Ubiquitinome Analysis Uncovers Alterations in Synaptic Proteins and Glucose Metabolism Enzymes in the Hippocampi of Adolescent Mice Following Cold Exposure.

Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 (Ndufv1) and Ndufv2. In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

Effects of Mild Closed-Head Injury and Subanesthetic Ketamine Infusion on Microglia, Axonal Injury, and Synaptic Density in Sprague-Dawley Rats.

Mild traumatic brain injury (mTBI) affects millions of people in the U.S. Approximately 20-30% of those individuals develop adverse symptoms lasting at least 3 months. In a rat mTBI study, the closed-head impact model of engineered rotational acceleration (CHIMERA) produced significant axonal injury in the optic tract (OT), indicating white-matter damage. Because retinal ganglion cells project to the lateral geniculate nucleus (LGN) in the thalamus through the OT, we hypothesized that synaptic density may be reduced in the LGN of rats following CHIMERA injury. A modified SEQUIN (synaptic evaluation and quantification by imaging nanostructure) method, combined with immunofluorescent double-labeling of pre-synaptic (synapsin) and post-synaptic (PSD-95) markers, was used to quantify synaptic density in the LGN. Microglial activation at the CHIMERA injury site was determined using Iba-1 immunohistochemistry. Additionally, the effects of ketamine, a potential neuroprotective drug, were evaluated in CHIMERA-induced mTBI. A single-session repetitive (ssr-) CHIMERA (3 impacts, 1.5 joule/impact) produced mild effects on microglial activation at the injury site, which was significantly enhanced by post-injury intravenous ketamine (10 mg/kg) infusion. However, ssr-CHIMERA did not alter synaptic density in the LGN, although ketamine produced a trend of reduction in synaptic density at post-injury day 4. Further research is necessary to characterize the effects of ssr-CHIMERA and subanesthetic doses of intravenous ketamine on different brain regions and multiple time points post-injury. The current study demonstrates the utility of the ssr-CHIMERA as a rodent model of mTBI, which researchers can use to identify biological mechanisms of mTBI and to develop improved treatment strategies for individuals suffering from head trauma.

Changes in SLITRK1 Level in the Amygdala Mediate Chronic Neuropathic Pain-Induced Anxio-Depressive Behaviors in Mice.

BACKGROUND: Comorbid chronic neuropathic pain (NPP) and anxio-depressive disorders (ADD) have become a serious global public-health problem. The SLIT and NTRK-like 1 (SLITRK1) protein is important for synaptic remodeling and is highly expressed in the amygdala, an important brain region involved in various emotional behaviors. We examined whether SLITRK1 protein in the amygdala participates in NPP and comorbid ADD. METHODS: A chronic NPP mouse model was constructed by L5 spinal nerve ligation; changes in chronic pain and ADD-like behaviors were measured in behavioral tests. Changes in SLITRK1 protein and excitatory synaptic functional proteins in the amygdala were measured by immunofluorescence and Western blot. Adeno-associated virus was transfected into excitatory synaptic neurons in the amygdala to up-regulate the expression of SLITRK1. RESULTS: Chronic NPP-related ADD-like behavior was successfully produced in mice by L5 ligation. We found that chronic NPP and related ADD decreased amygdalar expression of SLITRK1 and proteins important for excitatory synaptic function, including Homer1, postsynaptic density protein 95 (PSD95), and synaptophysin. Virally-mediated SLITRK1 overexpression in the amygdala produced a significant easing of chronic NPP and ADD, and restored the expression levels of Homer1, PSD95, and synaptophysin. CONCLUSION: Our findings indicated that SLITRK1 in the amygdala plays an important role in chronic pain and related ADD, and may prove to be a potential therapeutic target for chronic NPP-ADD comorbidity.

Increased glutamatergic neurotransmission between the retinohypothalamic tract and the suprachiasmatic nucleus of old mice.

