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.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

27-Hydroxycholesterol Induces Aberrant Morphology and Synaptic Dysfunction in Hippocampal Neurons.

Hypercholesterolemia is a risk factor for neurodegenerative diseases, but how high blood cholesterol levels are linked to neurodegeneration is still unknown. Here, we show that an excess of the blood-brain barrier permeable cholesterol metabolite 27-hydroxycholesterol (27-OH) impairs neuronal morphology and reduces hippocampal spine density and the levels of the postsynaptic protein PSD95. Dendritic spines are the main postsynaptic elements of excitatory synapses and are crucial structures for memory and cognition. Furthermore, PSD95 has an essential function for synaptic maintenance and plasticity. PSD95 synthesis is controlled by the REST-miR124a-PTBP1 axis. Here, we report that high levels of 27-OH induce REST-miR124a-PTBP1 axis dysregulation in a possible RxRγ-dependent manner, suggesting that 27-OH reduces PSD95 levels through this mechanism. Our results reveal a possible molecular link between hypercholesterolemia and neurodegeneration. We discuss the possibility that reduction of 27-OH levels could be a useful strategy for preventing memory and cognitive decline in neurodegenerative disorders.

In vitro and in vivo effects of a novel dimeric inhibitor of PSD-95 on excitotoxicity and functional recovery after experimental traumatic brain injury.

PSD-95 inhibitors have been shown to be neuroprotective in stroke, but have only to a very limited extent been evaluated in the treatment of traumatic brain injury (TBI) that has pathophysiological mechanisms in common with stroke. The aims of the current study were to assess the effects of a novel dimeric inhibitor of PSD-95, UCCB01-147, on histopathology and long-term cognitive outcome after controlled cortical impact (CCI) in rats. As excitotoxic cell death is thought to be a prominent part of the pathophysiology of TBI, we also investigated the neuroprotective effects of UCCB01-147 and related compounds on NMDA-induced cell death in cultured cortical neurons. Anesthetized rats were given a CCI or sham injury, and were randomized to receive an injection of either UCCB01-147 (10 mg/kg), the non-competitive NMDAR-receptor antagonist MK-801 (1 mg/kg) or saline immediately after injury. At 2 and 4 weeks post-trauma, spatial learning and memory were assessed in a water maze, and at 3 months, brains were removed for estimation of lesion volumes. Overall, neither treatment with UCCB01-147 nor MK-801 resulted in significant improvements of cognition and histopathology after CCI. Although MK-801 provided robust neuroprotection against NMDA-induced toxicity in cultured cortical neurons, UCCB01-147 failed to reduce cell death and became neurotoxic at high doses. The data suggest potential differential effects of PSD-95 inhibition in stroke and TBI that should be investigated further in future studies taking important experimental factors such as timing of treatment, dosage, and anesthesia into consideration.

Regional Specific Modulation of Stress-Induced Neuronal Activation Associated with the PSD95/NOS Interaction Inhibitor ZL006 in the Wistar Kyoto Rat.

Background: To determine brain areas involved in the antidepressant-related behavioral effects of the selective neuronal nitric oxide synthase inhibitor 1-(2-Trifluoro-methyl-phenyl) imidazole (TRIM) and experimental test compound 4-((3,5-dichloro-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid (ZL006), an inhibitor of the PSD of 95 kDa/neuronal nitric oxide synthase interaction in the N-methyl-D-aspartic acid receptor signalling pathway, regional specific expression of the neuronal activation marker c-FOS was assessed following exposure to the forced swimming test in the Wistar Kyoto rat. Methods: Wistar Kyoto rats were subjected to a 15-minute swim pretest (pre-forced swimming test) period on day 1. At 24, 5, and 1 hour prior to the 5-minute test, which took place 24 hours following the pre-forced swimming test, animals were treated with TRIM (50 mg/kg; i.p.), ZL006 (10 mg/kg; i.p.), or saline vehicle (1 mL/kg i.p). Behavior was recorded during both pretest and test periods. Results: Both TRIM and ZL006 decreased immobility time in Wistar Kyoto rats in the forced swimming test. Exposure to the forced swimming test increased c-FOS immunoreactivity in the lateral septum, paraventricular nucleus of the hypothalamus, periaqueductal grey, dentate gyrus, and ventral CA1 of the hippocampus compared with non-forced swimming test-exposed controls. Forced swimming test-induced c-FOS immunoreactivity was further increased in the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus following treatment with TRIM or ZL006. By contrast, forced swimming test-induced c-FOS immunoreactivity was reduced in dorsal dentate gyrus and ventral CA1 following treatment with TRIM or ZL006. Exposure to the forced swimming test resulted in an increase in NADPH diaphorase staining in the paraventricular nucleus of the hypothalamus. This forced swimming test-induced increase was attenuated following treatment with ZL006 and points to the paraventricular nucleus as a brain region where ZL006 acts to attenuate forced swimming test-induced neuronal nitric oxide synthase activity while concomitantly regulating region specific neuronal activation associated with an antidepressant-related response. Conclusions: This study identified a pattern of enhanced and reduced forced swimming test-related c-FOS immunoreactivity indicative of a regulated network where inhibition of nitric oxide coupled to the N-methyl-D-aspartic acid receptor leads to activation of the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus with concomitant inhibition of the hippocampus.

