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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.

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

The critical role of AMPA receptor (AMPAR) trafficking in long-term potentiation (LTP) of excitatory synaptic transmission is now well established, but the underlying molecular mechanism is still uncertain. Recent research suggests that PSD-95 captures AMPARs via an interaction with the AMPAR auxiliary subunits-transmembrane AMPAR regulatory proteins (TARPs). To determine if such interaction is a core minimal component of the AMPAR trafficking and LTP mechanism, we engineered artificial binding partners, which individually were biochemically and functionally dead but which, when expressed together, rescue binding and both basal synaptic transmission and LTP. These findings establish the TARP/PSD-95 complex as an essential interaction underlying AMPAR trafficking and LTP.

Chemical synaptic transmission represents the most sophisticated dynamic process and is highly regulated with optimized neurotransmitter balance. Imbalanced transmitters can lead to transmission impairments, for example, intracellular zinc accumulation is a hallmark of degenerating neurons. However, the underlying mechanisms remain elusive. Postsynaptic density protein-95 (PSD-95) is a primary postsynaptic membrane-associated protein and the major scaffolding component in the excitatory postsynaptic densities, which performs substantial functions in synaptic development and maturation. Its membrane association induced by palmitoylation contributes largely to its regulatory functions at postsynaptic sites. Unlike other structural domains in PSD-95, the N-terminal region (PSD-95NT) is flexible and interacts with various targets, which modulates its palmitoylation of two cysteines (C3/C5) and glutamate receptor distributions in postsynaptic densities. PSD-95NT contains a putative zinc-binding motif (C2H2) with undiscovered functions. This study is the first effort to investigate the interaction between Zn2+ and PSD-95NT. The NMR titration of 15 N-labeled PSD-95NT by ZnCl2 was performed and demonstrated Zn2+ binds to PSD-95NT with a binding affinity (Kd ) in the micromolar range. The zinc binding was confirmed by fluorescence and mutagenesis assays, indicating two cysteines and two histidines (H24, H28) are critical residues for the binding. These results suggested the concentration-dependent zinc binding is likely to influence PSD-95 palmitoylation since the binding site overlaps the palmitoylation sites, which was verified by the mimic PSD-95 palmitoyl modification and intact cell palmitoylation assays. This study reveals zinc as a novel modulator for PSD-95 postsynaptic membrane association by chelating its N-terminal region, indicative of its importance in postsynaptic signaling.

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

Beta-amyloid (Aβ) 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 Aβ on synapses. The protective effect persists in tissue lacking the AMPA receptor subunit GluA1, which prevents the confounding synaptic potentiation by increased PSD-95. Aβ 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 Aβ-induced effects on the NMDAR CTD conformation, its interaction with PP1, and synaptic weakening. Our results indicate that increased PSD-95 protects synapses from Aβ toxicity, suggesting that low levels of synaptic PSD-95 may be a molecular sign indicating synapse vulnerability to Aβ. Importantly, pharmacological inhibition of its depalmitoylation increases PSD-95 at synapses and rescues deficits caused by Aβ, possibly opening a therapeutic avenue against Alzheimer's disease.

Synaptic disturbances in excitatory to inhibitory (E/I) balance in forebrain circuits are thought to contribute to the progression of Alzheimer's disease (AD) and dementia, although direct evidence for such imbalance in humans is lacking. We assessed anatomical and electrophysiological synaptic E/I ratios in post-mortem parietal cortex samples from middle-aged individuals with AD (early-onset) or Down syndrome (DS) by fluorescence deconvolution tomography and microtransplantation of synaptic membranes. Both approaches revealed significantly elevated E/I ratios for AD, but not DS, versus controls. Gene expression studies in an independent AD cohort also demonstrated elevated E/I ratios in individuals with AD as compared to controls. These findings provide evidence of a marked pro-excitatory perturbation of synaptic E/I balance in AD parietal cortex, a region within the default mode network that is overly active in the disorder, and support the hypothesis that E/I imbalances disrupt cognition-related shifts in cortical activity which contribute to the intellectual decline in AD.

