Neurodegenerative Disease-Associated TDP-43 Fragments Are Extracellularly Secreted with CASA Complex Proteins

Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuronal EVs also contained the autophagy-related MAP1LC3B-II protein. Notably, we found that, under PQC inhibition, HSPB8, BAG3 and MAP1LC3B-II secretion paralleled that of TDP-43 species. Taken together, our data highlight the role of EVs, particularly of LVs, in the disposal of disease-associated TDP-43 species, and suggest a possible new role for the CASA complex in NDs

Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins

Genetic correlation between amyotrophic lateral sclerosis and schizophrenia

A. Palotie on työryhmän Schizophrenia Working Grp Psychiat jäsen.We have previously shown higher-than-expected rates of schizophrenia in relatives of patients with amyotrophic lateral sclerosis (ALS), suggesting an aetiological relationship between the diseases. Here, we investigate the genetic relationship between ALS and schizophrenia using genome-wide association study data from over 100,000 unique individuals. Using linkage disequilibrium score regression, we estimate the genetic correlation between ALS and schizophrenia to be 14.3% (7.05-21.6; P = 1 x 10(-4)) with schizophrenia polygenic risk scores explaining up to 0.12% of the variance in ALS (P = 8.4 x 10(-7)). A modest increase in comorbidity of ALS and schizophrenia is expected given these findings (odds ratio 1.08-1.26) but this would require very large studies to observe epidemiologically. We identify five potential novel ALS-associated loci using conditional false discovery rate analysis. It is likely that shared neurobiological mechanisms between these two disorders will engender novel hypotheses in future preclinical and clinical studies.Peer reviewe

NEDD8-activating enzyme inhibition potentiates the anti-myeloma activity of natural killer cells

Abstract Natural Killer (NK) cells act as important regulators in the development and progression of hematological malignancies and their suppressor activity against Multiple Myeloma (MM) cells has been confirmed in many studies. Significant changes in the distribution of NK cell subsets and dysfunctions of NK cell effector activities were described in MM patients and correlated with disease staging. Thus, restoring or enhancing the functionality of these effectors for the treatment of MM represents a critical need. Neddylation is a post-translational modification that adds a ubiquitin-like molecule, NEDD8, to the substrate protein. One of the outcomes is the activation of the Cullin Ring Ligases (CRLs), a class of ubiquitin-ligases that controls the degradation of about 20% of proteasome-regulated proteins. Overactivation of CRLs has been described in cancer and can lead to tumor growth and progression. Thus, targeting neddylation represents an attractive approach for cancer treatment. Our group has recently described how pharmacologic inhibition of neddylation increases the expression of the NKG2D activating receptor ligands, MICA and MICB, in MM cells, making these cells more susceptible to NK cell degranulation and killing. Here, we extended our investigation to the direct role of neddylation on NK cell effector functions exerted against MM. We observed that inhibition of neddylation enhanced NK cell-mediated degranulation and killing against MM cells and improved Daratumumab/Elotuzumab-mediated response. Mechanistically, inhibition of neddylation increased the expression of Rac1 and RhoA GTPases in NK cells, critical mediators for an efficient degranulation at the immunological synapse of cytotoxic lymphocytes, and augmented the levels of F-actin and perforin polarization in NK cells contacting target cells. Moreover, inhibition of neddylation partially abrogated TGFβ-mediated repression of NK cell effector activity. This study describes the role of neddylation on NK cell effector functions and highlights the positive immunomodulatory effects achieved by the inhibition of this pathway in MM

The role of Extracellular Vesicles (EVs) in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD)