Circadian rhythms synchronize to light through the retinohypothalamic tract (RHT), which is a bundle of axons coming from melanopsin retinal ganglion cells, whose synaptic terminals release glutamate to the ventral suprachiasmatic nucleus (SCN). Activation of AMPA-kainate and NMDA postsynaptic receptors elicits the increase in intracellular calcium required for triggering the signaling cascade that ends in phase shifts. During aging, there is a decline in the synchronization of circadian rhythms to light. With electrophysiological (whole-cell patch-clamp) and immunohistochemical assays, in this work, we studied pre- and postsynaptic properties between the RHT and ventral SCN neurons in young adult (P90-120) and old (P540-650) C57BL/6J mice. Incremental stimulation intensities (applied on the optic chiasm) induced much lesser AMPA-kainate postsynaptic responses in old animals, implying a lower recruitment of RHT fibers. Conversely, a higher proportion of old SCN neurons exhibited synaptic facilitation, and variance-mean analysis indicated an increase in the probability of release in RHT terminals. Moreover, both spontaneous and miniature postsynaptic events displayed larger amplitudes in neurons from aged mice, whereas analysis of the NMDA and AMPA-kainate components (evoked by RHT electrical stimulation) disclosed no difference between the two ages studied. Immunohistochemistry revealed a bigger size in the puncta of vGluT2, GluN2B, and GluN2A of elderly animals, and the number of immunopositive particles was increased, but that of PSD-95 was reduced. All these synaptic adaptations could be part of compensatory mechanisms in the glutamatergic signaling to ameliorate the loss of RHT terminals in old animals.

Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing.

Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here, we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining, and whole-cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered 3 d after slices were released from 5 d of deprivation, they were less able to recover after 10 d of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require activation of Hebbian plasticity. In single-neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39 and 56% following 5 and 10 d of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55 and 73%, respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.

High-frequency rTMS alleviates cognitive impairment and regulates synaptic plasticity in the hippocampus of rats with cerebral ischemia.

Poststroke cognitive impairment (PSCI) is a common complication of stroke, but effective treatments are currently lacking. Repetitive transcranial magnetic stimulation (rTMS) is gradually being applied to treat PSCI, but there is limited evidence of its efficacy. To determine rTMS effects on PSCI, we constructed a transient middle cerebral artery occlusion (tMCAO) rat model. Rats were then grouped by random digital table method: the sham group (n = 10), tMCAO group (n = 10) and rTMS group (n = 10). The shuttle box and Morris water maze (MWM) tests were conducted to detect the cognitive functions of the rats. In addition, synaptic density and synaptic ultrastructural parameters, including the active zone length, synaptic cleft width, and postsynaptic density (PSD) thickness, were quantified and analyzed using an electron microscope. What's more, synaptic associated proteins, including PSD95, SYN, and BDNF were detected by western blot. According to the shuttle box and MWM tests, rTMS improved tMCAO rats' cognitive functions, including spatial learning and memory and decision-making abilities. Electron microscopy revealed that rTMS significantly increased the synaptic density, synaptic active zone length and PSD thickness and decreased the synaptic cleft width. The western blot results showed that the expression of PSD95, SYN, and BDNF was markedly increased after rTMS stimulation. Based on these results, we propose that 20 Hz rTMS can significantly alleviate cognitive impairment after stroke. The underlying mechanism might be modulating the synaptic plasticity and up-regulating the expression PSD95, SYN, and BDNF in the hippocampus.

Targeting the postsynaptic scaffolding protein PSD-95 enhances BDNF signaling to mitigate depression-like behaviors in mice.

