An integrated approach to design novel therapeutic interventions for demyelinating disorders

Therapeutic strategies are often based on two general principles: interference with the pathogenic process and repair of the damaged tissues. Recent studies, however, have suggested that several pathological conditions may result from the interplay between genetic susceptibility traits and environmental influences that, by modulating the epigenome, also affect disease onset and progression. Based on lessons from neural development, it is conceivable that new lines of preventive and possibly therapeutic intervention might be developed to modulate disease onset or decrease the severity of the symptoms. This review will discuss these concepts within the context of multiple sclerosis, the most common demyelinating disease of the central nervous system, and the leading cause of progressive neurological disability in young adults

Combined calpain-induced downregulation of TrkB-FL and TrkB-T1 upregulation causes neuronal death in excitotoxicity and ischemia

Electronic response to "Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons" Gomes, et al., 32(13): 4610-4622; doi: 10.1523/JNEUROSCI.0374-12.2012Peer Reviewe

Thyroid hormone receptor-β (TRβ1) impairs cell proliferation by the transcriptional inhibition of cyclins D1, E and A2

Thyroid hormone receptor-β1 (TRβ1) belongs to the ligand-inducible transcription factor superfamily. We have previously described that stable TRβ1 expression impairs fibroblast proliferation diminishing levels and activity of the main regulators of the G1/S transition. To unmask the underlying molecular mechanism of this action, we have investigated the expression of cyclin D1, E and A2 upon serum stimulation in TRβ1 expressing cells, finding a strong downregulation of their mRNAs, concomitant with low protein levels. The inhibition of the transcriptional activation in response to serum of these cyclins is differently exerted. For cyclin D1, we demonstrate that TRβ1 represses its promoter as a consequence of the downregulation of c-jun levels, diminished AP-1 activation and loss of c-jun recruitment to its binding sites on cyclin D1 promoter. For cyclin E and A2, it is the impairment of the cyclinD/Rb/E2F pathway by TRβ1 that prevents the activation of these two E2F target genes. Indeed, recruitment of E2F-1 to cyclin A2 promoter could not be detected. In summary, we propose that apo-TRβ1 exerts its antiproliferative action through a mechanism that could constitute a model by which other nuclear receptors may control cell division. © 2008 Nature Publishing Group All rights reserved.This work was supported by grants to AR-P from Dirección General de Investigación Científica y Técnica (MEC-BFU2004-01498). EP is supported by a grant-associated contract, and OGV is recipient of a pre-doctoral fellowship from Ministerio de Educación y Ciencia associated to the MEC grant.Peer Reviewe

Kidins220/ARMS downregulation by excitotoxic activation of NMDARs reveals its involvement in neuronal survival and death pathways

12 pages, 7 figures.Functional and protein interactions between the N-methyl-D-aspartate type of glutamate receptor (NMDAR) and neurotrophin or ephrin receptors play essential roles in neuronal survival and differentiation. A shared downstream effector for neurotrophin- and ephrin-receptor signaling is kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS). Because this molecule is obligatory for neurotrophin-induced differentiation, we investigated whether Kidins220/ARMS and NMDAR functions were related. Here, we identify an association between these proteins and discover that excitotoxicity, a specific form of neuronal death induced by NMDAR overstimulation, dramatically decreases Kidins220/ARMS levels in cortical neurons and in a model of cerebral ischemia. Kidins220/ARMS downregulation is triggered by overactivation of NMDARs containing NR2B subunits and subsequent Ca2+ influx, and involves a dual mechanism: rapid cleavage by the Ca2+-dependent protease calpain and calpain-independent silencing of Kidins220/Arms gene transcription. Additionally, Kidins220/ARMS knockdown decreases ERK activation and basal neuronal viability, and enhances neuronal death under excitotoxic conditions. Our results demonstrate Kidins220/ARMS participation in neuronal life and death pathways, and constitute the first report of its regulation under pathological conditions.This work was supported by grants BFU2007-67695 from `Ministerio de Ciencia e Innovación and `Fundación Mutua Madrileña' to M.D.-G.; and SAF2008-01951 from `Ministerio de Ciencia e Innovación', CAM S-SAL-0202-2006-01 from `Comunidad de Madrid' and CIBERNED from `Instituto de Salud Carlos III' to T.I. C.L.-M. and S.G. were recipients of a research contract and a predoctoral fellowship, respectively, funded by `Comunidad de Madrid'.Peer reviewe

A novel cell-penetrating peptide targeting calpain-cleavage of PSD-95 induced by excitotoxicity improves neurological outcome after stroke

