Repositorio Institucional Académico. Universidad Andrés Bello. Chile :: Examinando por Autor "Bustos, Fernando J."

Examinando por Autor "Bustos, Fernando J."

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ALS-linked protein disulfide isomerase variants cause motor dysfunction
(EMBO Journal. Volume 35, Issue 8, Pages 845 - 865. 15 April 2016, 2016-04) Woehlbier, Ute; Colombo, Alicia; Saaranen, Mirva J; Pérez, Viviana; Ojeda, Jorge; Bustos, Fernando J.; Andreu, Catherine I.; Torres, Mauricio; Valenzuela, Vicente; Medinas, Danilo B; Rozas, Pablo; Vidal, Rene L.; Lopez-Gonzalez, Rodrigo; Salameh, Johnny; Fernandez-Collemann, Sara; Muñoz, Natalia; Matus, Soledad; Armisen, Ricardo; Sagredo, Alfredo; Palma, Karina; Irrazabal, Thergiory; Almeida, Sandra; Gonzalez-Perez, Paloma; Campero, Mario; Gao, Fen-Biao; Henny, Pablo; Van Zundert, Brigitte; Ruddock, Lloyd W; Concha, Miguel L; Henriquez, Juan P.; Brown, Robert H.; Hetz, Claudio
Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS-linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.
Antiviral Activity of an Endogenous Parvoviral Element
(MDPI, 2023-07) Bravo, Angelica; Fernández-García, Leandro; Ibarra-Karmy, Rodrigo; Mardones, Gonzalo A.; Mercado, Luis; Bustos, Fernando J.; Gifford, Robert J.; Arriagada, Gloria
Endogenous viral elements (EVEs) are genomic DNA sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express proteins with physiological roles in their host. Furthermore, some EVEs exhibit a protective role against exogenous viral infection in their host. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and although some of them contain ORFs, their function is unknown. We have shown that the locus EPV-Dependo.43-ODegus, an EPV with an intact ORF, is transcribed in Octodon degus (degu). Here we examine the antiviral activity of the protein encoded in this EPV, named DeRep. DeRep was produced in bacteria and used to generate antibodies that recognize DeRep in western blots of degu tissue. To test if DeRep could protect against exogenous parvovirus, we challenged cells with the minute virus of mice (MVM), a model autonomous parvovirus. We observed that MVM protein expression, DNA damage induced by replication, viral DNA, and cytopathic effects are reduced when DeRep is expressed in cells. The results of this study demonstrate that DeRep is expressed in degu and can inhibit parvovirus replication. This is the first time that an EPV has been shown to have antiviral activity against an exogenous virus. © 2023 by the authors.
Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior
(BMC Biology,Volume 22, Issue 1 December 2024, Article number 142, 2024-12) Ahumada-Marchant, Constanza; Ancatén-Gonzalez, Carlos; Haensgen, Henny; Brauer, Bastian; Merino-Veliz, Nicolas; Droste, Rita; Arancibia, Felipe; Horvitz, H. Robert; Constantine-Paton, Martha; Arriagada, Gloria; Chávez, Andrés E.; Bustos, Fernando J.
Background: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. Results: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. Conclusions: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions. © The Author(s) 2024.
Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice
(BRAIA, 2017-12) Bustos, Fernando J.; Ampuero, Estibaliz; Jury, Nur; Aguilar, Rodrigo; Falahi, Fahimeh; Toledo, Jorge; Ahumada, Juan; Lata, Jaclyn; Cubillos, Paula; Henríquez, Berta; Guerra, Miguel V.; Stehberg, Jimmy; Neve, Rachael L.; Inestrosa, Nibaldo C.; Wyneken, Ursula; Fuenzalida, Marco; Härtel, Steffen; Sena-Esteves, Miguel; Varela-Nallar, Lorena; Rots, Marianne G.; Montecino, Martin; Van Zundert, Brigitte
The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer's disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders. © 2017 The Author. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons
(Cell Press, 2022-05-18) Arredondo, Cristian; Cefaliello, Carolina; Dyrda, Agnieszka; Jury, Nur; Martinez, Pablo; Díaz, Iván; Amaro, Armando; Tran, Helene; Morales, Danna; Pertusa, Maria; Stoica, Lorelei; Fritz, Elsa; Corvalán, Daniela; Abarzúa, Sebastián; Méndez-Ruette, Maxs; Fernández, Paola; Rojas, Fabiola; Kumar, Meenakshi Sundaram; Aguilar, Rodrigo; Almeida, Sandra; Weiss, Alexandra; Bustos, Fernando J.; González-Nilo, Fernando; Otero, Carolina; Tevy, Maria Florencia; Bosco, Daryl A.; Sáez, Juan C.; Kähne, Thilo; Gao, Fen-Biao; Berry, James D.; Nicholson, Katharine; Sena-Esteves, Miguel; Madrid, Rodolfo; Varela, Diego; Montecino, Martin; Brown, Robert H.; van Zundert, Brigitte
Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD. © 2022 Elsevier Inc.