We recently demonstrated that NMDA receptors (NMDARs) are capable of ion-flux independent signaling through conformational change in the NMDAR intracellular domain resulting in long-term depression of synaptic transmission (LTD). Here we show that PSD-95 overexpression blocks agonist induced conformational movement in the NMDAR intracellular domain as well as LTD that is NMDAR-dependent and ion-flux independent. Interestingly, previous studies indicate that overexpressed PSD-95 does not block NMDAR-dependent LTD. These data support a model where ion-flux independent LTD is predominant in young animals, which have synapses with low amounts of PSD-95, whereas only ion flux dependent LTD occurs at more mature synapses, which have more PSD-95 that would block ion-flux independent LTD. These results may reconcile different findings regarding ion-flux independent LTD.

Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules. Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy. Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

The proposed deep space exploration to the moon and later to Mars will result in astronauts receiving significant chronic exposures to space radiation (SR). SR exposure results in multiple neurocognitive impairments. Recently, our cross-species (mouse/rat) studies reported impaired associative memory formation in both species following a chronic 6-month low dose exposure to a mixed field of neutrons (1 mGy/day for a total dose pf 18 cGy). In the present study, we report neutron exposure induced synaptic plasticity in the medial prefrontal cortex, accompanied by microglial activation and significant synaptic loss in the hippocampus. In a parallel study, neutron exposure was also found to alter fluorescence assisted single synaptosome LTP (FASS-LTP) in the hippocampus of rats, that may be related to a reduced ability to insert AMPAR into the post-synaptic membrane, which may arise from increased phosphorylation of the serine 845 residue of the GluA1 subunit. Thus, we demonstrate for the first time, that low dose chronic neutron irradiation impacts homeostatic synaptic plasticity in the hippocampal-cortical circuit in two rodent species, and that the ability to successfully encode associative recognition memory is a dynamic, multicircuit process, possibly involving compensatory changes in AMPAR density on the synaptic surface.

Astrocytes are implicated in synapse formation and elimination, which are associated with developmental refinements of neuronal circuits. Astrocyte dysfunctions are also linked to synapse pathologies associated with neurodevelopmental disorders and neurodegenerative diseases. Although several astrocyte-derived secreted factors are implicated in synaptogenesis, the role of contact-mediated glial-neuronal interactions in synapse formation and elimination during development is still unknown. In this study, we examined whether the loss or overexpression of the membrane-bound ephrin-B1 in astrocytes during postnatal day (P) 14-28 period would affect synapse formation and maturation in the developing hippocampus. We found enhanced excitation of CA1 pyramidal neurons in astrocyte-specific ephrin-B1 KO male mice, which coincided with a greater vGlut1/PSD95 colocalization, higher dendritic spine density, and enhanced evoked AMPAR and NMDAR EPSCs. In contrast, EPSCs were reduced in CA1 neurons neighboring ephrin-B1-overexpressing astrocytes. Overexpression of ephrin-B1 in astrocytes during P14-28 developmental period also facilitated evoked IPSCs in CA1 neurons, while evoked IPSCs and miniature IPSC amplitude were reduced following astrocytic ephrin-B1 loss. Lower numbers of parvalbumin-expressing cells and a reduction in the inhibitory VGAT/gephyrin-positive synaptic sites on CA1 neurons in the stratum pyramidale and stratum oriens layers of KO hippocampus may contribute to reduced inhibition and higher excitation. Finally, dysregulation of excitatory/inhibitory balance in KO male mice is most likely responsible for impaired sociability observed in these mice. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits.SIGNIFICANCE STATEMENT This report establishes a link between astrocytes and the development of excitatory and inhibitory balance in the mouse hippocampus during early postnatal development. We provide new evidence that astrocytic ephrin-B1 differentially regulates development of excitatory and inhibitory circuits in the hippocampus during early postnatal development using a multidisciplinary approach. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits and associated behaviors. Given widespread and growing interest in the astrocyte-mediated mechanisms that regulate synapse development, and the role of EphB receptors in neurodevelopmental disorders, these findings establish a foundation for future studies of astrocytes in clinically relevant conditions.

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses.

We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages-P0, P7, and P14-when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from ∼7300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynx1, Nrn1, Cntnap1 over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbp1b. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.