Effects of Dimeric PSD-95 Inhibition on Excitotoxic Cell Death and Outcome After Controlled Cortical Impact in Rats.

Therapeutic effects of PSD-95 inhibition have been demonstrated in numerous studies of stroke; however only few studies have assessed the effects of PSD-95 inhibitors in traumatic brain injury (TBI). As the pathophysiology of TBI partially overlaps with that of stroke, PSD-95 inhibition may also be an effective therapeutic strategy in TBI. The objectives of the present study were to assess the effects of a dimeric inhibitor of PSD-95, UCCB01-144, on excitotoxic cell death in vitro and outcome after experimental TBI in rats in vivo. In addition, the pharmacokinetic parameters of UCCB01-144 were investigated in order to assess uptake of the drug into the central nervous system of rats. After a controlled cortical impact rats were randomized to receive a single injection of either saline or two different doses of UCCB01-144 (10 or 20 mg/kg IV) immediately after injury. Spatial learning and memory were assessed in a water maze at 2 weeks post-trauma, and at 4 weeks lesion volumes were estimated. Overall, UCCB01-144 did not protect against NMDA-toxicity in neuronal cultures or experimental TBI in rats. Important factors that should be investigated further in future studies assessing the effects of PSD-95 inhibitors in TBI are discussed.

Sevoflurane Acts on Ubiquitination-Proteasome Pathway to Reduce Postsynaptic Density 95 Protein Levels in Young Mice.

BACKGROUND: Children with multiple exposures to anesthesia and surgery may have an increased risk of developing cognitive impairment. Sevoflurane, a commonly used anesthetic in children, has been reported to decrease levels of postsynaptic density 95 protein. However, the upstream mechanisms and downstream consequences of the sevoflurane-induced reduction in postsynaptic density 95 protein levels remains largely unknown. We therefore set out to assess whether sevoflurane acts on ubiquitination-proteasome pathway to facilitate postsynaptic density 95 protein degradation. METHODS: Six-day-old wild-type mice received anesthesia with 3% sevoflurane 2 h daily for 3 days starting on postnatal day 6. We determined the effects of the sevoflurane anesthesia on mRNA, protein and ubiquitinated levels of postsynaptic density 95 protein in neurons, and synaptosomes and hippocampus of young mice. Cognitive function in the mice was determined at postnatal day 31 by using a Morris water maze. Proteasome inhibitor MG132 and E3 ligase mouse double mutant 2 homolog inhibitor Nutlin-3 were used for the interaction studies. RESULTS: The sevoflurane anesthesia decreased protein, but not mRNA, levels of postsynaptic density 95, and reduced ubiquitinated postsynaptic density 95 protein levels in neurons, synaptosomes, and hippocampus of young mice. Both MG132 and Nutlin-3 blocked these sevoflurane-induced effects. Sevoflurane promoted the interaction of mouse double mutant 2 homolog and postsynaptic density 95 protein in neurons. Finally, MG132 and Nutlin-3 ameliorated the sevoflurane-induced cognitive impairment in the mice. CONCLUSIONS: These data suggest that sevoflurane acts on the ubiquitination-proteasome pathway to facilitate postsynaptic density 95 protein degradation, which then decreases postsynaptic density 95 protein levels, leading to cognitive impairment in young mice. These studies would further promote the mechanistic investigation of anesthesia neurotoxicity in the developing brain.

Targeting PSD95/nNOS by ZL006 alleviates social isolation-induced heightened attack behavior in mice.

