Different Chronic Stress Paradigms Converge on Endogenous TDP43 Cleavage and Aggregation.

The TAR-DNA binding protein (TDP43) is a nuclear protein whose cytoplasmic inclusions are hallmarks of Amyotrophic Lateral Sclerosis (ALS). Acute stress in cells causes TDP43 mobilization to the cytoplasm and its aggregation through different routes. Although acute stress elicits a strong phenotype, is far from recapitulating the years-long aggregation process. We applied different chronic stress protocols and described TDP43 aggregation in a human neuroblastoma cell line by combining solubility assays, thioflavin-based microscopy and flow cytometry. This approach allowed us to detect, for the first time to our knowledge in vitro, the formation of 25 kDa C-terminal fragment of TDP43, a pathogenic hallmark of ALS. Our results indicate that chronic stress, compared to the more common acute stress paradigm, better recapitulates the cell biology of TDP43 proteinopathies. Moreover, we optimized a protocol for the detection of bona fide prions in living cells, suggesting that TDP43 may form amyloids as a stress response.

Pattern of Mitochondrial Respiration in Peripheral Blood Cells of Patients with Parkinson's Disease.

Mitochondria are central in the pathogenesis of Parkinson's disease (PD), as they are involved in oxidative stress, synaptopathy, and other immunometabolic pathways. Accordingly, they are emerging as a potential neuroprotection target, although further human-based evidence is needed for therapeutic advancements. This study aims to shape the pattern of mitochondrial respiration in the blood leukocytes of PD patients in relation to both clinical features and the profile of cerebrospinal fluid (CSF) biomarkers of neurodegeneration. Mitochondrial respirometry on the peripheral blood mononucleate cells (PBMCs) of 16 PD patients and 14 controls was conducted using Seahorse Bioscience technology. Bioenergetic parameters were correlated either with standard clinical scores for motor and non-motor disturbances or with CSF levels of α-synuclein, amyloid-ß peptides, and tau proteins. In PD, PBMC mitochondrial basal respiration was normal; maximal and spare respiratory capacities were both increased; and ATP production was higher, although not significantly. Maximal and spare respiratory capacity was directly correlated with disease duration, MDS-UPDRS part III and Hoehn and Yahr motor scores; spare respiratory capacity was correlated with the CSF amyloid-ß-42 to amyloid-ß-42/40 ratio. We provided preliminary evidence showing that mitochondrial respiratory activity increases in the PBMCs of PD patients, probably following the compensatory adaptations to disease progression, in contrast to the bases of the neuropathological substrate.

Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells.

Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications.

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable ß-sheet enriched intermediates, which are stabilized by intermolecular interactions with other ß-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.

Emerging Role for Linear and Circular Spermine Oxidase RNAs in Skeletal Muscle Physiopathology.

Skeletal muscle atrophy is a pathological condition so far without effective treatment and poorly understood at a molecular level. Emerging evidence suggest a key role for circular RNAs (circRNA) during myogenesis and their deregulation has been reported to be associated with muscle diseases. Spermine oxidase (SMOX), a polyamine catabolic enzyme plays a critical role in muscle differentiation and the existence of a circRNA arising from SMOX gene has been recently identified. In this study, we evaluated the expression profile of circular and linear SMOX in both C2C12 differentiation and dexamethasone-induced myotubes atrophy. To validate our findings in vivo their expression levels were also tested in two murine models of amyotrophic lateral sclerosis: SOD1G93A and hFUS+/+, characterized by progressive muscle atrophy. During C2C12 differentiation, linear and circular SMOX show the same trend of expression. Interestingly, in atrophy circSMOX levels significantly increased compared to the physiological state, in both in vitro and in vivo models. Our study demonstrates that SMOX represents a new player in muscle physiopathology and provides a scientific basis for further investigation on circSMOX RNA as a possible new therapeutic target for the treatment of muscle atrophy.