Similar to the pattern observed in people with substance abuse disorders, laboratory animals will exhibit escalation of cocaine intake when the drug is available over prolonged periods of time. Here, we investigated the contribution of behavioral contingency of cocaine administration on escalation of cocaine intake and gene expression in the dorsal medial prefrontal cortex (dmPFC) in adult male rats. Rats were allowed to self-administer intravenous cocaine (0.25 mg/infusion) under either limited cocaine-(1 h/day), prolonged cocaine-(6 h/day), or limited cocaine-(1 h/day) plus yoked cocaine-access (5 h/day); a control group received access to saline (1 h/day). One day after the final self-administration session, the rats were euthanized and the dmPFC was removed for quantification of mRNA expression of critical glutamatergic signaling genes, Homer2, Grin1, and Dlg4, as these genes and brain region have been previously implicated in addiction, learning, and memory. All groups with cocaine-access showed escalated cocaine intake during the first 10 min of each daily session, and within the first 1 h of cocaine administration. Additionally, the limited-access + yoked group exhibited more non-reinforced lever responses during self-administration sessions than the other groups tested. Lastly, Homer2, Grin1, and Dlg4 mRNA were impacted by both duration and mode of cocaine exposure. Only prolonged-access rats exhibited increases in mRNA expression for Homer2, Grin1, and Dlg4 mRNA. Taken together, these findings indicate that both contingent and non-contingent "excessive" cocaine exposure supports escalation behavior, but the behavioral contingency of cocaine-access has distinct effects on the patterning of operant responsiveness and changes in mRNA expression.

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.

Postsynaptic density protein-95 (PSD-95) localizes AMPA-type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca2+ influx, Ca2+/calmodulin (Ca2+/CaM) binding to the N-terminus of PSD-95 mediates postsynaptic loss of PSD-95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD-95 N-terminus as important for binding to Ca2+/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaMR126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca2+/CaM to PSD-95 induced by a chronic increase in Ca2+ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.

Humans routinely use past experience with complexity to deal with novel, challenging circumstances. This fundamental aspect of real-world behavior has received surprisingly little attention in animal studies, and the underlying brain mechanisms are unknown. The present experiments tested for transfer from past experience in rats and then used quantitative imaging to localize synaptic modifications in hippocampus. Six daily exposures to an enriched environment (EE) caused a marked enhancement of short- and long-term memory encoded during a 30-min session in a different and complex environment relative to rats given extensive handling or access to running wheels. Relatedly, the EE animals investigated the novel environment in a different manner than the other groups, suggesting transfer of exploration strategies acquired in earlier interactions with complexity. This effect was not associated with changes in the number or size of excitatory synapses in hippocampus. Maps of synapses expressing a marker for long-term potentiation indicated that encoding in the EE group, relative to control animals, was concentrated in hippocampal field CA1. Importantly,

Stress exposure can lead to post-traumatic stress disorder (PTSD) in male and female rats. Social-Single Prolonged Stress (SPS) protocol has been considered a potential PTSD model. This study aimed to pharmacologically validate the Social-SPS as a PTSD model in male and female rats. Male and female Wistar rats (60-day-old) were exposed to Social-SPS protocol and treated with fluoxetine (10 mg/Kg) or saline solution intraperitoneally 24 h before euthanasia. Two cohorts of animals were used; for cohort 1, male and female rats were still undisturbed until day 7 post-Social-SPS exposure, underwent locomotor and conditioned fear behaviors, and were euthanized on day 9. Animals of cohort 2 were subjected to the same protocol but were re-exposed to contextual fear behavior on day 14. Results showed that fluoxetine-treated rats gained less body weight than control and Social-SPS in both sexes. Social-SPS effectively increased the freezing time in male and female rats on day eight but not on day fourteen. Fluoxetine blocked the increase of freezing in male and female rats on day 8. Different mechanisms for fear behavior were observed in males, such as Social-SPS increased levels of glucocorticoid receptors and Beclin-1 in the amygdala. Social-SPS was shown to increase the levels of NMDA2A, GluR-1, PSD-95, and CAMKII in the amygdala of female rats. No alterations were observed in the amygdala of rats on day fourteen. The study revealed that Social-SPS is a potential PTSD protocol applicable to both male and female rats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abnormalities in dendritic spines are manifestations of several neurodevelopmental and psychiatric diseases. TAOK2 is one of the genes in the 16p11.2 locus, copy number variations of which are associated with autism and schizophrenia. Here, we show that the kinase activity of the serine/threonine kinase encoded by TAOK2 is required for spine maturation. TAOK2 depletion results in unstable dendritic protrusions, mislocalized shaft-synapses, and loss of compartmentalization of NMDA receptor-mediated calcium influx. Using chemical-genetics and mass spectrometry, we identified several TAOK2 phosphorylation targets. We show that TAOK2 directly phosphorylates the cytoskeletal GTPase Septin7, at an evolutionary conserved residue. This phosphorylation induces translocation of Septin7 to the spine, where it associates with and stabilizes the scaffolding protein PSD95, promoting dendritic spine maturation. This study provides a mechanistic basis for postsynaptic stability and compartmentalization via TAOK2-Sept7 signaling, with implications toward understanding the potential role of TAOK2 in neurological deficits associated with the 16p11.2 region.