RATIONALE: Deregulated attack behaviors have devastating social consequences; however, satisfactory clinical management for the behavior is still an unmet need so far. Social isolation (SI) has been common during the COVID-19 pandemic and may have detrimental effects on mental health, including eliciting heightened attack behavior. OBJECTIVES: This study aims to explore whether injection of ZL006 can alleviate SI-induced escalation of attack behavior in mice. METHODS: Pharmacological tools, biochemical methods, and behavioral tests were used to explore the potential therapeutic effects of ZL006 targeting postsynaptic density 95 (PSD95)/neuronal nitric oxide synthase (nNOS) pathway on escalation of attack behavior induced by SI in mice. RESULTS: ZL006 mitigated SI-induced escalated attack behaviors and elevated nitric oxide (NO) level in the cortex of the SI mice. The beneficial effects of ZL006 lasted for at least 72 h after a single injection of ZL006. Potentiation of NO levels by L-arginine blocked the effects of ZL006. Moreover, a sub-effective dose of 7-NI in combination with a sub-effective dose of ZL006 decreased both SI-induced escalated attack behaviors and NO levels in mice subjected to SI. CONCLUSIONS: Our study highlights the importance of the PSD95/nNOS pathway in mediating SI-induced escalation of attack behavior. ZL006 may be a promising therapeutic strategy for treating aggressive behaviors.

Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke.

Neuroprotection for acute ischemic stroke is achievable with the eicosapeptide nerinetide, an inhibitor of the protein-protein interactions of the synaptic scaffolding protein PSD-95. However, nerinetide is subject to proteolytic cleavage if administered after alteplase, a standard-of-care thrombolytic agent that nullifies nerinetide's beneficial effects. Here, we showed, on the basis of pharmacokinetic data consistent between rats, primates, and humans, that in a rat model of embolic middle cerebral artery occlusion (eMCAO), nerinetide maintained its effectiveness when administered before alteplase. Because of its short plasma half-life, it can be followed by alteplase within minutes without reducing its neuroprotective effectiveness. In addition, the problem of protease sensitivity is solved by substituting cleavage-prone amino acids from their l- to their d-enantiomeric form. Treatment of rats subjected to eMCAO with such an agent, termed d-Tat-l-2B9c, eliminated protease sensitivity and maintained neuroprotective effectiveness. Our data suggest that both the clinical-stage PSD-95 inhibitor nerinetide and protease-resistant agents such as d-Tat-l-2B9c may be practically integrated into existing stroke care workflows and standards of care.

Identification of Novel Fragments Binding to the PDZ1-2 Domain of PSD-95.

Inhibition of PSD-95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide-based compounds that target the PDZ domains of PSD-95. In contrast, developing potent and drug-like small molecules against the PSD-95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD-95 PDZ1-2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter-test, and found four promising fragments. Three ligand efficient fragments were shown by 1 H,15 N HSQC NMR to bind in the small hydrophobic P0 pockets of PDZ1-2, and one of them underwent structure-activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1-2 of PSD-95 and disclose novel fragments that can serve as starting points for optimization towards small-molecule PDZ domain inhibitors.

Development of Peptide-Based PDZ Domain Inhibitors.

Over the past decades, peptide-based drugs have gained increasing interest in a wide range of treatment applications, primarily because of high potency and selectivity, as well as good efficacy, tolerability, and safety often achieved with peptides. Attempts to target postsynaptic density protein of 95 (PSD-95) PSD-95/Discs large/Zonula occludens-1 (PDZ) domains, which mediate the formation of a ternary complex with the N-methyl-D-aspartate (NMDA) receptor and neuronal nitric oxide synthase (nNOS) responsible for excitotoxicity in ischemic stroke, by high-affinity small molecules have failed in the past. In this chapter, we focus on the discovery of peptide-based drugs targeting PSD-95, using AVLX-144 as an example, from the synthesis, over binding assays to its target, to further in vitro experiments based on the development of AVLX-144, a potential stroke treatment, which is planned to enter clinical trials in 2020.

PSD-95 protects synapses from ß-amyloid.

Beta-amyloid (Aß) depresses excitatory synapses by a poorly understood mechanism requiring NMDA receptor (NMDAR) function. Here, we show that increased PSD-95, a major synaptic scaffolding molecule, blocks the effects of Aß on synapses. The protective effect persists in tissue lacking the AMPA receptor subunit GluA1, which prevents the confounding synaptic potentiation by increased PSD-95. Aß modifies the conformation of the NMDAR C-terminal domain (CTD) and its interaction with protein phosphatase 1 (PP1), producing synaptic weakening. Higher endogenous levels or overexpression of PSD-95 block Aß-induced effects on the NMDAR CTD conformation, its interaction with PP1, and synaptic weakening. Our results indicate that increased PSD-95 protects synapses from Aß toxicity, suggesting that low levels of synaptic PSD-95 may be a molecular sign indicating synapse vulnerability to Aß. Importantly, pharmacological inhibition of its depalmitoylation increases PSD-95 at synapses and rescues deficits caused by Aß, possibly opening a therapeutic avenue against Alzheimer's disease.