Signaling mediated by brain-derived neurotrophic factor (BDNF), which is supported by the postsynaptic scaffolding protein PSD-95, has antidepressant effects. Conversely, clinical depression is associated with reduced BDNF signaling. We found that peptidomimetic compounds that bind to PSD-95 promoted signaling by the BDNF receptor TrkB in the hippocampus and reduced depression-like behaviors in mice. The compounds CN2097 and Syn3 both bind to the PDZ3 domain of PSD-95, and Syn3 also binds to an α-helical region of the protein. Syn3 reduced depression-like behaviors in two mouse models of stress-induced depression; CN2097 had similar but less potent effects. In hippocampal neurons, application of Syn3 enhanced the formation of TrkB-Gαi1/3-PSD-95 complexes and potentiated downstream PI3K-Akt-mTOR signaling. In mice subjected to chronic mild stress (CMS), systemic administration of Syn3 reversed the CMS-induced, depression-associated changes in PI3K-Akt-mTOR signaling, dendrite complexity, spine density, and autophagy in the hippocampus and reduced depression-like behaviors. Knocking out Gαi1/3 in hippocampal neurons prevented the therapeutic effects of Syn3, indicating dependence of these effects on the TrkB pathway. The findings suggest that compounds that induce the formation of PSD-95-TrkB complexes have therapeutic potential to alleviate depression.

Differential nanoscale organization of excitatory synapses onto excitatory vs. inhibitory neurons.

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters (NCs) whose precise alignment across the cleft in a transsynaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses-those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses, presynaptic Munc13-1 and postsynaptic PSD-95 both form NCs that demonstrate alignment, underscoring synaptic nanostructure and the transsynaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell was also represented in Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of transsynaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses.

Bergamot essential oil improves CUMS-induced depression-like behaviour in rats by protecting the plasticity of hippocampal neurons.

Bergamot essential oil (BEO) is an extract of the bergamot fruit with significant neuroprotective effect. This study was to investigate the effects and the underlying mechanism of BEO in mitigating depression. GC-MS were used to identify its constituents. Antidepressive properties of BEO were evaluated by sucrose preference test (SPT), force swimming test (FST) and open field test (OFT). Nissl staining was used to determine the number of Nissl bodies in hippocampus (HIPP) of rats. Changes in HIPP dendritic length and dendritic spine density were detected by Golgi-Cox staining. Immunohistochemistry and Western blot were used to detect the postsynaptic density protein-95 (PSD-95) and synaptophysin (SYP) in the HIPP of rats. The enzyme-linked immunosorbent assay was used to determine the 5-hydroxytryptamine (5-HT), insulin-like growth factor 1 (IGF-1) and interleukin-1ß (IL-1ß) in the HIPP, serum and cerebrospinal fluid (CSF) of rats. Inhaled BEO significantly improved depressive behaviour in chronic unpredictable mild stress (CUMS) rats. BEO increased Nissl bodies, dendritic length and spine density, PSD-95 and SYP protein in the HIPP. Additionally, BEO upregulated serum 5-HT, serum and CSF IGF-1, while downregulating serum IL-1ß. Collectively, inhaled BEO mitigates depression by protecting the plasticity of hippocampal neurons, hence, providing novel insights into treatment of depression.

Tanshinone IIA Regulates Synaptic Plasticity in Mg2+-Free-Induced Epileptic Hippocampal Neurons via the PI3K/Akt Signaling Pathway.

BACKGROUND: Tanshinone IIA (TSIIA) is an element of the effective ingredients of Salvia miltiorrhiza Bunge (Labiatae), exhibits a significant therapeutic effect in brain neuroprotection. The focus of this study was the examination of synaptic plasticity of in Mg2+-free-induced epileptic hippocampus neurons and how TSIIA protects against it. METHODS: The purity of the primary hippocampal neurons extracted from Sprague Dawley rats was assessed within 24 hours by microtubule-associated protein (MAP2) immunofluorescence staining. A hippocampal neuron model for Mg2+-free-induced spontaneous recurrent epileptiform discharge was developed, five experimental groups were then randomized: blank (Blank), model (Model), TSIIA (TSIIA, 20 µM), LY294002 (LY294002, 25 µM), and TSIIA+LY294002 (TSIIA+LY294002, 20 µM+25 µM). FIJI software was used to examine variations of neurite complexity, total length of hippocampal neurons, number of primary dendrites and density of dendritic spines. Developmental regulation brain protein (Drebrin) and brain-derived neurotrophic factor (BDNF) expression was evaluated using immunofluorescence staining and the relative expression of phospho-protein kinase B (p-Akt)/Akt, BDNF, synaptophysin (SYN) and postsynaptic density 95 (PSD-95) determined by Western blot. RESULTS: In contrast to the model group, TSIIA drastically reduced damage to synaptic plasticity of hippocampal neurons caused by epilepsy (p < 0.05). The TSIIA group showed a significant increase in the relative expression of PSD-95, SYN, BDNF, and p-Akt/Akt (p < 0.01). CONCLUSIONS: TSIIA was effective in reducing harm to the synaptic plasticity of hippocampal neurons induced by persistent status epilepticus, with the possible mechanism being regulation of the phosphatidylinositol 3-kinase 56 (PI3K)/Akt signaling pathway.

Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases.

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

Targeting postsynaptic glutamate receptor scaffolding proteins PSD-95 and PICK1 for obesity treatment.

Human genome-wide association studies (GWAS) suggest a functional role for central glutamate receptor signaling and plasticity in body weight regulation. Here, we use UK Biobank GWAS summary statistics of body mass index (BMI) and body fat percentage (BF%) to identify genes encoding proteins known to interact with postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors. Loci in/near discs large homolog 4 (DLG4) and protein interacting with C kinase 1 (PICK1) reached genome-wide significance (P < 5 × 10-8) for BF% and/or BMI. To further evaluate the functional role of postsynaptic density protein-95 (PSD-95; gene name: DLG4) and PICK1 in energy homeostasis, we used dimeric PSD-95/disc large/ZO-1 (PDZ) domain-targeting peptides of PSD-95 and PICK1 to demonstrate that pharmacological inhibition of PSD-95 and PICK1 induces prolonged weight-lowering effects in obese mice. Collectively, these data demonstrate that the glutamate receptor scaffolding proteins, PICK1 and PSD-95, are genetically linked to obesity and that pharmacological targeting of their PDZ domains represents a promising therapeutic avenue for sustained weight loss.

FAM81A is a postsynaptic protein that regulates the condensation of postsynaptic proteins via liquid-liquid phase separation.

Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.

Cisplatin induces BDNF downregulation in middle-aged female rat model while BDNF enhancement attenuates cisplatin neurotoxicity.

Cancer-related cognitive impairments (CRCI) are neurological complications associated with cancer treatment, and greatly affect cancer survivors' quality of life. Brain-derived neurotrophic factor (BDNF) plays an essential role in neurogenesis, learning and memory. The reduction of BDNF is associated with the decrease in cognitive function in various neurological disorders. Few pre-clinical studies have reported on the effects of chemotherapy and medical stress on BDNF levels and cognition. The present study aimed to compare the effects of medical stress and cisplatin on serum BDNF levels and cognitive function in 9-month-old female Sprague Dawley rats to age-matched controls. Serum BDNF levels were collected longitudinally during cisplatin treatment, and cognitive function was assessed by novel object recognition (NOR) 14 weeks post-cisplatin initiation. Terminal BDNF levels were collected 24 weeks after cisplatin initiation. In cultured hippocampal neurons, we screened three neuroprotective agents, riluzole (an approved treatment for amyotrophic lateral sclerosis), as well as the ampakines CX546 and CX1739. We assessed dendritic arborization by Sholl analysis and dendritic spine density by quantifying postsynaptic density-95 (PSD-95) puncta. Cisplatin and exposure to medical stress reduced serum BDNF levels and impaired object discrimination in NOR compared to age-matched controls. Pharmacological BDNF augmentation protected neurons against cisplatin-induced reductions in dendritic branching and PSD-95. Ampakines (CX546 and CX1739) and riluzole did not affect the antitumor efficacy of cisplatin in vitro. In conclusion, we established the first middle-aged rat model of cisplatin-induced CRCI, assessing the contribution of medical stress and longitudinal changes in BDNF levels on cognitive function, although future studies are warranted to assess the efficacy of BDNF enhancement in vivo on synaptic plasticity. Collectively, our results indicate that cancer treatment exerts long-lasting changes in BDNF levels, and support BDNF enhancement as a potential preventative approach to target CRCI with therapeutics that are FDA approved and/or in clinical study for other indications.

