate Neu2 expression during muscle atrophy, we employed an in vitro model consisting of terminally differentiated C2C12 myotubes exposed to different pro-atrophic stimuli that triggered catabolic pathways involved in proteasome activation or autophagy. RESULTS Neu2 expression was unchanged in myotubes treated with TNF-alpha, a cytokine known to activate the proteasome. However, Neu2 transcript levels and enzymatic activity were downregulated in starved or dexamethasone-treated myotubes that showed proteosomal activation and several hallmarks of macroautophagy, such as formation of autophagosomes, the accumulation of LC3 dots and bulk degradation of long-lived proteins. Neu2 activity and protein levels were rescued upon cotreatment with the lysosomotropic agent NH4Cl, the autophagy inhibitor 3-methyladenine or cathepsin inhibitors, as well as by insulin administration, but were unaffected upon pharmacological inhibition of the proteasome. Moreover, HA- or GST-Neu2 recombinant fusion proteins were rapidly degraded in vitro by purified cathepsin L and B. Overall, we may conclude that Neu2 is degraded by lysosomal enzymes in myotubes undergoing autophagy-mediated atrophy. GENERAL SIGNIFICANCE This study demonstrates that Neu2 enzyme degradation occurs in atrophic myotubes via macroautophagy and independently of proteasome activation.

The enzymatic activity of sialidase Neu2 is inversely regulated during in vitro myoblast hypertrophy and atrophy

Sialidase Neu2 is an exoglycosidase that removes terminal sialic acids from glycolipids and glycoproteins. In this study, we investigated Neu2 expression during muscle hypertrophy and atrophy. Neu2 mRNA and enzymatic activity were significantly increased in hypertrophic myofibers. A rise in Neu2 activity was observed after constitutive activation of AKT or Igf-1 treatment as well as in myoblasts treated with vasopressin or trichostatin, an inhibitor of histone deacetylases. In contrast, myofiber atrophy obtained by dexamethasone treatment or starvation triggered a significant loss of Neu2 activity and was paralleled by downregulation of Neu2 transcript levels. Overall, we may conclude that Neu2 enzymatic activity is causally linked to proper muscle differentiation and growth.

A window into the heterogeneity of human cerebrospinal fluid Aβ peptides.

The initiating event in Alzheimers disease (AD) is an imbalance in the production and clearance of amyloid beta (Aβ) peptides leading to the formation of neurotoxic brain Aβ assemblies. Cerebrospinal Fluid (CSF), which is a continuum of the brain, is an obvious source of markers reflecting central neuropathologic features of brain diseases. In this review, we provide an overview and update on our current understanding of the pathobiology of human CSF Aβ peptides. Specifically, we focused our attention on the heterogeneity of the CSF Aβ world discussing (1) basic research studies and what has been translated to clinical practice, (2) monomers and other soluble circulating Aβ assemblies, and (3) communication modes for Aβ peptides and their microenvironment targets. Finally, we suggest that Aβ peptides as well as other key signals in the central nervous system (CNS), mainly involved in learning and hence plasticity, may have a double-edged sword action on neuron survival and function.

Defective myogenic differentiation of human rhabdomyosarcoma cells is characterized by sialidase Neu2 loss of expression

Sialidase Neu2 is a glycohydrolytic enzyme whose tissue distribution has been detected principally in differentiated skeletal muscle. In this study we show that Neu2 expression is absent in different embryonal and alveolar human tumor rhabdomyosarcoma (RMS) cells, which are genetically committed myoblasts characterized by delayed differentiation. Forced myogenic differentiation of an embryonal RMS cell line, as obtained via pharmacological and genetic p38 activation or via follistatin overexpression, was characterized by Neu2 loss of expression despite the significant rise of different muscle‐specific markers, suggesting therefore that the defective myogenic program of RMS cells is accompanied by Neu2 suppression.

L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF- Members Are Candidates to Developmentally Control Their Fiber Formation

This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs) and follistatin as well as the highly related TGF-β members Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF-β ligands involved in developmental regulation of fiber size.