Metabolic reprogramming of fibro/adipogenic progenitors facilitates muscle regeneration.

In Duchenne muscular dystrophy (DMD), the absence of the dystrophin protein causes a variety of poorly understood secondary effects. Notably, muscle fibers of dystrophic individuals are characterized by mitochondrial dysfunctions, as revealed by a reduced ATP production rate and by defective oxidative phosphorylation. Here, we show that in a mouse model of DMD (mdx), fibro/adipogenic progenitors (FAPs) are characterized by a dysfunctional mitochondrial metabolism which correlates with increased adipogenic potential. Using high-sensitivity mass spectrometry-based proteomics, we report that a short-term high-fat diet (HFD) reprograms dystrophic FAP metabolism in vivo. By combining our proteomic dataset with a literature-derived signaling network, we revealed that HFD modulates the ß-catenin-follistatin axis. These changes are accompanied by significant amelioration of the histological phenotype in dystrophic mice. Transplantation of purified FAPs from HFD-fed mice into the muscles of dystrophic recipients demonstrates that modulation of FAP metabolism can be functional to ameliorate the dystrophic phenotype. Our study supports metabolic reprogramming of muscle interstitial progenitor cells as a novel approach to alleviate some of the adverse outcomes of DMD.

P2X7 activation enhances skeletal muscle metabolism and regeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis.

Muscle weakness plays an important role in neuromuscular disorders comprising amyotrophic lateral sclerosis (ALS). However, it is not established whether muscle denervation originates from the motor neurons, the muscles or more likely both. Previous studies have shown that the expression of the SOD1G93A mutation in skeletal muscles causes denervation of the neuromuscular junctions, inability to regenerate and consequent atrophy, all clear symptoms of ALS. In this work, we used SOD1G93A mice, a model that best mimics some pathological features of both familial and sporadic ALS, and we investigated some biological effects induced by the activation of the P2X7 receptor in the skeletal muscles. The P2X7, belonging to the ionotropic family of purinergic receptors for extracellular ATP, is abundantly expressed in the healthy skeletal muscles, where it controls cell duplication, differentiation, regeneration or death. In particular, we evaluated whether an in vivo treatment in SOD1G93A mice with the P2X7 specific agonist 2'(3')-O-(4-Benzoylbenzoyl) adenosine5'-triphosphate (BzATP) just before the onset of a pathological neuromuscular phenotype could exert beneficial effects in the skeletal muscles. Our findings indicate that stimulation of P2X7 improves the innervation and metabolism of myofibers, moreover elicits the proliferation/differentiation of satellite cells, thus preventing the denervation atrophy of skeletal muscles in SOD1G93A mice. Overall, this study suggests that a P2X7-targeted and site-specific modulation might be a strategy to interfere with the complex multifactorial and multisystem nature of ALS.

Role of LRRK2 in the regulation of dopamine receptor trafficking.

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

Cysteine Modifications in the Pathogenesis of ALS.

Several proteins are found misfolded and aggregated in sporadic and genetic forms of amyotrophic lateral sclerosis (ALS). These include superoxide dismutase (SOD1), transactive response DNA-binding protein (TDP-43), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), p62, vasolin-containing protein (VCP), Ubiquilin-2 and dipeptide repeats produced by unconventional RAN-translation of the GGGGCC expansion in C9ORF72. Up to date, functional studies have not yet revealed a common mechanism for the formation of such diverse protein inclusions. Consolidated studies have demonstrated a fundamental role of cysteine residues in the aggregation process of SOD1 and TDP43, but disturbance of protein thiols homeostatic factors such as protein disulfide isomerases (PDI), glutathione, cysteine oxidation or palmitoylation might contribute to a general aberration of cysteine residues proteostasis in ALS. In this article we review the evidence that cysteine modifications may have a central role in many, if not all, forms of this disease.

c-MYC inhibition impairs hypoxia response in glioblastoma multiforme.

