Publications by authors named "Emiliano Trias"

20 Publications

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Olive oil-derived nitro-fatty acids: protection of mitochondrial function in non-alcoholic fatty liver disease.

J Nutr Biochem 2021 Apr 7:108646. Epub 2021 Apr 7.

Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CENIBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. Electronic address:

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive liver fat deposition in the absence of significant alcohol intake. Since extra virgin olive oil (EVOO) reduces fat accumulation, we analyzed the involvement of nitro-fatty acids (NO-FA) on the beneficial effects of EVOO consumption on NAFLD. Nitro-fatty acids formation was observed during digestion in mice supplemented with EVOO and nitrite. Mice fed with a high-fat diet (HF) presented lower plasma NO-FA levels than normal chow, and circulating concentrations recovered when the HF diet was supplemented with 10% EVOO plus nitrite. Under NO-FA formation conditions, liver hemoxygenase-1 expression significantly increased while decreased body weight and fat liver accumulation. Mitochondrial dysfunction plays a central role in the pathogenesis of NAFLD while NO-FA has been shown to protect from mitochondrial oxidative damage. Accordingly, an improvement of respiratory indexes was observed when mice were supplemented with both EVOO plus nitrite. Liver mitochondrial complexes II and V activities were greater in mice with EVOO supplementation and further improved in the presence of nitrite. Overall, our results strongly suggest a positive correlation between NO-OA formation from EVOO and the observed improvement of mitochondrial function in NAFLD. The formation of NO-FA can account for the health benefits associated with EVOO consumption.
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http://dx.doi.org/10.1016/j.jnutbio.2021.108646DOI Listing
April 2021

Sunitinib-Containing Carborane Pharmacophore with the Ability to Inhibit Tyrosine Kinases Receptors FLT3, KIT and PDGFR-β, Exhibits Powerful In Vivo Anti-Glioblastoma Activity.

Cancers (Basel) 2020 Nov 18;12(11). Epub 2020 Nov 18.

Grupo de Química Orgánica Medicinal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay.

Malignant gliomas are the most common malignant and aggressive primary brain tumors in adults, the prognosis being-especially for glioblastomas-extremely poor. There are no effective treatments yet. However, tyrosine kinase receptor (TKR) inhibitors and boron neutron capture therapy (BNCT), together, have been proposed as future therapeutic strategies. In this sense in our ongoing project of developing new anti-glioblastoma drugs, we identified a sunitinib-carborane hybrid agent, , with both in vitro selective cytotoxicity and excellent BNCT-behavior. Consequently, we studied the ability of compound to inhibit TKRs, its promotion of cellular death processes, and its effects on the cell cycle. Moreover, we analyzed some relevant drug-like properties of , i.e., mutagenicity and ability to cross the blood-brain barrier. These results encouraged us to perform an in vivo anti-glioblastoma proof of concept assay. It turned out to be a selective FLT3, KIT, and PDGFR-β inhibitor and increased the apoptotic glioma-cell numbers and arrested sub-G1-phase cell cycle. Its in vivo activity in immunosuppressed mice bearing U87 MG human glioblastoma evidenced excellent anti-tumor behavior.
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http://dx.doi.org/10.3390/cancers12113423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698965PMC
November 2020

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition.

Neurotherapeutics 2020 Oct 28. Epub 2020 Oct 28.

Neurodegeneration Laboratory, Institut Pasteur de Montevideo, Mataojo, 2020, Montevideo, Uruguay.

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1 rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1 rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1 rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.
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http://dx.doi.org/10.1007/s13311-020-00953-zDOI Listing
October 2020

-Carboranyl- and Metallacarboranyl [1,2,3]triazolyl-Decorated Lapatinib-Scaffold for Cancer Therapy Combining Tyrosine Kinase Inhibition and Boron Neutron Capture Therapy.

Cells 2020 06 5;9(6). Epub 2020 Jun 5.

Grupo de Química Orgánica Medicinal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.