Impact of KDM6B mosaic brain knockout on synaptic function and behavior
(Scientific Reports, Volume 14, Issue 1 December 2024, Article number 20416, 2024-11) Brauer, Bastian; Ancatén-González, Carlos; Ahumada-Marchant, Constanza; Meza, Rodrigo C.; Merino-Veliz, Nicolas; Nardocci, Gino; Varela-Nallar, Lorena; Arriagada, Gloria; Chávez, Andrés E.; Bustos, Fernando J.
Autism spectrum disorders (ASD) are complex neurodevelopmental conditions characterized by impairments in social communication, repetitive behaviors, and restricted interests. Epigenetic modifications serve as critical regulators of gene expression playing a crucial role in controlling brain function and behavior. Lysine (K)-specific demethylase 6B (KDM6B), a stress-inducible H3K27me3 demethylase, has emerged as one of the highest ASD risk genes, but the precise effects of KDM6B mutations on neuronal activity and behavioral function remain elusive. Here we show the impact of KDM6B mosaic brain knockout on the manifestation of different autistic-like phenotypes including repetitive behaviors, social interaction, and significant cognitive deficits. Moreover, KDM6B mosaic knockout display abnormalities in hippocampal excitatory synaptic transmission decreasing NMDA receptor mediated synaptic transmission and plasticity. Understanding the intricate interplay between epigenetic modifications and neuronal function may provide novel insights into the pathophysiology of ASD and potentially inform the development of targeted therapeutic interventions. © The Author(s) 2024.
KMT2C knockout generates ASD-like behaviors in mice
(Frontiers Media SA, 2023) Brauer, Bastian; Merino-Veliz, Nicolas; Ahumada-Marchant, Constanza; Arriagada, Gloria; Bustos, Fernando J.
Neurodevelopmental disorders have been associated with genetic mutations that affect cellular function, including chromatin regulation and epigenetic modifications. Recent studies in humans have identified mutations in KMT2C, an enzyme responsible for modifying histone tails and depositing H3K4me1 and H3K4me3, as being associated with Kleefstra syndrome 2 and autism spectrum disorder (ASD). However, the precise role of KMT2C mutations in brain disorders remains poorly understood. Here we employed CRISPR/Cas9 gene editing to analyze the effects of KMT2C brain specific knockout on animal behavior. Knocking out KMT2C expression in cortical neurons and the mouse brain resulted in decreased KMT2C levels. Importantly, KMT2C brain specific knockout animals exhibited repetitive behaviors, social deficits, and intellectual disability resembling ASD. Our findings shed light on the involvement of KMT2C in neurodevelopmental processes and establish a valuable model for elucidating the cellular and molecular mechanisms underlying KMT2C mutations and their relationship to Kleefstra syndrome 2 and ASD. Copyright © 2023 Brauer, Merino-Veliz, Ahumada-Marchant, Arriagada and Bustos.
Myosin va brain-specific mutation alters mouse behavior and disrupts hippocampal synapses
(Society for Neuroscience, 2020-11) Pandian, Swarna; Zhao, Jian-Ping; Murata, Yasunobu; Bustos, Fernando J.; Tunca, Cansu; Almeida, Ramiro D.; Constantine-Paton, Martha
Myosin Va (MyoVa) is a plus-end filamentous-actin motor protein that is highly and broadly expressed in the vertebrate body, including in the nervous system. In excitatory neurons, MyoVa transports cargo toward the tip of the dendritic spine, where the postsynaptic density (PSD) is formed and maintained. MyoVa mutations in humans cause neurologic dysfunction, intellectual disability, hypomelanation, and death in infancy or child-hood. Here, we characterize the Flailer (Flr) mutant mouse, which is homozygous for a myo5a mutation that drives high levels of mutant MyoVa (Flr protein) specifically in the CNS. Flr protein functions as a dominant-negative MyoVa, sequestering cargo and blocking its transport to the PSD. Flr mice have early seizures and mild ataxia but mature and breed normally. Flr mice display several abnormal behaviors known to be associated with brain regions that show high expression of Flr protein. Flr mice are defective in the transport of synaptic components to the PSD and in mGluR-dependent long-term depression (LTD) and have a reduced number of mature dendritic spines. The synaptic and behavioral abnormalities of Flr mice result in anxiety and memory deficits similar to that of other mouse mutants with obsessive-compulsive disorder and autism spectrum disorder (ASD). Because of the dominant-negative nature of the Flr protein, the Flr mouse offers a power-ful system for the analysis of how the disruption of synaptic transport and lack of LTD can alter synaptic function, development and wiring of the brain and result in symptoms that characterize many neuropsychiatric disorders. © 2020 Pandian et al.
PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling
(16625099, 2024-04-04) Moya-Alvarado, Guillermo; Valero-Peña, Xavier; Aguirre-Soto, Alejandro; Bustos, Fernando J.; Lazo, Oscar M.; Bronfman, Francisca C.
Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.
Polycomb PRC2 complex mediates epigenetic silencing of a critical osteogenic master regulator in the hippocampus
(Elsevier B.V., 2016-08) Aguilar, Rodrigo; Bustos, Fernando J.; Saez, Mauricio; Rojas, Adriana; Allende, Miguel L.; van Wijnen, Andre J.; van Zundert, Brigitte; Montecino, Martin
During hippocampal neuron differentiation, the expression of critical inducers of non-neuronal cell lineages must be efficiently silenced. Runx2 transcription factor is the master regulator of mesenchymal cells responsible for intramembranous osteoblast differentiation and formation of the craniofacial bone tissue that surrounds and protects the central nervous system (CNS) in mammalian embryos. The molecular mechanisms that mediate silencing of the Runx2 gene and its downstream target osteogenic-related genes in neuronal cells have not been explored. Here, we assess the epigenetic mechanisms that mediate silencing of osteoblast-specific genes in CNS neurons. In particular, we address the contribution of histone epigenetic marks and histone modifiers on the silencing of the Runx2/p57 bone-related isoform in rat hippocampal tissues at embryonic to adult stages. Our results indicate enrichment of repressive chromatin histone marks and of the Polycomb PRC2 complex at the Runx2/p57 promoter region. Knockdown of PRC2 H3K27-methyltransferases Ezh2 and Ezh1, or forced expres sion of the Trithorax/COMPASS subunit Wdr5 activates Runx2/p57 mRNA expression in both immature and mature hippocampal cells. Together these results indicate that complementary epigenetic mechanisms progressively and efficiently silence critical osteoblastic genes during hippocampal neuron differentiation. © 2016 Elsevier B.V. All rights reserved.
PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors
(Public Library of Science, 2014-04) Bustos, Fernando J.; Varela-Nallar, Lorena; Campos, Matias; Henriquez, Berta; Phillips, Marnie; Opazo, Carlos; Aguayo, Luis G.; Montecino, Martin; Constantine-Paton, Martha; Inestrosa, Nibaldo C.; Van Zundert, Brigitte
Considerable evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B are critical mediators of synaptic plasticity and dendritogenesis; however, how they differentially regulate these processes is unclear. Here we investigate the roles of the NR2A and NR2B subunits, and of their scaffolding proteins PSD-95 and SAP102, in remodeling the dendritic architecture of developing hippocampal neurons (2–25 DIV). Analysis of the dendritic architecture and the temporal and spatial expression patterns of the NMDARs and anchoring proteins in immature cultures revealed a strong positive correlation between synaptic expression of the NR2B subunit and dendritogenesis. With maturation, the pruning of dendritic branches was paralleled by a strong reduction in overall and synaptic expression of NR2B, and a significant elevation in synaptic expression of NR2A and PSD95. Using constructs that alter the synaptic composition, we found that either over-expression of NR2B or knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature neurons. Reactivation of dendritogenesis could also be achieved in mature cultured neurons, but required both manipulations simultaneously, and was accompanied by increased dendritic clustering of NR2B. Our results indicate that the developmental increase in synaptic expression of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby restricts reactivation of dendritic branching. Experiments with shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that C-terminus of the NR2B subunit (tail) was sufficient to induce robust dendritic branching in mature hippocampal neurons, and suggest that the NR2B tail is important in recruiting calcium-dependent signaling proteins and scaffolding proteins necessary for dendritogenesis.
Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer
(BioMed Central Ltd, 2023-12) Bustos, Fernando J.; Pandian, Swarna; Haensgen, Henny; Zhao, Jian-Ping; Strouf, Haley; Heidenreich, Matthias; Swiech, Lukasz; Deverman, Benjamin E.; Gradinaru, Viviana; Zhang, Feng; Constantine-Paton, Martha
Background: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Results: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. Conclusions: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases. © 2023, BioMed Central Ltd., part of Springer Nature.
The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK
(MDPI, 2024-03) Fernández-García, Leandro; Ahumada-Marchant, Constanza; Lobos-Ávila, Pablo; Brauer, Bastián; Bustos, Fernando J.; Arriagada, Gloria
LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5′ and 3′LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5′UTR sense is unable to initiate translation, whereas the antisense 5′UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5′UTR IRES activity was tested using bicistronic reporters. The antisense 5′UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon. © 2024 by the authors.

Examinando por Autor "Bustos, Fernando J."

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