Gens PSD-95 and GSK-3ß expression improved by hair follicular stem cells-conditioned medium enhances synaptic transmission and cognitive abilities in the rat model of vascular dementia.

INTRODUCTION: Vascular dementia (VaD) is a common type of dementia. The aim of this study was to investigate the cellular and molecular mechanism of conditioned medium (CM) in VaD. MATERIAL AND METHODS: The rats were divided into four groups of control (n = 9), sham-operation (n = 10), VaD with vehicle (n = 9), and VaD with CM (n = 12) that received CM on days 4, 14, and 24 after 2VO. Before sacrificing the rats, cognitive performance was assessed through the open-field (OP), passive-avoidance, and Morris-water maze. The field-potential recording was used to investigate basal synaptic transmission (BST) and long-term potentiation (LTP). Subsequently, the hippocampus was dissected, and real-time PCR was used to quantify the expression levels of ß1-catenin, insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-ß), glycogen synthase kinase-3ß (GSK-3ß), postsynaptic density protein 95 (PSD-95), and NR2B genes. RESULTS: The results indicated impaired performance in behavioral tests in 2VO rats, coupled with reductions in BST and LTP induction. The expression levels of ß1-catenin, IGF-1, PSD-95, and TGF-ß genes decreased, whereas NR2B and GSK-3ß expression increased. Treatment with CM restores the expression of PSD-95 and GSK-3ß as well as fear-memory, spatial learning, and grooming number without a positive effect on memory retrieval, time spent on the periphery and center of OP. The BST recovered upon administration of CM but, the LTP induction was still impaired. CONCLUSION: The recovery of BST in VaD rats appears to be the most important outcome of this study which is caused by the improvement of gene expression and leads to the restoration of fear memory.

Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Structure-based design of peptidomimetic inhibitors of PSD-95 with improved affinity for the PDZ3 domain.

Aberrant brain-derived neurotrophic factor (BDNF) signaling has been proposed to contribute to the pathophysiology of depression and other neurological disorders such as Angelman syndrome. We have previously shown that targeting the tropomyosin receptor kinase B/postsynaptic density protein-95 (PSD-95) nexus in the BDNF signaling pathway by peptidomimetic inhibitors is a promising approach for therapeutic intervention. Here, we used structure-based knowledge to develop a new Syn3 peptidomimetic compound series that fuses peptides derived from the PSD-95-binding protein SynGAP to our prototype compound CN2097. The new compounds target the PSD-95 PDZ3 domain and adjoining αC helix to achieve bivalent binding that results in up to 7-fold stronger affinity compared to CN2097. These compounds were designed to improve CN2097 specificity for the PSD-95 PDZ3 domain, and structure-activity relationship studies were performed to improve their resistance to proteolysis.

PSD-95 inhibitor Tat-NR2B9c (NA-1) protects the integrity of the blood-brain barrier after transient middle artery occlusion in rats by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