The c-MYC oncoprotein is a DNA binding transcription factor that enhances the expression of many active genes. c-MYC transcriptional signatures vary according to the transcriptional program defined in each cell type during differentiation. Little is known on the involvement of c-MYC in regulation of gene expression programs that are induced by extracellular cues such as a changing microenvironment. Here we demonstrate that inhibition of c-MYC in glioblastoma multiforme cells blunts hypoxia-dependent glycolytic reprogramming and mitochondria fragmentation in hypoxia. This happens because c-MYC inhibition alters the cell transcriptional response to hypoxia and finely tunes the expression of a subset of Hypoxia Inducible Factor 1-regulated genes. We also show that genes whose expression in hypoxia is affected by c-MYC inhibition are able to distinguish the Proneural subtype of glioblastoma multiforme, thus potentially providing a molecular signature for this class of tumors that are the least tractable among glioblastomas.

Glutaredoxin 1 is a major player in copper metabolism in neuroblastoma cells.

BACKGROUND: Glutaredoxin 1 (Grx1), a small protein belonging to the thioredoxin family, is involved in redox-regulation since it catalyzes the reduction of protein disulfides and that of mixed disulfides. It was reported to modulate active copper extrusion from cells, by affecting the function of the pumps ATP7A and B. These are components of the network of protein chaperones involved in the control of the homeostasis of copper, an essential, though harmful, metal. However, the effect of Grx1 on copper levels, copper chaperones and copper-elicited cell toxicity was never investigated. METHODS: In order to investigate the effect of Grx1 on copper metabolism, we constitutively overexpressed Grx1 in human neuroblastoma SH-SY5Y cells (SH-Grx1 cells) and assessed a number of copper-related parameters. RESULTS: SH-Grx1 cells show a basal intracellular copper level higher than control cells, accumulate more copper upon CuSO4 treatment, but are more resistant to copper-induced toxicity. Grx1 shows copper-binding properties and copper overload produces a decrease of Grx1 enzyme activity in SH-Grx1 cells. Finally, Grx1 overexpression decreases copper accumulation in mitochondria upon copper overload and modulates the expression of copper transporter 1 (Ctr1). CONCLUSION: Altogether, these data demonstrate that Grx1 is a major player in copper metabolism in neuronal cells.

Mitochondria and ALS: implications from novel genes and pathways.

Evidence from patients with sporadic and familiar amyotrophic lateral sclerosis (ALS) and from models based on the overexpression of mutant SOD1 found in a small subset of patients, clearly point to mitochondrial damage as a relevant facet of this neurodegenerative condition. In this mini-review we provide a brief update on the subject in the light of newly discovered genes (such as TDP-43 and FUS/TLS) associated to familial ALS and of a deeper knowledge of the mechanisms of derangement of mitochondria. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity.

Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.

Genetic analysis of decreased praziquantel sensitivity in a laboratory strain of Schistosoma mansoni.

A laboratory strain of Schistosoma mansoni subjected to repeated in vivo praziquantel (PZQ) treatments for several generations has been previously found to have lesser sensitivity to the drug than the original unselected strain. In this study we have collected evidence on the mode of inheritance of the partial insensitivity exhibited by the PZQ-selected schistosomes. A single male and a single female worm of the two strains, assorted in the four possible combinations, were introduced into the mesenteric veins of mice and the eggs produced by each pair were used as the source of the F(1) progeny. PZQ sensitivity was assessed using both in vivo and in vitro methods. In the first approach, the PZQ ED(50) was determined by infecting mice with cercariae of the strains to be tested, treating at seven weeks with different drug doses and counting the number of surviving worms three weeks later. For the in vitro approach, adult schistosomes kept in culture were exposed overnight to different PZQ concentrations and their survival was monitored during the subsequent 7 days. Results from both approaches lead to the conclusion that hybrid schistosomes of the F(1) generation have a drug sensitivity intermediate between those of the two parental strains and are thus suggestive of a pattern of partial dominance for the trait under study.