© The author(s).Postsynaptic density protein-95 (PSD-95) is a multidomain protein critical to the assembly of signaling complexes at excitatory synapses, required for neuronal survival and function. However, calpain-processing challenges PSD-95 function after overactivation of excitatory glutamate receptors (excitotoxicity) in stroke, a leading cause of death, disability and dementia in need of efficient pharmacological treatments. A promising strategy is neuroprotection of the infarct penumbra, a potentially recoverable area, by promotion of survival signaling. Interference of PSD-95 processing induced by excitotoxicity might thus be a therapeutic target for stroke and other excitotoxicity-associated pathologies. [Methods]: The nature and stability of PSD-95 calpain-fragments was analyzed using in vitro assays or excitotoxic conditions induced in rat primary neuronal cultures or a mouse model of stroke. We then sequenced PSD-95 cleavage-sites and rationally designed three cell-penetrating peptides (CPPs) containing these sequences. The peptides effects on PSD-95 stability and neuronal viability were investigated in the cultured neurons, subjected to acute or chronic excitotoxicity. We also analyzed the effect of one of these peptides in the mouse model of stroke by measuring infarct size and evaluating motor coordination and balance. [Results]: Calpain cleaves three interdomain linker regions in PSD-95 and produces stable fragments corresponding to previously described PSD-95 supramodules (PDZ1-2 and P-S-G) as well as a truncated form SH3-GK. Peptide TP95414, containing the cleavage site in the PDZ3-SH3 linker, is able to interfere PSD-95 downregulation and reduces neuronal death by excitotoxicity. Additionally, TP95414 is delivered to mice cortex and, in a severe model of permanent ischemia, significantly improves the neurological outcome after brain damage. [Conclusions]: Interference of excitotoxicity-induced PSD-95-processing with specific CPPs constitutes a novel and promising therapeutic approach for stroke treatment.We acknowledge funding from Ministerio de Economía y Competitividad (BFU2013-43808-R and BFU2016-75973-R) and from Agencia Estatal de Investigación (PID2019-105784RB-100/AEI/10.13039/501100011033). The cost of publication has been paid in part by FEDER funds. Contracts were funded associated to projects BFU2013-43808-R (G.M. E-O and S.A-D) and BFU2016-75973-R (G.M.E-O). We are grateful to Dr. Sobrado (HU La Princesa) and Drs. Avendaño and Negredo (Departamento de Anatomía, Histología y Neurociencia, UAM, Spain) for technical advice with the ischemia model, and Dr. Scheiffele for sharing plasmid pNice-PSD-95-YF

An integrated approach to design novel therapeutic interventions for demyelinating disorders

Therapeutic strategies are often based on two general principles: interference with the pathogenic process and repair of the damaged tissues. Recent studies, however, have suggested that several pathological conditions may result from the interplay between genetic susceptibility traits and environmental influences that, by modulating the epigenome, also affect disease onset and progression. Based on lessons from neural development, it is conceivable that new lines of preventive and possibly therapeutic intervention might be developed to modulate disease onset or decrease the severity of the symptoms. This review will discuss these concepts within the context of multiple sclerosis, the most common demyelinating disease of the central nervous system, and the leading cause of progressive neurological disability in young adults

Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection

© 2019 The Authors.Neuroprotective strategies aimed to pharmacologically treat stroke, a prominent cause of death, disability, and dementia, have remained elusive. A promising approach is restriction of excitotoxic neuronal death in the infarct penumbra through enhancement of survival pathways initiated by brain‐derived neurotrophic factor (BDNF). However, boosting of neurotrophic signaling after ischemia is challenged by downregulation of BDNF high‐affinity receptor, full‐length tropomyosin‐related kinase B (TrkB‐FL), due to calpain‐degradation, and, secondarily, regulated intramembrane proteolysis. Here, we have designed a blood–brain barrier (BBB) permeable peptide containing TrkB‐FL sequences (TFL457) which prevents receptor disappearance from the neuronal surface, early induced after excitotoxicity. In this way, TFL457 interferes TrkB‐FL cleavage by both proteolytic systems and increases neuronal viability via a PLCγ‐dependent mechanism. By preserving downstream CREB and MEF2 promoter activities, TFL457 initiates a feedback mechanism favoring increased levels in excitotoxic neurons of critical prosurvival mRNAs and proteins. This neuroprotective peptide could be highly relevant for stroke therapy since, in a mouse ischemia model, it counteracts TrkB‐FL downregulation in the infarcted brain, efficiently decreases infarct size, and improves neurological outcome.We acknowledge funding from Ministerio de Economía y Competitividad (BFU2013-43808-R and BFU2016-75973-R). The cost of publication has been paid in part by FEDER funds. Contracts were funded associated with projects BFU2013-43808-R (G.S.T and G.M.E-O) and BFU2016-75973-R (G.M.E-O and E.SA)