One of the driving forces of carcinogenesis in humans is the aberrant activation of receptors; consequently, one of the most promising mechanisms for cancer treatment is receptor inhibition by chemotherapy. Although a variety of cancers are initially susceptible to chemotherapy, they eventually develop multi-drug resistance. Anti-tumor agents overcoming resistance and acting through two or more ways offer greater therapeutic benefits over single-mechanism entities. In this study, we report on a new family of bifunctional compounds that, offering the possibility of dual action (drug + radiotherapy combinations), may result in significant clinical benefits. This new family of compounds combines two fragments: the drug fragment is a lapatinib group, which inhibits the tyrosine kinase receptor activity, and an icosahedral boron cluster used as agents for neutron capture therapy (BNCT). The developed compounds were evaluated in vitro against different tyrosine kinase receptors (TKRs)-expressing tumoral cells, and in vitro-BNCT experiments were performed for two of the most promising hybrids, and . We identified hybrid with excellent selectivity to inhibit cell proliferation and ability to induce necrosis/apoptosis of glioblastoma U87 MG cell line. Furthermore, derivative , bearing a water-solubility-enhancing moiety, showed moderate inhibition of cell proliferation in both U87 MG and colorectal HT-29 cell lines. Additionally, the HT-29 cells accumulated adequate levels of boron after hybrids and incubations rendering, and after neutron irradiation, higher BNCT-effects than . The attractive profile of developed hybrids makes them interesting agents for combined therapy.
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http://dx.doi.org/10.3390/cells9061408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349914PMC
June 2020

Schwann cells orchestrate peripheral nerve inflammation through the expression of CSF1, IL-34, and SCF in amyotrophic lateral sclerosis.

Glia 2020 06 20;68(6):1165-1181. Epub 2019 Dec 20.

Institut Pasteur de Montevideo, Montevideo, Uruguay.

Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian-like degeneration characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1 rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100β, GFAP, isolectin and/or p75 immunoreactivity. Different subsets of reactive SCs expressed colony-stimulating factor-1 (CSF1) and Interleukin-34 (IL-34) and closely interacted with numerous endoneurial CSF-1R-expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c-Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c-Kit in the sciatic nerves of SOD1 rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c-Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF-1R and c-Kit with masitinib in SOD1 rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies.
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http://dx.doi.org/10.1002/glia.23768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269115PMC
June 2020

CD34 Identifies a Subset of Proliferating Microglial Cells Associated with Degenerating Motor Neurons in ALS.

Int J Mol Sci 2019 Aug 9;20(16). Epub 2019 Aug 9.

Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1 mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1 rats, but not in controls. Most CD34 cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34 cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34 cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1 rat spinal cords yielded a large number of CD34 cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34 cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.
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http://dx.doi.org/10.3390/ijms20163880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720880PMC
August 2019

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS.

Front Aging Neurosci 2019 28;11:42. Epub 2019 Feb 28.

Institut Pasteur de Montevideo, Montevideo, Uruguay.

Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1 mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1 rats, numerous cells displayed nuclear p16 as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1 rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days and subsequent passages. Senescent SOD1 microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-β-Gal) activity as well as positive labeling for p16, p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16 staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1 rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS.
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http://dx.doi.org/10.3389/fnagi.2019.00042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403180PMC
February 2019

Phenotypic heterogeneity of astrocytes in motor neuron disease.

Clin Exp Neuroimmunol 2018 Nov 4;9(4):225-234. Epub 2018 Oct 4.

Department of Neuroscience and Pathobiology Research Institute of Environmental Medicine Nagoya University Nagoya Japan.

Accumulating evidence has shown that astrocytes do not just support the function of neurons, but play key roles in maintaining the brain environment in health and disease. Contrary to the traditional understanding of astrocytes as static cells, reactive astrocytes possess more diverse functions and phenotypes than previously predicted. In the present focused review, we summarize the evidence showing that astrocytes are playing profound roles in the disease process of amyotrophic lateral sclerosis. Aberrantly activated astrocytes in amyotrophic lateral sclerosis rodents express microglial molecular markers and provoke toxicities to accelerate disease progression. In addition, TIR domain-containing adapter protein-inducing interferon-β-dependent innate immune pathway in astrocytes also has a novel function in terminating glial activation and neuroinflammation. Furthermore, heterogeneity in phenotypes and functions of astrocytes are also observed in various disease conditions, such as other neurodegenerative diseases, ischemia, aging and acute lesions in the central nervous system. Through accumulating knowledge of the phenotypic and functional diversity of astrocytes, these cells will become more attractive therapeutic targets for neurological diseases.
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http://dx.doi.org/10.1111/cen3.12476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282976PMC
November 2018

Neuroimmune and Inflammatory Signals in Complex Disorders of the Central Nervous System.

Neuroimmunomodulation 2018 5;25(5-6):246-270. Epub 2018 Dec 5.

Neuroscience Program, Federal Fluminense University, Niterói, Brazil.