PurposeMetabolic syndrome (MetS) is associated with several degenerative diseases, including retinal degeneration. Previously, we reported on progressive retinal degeneration in a spontaneous obese rat (WNIN/Ob) model. In this study, we investigated the additional effect of impaired glucose tolerance (IGT), an essential component of MetS, on retinal degeneration using the WNIN/GR-Ob rat model.MethodsThe retinal morphology and ultrastructure of WNIN/GR-Ob and age-matched littermate lean rats were studied by microscopy and immunohistochemistry. The retinal transcriptome of WNIN/GR-Ob was compared with the respective lean controls and with the WNIN/Ob model using microarray analysis. Expression of selected retinal marker genes was studied via real-time PCR.ResultsProgressive loss of photoreceptor cells was observed in WNIN/GR-Ob rats with an onset as early as 3 months. Similarly, thinning of the inner nuclear layer was observed from 6 months in these rats. Immunohistochemical analysis showed decreased levels of rhodopsin and postsynaptic density protein-95 (PSD-95) proteins and increased levels of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and calretinin in WNIN/GR-Ob rats compared with the age-matched lean controls, further supporting cellular stress/damage and retinal degeneration. The retinal transcriptome analysis indicated altered expression profiles in both the WNIN/GR-Ob and WNIN/Ob rat models compared to their respective lean controls; these pathways are associated with activation of pathways like cellular oxidative stress response, inflammation, apoptosis, and phototransduction, although the changes were more prominent in WNIN/GR-Ob than in WNIN/Ob animals.ConclusionsWNIN/GR-Ob rats with added glucose intolerance developed retinal degeneration similar to the parent line WNIN/Ob. The severity of retinal degeneration was greater in WNIN/GR-Ob rats compared to WNIN/Ob, suggesting a possible role for IGT in this model. Hence, the WNIN/GR-Ob model could be a valuable tool for investigating the impact of MetS on retinal degeneration pathology.

The Mars mission will result in an inevitable exposure to cosmic radiation that has been shown to cause cognitive impairments in rodent models, and possibly in astronauts engaged in deep space travel. Of particular concern is the potential for cosmic radiation exposure to compromise critical decision making during normal operations or under emergency conditions in deep space. Rodents exposed to cosmic radiation exhibit persistent hippocampal and cortical based performance decrements using six independent behavioral tasks administered between separate cohorts 12 and 24 weeks after irradiation. Radiation-induced impairments in spatial, episodic and recognition memory were temporally coincident with deficits in executive function and reduced rates of fear extinction and elevated anxiety. Irradiation caused significant reductions in dendritic complexity, spine density and altered spine morphology along medial prefrontal cortical neurons known to mediate neurotransmission interrogated by our behavioral tasks. Cosmic radiation also disrupted synaptic integrity and increased neuroinflammation that persisted more than 6 months after exposure. Behavioral deficits for individual animals correlated significantly with reduced spine density and increased synaptic puncta, providing quantitative measures of risk for developing cognitive impairment. Our data provide additional evidence that deep space travel poses a real and unique threat to the integrity of neural circuits in the brain.