Effects of brazilein on PSD-95 protein expression and neurological recovery in mice after sciatic nerve injury.

PURPOSE: To evaluate the local nerve myelin recovery and the expression of PSD-95 protein and mRNA in the L4-L6 segment of the spinal cord after applying Brazilein to sciatic nerve injury BALB/c mice model and investigate the regulatory effects of Brazilein on myelin recovery after peripheral nerve injury. METHODS: A total of 160 BALB/c mice were selected to establish the unilateral sciatic nerve injury model and randomly divided into four groups: saline blank control, Brazilein high-dose, medium-dose, and low-dose. Mice were assessed at different time points (1 w, 2 w, 4 w, 8 w) after sciatic nerve injury for the sciatic functional index (SFI) and sciatic nerve function recovery of the injured side by myelin Luxol Fast Blue (LFB) staining of the sciatic nerve. In addition, immunohistochemistry, real time-PCR, and Western blot were used to detect the PSD-95 expression in the spinal cord L4-L6 segments of the injured sciatic nerve at each time point. RESULTS: The results of SFI and sciatic nerve function recovery, as well as, myelin LFB staining of the injured side indicated that all indexes of the Brazilein middle- and high-dose groups were significantly better than the low-dose and blank control groups at each time point. The PSD-95 expression in the L4-L6 segment of the spinal cord was statistically lower in the high- and medium-dose groups than in the low-dose and blank control groups at 1 w, 2 w, and 4 w, while the differences between the groups were not significant at 8 w. CONCLUSION: Brazilein inhibits PSD-95 activation in the corresponding segment of sciatic nerve spinal cord in BALB/c mice after sciatic nerve injury, thereby inhibiting the excessive expression of free radicals and promoting myelin regeneration.

In silico identification and design of potent peptide inhibitors against PDZ-3 domain of Postsynaptic Density Protein (PSD-95).

Unique intrinsic properties of peptides like low toxicity, high biological activity, and specificity make them attractive therapeutic agents. PDZ-binding peptide inhibitors have been demonstrated for curing of Alzheimer, Parkinson, Dementia, and other central nervous system ailments. In this article, we report the successful use of an integrated computational protocol to analyze the structural basis of how peptides bind to the shallow groove of the third PDZ domain (PDZ-3) from the postsynaptic density (PSD-95) protein. This protocol employs careful and precise computational techniques for design of new strategy for predicting novel and potent peptides against PDZ protein. We attempted to generate a pharmacophore model using crystal structure of peptide inhibitor bound to the PDZ-3. A highly specific and sensitive generated pharmacophore model was used for screening virtual database generated using different combination of amino acid substitutions as well as decoy peptide database for its sensitivity and specificity. Identified hit peptides were further analyzed by docking studies, and their stability analyzed using solvated molecular dynamics. Quantum Mechanics/Molecular Mechanics (QM/MM) interaction energy and GMX-PBSA scoring schemes were used for ranking of stable peptides. Computational approach applied here generated encouraging results for identifying peptides against PDZ interaction model. The workflow can be further exercised as a virtual screening technique for reducing the search space for candidate target peptides against PDZ domains.

Long-term social isolation inhibits autophagy activation, induces postsynaptic dysfunctions and impairs spatial memory.

Social isolation in adolescence leads to lasting deficits in hippocampal-dependent tasks. The reported effects of isolation on learning and memory in the Morris water maze and synaptic-related proteins have been inconsistent. Moreover, the autophagy level and its effect on cognition in the isolation model are also not clear. In the present study, we did an extended isolation period up to six months to establish a stable and appropriate isolation model to investigate the cognitive changes associated with it. The mTOR inhibitor rapamycin was systemically administered to mice to determine the roles of autophagy activation on cognitive changes. We discovered that long-term post-weaning social isolation (L-PWSI) produced marked deficits in spatial learning and memory and inhibited CA1 long-term potentiation (LTP), but paired-pulse facilitation (PPF) and input/output (I/O) curve were unaffected. The results further showed that the L-PWSI significantly decreased the protein expression levels of PSD-95, GluA1, NR1 and NR2B in the hippocampus, and no significant changes in the extracellular release of glutamate and the protein expression levels of synaptophysin, synapsin I, GAP-43, NR2A and GABAA. Moreover, we found that L-PWSI increased the protein expression of p-AKT/AKT, p-mTOR/mTOR and p62, whereas the protein levels of LC3B and Beclin1 were decreased indicating an inhibition in autophagy activity. Intraperitoneal injection of rapamycin significantly potentiated fEPSP slope and cognition-related proteins expression in the L-PWSI mice. These results therefore suggest that L-PWSI induces postsynaptic dysfunction by disrupting the interaction between AMPAR, NMDAR and PSD-95, and inhibit the autophagy activity which led to impaired spatial memory and cognitive function.

