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.

The Role of NMDAR and BDNF in Cognitive Dysfunction Induced by Different Microwave Radiation Conditions in Rats

Background: To investigate the effects of different levels of microwave radiation on learning and memory in Wistar rats and explore the underlying mechanisms of N-methyl-D-aspartate receptor (NMDAR/NR) and Brain-derived neurotropic factor (BDNF); Methods: A total of 140 Wistar rats were exposed to microwave radiation levels of 0, 10, 30 or 50 mW/cm2 for 6 min. Morris Water Maze Test, high-performance liquid chromatography, Transmission Electron Microscope and Western blotting were used; Results: The 30 and 50 mW/cm2 groups exhibited longer average escape latencies and fewer platform crossings than the 0 mW/cm2 group from 6 h to 3 d after microwave radiation. Alterations in the amino acid neurotransmitters of the hippocampi were shown at 6 h, 3 d and 7 d after exposure to 10, 30 or 50 mW/cm2 microwave radiation. The length and width of the Postsynaptic density were increased. The expression of NR1, NR2A and NR2B increased from day 1 to day 7; Postsynaptic density protein-95 and cortactin expression increased from day 3 to day 7; BDNF and Tyrosine kinase receptor B (TrkB) expression increased between 6 h and 1 d after 30 mW/cm2 microwave radiation exposure, but they decreased after 50mW/cm2 exposure. Conclusions: Microwave exposure (30 or 50 mW/cm2, for 6 min) may cause abnormalities in neurotransmitter release and synaptic structures, resulting in impaired learning and memory; BDNF and NMDAR-related signaling molecules might contribute differently to these alterations.

Pengaruh Pajanan Auditorik Kombinasi Gelombang Monoton dan Ritmis terhadap Ekspresi Postsynapticdensity-95 Hipokampus Neonatus Ayam

Ibu hamil yang terpapar bising akan memengaruhi janin yang dikandungnya bahkan memiliki efek negatif terhadap neurogenesis. Suara dari gelombang ritmis (musik) dapat meningkatkan neurogenesis yang berpengaruh langsung terhadap fungsi memori. Penelitian ini bertujuan untuk mengetahui efek positif gelombang ritmis untuk meredam atau menjadi terapi bagi bagian otak yang sebelumnya mendapat efek negatif gelombang monoton, dilihat dari peningkatan ekspresi densitas optik protein postsynapticdensity-95 dan waktu uji labirin T. Penelitian eksperimental ini dilakukan di laboratorium Developmental Biologi dan Patologi Anatomik Fakultas Kedokteran Universitas Indonesia pada bulan September 2018. Penelitian menggunakan 24 telur ayam yang difertilisasi dan dibagi empat kelompok: kelompok suara ritmis, monoton, gabungan (kombinasi suara monoton dan ritmis), dan kontrol. Telur yang sudah difertilisasi diinkubasi selama 21 hari dan dimiringkan secara otomatis tiap 4 jam. Dilakukan proses candling hari ke-3, ke-5, ke-10 dan ke-15 untuk menilai perkembangan embrio ayam. Hasil penelitian ini, terdapat perbedaan bermakna densitas optik protein postsynapticdensity 95 dan waktu uji labirin T antara kelompok gabungan dengan kelompok ritmis dan monoton, namun tidak terjadi perbedaan bermakna dengan kelompok kontrol. Gelombang ritmis dapat mengurangi efek buruk gelombang monoton dan mengembalikannya menjadi sama dengan normal (kelompok kontrol). Kata kunci: ritmis, monoton, kombinasi suara, memori, postsynapticdensity-95. Effect of Auditory Exposure combination of Monotonous and Rhythmic Waves on Postsynapticdensity-95 Expression on Neonatal Hippocampus Abstract Pregnant women who are exposed to noise will affect the fetus they contain and even have a negative effect on neurogenesis. Sound from rhythmic waves (music) can increase neurogenesis which directly affects memory function. This study aims to determine the positive effect of rhythmic waves to dampen or become therapy for parts of the brain that previously received the negative effects of monotonous waves, seen from the increase in the expression of the optical density of the postsynapticdensity-95 protein and the labyrint T test time. This experimental study was conducted in the Developmental Biology laboratory and Anatomic Pathology, Faculty of Medicine, University of Indonesia in September 2018. The study used 24 fertilized chicken eggs and divided into four groups: rhythmic, monotonous, combined (a combination of monotonous and rhythmic sounds), and control. Fertilized eggs are incubated for 21 days and automatically tilted every 4 hours. The candling process was carried out on the 3rd, 5th, 10th and 15th days to assess the development of chicken embryos. The results of this study, there was a significant difference in the optical density of the postsynaptic density protein 95 and the time of the T labyrint test between the combined group and the rhythmic and monotonous group, but there was no significant difference with the control group. Rhythmic waves can reduce the bad effects of monotonous waves and return them to normal (control group). Keywords: rhythmic, monotone, sound combination, memory, postsynaptic density-95.