BACKGROUND: Protection against ischemic stroke may be most effective when multiple components of the neurovascular unit are protected, yet current treatments target mainly neurons. Here we explored whether the PSD-95 inhibitor Tat-NR2B9c (NA-1) can protect not only neurons but also the blood-brain barrier. METHODS: Adult male Sprague-Dawley rats were randomly divided into three groups, which were subjected to either sham surgery or transient cerebral ischemia-reperfusion, after which some animals were treated with Tat-NR2B9c. The therapeutic efficacy of Tat-NR2B9c was assessed in terms of the degree of neurological deficit and cerebral infarction, integrity of the blood-brain barrier, cerebral water content, as well as expression of PSD-95, nitric oxide synthase, and matrix metalloprotease-9. RESULTS: Tat-NR2B9c (NA-1) ameliorated neurofunctional deficit, reduced cerebral infarction, mitigated blood-brain barrier injury and improved its integrity following ischemia-reperfusion, leading to less cerebral edema. These improvements were associated with upregulation of tight junction proteins in the blood-brain barrier. At the same time, Tat-NR2B9c (NA-1) downregulated neuronal nitric oxide synthase and matrix metalloprotease-9, while reversing the ischemia-induced downregulation of endothelial nitric oxide synthase in brain. We report here the first evidence that PSD-95 is expressed in vascular endothelial cells in the brain. CONCLUSION: Our experiments in a rat model of transient occlusion of the middle cerebral artery suggest that Tat-NR2B9c (NA-1) can mitigate ischemic injury to the blood-brain barrier, and that it may do so by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

Developmental epileptic encephalopathy in DLG4-related synaptopathy.

OBJECTIVE: The postsynaptic density protein of excitatory neurons PSD-95 is encoded by discs large MAGUK scaffold protein 4 (DLG4), de novo pathogenic variants of which lead to DLG4-related synaptopathy. The major clinical features are developmental delay, intellectual disability (ID), hypotonia, sleep disturbances, movement disorders, and epilepsy. Even though epilepsy is present in 50% of the individuals, it has not been investigated in detail. We describe here the phenotypic spectrum of epilepsy and associated comorbidities in patients with DLG4-related synaptopathy. METHODS: We included 35 individuals with a DLG4 variant and epilepsy as part of a multicenter study. The DLG4 variants were detected by the referring laboratories. The degree of ID, hypotonia, developmental delay, and motor disturbances were evaluated by the referring clinician. Data on awake and sleep electroencephalography (EEG) and/or video-polygraphy and brain magnetic resonance imaging were collected. Antiseizure medication response was retrospectively assessed by the referring clinician. RESULTS: A large variety of seizure types was reported, although focal seizures were the most common. Encephalopathy related to status epilepticus during slow-wave sleep (ESES)/developmental epileptic encephalopathy with spike-wave activation during sleep (DEE-SWAS) was diagnosed in >25% of the individuals. All but one individual presented with neurodevelopmental delay. Regression in verbal and/or motor domains was observed in all individuals who suffered from ESES/DEE-SWAS, as well as some who did not. We could not identify a clear genotype-phenotype relationship even between individuals with the same DLG4 variants. SIGNIFICANCE: Our study shows that a subgroup of individuals with DLG4-related synaptopathy have DEE, and approximately one fourth of them have ESES/DEE-SWAS. Our study confirms DEE as part of the DLG4-related phenotypic spectrum. Occurrence of ESES/DEE-SWAS in DLG4-related synaptopathy requires proper investigation with sleep EEG.

A deep intronic DLG4 variant resulting in DLG4-related synaptopathy.

The rare autosomal dominant brain disorder DLG4-related synaptopathy is caused by de novo variants in DLG4 (encoding PSD-95), the majority of which are predicted to be protein-truncating. In addition to splice site variants, a number of synonymous and missense DLG4 variants are predicted to exert their effect through altered RNA splicing, although the pathogenicity of these variants is uncertain without functional RNA studies. Here, we describe a young boy with a deep intronic DLG4 variant (c.2105+235C>T) identified using whole genome sequencing. By using reverse-transcription PCR on RNA derived from peripheral blood, we demonstrate that DLG4 mRNA expression is detectable in blood and the deep intronic variant gives rise to two alternative DLG4 transcripts, one of which includes a pseudoexon. Both alternative transcripts are out-of-frame and predicted to result in protein-truncation, thereby establishing the genetic diagnosis for the proband. This adds to the evidence concerning the pathogenic potential of deep intronic variants and underlines the importance of functional studies, even in cases where reported tissue-specific gene expression might suggest otherwise.