An extensive microglial-astrocyte-monocyte-neuronal cross talk seems to be crucial for normal brain function, development, and recovery. However, under certain conditions neuroinflammatory interactions between brain cells and neuroimmune cells influence disease outcome and brain pathology. Microglial cells express a range of functional states with dynamically pleomorphic profiles from a surveilling status of synaptic transmission to an active player in major events of development such as synaptic elimination, regeneration, and repair. Also, inflammation mediates a series of neurotoxic roles in neuropsychiatric conditions and neurodegenerative diseases. The present review discusses data on the involvement of neuroinflammatory conditions that alter neuroimmune interactions in four different pathologies. In the first section of this review, we discuss the ability of the early developing brain to respond to a focal lesion with a rapid compensatory plasticity of intact axons and the role of microglial activation and proinflammatory cytokines in brain repair. In the second section, we present data of neuroinflammation and neurodegenerative disorders and discuss the role of reactive astrocytes in motor neuron toxicity and the progression of amyotrophic lateral sclerosis. In the third section, we discuss major depressive disorders as the consequence of dysfunctional interactions between neural and immune signals that result in increased peripheral immune responses and increase proinflammatory cytokines. In the last section, we discuss autism spectrum disorders and altered brain circuitries that emerge from abnormal long-term responses of innate inflammatory cytokines and microglial phenotypic dysfunctions.
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http://dx.doi.org/10.1159/000494761DOI Listing
October 2019

Mast cells and neutrophils mediate peripheral motor pathway degeneration in ALS.

JCI Insight 2018 10 4;3(19). Epub 2018 Oct 4.

Institut Pasteur de Montevideo, Uruguay.

Neuroinflammation is a recognized pathogenic mechanism underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the inflammatory mechanisms influencing peripheral motor axon degeneration remain largely unknown. A recent report showed a pathogenic role for c-Kit-expressing mast cells mediating inflammation and neuromuscular junction denervation in muscles from SOD1G93A rats. Here, we have explored whether mast cells infiltrate skeletal muscles in autopsied muscles from ALS patients. We report that degranulating mast cells were abundant in the quadriceps muscles from ALS subjects but not in controls. Mast cells were associated with myofibers and motor endplates and, remarkably, interacted with neutrophils forming large extracellular traps. Mast cells and neutrophils were also abundant around motor axons in the extensor digitorum longus muscle, sciatic nerve, and ventral roots of symptomatic SOD1G93A rats, indicating that immune cell infiltration extends along the entire peripheral motor pathway. Postparalysis treatment of SOD1G93A rats with the tyrosine kinase inhibitor drug masitinib prevented mast cell and neutrophil infiltration, axonal pathology, secondary demyelination, and the loss of type 2B myofibers, compared with vehicle-treated rats. These findings provide further evidence for a yet unrecognized contribution of immune cells in peripheral motor pathway degeneration that can be therapeutically targeted by tyrosine kinase inhibitors.
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http://dx.doi.org/10.1172/jci.insight.123249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237484PMC
October 2018

Focal Transplantation of Aberrant Glial Cells Carrying the SOD1G93A Mutation into Rat Spinal Cord Induces Extensive Gliosis.

Neuroimmunomodulation 2017 1;24(3):143-153. Epub 2017 Nov 1.

Laboratorio de Neurodegeneración, Institut Pasteur de Montevideo, Montevideo, Uruguay.

Objective: We aimed to determine the potential of aberrant glial cells (AbAs) isolated from the spinal cord of adult SOD1G93A symptomatic rats to induce gliosis and neuronal damage following focal transplantation into the lumbar spinal cord of wild-type rats.

Methods: AbAs were obtained from the spinal cords of SOD1G93A symptomatic rats. One hundred thousand cells were injected using a glass micropipette into the lumbar spinal cords (L3-L5) of syngeneic wild-type adult rats. Equal volumes of culture medium or wild-type neonatal microglia were used as controls. Seven days after transplantation, immunohistochemistry analysis was carried out using astrocytic and microglia cell markers. Transplanted SOD1G93A AbAs were recognized by specific antibodies to human SOD1 (hSOD1) or misfolded human SOD1.

Results: Seven days after transplantation, AbAs were mainly detected in the medial region of the lumbar ventral horn as a well-limited cell cluster formed at the site of injection by their immunoreactivity to either misfolded SOD1 or normally folded hSOD1. Compared with controls, transplanted AbAs were surrounded by marked microgliosis and reactive astrocytes. Marked microgliosis was observed to extend bilaterally up to the cervical cord. Motor neurons close to AbA transplants were surrounded by activated glial cells and displayed ubiquitin aggregation.

Conclusions: AbAs bearing mutant SOD1G93A have the potential to induce neuroinflammation along the spinal cord and incipient damage to the motor neurons. The emergence of AbAs during amyotrophic lateral sclerosis pathogenesis may therefore be a mechanism to boost neuroinflammation and spread motor neuron damage along the neuroaxis.
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http://dx.doi.org/10.1159/000480639DOI Listing
July 2018

Evidence for mast cells contributing to neuromuscular pathology in an inherited model of ALS.

JCI Insight 2017 10 19;2(20). Epub 2017 Oct 19.

Institut Pasteur de Montevideo, Uruguay.

Evidence indicates that neuroinflammation contributes to motor neuron degeneration in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease leading to progressive muscular paralysis. However, it remains elusive whether inflammatory cells can interact with degenerating distal motor axons, influencing the progressive denervation of neuromuscular junctions (NMJs). By analyzing the muscle extensor digitorum longus (EDL) following paralysis onset in the SOD1G93A rat model, we have observed a massive infiltration and degranulation of mast cells, starting after paralysis onset and correlating with progressive NMJ denervation. Remarkably, mast cells accumulated around degenerating motor axons and NMJs, and were also associated with macrophages. Mast cell accumulation and degranulation in paralytic EDL muscle was prevented by systemic treatment over 15 days with masitinib, a tyrosine kinase inhibitor currently in clinical trials for ALS exhibiting pharmacological activity affecting mast cells and microglia. Masitinib-induced mast cell reduction resulted in a 35% decrease in NMJ denervation and reduced motor deficits as compared with vehicle-treated rats. Masitinib also normalized macrophage infiltration, as well as regressive changes in Schwann cells and capillary networks observed in advanced paralysis. These findings provide evidence for mast cell contribution to distal axonopathy and paralysis progression in ALS, a mechanism that can be therapeutically targeted by masitinib.
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http://dx.doi.org/10.1172/jci.insight.95934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846907PMC
October 2017

Significance of aberrant glial cell phenotypes in pathophysiology of amyotrophic lateral sclerosis.

Neurosci Lett 2017 01 26;636:27-31. Epub 2016 Jul 26.

Laboratorio de Neurodegeneración, Institut Pasteur de Montevideo, Uruguay. Electronic address:

Amyotrophic Lateral Sclerosis (ALS) is a paradigmatic neurodegenerative disease, characterized by progressive paralysis of skeletal muscles associated with motor neuron degeneration. It is well-established that glial cells play a key role in ALS pathogenesis. In transgenic rodent models for familial ALS reactive astrocytes, microglia and oligodendrocyte precursors accumulate in the degenerating spinal cord and appear to contribute to primary motor neuron death through a non-cell autonomous pathogenic mechanism. Furthermore in rats expressing the ALS-linked SOD1 mutation, rapid spread of paralysis coincides with emergence of neurotoxic and proliferating aberrant glia cells with an astrocyte-like phenotype (AbA cells) that are found surrounding damaged motor neurons. AbAs simultaneously express astrocytic markers GFAP, S100β and Connexin-43 along with microglial markers Iba-1, CD11b and CD163. Studies with cell cultures have shown that AbAs originate from inflammatory microglial cells that undergo phenotypic transition. Because AbAs appear only after paralysis onset and exponentially increase in parallel with disease progression, they appear to actively contribute to ALS progression. While several reviews have been published on the pathogenic role of glial cells in ALS, this review focuses on emergence and pro-inflammatory activity of AbAs as part of an increasingly complex neurodegenerative microenvironment during ALS disease development.
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http://dx.doi.org/10.1016/j.neulet.2016.07.052DOI Listing
January 2017

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis.

J Neuroinflammation 2016 07 11;13(1):177. Epub 2016 Jul 11.

Institut Pasteur de Montevideo, Mataojo 2020, Montevideo, 11.400, Uruguay.

Background: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS.

Methods: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset.

Results: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %.

Conclusions: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.
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http://dx.doi.org/10.1186/s12974-016-0620-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4940876PMC
July 2016

Electrophilic nitro-fatty acids prevent astrocyte-mediated toxicity to motor neurons in a cell model of familial amyotrophic lateral sclerosis via nuclear factor erythroid 2-related factor activation.

Free Radic Biol Med 2016 06 22;95:112-20. Epub 2016 Mar 22.

Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. Electronic address:

Nitro-fatty acids (NO2-FA) are electrophilic signaling mediators formed in tissues during inflammation, which are able to induce pleiotropic cytoprotective and antioxidant pathways including up regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) responsive genes. Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons associated to an inflammatory process that usually aggravates the disease progression. In ALS animal models, the activation of the transcription factor Nrf2 in astrocytes confers protection to neighboring neurons. It is currently unknown whether NO2-FA can exert protective activity in ALS through Nrf2 activation. Herein we demonstrate that nitro-arachidonic acid (NO2-AA) or nitro-oleic acid (NO2-OA) administrated to astrocytes expressing the ALS-linked hSOD1(G93A) induce antioxidant phase II enzyme expression through Nrf2 activation concomitant with increasing intracellular glutathione levels. Furthermore, treatment of hSOD1(G93A)-expressing astrocytes with NO2-FA prevented their toxicity to motor neurons. Transfection of siRNA targeted to Nrf2 mRNA supported the involvement of Nrf2 activation in NO2-FA-mediated protective effects. Our results show for the first time that NO2-FA induce a potent Nrf2-dependent antioxidant response in astrocytes capable of preventing motor neurons death in a culture model of ALS.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867302PMC
June 2016

Copper delivery to the CNS by CuATSM effectively treats motor neuron disease in SOD(G93A) mice co-expressing the Copper-Chaperone-for-SOD.

Neurobiol Dis 2016 May 27;89:1-9. Epub 2016 Jan 27.

Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University, United States. Electronic address:

Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice.
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http://dx.doi.org/10.1016/j.nbd.2016.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785045PMC
May 2016

Striatal neuronal death mediated by astrocytes from the Gcdh-/- mouse model of glutaric acidemia type I.

Hum Mol Genet 2015 Aug 12;24(16):4504-15. Epub 2015 May 12.

Institut Pasteur Montevideo, Mataojo 2020, Montevideo CIP 11400, Uruguay

Glutaric acidemia type I (GA-I) is an inherited neurometabolic childhood disorder caused by defective activity of glutaryl CoA dehydrogenase (GCDH) which disturb lysine (Lys) and tryptophan catabolism leading to neurotoxic accumulation of glutaric acid (GA) and related metabolites. However, it remains unknown whether GA toxicity is due to direct effects on vulnerable neurons or mediated by GA-intoxicated astrocytes that fail to support neuron function and survival. As damaged astrocytes can also contribute to sustain high GA levels, we explored the ability of Gcdh-/- mouse astrocytes to produce GA and induce neuronal death when challenged with Lys. Upon Lys treatment, Gcdh-/- astrocytes synthetized and released GA and 3-hydroxyglutaric acid (3HGA). Lys and GA treatments also increased oxidative stress and proliferation in Gcdh-/- astrocytes, both prevented by antioxidants. Pretreatment with Lys also caused Gcdh-/- astrocytes to induce extensive death of striatal and cortical neurons when compared with milder effect in WT astrocytes. Antioxidants abrogated the neuronal death induced by astrocytes exposed to Lys or GA. In contrast, Lys or GA direct exposure on Gcdh-/- or WT striatal neurons cultured in the absence of astrocytes was not toxic, indicating that neuronal death is mediated by astrocytes. In summary, GCDH-defective astrocytes actively contribute to produce and accumulate GA and 3HGA when Lys catabolism is stressed. In turn, astrocytic GA production induces a neurotoxic phenotype that kills striatal and cortical neurons by an oxidative stress-dependent mechanism. Targeting astrocytes in GA-I may prompt the development of new antioxidant-based therapeutical approaches.
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http://dx.doi.org/10.1093/hmg/ddv175DOI Listing
August 2015

Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS.

Front Cell Neurosci 2013 24;7:274. Epub 2013 Dec 24.

Institut Pasteur de Montevideo Montevideo, Uruguay.

Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1 (G93A) mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67 (+)AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1 (G93A) rats yielded large numbers of microglia expressing Iba1, CD11b, and CD68. Cells sorted for CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1 (G93A) rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS.
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http://dx.doi.org/10.3389/fncel.2013.00274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871969PMC
January 2014

Modulation of astrocytic mitochondrial function by dichloroacetate improves survival and motor performance in inherited amyotrophic lateral sclerosis.

PLoS One 2012 3;7(4):e34776. Epub 2012 Apr 3.

Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.

Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1(G93A) mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1(G93A) mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1(G93A) astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1(G93A) mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1(G93A) mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1(G93A) mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318006PMC
August 2012

Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited amyotrophic lateral sclerosis.

Proc Natl Acad Sci U S A 2011 Nov 18;108(44):18126-31. Epub 2011 Oct 18.

Instituto de Investigaciones Biológicas Clemente Estable, Montevideo 11600, Uruguay.

Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as "AbA cells") from primary spinal cord cultures of symptomatic rats expressing the SOD1(G93A) mutation. Isolation was based on AbA cells' marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100β protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1(G93A) astrocytes. AbA-like aberrant astrocytes expressing S100β and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression.
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http://dx.doi.org/10.1073/pnas.1110689108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207668PMC
November 2011