Non-microtubule tubulin-based backbone and subordinate components of postsynaptic density lattices

A purification protocol was developed to identify and analyze the component proteins of a postsynaptic density (PSD) lattice, a core structure of the PSD of excitatory synapses in the central nervous system. “Enriched”- and “lean”-type PSD lattices were purified by synaptic plasma membrane treatment to identify the protein components by comprehensive shotgun mass spectrometry and group them into minimum essential cytoskeleton (MEC) and non-MEC components. Tubulin was found to be a major component of the MEC, with non-microtubule tubulin widely distributed on the purified PSD lattice. The presence of tubulin in and around PSDs was verified by post-embedding immunogold labeling EM of cerebral cortex. Non-MEC proteins included various typical scaffold/adaptor PSD proteins and other class PSD proteins. Thus, this study provides a new PSD lattice model consisting of non-microtubule tubulin-based backbone and various non-MEC proteins. Our findings suggest that tubulin is a key component constructing the backbone and that the associated components are essential for the versatile functions of the PSD.

Chemical shift assignments of the N-terminal domain of PSD95 (PSD95-NT)

Postsynaptic density protein-95 (PSD95) contributes to the postsynaptic architecture of neuronal synapses and plays an important role in controlling synaptic plasticity. The N-terminal domain of PSD95 (residues 1–71, called PSD95-NT) interacts with target proteins (calmodulin, α-actinin-1 and CDKL5), which regulate the Ca2+-dependent degradation of glutamate receptors. We report complete backbone NMR chemical shift assignments of PSD95-NT (BMRB No. 50752).

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The relevance of missense mutations for these pathologies is unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (i) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (ii) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.

Hippocampal overexpression of Nos1ap promotes endophenotypes related to mental disorders

Nitric oxide synthase 1 adaptor protein (NOS1AP; previously named CAPON) is linked to the glutamatergic postsynaptic density through interaction with neuronal nitric oxide synthase (nNOS). NOS1AP and its interaction with nNOS have been associated with several mental disorders. Despite the high levels of NOS1AP expression in the hippocampus and the relevance of this brain region in glutamatergic signaling as well as mental disorders, a potential role of hippocampal NOS1AP in the pathophysiology of these disorders has not been investigated yet. To uncover the function of NOS1AP in hippocampus, we made use of recombinant adeno-associated viruses to overexpress the murine Nos1ap isoform in the hippocampus of mice. We investigated these mice for changes in gene expression, electrophysiology, neuronal morphology, and relevant behavioral phenotypes. We found that overexpression of hippocampal Nos1ap markedly increased the interaction of nNOS with PSD-95, reduced dendritic spine density and changed dendritic spine morphology without affecting basic synaptic signaling properties at CA1 synapses. At the behavioral level, we observed an impairment in social interaction and social memory, as well as decreased spatial working memory capacity. Our data provide a mechanistic explanation for a highly selective and specific contribution of hippocampal NOS1AP and its interaction with the glutamatergic postsynaptic density to cross-disorder pathophysiology. Our findings allude to therapeutic relevance due to the druggability of this molecule.