Targeting PSD95-nNOS interaction by Tat-N-dimer peptide during status epilepticus is neuroprotective in MAM-pilocarpine rat model.

Glutamate receptors play a crucial pathogenic role in brain damage induced by status epilepticus (SE). SE may initiate NMDAR-dependent excitotoxicity through the production of oxidative damage mediated by the activation of a ternary complex formed by the NMDA receptor, the post-synaptic density scaffolding protein 95 (PSD95) and the neuronal NO synthase (nNOS). The inhibition of the protein-protein-interaction (PPI) of the NMDAR-PSD95-nNOS complex is one of the most intriguing challenges recently developed to reduce neuronal death in both animal models and in patients with cerebral ischemia. We took advantage of this promising approach to verify whether early administration of a neuroprotective NMDAR-PSD95-nNOS PPI inhibitor preserves the brain from SE-induced damage in a model of acquired cortical dysplasia, the methylazoxymethanol (MAM)/pilocarpine rat. Pilocarpine-induced SE rapidly determined neurodegenerative changes mediated by a NMDAR-downstream neurotoxic pathway in MAM rats. We demonstrated that SE rapidly induces NMDAR activation, nNOS membrane translocation, PSD95-nNOS molecular interaction associated with neuronal and glial peroxynitrite accumulation in the neocortex of MAM-pilocarpine rats. These changes were paralleled by rapid c-fos overexpression and by progressive spectrin proteolysis, suggestive of calpain activity and irreversible cytoskeletal damage. Early administration of a cell-penetrating Tat-N-dimer peptide inhibitor of NMDAR-PSD95-nNOS PPI during SE significantly rescued the MAM-pilocarpine rats from SE-induced mortality, reduced the number of degenerating neurons, decreased neuronal c-fos activation, peroxynitrite formation and cytoskeletal degradation and prevented astrogliosis. Our findings suggest an overall neuroprotective effect of blocking PSD95-nNOS protein-protein-interaction against SE insult.

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules.

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor.

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

N-methyl D-aspartate receptor subtype 2B antagonist, Ro 25-6981, attenuates neuropathic pain by inhibiting postsynaptic density 95 expression.

Postsynaptic density-95 (PSD-95) is a synaptic scaffolding protein that plays a crucial role in the development of neuropathic pain. However, the underlying mechanism remains unclear. To address the role of PSD-95 in N-methyl-D-aspartate receptor subtype 2B (NR2B) -mediated chronic pain, we investigated the relationship between PSD-95 activation and NR2B function in the spinal cord, by using a rat model of sciatic nerve chronic constriction injury (CCI). We demonstrate that the expression levels of total PSD-95 and cAMP response element binding protein (CREB), as well as phosphorylated NR2B, PSD-95, and CREB, in the spinal dorsal horn, and the interaction of NR2B with PSD-95 were increased in the CCI animals. Intrathecal injection of the selective NR2B antagonist Ro 25-6981 increased paw withdrawal latency, in a thermal pain assessment test. Moreover, repeated treatment with Ro 25-6981 markedly attenuated the thermal hypersensitivity, and inhibited the CCI-induced upregulation of PSD-95 in the spinal dorsal horn. Furthermore, intrathecal injection of the PSD-95 inhibitor strikingly reversed the thermal and mechanical hyperalgesia. Our results suggest that blocking of NR2B signaling in the spinal cord could be used as a therapeutic candidate for treating neuropathic pain.

Small molecule inhibitors of PSD95-nNOS protein-protein interactions suppress formalin-evoked Fos protein expression and nociceptive behavior in rats.

Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide. NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein-protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95-nNOS protein-protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within the lumbar spinal cord of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95-nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95-nNOS protein-protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.