ALS and FTLD are neurodegenerative diseases characterized by pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. In 98% of ALS and in the majority of Tau-negative FTLD cases the main component is the TAR DNA-binding protein of 43 KDa (TDP-43) together with its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25). TDP-inclusions are mainly removed from cells via the protein quality control (PQC) system, but they could also be secreted within extracellular vesicles (EVs). In our work we first analysed the TDP-content of the EVs, by comparing large (LVs) with small vesicles (SVs); then, we evaluated the presence of some PQC-members. Finally, we investigated the effect of PQC blockage on EVs secretion and content. Methods. We isolated EVs produced by NSC34 cells untreated or treated with MG132 or NH4Cl (proteasome and autophagy inhibitors). To isolate EVs we used the differential ultracentrifugation method. We analysed EVs size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and their protein content through western blot analysis. Results. We showed that both TDP-43 and its C-terminal fragments (especially TDP-35) are secreted in EVs, mainly in LVs. Interestingly, in cells TDPs are present as soluble forms, instead the secreted TDPs are mainly insoluble. We found that many PQC-components are secreted in EVs and PQC modulation resulted in a significant increase in EVs numbers, that is paralleled by a slight increase in TDP-content. Summary/Conclusion. EVs may positively contribute to the clearance of insoluble TDPs species by cooperating with PQC, having a protective role for affected cells. However, they may also contribute to the prion-like distribution of TDP-neurotoxic forms in neighboring and more distant cells

Encephalopathies with intracranial calcification in children: Clinical and genetic characterization

none42noBackground: We present a group of patients affected by a paediatric onset genetic encephalopathy with cerebral calcification of unknown aetiology studied with Next Generation Sequencing (NGS) genetic analyses. Methods: We collected all clinical and radiological data. DNA samples were tested by means of a customized gene panel including fifty-nine genes associated with known genetic diseases with cerebral calcification. Results: We collected a series of fifty patients. All patients displayed complex and heterogeneous phenotypes mostly including developmental delay and pyramidal signs and less frequently movement disorder and epilepsy. Signs of cerebellar and peripheral nervous system involvement were occasionally present. The most frequent MRI abnormality, beside calcification, was the presence of white matter alterations; calcification was localized in basal ganglia and cerebral white matter in the majority of cases. Sixteen out of fifty patients tested positive for mutations in one of the fifty-nine genes analyzed. In fourteen cases the analyses led to a definite genetic diagnosis while results were controversial in the remaining two. Conclusions: Genetic encephalopathies with cerebral calcification are usually associated to complex phenotypes. In our series, a molecular diagnosis was achieved in 32% of cases, suggesting that the molecular bases of a large number of disorders are still to be elucidated. Our results confirm that cerebral calcification is a good criterion to collect homogeneous groups of patients to be studied by exome or whole genome sequencing; only a very close collaboration between clinicians, neuroradiologists and geneticists can provide better results from these new generation molecular techniques.noneTonduti D.; Panteghini C.; Pichiecchio A.; Decio A.; Carecchio M.; Reale C.; Moroni I.; Nardocci N.; Campistol J.; Garcia-Cazorla A.; Perez Duenas B.; Zorzi G.; Ardissone A.; Granata T.; Freri E.; Zibordi F.; Ragona F.; D'Arrigo S.; Saletti V.; Esposito S.; Pantaleoni C.; Riva D.; De Giorgis V.; Cereda C.; Valente M.L.; Sproviero D.; Poo Arguelles M.P.; Estupina C.F.; Sans Fito A.M.; Martorell Sampol L.; Del Mar O'Callaghan Gordo M.; Ortez Gonzalez C.I.; Gonzalez Alvarez V.; Garcia-Segarra N.; Fusco C.; Bertini E.; Diodato D.; Fazzi E.; Galli J.; Chiapparini L.; Garavaglia B.; Orcesi S.Tonduti, D.; Panteghini, C.; Pichiecchio, A.; Decio, A.; Carecchio, M.; Reale, C.; Moroni, I.; Nardocci, N.; Campistol, J.; Garcia-Cazorla, A.; Perez Duenas, B.; Zorzi, G.; Ardissone, A.; Granata, T.; Freri, E.; Zibordi, F.; Ragona, F.; D'Arrigo, S.; Saletti, V.; Esposito, S.; Pantaleoni, C.; Riva, D.; De Giorgis, V.; Cereda, C.; Valente, M. L.; Sproviero, D.; Poo Arguelles, M. P.; Estupina, C. F.; Sans Fito, A. M.; Martorell Sampol, L.; Del Mar O'Callaghan Gordo, M.; Ortez Gonzalez, C. I.; Gonzalez Alvarez, V.; Garcia-Segarra, N.; Fusco, C.; Bertini, E.; Diodato, D.; Fazzi, E.; Galli, J.; Chiapparini, L.; Garavaglia, B.; Orcesi, S

Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins