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

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.

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

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.

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 ß2 (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.

Subcellular Distribution of HDAC1 in Neurotoxic Conditions Is Dependent on Serine Phosphorylation.

Calcium-dependent nuclear export of histone deacetylase 1 (HDAC1) was shown previously to precede axonal damage in culture, but the in vivo relevance of these findings and the potential posttranslational modifications of HDAC1 remained elusive. Using acute hippocampal slices from mice of either sex with genetic conditional ablation of Hdac1 in CA1 hippocampal neurons (i.e., Camk2a-cre;Hdac1fl/fl), we show significantly diminished axonal damage in response to neurotoxic stimuli. The protective effect of Hdac1 ablation was detected also in CA3 neurons in Grik4-cre;Hdac1fl/f mice, which were more resistant to the excitotoxic damage induced by intraventricular injection of kainic acid. The amino acid residues modulating HDAC1 subcellular localization were identified by site-directed mutagenesis, which identified serine residues 421 and 423 as critical for its nuclear localization. The physiological phosphorylation of HDAC1 was decreased by neurotoxic stimuli, which stimulated the phosphatase enzymatic activity of calcineurin. Treatment of neurons with the calcineurin inhibitors FK506 or cyclosporin A resulted in nuclear accumulation of phospho-HDAC1 and was neuroprotective. Together, our data identify HDAC1 and the phosphorylation of specific serine residues in the molecule as potential targets for neuroprotection.SIGNIFICANCE STATEMENT The importance of histone deacetylation in normal brain functions and pathological conditions is unquestionable, yet the molecular mechanisms responsible for the neurotoxic potential of histone deacetylase 1 (HDAC1) and its subcellular localization are not fully understood. Here, we use transgenic lines to define the in vivo relevance of HDAC1 and identify calcineurin-dependent serine dephosphorylation as the signal modulating the neurotoxic role of HDAC1 in response to neurotoxic stimuli.

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.

Multiple sclerosis patient-derived CSF induces transcriptional changes in proliferating oligodendrocyte progenitors.

BACKGROUND: Cerebrospinal fluid (CSF) is in contact with brain parenchyma and ventricles, and its composition might influence the cellular physiology of oligodendrocyte progenitor cells (OPCs) thereby contributing to multiple sclerosis (MS) disease pathogenesis. OBJECTIVE: To identify the transcriptional changes that distinguish the transcriptional response induced in proliferating rat OPCs upon exposure to CSF from primary progressive multiple sclerosis (PPMS) or relapsing remitting multiple sclerosis (RRMS) patients and other neurological controls. METHODS: We performed gene microarray analysis of OPCs exposed to CSF from neurological controls, or definitive RRMS or PPMS disease course. Results were confirmed by quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry and western blot of cultured cells, and validated in human brain specimens. RESULTS: We identified common and unique oligodendrocyte genes for each treatment group. Exposure to CSF from PPMS uniquely induced branching of cultured progenitors and related transcriptional changes, including upregulation (P<0.05) of the adhesion molecule GALECTIN-3/Lgals3, which was also detected at the protein level in brain specimens from PPMS patients. This pattern of gene expression was distinct from the transcriptional programme of oligodendrocyte differentiation during development. CONCLUSIONS: Despite evidence of morphological differentiation induced by exposure to CSF of PPMS patients, the overall transcriptional response elicited in cultured OPCs was consistent with the activation of an aberrant transcriptional programme.

Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination.

Axonal damage has been associated with aberrant protein trafficking. We examined a newly characterized class of compounds that target nucleo-cytoplasmic shuttling by binding to the catalytic groove of the nuclear export protein XPO1 (also known as CRM1, chromosome region maintenance protein 1). Oral administration of reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but was also observed in another mouse model of axonal damage (that is, kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection.

Cerebrospinal fluid ceramides from patients with multiple sclerosis impair neuronal bioenergetics.

Axonal damage is a prominent cause of disability and yet its pathogenesis is incompletely understood. Using a xenogeneic system, here we define the bioenergetic changes induced in rat neurons by exposure to cerebrospinal fluid samples from patients with multiple sclerosis compared to control subjects. A first discovery cohort of cerebrospinal fluid from 13 patients with multiple sclerosis and 10 control subjects showed that acute exposure to cerebrospinal fluid from patients with multiple sclerosis induced oxidative stress and decreased expression of neuroprotective genes, while increasing expression of genes involved in lipid signalling and in the response to oxidative stress. Protracted exposure of neurons to stress led to neurotoxicity and bioenergetics failure after cerebrospinal fluid exposure and positively correlated with the levels of neurofilament light chain. These findings were validated using a second independent cohort of cerebrospinal fluid samples (eight patients with multiple sclerosis and eight control subjects), collected at a different centre. The toxic effect of cerebrospinal fluid on neurons was not attributable to differences in IgG content, glucose, lactate or glutamate levels or differences in cytokine levels. A lipidomic profiling approach led to the identification of increased levels of ceramide C16:0 and C24:0 in the cerebrospinal fluid from patients with multiple sclerosis. Exposure of cultured neurons to micelles composed of these ceramide species was sufficient to recapitulate the bioenergetic dysfunction and oxidative damage induced by exposure to cerebrospinal fluid from patients with multiple sclerosis. Therefore, our data suggest that C16:0 and C24:0 ceramides are enriched in the cerebrospinal fluid of patients with multiple sclerosis and are sufficient to induce neuronal mitochondrial dysfunction and axonal damage.

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.

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

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 Ca(2+) influx, and involves a dual mechanism: rapid cleavage by the Ca(2+)-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.