Estradiol (E2) perfusion rapidly increases the strength of fast excitatory transmission and facilitates long-term potentiation in the hippocampus, two effects likely related to its memory-enhancing properties. Past studies showed that E2s facilitation of transmission involves activation of RhoA signaling leading to actin polymerization in dendritic spines. Here we report that brief exposure of adult male hippocampal slices to 1 nM E2 increases the percentage of postsynaptic densities associated with high levels of immunoreactivity for activated forms of the BDNF receptor TrkB and β1-integrins, two synaptic receptors that engage actin regulatory RhoA signaling. The effects of E2 on baseline synaptic responses were unaffected by pretreatment with the TrkB-Fc scavenger for extracellular BDNF or TrkB antagonism, but were eliminated by neutralizing antisera for β1-integrins. E2 effects on synaptic responses were also absent in conditional β1-integrin knockouts, and with inhibition of matrix metalloproteinases, extracellular enzymes that generate integrin ligands. We propose that E2, acting through estrogen receptor-β, transactivates synaptic TrkB and β1-integrin, and via mechanisms dependent on integrin activation and signaling, reversibly reorganizes the spine cytoskeleton and thereby enhances synaptic responses in adult hippocampus.

Modification of the strength of excitatory synaptic connections is a fundamental mechanism by which neural circuits are refined during development and learning. Synapse Differentiation Induced Gene 1 (SynDIG1) has been shown to play a key role in regulating synaptic strength in vitro. Here, we investigated the role of SynDIG1 in vivo in mice with a disruption of the SynDIG1 gene rather than use an alternate loxP-flanked conditional mutant that we find retains a partial protein product. The gene-trap insertion with a reporter cassette mutant mice shows that the SynDIG1 promoter is active during embryogenesis in the retina with some activity in the brain, and postnatally in the mouse hippocampus, cortex, hindbrain, and spinal cord. Ultrastructural analysis of the hippocampal CA1 region shows a decrease in the average PSD length of synapses and a decrease in the number of synapses with a mature phenotype. Intriguingly, the total synapse number appears to be increased in SynDIG1 mutant mice. Electrophysiological analyses show a decrease in AMPA and NMDA receptor function in SynDIG1-deficient hippocampal neurons. Glutamate stimulation of individual dendritic spines in hippocampal slices from SynDIG1-deficient mice reveals increased short-term structural plasticity. Notably, the overall levels of PSD-95 or glutamate receptors enriched in postsynaptic biochemical fractions remain unaltered; however, activity-dependent synapse development is strongly compromised upon the loss of SynDIG1, supporting its importance for excitatory synapse maturation. Together, these data are consistent with a model in which SynDIG1 regulates the maturation of excitatory synapse structure and function in the mouse hippocampus in vivo.

UnlabelledKey issues concerning ganglion cell type-specific loss and synaptic changes in animal models of experimental glaucoma remain highly debated. Importantly, changes in the structure and function of various RGC types that occur early, within 14 d after acute, transient intraocular pressure elevation, have not been previously assessed. Using biolistic transfection of individual RGCs and multielectrode array recordings to measure light responses in mice, we examined the effects of laser-induced ocular hypertension on the structure and function of a subset of RGCs. Among the α-like RGCs studied, αOFF-transient RGCs exhibited higher rates of cell death, with corresponding reductions in dendritic area, dendritic complexity, and synapse density. Functionally, OFF-transient RGCs displayed decreases in spontaneous activity and receptive field size. In contrast, neither αOFF-sustained nor αON-sustained RGCs displayed decreases in light responses, although they did exhibit a decrease in excitatory postsynaptic sites, suggesting that synapse loss may be one of the earliest signs of degeneration. Interestingly, presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer, corroborating the hypothesis that RGCs with dendrites stratifying in the OFF sublamina may be damaged early. Indeed, OFF arbors of ON-OFF RGCs lose complexity more rapidly than ON arbors. Our results reveal type-specific differences in RGC responses to injury with a selective vulnerability of αOFF-transient RGCs, and furthermore, an increased susceptibility of synapses in the OFF sublamina. The selective vulnerability of specific RGC types offers new avenues for the design of more sensitive functional tests and targeted neuroprotection.Significance statementConflicting reports regarding the selective vulnerability of specific retinal ganglion cell (RGC) types in glaucoma exist. We examine, for the first time, the effects of transient intraocular pressure elevation on the structure and function of various RGC types. Among the α-like RGCs studied, αOFF-transient RGCs are the most vulnerable to transient transient intraocular pressure elevation as measured by rates of cell death, morphologic alterations in dendrites and synapses, and physiological dysfunction. Specifically, we found that presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer. Our results suggest selective vulnerability both of specific types of RGCs and of specific inner plexiform layer sublaminae, opening new avenues for identifying novel diagnostic and treatment targets in glaucoma.

The majority of excitatory synapses in the brain exist on dendritic spines. Accordingly, the regulation of dendritic spine density in the hippocampus is thought to play a central role in learning and memory. The development of novel methods to control spine density could, therefore, have important implications for treatment of a host of neurodegenerative and developmental cognitive disorders. Herein, we report the design and evaluation of a new class of benzothiazole amphiphiles that exhibit a dose-dependent response leading to an increase in dendritic spine density in primary hippocampal neurons. Cell exposure studies reveal that the increase in spine density can persist for days in the presence of these compounds, but returns to normal spine density levels within 24 h when the compounds are removed, demonstrating the capability to reversibly control spinogenic activity. Time-lapse imaging of dissociated hippocampal neuronal cultures shows that these compounds promote a net increase in spine density through the formation of new spines. Biochemical studies support that promotion of spine formation by these compounds is accompanied by Ras activation. These spinogenic molecules were also capable of inhibiting a suspected mechanism for dendritic spine loss induced by Alzheimer-related aggregated amyloid-β peptides in primary neurons. Evaluation of this new group of spinogenic agents reveals that they also exhibit relatively low toxicity at concentrations displaying activity. Collectively, these results suggest that small molecules that promote spine formation could be potentially useful for ameliorating cognitive deficiencies associated with spine loss in neurodegenerative diseases such as Alzheimer disease, and may also find use as general cognitive enhancers.

The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate uncaging, two-photon Ca(2+) imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca(2+) transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction-selective Ca(2+) transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.

BackgroundThe molecular mechanisms underlying autistic behaviors remain to be elucidated. Mutations in genes linked to autism adversely affect molecules regulating dendritic spine formation, function and plasticity, and some increase the mammalian target of rapamycin, mTOR, a regulator of protein synthesis at spines. Here, we investigated whether the Akt/mTOR pathway is disrupted in idiopathic autism and in rats exposed to valproic acid, an animal model exhibiting autistic-like behavior.MethodsComponents of the mTOR pathway were assayed by Western blotting in postmortem fusiform gyrus samples from 11 subjects with idiopathic autism and 13 controls and in valproic acid versus saline-exposed rat neocortex. Additionally, protein levels of brain-derived neurotrophic factor receptor (TrkB) isoforms and the postsynaptic organizing molecule PSD-95 were measured in autistic versus control subjects.ResultsFull-length TrkB, PI3K, Akt, phosphorylated and total mTOR, p70S6 kinase, eIF4B and PSD-95 were reduced in autistic versus control fusiform gyrus. Similarly, phosphorylated and total Akt, mTOR and 4E-BP1 and phosphorylated S6 protein were decreased in valproic acid- versus saline-exposed rats. However, no changes in 4E-BP1 or eIF4E were found in autistic brains.ConclusionsIn contrast to some monogenic disorders with high rates of autism, our data demonstrate down-regulation of the Akt/mTOR pathway, specifically via p70S6K/eIF4B, in idiopathic autism. These findings suggest that disruption of this pathway in either direction is widespread in autism and can have adverse consequences for synaptic function. The use of valproic acid, a histone deacetylase inhibitor, in rats successfully modeled these changes, implicating an epigenetic mechanism in these pathway disruptions.

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., Competing Interests: Declaration of competing interest The authors do not have any potential conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The mechanisms controlling synapse growth and maintenance are of critical importance for learning and memory. The MAGUK family of synaptic scaffolding proteins is abundantly expressed at glutamatergic central synapses, but their importance in controlling the synaptic content of glutamate receptors is poorly understood. Here, we use a chained RNAi-mediated knockdown approach to simultaneously remove PSD-93, PSD-95, and SAP102, the MAGUKs previously shown to be responsible for synaptic localization of glutamate receptors. We find that MAGUKs are specifically responsible for creating functional synapses after initial spine formation by filling functionally silent spines with glutamate receptors. Removal of the MAGUKs causes a transient reduction in AMPA receptor quantal size followed by synaptic consolidation resulting in a normalization of quantal size at the few remaining functional synapses. Consolidation requires signaling through L-type calcium channels, CaM kinase kinase, and the GluA2 AMPA receptor subunit, akin to a homeostatic process.

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

RationaleAn abundance of genetic and epidemiologic evidence as well as longitudinal neuroimaging data point to developmental origins for schizophrenia and other mental health disorders. Recent clinical studies indicate that microduplications of VIPR2, encoding the vasoactive intestinal peptide (VIP) receptor VPAC2, confer significant risk for schizophrenia and autism spectrum disorder. Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP responsiveness (cAMP induction), but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown.ObjectivesWe subcutaneously administered the highly selective VPAC2 receptor agonist Ro 25-1553 to C57BL/6 mice from postnatal day 1 (P1) to P14 to determine if overactivation of VPAC2 receptor signaling during postnatal brain maturation affects synaptogenesis and selected behaviors.ResultsWestern blot analyses on P21 revealed significant reductions of synaptophysin and postsynaptic density protein 95 (PSD-95) in the prefrontal cortex, but not in the hippocampus in Ro 25-1553-treated mice. The same postnatally restricted treatment resulted in a disruption in prepulse inhibition of the acoustic startle measured in adult mice. No effects were observed in open-field locomotor activity, sociability in the three-chamber social interaction test, or fear conditioning or extinction.ConclusionOveractivation of the VPAC2 receptor in the postnatal mouse results in a reduction in synaptic proteins in the prefrontal cortex and selective alterations in prepulse inhibition. These findings suggest that the VIPR2-linkage to mental health disorders may be due in part to overactive VPAC2 receptor signaling during a critical time of synaptic maturation.

The Tat-NR2B9c peptide has shown clinical efficacy as a neuroprotective agent in acute stroke. Tat-NR2B9c is designed to prevent nitric oxide (NO) production by preventing postsynaptic density protein 95 (PSD-95) binding to N-methyl-D-aspartate (NMDA) receptors and neuronal nitric oxide synthase; however, PSD-95 is a scaffolding protein that also couples NMDA receptors to other downstream effects. Here, using neuronal cultures, we show that Tat-NR2B9c also prevents NMDA-induced activation of neuronal NADPH oxidase, thereby blocking superoxide production. Given that both superoxide and NO are required for excitotoxic injury, the neuroprotective effect of Tat-NR2B9c may alternatively be attributable to uncoupling neuronal NADPH oxidase from NMDA receptor activation.

Docosahexaenoic acid (DHA, 22:6n-3) is an essential component of the nervous system, and maternal n-3 polyunsaturated fatty acids (PUFAs) are an important source for brain development. Here, the impact of DHA on developing central neurons was examined using an accessible in vivo model. Xenopus laevis embryos from adult female frogs fed n-3 PUFA-adequate or deficient diets were analyzed every 10 weeks for up to 60 weeks, when frogs were then switched to a fish oil-supplemented diet. Lipid analysis showed that DHA was significantly reduced both in oocytes and tadpoles 40 weeks after deprivation, and brain DHA was reduced by 57% at 60 weeks. In vivo imaging of single optic tectal neurons coexpressing tdTomato and PSD-95-GFP revealed that neurons were morphologically simpler in tadpoles from frogs fed the deficient diet compared with the adequate diet. Tectal neurons had significantly fewer dendrite branches and shorter dendritic arbor over a 48 h imaging period. Postsynaptic cluster number and density were lower in neurons deprived of n-3 PUFA. Moreover, changes in neuronal morphology correlated with a 40% decrease in the levels of BDNF mRNA and mature protein in the brain, but not in TrkB. Importantly, switching to a fish oil-supplemented diet induced a recovery in DHA content in the frog embryos within 20 weeks and diminished the deprivation effects observed on tectal neurons of Stage 45 tadpoles. Consequently, our results indicate that DHA impacts dendrite maturation and synaptic connectivity in the developing brain, and it may be involved in neurotrophic support by BDNF.

