PSD-95: An Effective Target for Stroke Therapy Using Neuroprotective Peptides.

Therapies for stroke have remained elusive in the past despite the great relevance of this pathology. However, recent results have provided strong evidence that postsynaptic density protein-95 (PSD-95) can be exploited as an efficient target for stroke neuroprotection by strategies able to counteract excitotoxicity, a major mechanism of neuronal death after ischemic stroke. This scaffold protein is key to the maintenance of a complex framework of protein interactions established at the postsynaptic density (PSD) of excitatory neurons, relevant to neuronal function and survival. Using cell penetrating peptides (CPPs) as therapeutic tools, two different approaches have been devised and advanced to different levels of clinical development. First, nerinetide (Phase 3) and AVLX-144 (Phase 1) were designed to interfere with the coupling of the ternary complex formed by PSD-95 with GluN2B subunits of the N-methyl-D-aspartate type of glutamate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS). These peptides reduced neurotoxicity derived from NMDAR overactivation, decreased infarct volume and improved neurobehavioral results in different models of ischemic stroke. However, an important caveat to this approach was PSD-95 processing by calpain, a pathological mechanism specifically induced by excitotoxicity that results in a profound alteration of survival signaling. Thus, a third peptide (TP95414) has been recently developed to interfere with PSD-95 cleavage and reduce neuronal death, which also improves neurological outcome in a preclinical mouse model of permanent ischemia. Here, we review recent advancements in the development and characterization of PSD-95-targeted CPPs and propose the combination of these two approaches to improve treatment of stroke and other excitotoxicity-associated disorders.

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.

Excitotoxic targeting of Kidins220 to the Golgi apparatus precedes calpain cleavage of Rap1-activation complexes.

Excitotoxic neuronal death induced by high concentrations of glutamate is a pathological event common to multiple acute or chronic neurodegenerative diseases. Excitotoxicity is mediated through overactivation of the N-Methyl-D-aspartate type of ionotropic glutamate receptors (NMDARs). Physiological stimulation of NMDARs triggers their endocytosis from the neuronal surface, inducing synaptic activity and survival. However almost nothing is known about the internalization of overactivated NMDARs and their interacting proteins, and how this endocytic process is connected with neuronal death has been poorly explored. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a component of NMDAR complexes essential for neuronal viability by the control of ERK activation. Here we have investigated Kidins220 endocytosis induced by NMDAR overstimulation and the participation of this internalization step in the molecular mechanisms of excitotoxicity. We show that excitotoxicity induces Kidins220 and GluN1 traffic to the Golgi apparatus (GA) before Kidins220 is degraded by the protease calpain. We also find that excitotoxicity triggers an early activation of Rap1-GTPase followed by its inactivation. Kidins220 excitotoxic endocytosis and subsequent calpain-mediated downregulation governs this late inactivation of Rap1 that is associated to decreases in ERK activity preceding neuronal death. Furthermore, we identify the molecular mechanisms involved in the excitotoxic shutoff of Kidins220/Rap1/ERK prosurvival cascade that depends on calpain processing of Rap1-activation complexes. Our data fit in a model where Kidins220 targeting to the GA during early excitotoxicity would facilitate Rap1 activation and subsequent stimulation of ERK. At later times, activation of Golgi-associated calpain, would promote the degradation of GA-targeted Kidins220 and two additional components of the specific Rap1 activation complex, PDZ-GEF1, and S-SCAM. In this way, late excitotoxicity would turn off Rap1/ERK cascade and compromise neuronal survival.

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.

Integral Characterization of Defective BDNF/TrkB Signalling in Neurological and Psychiatric Disorders Leads the Way to New Therapies.

Enhancement of brain-derived neurotrophic factor (BDNF) signalling has great potential in therapy for neurological and psychiatric disorders. This neurotrophin not only attenuates cell death but also promotes neuronal plasticity and function. However, an important challenge to this approach is the persistence of aberrant neurotrophic signalling due to a defective function of the BDNF high-affinity receptor, tropomyosin-related kinase B (TrkB), or downstream effectors. Such changes have been already described in several disorders, but their importance as pathological mechanisms has been frequently underestimated. This review highlights the relevance of an integrative characterization of aberrant BDNF/TrkB pathways for the rational design of therapies that by combining BDNF and TrkB targets could efficiently promote neurotrophic signalling.

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.

Kidins220 accumulates with tau in human Alzheimer's disease and related models: modulation of its calpain-processing by GSK3ß/PP1 imbalance.

Failures in neurotrophic support and signalling play key roles in Alzheimer's disease (AD) pathogenesis. We previously demonstrated that downregulation of the neurotrophin effector Kinase D interacting substrate (Kidins220) by excitotoxicity and cerebral ischaemia contributed to neuronal death. This downregulation, triggered through overactivation of N-methyl-D-aspartate receptors (NMDARs), involved proteolysis of Kidins220 by calpain and transcriptional inhibition. As excitotoxicity is at the basis of AD aetiology, we hypothesized that Kidins220 might also be downregulated in this disease. Unexpectedly, Kidins220 is augmented in necropsies from AD patients where it accumulates with hyperphosphorylated tau. This increase correlates with enhanced Kidins220 resistance to calpain processing but no higher gene transcription. Using AD brain necropsies, glycogen synthase kinase 3-ß (GSK3ß)-transgenic mice and cell models of AD-related neurodegeneration, we show that GSK3ß phosphorylation decreases Kidins220 susceptibility to calpain proteolysis, while protein phosphatase 1 (PP1) action has the opposite effect. As altered activities of GSK3ß and phosphatases are involved in tau aggregation and constitute hallmarks in AD, a GSK3ß/PP1 imbalance may also contribute to Kidins220 decreased clearance, accumulation and hampered neurotrophin signalling from early stages of the disease pathogenesis. These results encourage searches for mutations in Kidins220 gene and their possible associations to dementias. Finally, our data support a model where the effects of excitotoxicity drastically differ when occurring in cerebral ischaemia versus progressively sustained toxicity along AD progression. The striking differences in Kidins220 stability resulting from chronic versus acute brain damage may also have important implications for the therapeutic intervention of neurodegenerative disorders.

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.

Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons.

Gene transfer methods for efficient co-expression of exogenous proteins in neurons are crucial tools towards the understanding of the molecular basis of the central nervous system. Lentiviruses are retroviral vectors that can transduce a wide variety of cells including differentiated neurons. In this work, we have generated lentiviral vectors containing dual promoters that allow efficient co-expression of exogenous proteins in neurons. We show that insertion of two copies of a human synapsin promoter/WPRE cassette in a single lentiviral vector directs robust co-expression of cDNAs in cultured neurons, while excluding expression in the surrounding glial cells. Furthermore, insertion of the tetracycline-inducible system (Tet-off) controlled by the synapsin promoter results in tightly regulated expression of EGFP when used as a transgene in cultured neurons. Transduction of primary neurons with this inducible system leads to a 100-fold increase in EGFP mRNA levels in the absence of doxycycline. In transduced cultures, EGFP transcription is inhibited within 24h upon addition of doxycycline. The viral systems we developed here provide neuron-specific and regulated expression mediated by single lentiviral vectors and will prove valuable tools for the study of neuronal function.

Endoplasmic reticulum-associated degradation of the NR1 but not the NR2 subunits of the N-methyl-D-aspartate receptor induced by inhibition of the N-glycosylation in cortical neurons.

The N-methyl-D-aspartate receptor (NMDAR) is fundamental to normal and pathological functioning of neurons. The receptor subunits are N-glycosylated proteins synthesized in the endoplasmic reticulum (ER) that fold, mature, and oligomerize as they transit through the secretory pathway. Although the early processes of biogenesis are fundamental to NMDAR expression and function, our knowledge of them is nevertheless limited. Additionally, the investigation of NMDAR synthesis is highly relevant, in that ER dysfunction, frequently associated with acute and degenerative brain diseases, might alter this process. We characterize here the effect of ER stress produced by inhibition of N-glycosylation on NMDAR synthesis and function. We use first heterologous systems of NMDAR expression in which NR1 and NR2A subunits are synthesized in nonneuronal cells. The function of these NMDARs as Ca2+ channels is repressed by tunicamycin, because of the inhibition of NR1, but no NR2A, synthesis. The regulation of NR1 is relevant to the central nervous system, in that a dramatic decrease in synthesis of this subunit and assembly of NMDARs is observed in cortical neurons treated with tunicamycin. The inhibition of NR1 synthesis is not due to changes in levels of mRNA but associated with the earliest stages in NMDAR biogenesis. The inhibition of N-glycosylation activates ER-specific stress responses in neurons, which include the ER-associated degradation (ERAD) mechanism responsible for differential and extremely efficient degradation of nonglycosylated NR1 by the proteasome after ubiquitination. Because this is an obligatory NMDAR component, the significant sensitivity of NR1 to ER stress will have important consequences on receptor function.

Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia.

The N-methyl-D-aspartate (NMDA) type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 microM NMDA or glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (< or = 10 microM) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h of stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of autoregulation of the receptor probably operates during cerebral ischemia, because NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Because NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning.

Characterization of regions in the GP5 protein of porcine reproductive and respiratory syndrome virus required to induce apoptotic cell death.

Expression of the GP5 protein of porcine reproductive and respiratory syndrome virus in mammalian cells using a recombinant vaccinia virus has been shown to induce strong cytotoxicity due to apoptotic death. We have now developed a transient expression system that allows the observation and quantitation of the cell death due to GP5 synthesis, taking advantage of the reduction that this protein induces in the expression of two different co-transfected reporter genes. In this way, we are able to study the regions in GP5 implicated in apoptosis induction. The first 119 aminoacids constitute a region capable of fully inducing apoptosis, aminoacids 90-119 having a fundamental role. On the contrary, the C-terminal region is unable by itself of cell death induction and, moreover, is dispensable for this phenotype. We have also observed that induction of apoptosis is independent of cleavage of the N-terminal putative signal sequence in GP5 or N-glycosylation of this protein.