Brain ischaemia induces shedding of a BDNF-scavenger ectodomain from TrkB receptors by excitotoxicity activation of metalloproteinases and γ-secretases

Stroke remains a leading cause of death and disability in the world with limited therapies available to restrict brain damage or improve functional recovery after cerebral ischaemia. A promising strategy currently under investigation is the promotion of brain‐derived neurotrophic factor (BDNF) signalling through tropomyosin‐related kinase B (TrkB) receptors, a pathway essential for neuronal survival and function. However, TrkB and BDNF‐signalling are impaired by excitotoxicity, a primary pathological process in stroke also associated with neurodegenerative diseases. Pathological imbalance of TrkB isoforms is critical in neurodegeneration and is caused by calpain processing of BDNF high affinity full‐length receptor (TrkB‐FL) and an inversion of the transcriptional pattern of the Ntrk2 gene, to favour expression of the truncated isoform TrkB‐T1 over TrkB‐FL. We report here that both TrkB‐FL and neuronal TrkB‐T1 also undergo ectodomain shedding by metalloproteinases activated after ischaemic injury or excitotoxic damage of cortical neurons. Subsequently, the remaining membrane‐bound C‐terminal fragments (CTFs) are cleaved by γ‐secretases within the transmembrane region, releasing their intracellular domains (ICDs) into the cytosol. Therefore, we identify TrkB‐FL and TrkB‐T1 as new substrates of regulated intramembrane proteolysis (RIP), a mechanism that highly contributes to TrkB‐T1 regulation in ischaemia but is minor for TrkB‐FL which is mainly processed by calpain. However, since the secreted TrkB ectodomain acts as a BDNF scavenger and significantly alters BDNF/TrkB signalling, the mechanism of RIP could contribute to neuronal death in excitotoxicity. These results are highly relevant since they reveal new targets for the rational design of therapies to treat stroke and other pathologies with an excitotoxic component

Bioenergetic Failure in Rat Oligodendrocyte Progenitor Cells Treated with Cerebrospinal Fluid Derived from Multiple Sclerosis Patients

In relapsing-remitting multiple sclerosis (RRMS) subtype, the patient’s brain itself is capable of repairing the damage, remyelinating the axon and recovering the neurological function. Cerebrospinal fluid (CSF) is in close proximity with brain parenchyma and contains a host of proteins and other molecules, which influence the cellular physiology, that may balance damage and repair of neurons and glial cells. The purpose of this study was to determine the pathophysiological mechanisms underpinning myelin repair in distinct clinical forms of MS and neuromyelitis optica (NMO) patients by studying the effect of diseased CSF on glucose metabolism and ATP synthesis. A cellular model with primary cultures of oligodendrocyte progenitor cells (OPCs) from rat cerebrum was employed, and cells were treated with CSF from distinct clinical forms of MS, NMO patients and neurological controls. Prior to comprehending mechanisms underlying myelin repair, we determine the best stably expressed reference genes in our experimental condition to accurately normalize our target mRNA transcripts. The GeNorm and NormFinder algorithms showed that mitochondrial ribosomal protein (Mrpl19), hypoxanthine guanine phosphoribosyl transferase (Hprt), microglobulin β2 (B2m), and transferrin receptor (Tfrc) were identified as the best reference genes in OPCs treated with MS subjects and were used for normalizing gene transcripts. The main findings on microarray gene expression profiling analysis on CSF treated OPCs cells revealed a disturbed carbohydrate metabolism and ATP synthesis in MS and NMO derived CSF treated OPCs. In addition, using STRING program, we investigate whether gene–gene interaction affected the whole network in our experimental conditions. Our findings revealed downregulated expression of genes involved in carbohydrate metabolism, and that glucose metabolism impairment and reduced ATP availability for cellular damage repair clearly differentiate more benign forms from the most aggressive forms and worst prognosis in MS patients

Cerebrospinal fluid ceramides from patients with multiple sclerosis impair neuronal bioenergetics

none20siopenVidaurre, Og; Haines, Jd; Katz Sand, I; Adula, Kp; Huynh, Jl; Mcgraw, Ca; Zhang, F; Varghese, M; Sotirchos, E; Bhargava, P; Bandaru, Vv; Pasinetti, G; Zhang, W; Inglese, M; Calabresi, Pa; Wu, G; Miller, Ae; Haughey, Nj; Lublin, Fd; Casaccia, P.Vidaurre, Og; Haines, Jd; Katz Sand, I; Adula, Kp; Huynh, Jl; Mcgraw, Ca; Zhang, F; Varghese, M; Sotirchos, E; Bhargava, P; Bandaru, Vv; Pasinetti, G; Zhang, W; Inglese, MARIA MATILDE; Calabresi, Pa; Wu, G; Miller, Ae; Haughey, Nj; Lublin, Fd; Casaccia, P