Will new antischistosomal drugs finally emerge?

It has been often observed that the chemotherapeutic armamentarium against an important disease such as schistosomiasis consists of just one drug, praziquantel. Thus, development of drug resistance is an impending danger, with serious implications for the health protection of many millions of people. This rational and legitimate concern might now begin to be relieved by the recent proposal of a new class of compounds that could represent a novel source of drugs against schistosomiasis.

Schistosoma mansoni: lack of correlation between praziquantel-induced intra-worm calcium influx and parasite death.

The schistosomicidal activity of praziquantel (PZQ) is accompanied by a large influx of calcium into the worms, suggesting that this phenomenon could be the source of the observed muscular contraction, surface disruption and eventual death of the parasite. We have incubated live adult schistosomes in a medium containing radioactive calcium and we were able to confirm that PZQ does indeed stimulate calcium entry into the parasite. An even higher calcium uptake, however, occurred in schistosomes exposed to PZQ after pre-incubation with cytochalasin D, a condition that suppresses PZQ schistosomicidal effects and allows the complete survival of the parasites. The calcium blockers nicardipine and nifedipine also failed to prevent the calcium influx induced by PZQ. Similarly, a large calcium influx occurred in 28-day-old worms exposed to PZQ, in spite of the fact that these immature worms are largely insensitive to the schistosomicidal effects of the drug. Schistosomes incubated overnight with radioactive calcium and PZQ and then returned to normal medium, retained a calcium content higher than worms pre-incubated with cytochalasin D, but the difference could be a consequence--rather than a cause--of schistosomicidal effects. These results suggest that calcium accumulation by itself, at least as measured in whole parasites maintained in vitro, may not represent an exhaustive explanation for the schistosomicidal effects of PZQ.

Cytochalasin D abolishes the schistosomicidal activity of praziquantel.

To test the hypothesis that calcium channels of schistosomes are the targets for the action of praziquantel, we subjected schistosomes in vitro to pharmacological agents capable of interfering with the functioning of calcium channels. After 1-h exposure to these agents, praziquantel was added and incubation continued overnight. Worms were then washed, resuspended in drug-free medium and observed during the following 7-10 days. About 50% of schistosomes pre-exposed to the calcium channel blockers nicardipine and nifedipine were able to survive a praziquantel concentration (3 microM) that normally killed the majority of adult male worms. Since the organization of the actin cytoskeleton controls the activity of calcium channels in a number of different systems, we also pre-exposed schistosomes to the actin depolymerizing agent cytochalasin D. This treatment rendered the parasites completely refractory to the effects of very high praziquantel levels (up to 36 microM). These results are consistent with the hypothesis that schistosome calcium channels are involved in the mechanism of action of praziquantel.

Sequence and level of endogenous expression of calcium channel beta subunits in Schistosoma mansoni displaying different susceptibilities to praziquantel.

Kohn et al. [J. Biol. Chem. 276 (2001) 36873] demonstrated that cells expressing the structurally unusual schistosome beta subunit SmCavbeta1 in their voltage-operated calcium channels, exhibit an increased current amplitude in the presence of praziquantel (PZQ). This suggests that the beta subunit is involved in PZQ activity and is consistent with the known pharmacological effects of the drug. If this is so, the low susceptibility to PZQ noted in some Schistosoma mansoni strains could be due to some mutation(s) in the gene coding for this protein. We have sequenced the cDNAs coding for the SmCavbeta1 and SmCavbeta2 subunits of different sensitive and resistant strains and we have not been able to detect any meaningful differences. As an alternative hypothesis, the different sensitivity of schistosomes to PZQ action could be due to the expression of different beta subunits in the parasite. This interpretation could also explain the low PZQ susceptibility of immature worms (28 days). We analyzed Northern blots of various strains and various developmental stages, but we were unable to demonstrate major quantitative differences in the expression of the beta subunits.