Cranial radiotherapy is used routinely to control the growth of primary and secondary brain tumors, but often results in serious and debilitating cognitive dysfunction. In part due to the beneficial dose depth distributions that may spare normal tissue damage, the use of protons to treat CNS and other tumor types is rapidly gaining popularity. Astronauts exposed to lower doses of protons in the space radiation environment are also at risk for developing adverse CNS complications. To explore the consequences of whole body proton irradiation, mice were subjected to 0.1 and 1 Gy and analyzed for morphometric changes in hippocampal neurons 10 and 30 days following exposure. Significant dose-dependent reductions (~33 %) in dendritic complexity were found, when dendritic length, branching and area were analyzed 30 days after exposure. At equivalent doses and times, significant reductions in the number (~30 %) and density (50-75 %) of dendritic spines along hippocampal neurons of the dentate gyrus were also observed. Immature spines (filopodia, long) exhibited the greatest sensitivity (1.5- to 3-fold) to irradiation, while more mature spines (mushroom) were more resistant to changes over a 1-month post-irradiation timeframe. Irradiated granule cell neurons spanning the subfields of the dentate gyrus showed significant and dose-responsive reductions in synaptophysin expression, while the expression of postsynaptic density protein (PSD-95) was increased significantly. These findings corroborate our past work using photon irradiation, and demonstrate for the first time, dose-responsive changes in dendritic complexity, spine density and morphology and synaptic protein levels following exposure to low-dose whole body proton irradiation.

Membrane/lipid rafts (MLR) are plasmalemmal microdomains that are essential for neuronal signaling and synaptic development/stabilization. Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the biosynthesis of mevalonic, a precursor to cholesterol via the mevalonate pathway. Because there has been controversy over the effects of statins on neuronal and cognitive function, we investigated the impact of long-term atorvastatin treatment (5mg/kg/d for 7 months by oral gavage) on behavior, cognition, and brain biochemistry in mice. We hypothesized that long-term statin treatment would alter lipid rafts and cognitive function. Atorvastatin treatment resulted in behavioral deficits as measured in paradigms for basic exploration (open field activity) and cognitive function (Barnes maze, startle response) without impairment in global motor function (Rotor Rod). Furthermore, significant changes in MLR-associated proteins (syntaxin-1α and synaptophysin) and a global change of post-synaptic density protein-95 (PSD95) were observed. The observed decreases in the MLR-localized pre-synaptic vesicle proteins syntaxin-1α and synaptophysin suggest a molecular mechanism for the statin-associated impairment of cognitive function that was observed and that has been suggested by the clinical literature.

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

A significant proportion of patients with autism exhibit some degree of intellectual disability. The BTBR T(+) Itpr3(tf)/J mouse strain exhibits behaviors that align with the major diagnostic criteria of autism. To further evaluate the BTBR strain's cognitive impairments, we quantified hippocampus-dependent object location memory (OLM) and found that one-third of the BTBR mice exhibited robust memory, whereas the remainder did not. Fluorescence deconvolution tomography was used to test whether synaptic levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), a protein that contributes importantly to plasticity, correlate with OLM scores in individual mice. In hippocampal field CA1, the BTBRs had fewer post-synaptic densities associated with high levels of phosphorylated (p-) ERK1/2 as compared with C57BL/6 mice. Although counts of p-ERK1/2 immunoreactive synapses did not correlate with OLM performance, the intensity of synaptic p-ERK1/2 immunolabeling was negatively correlated with OLM scores across BTBRs. Metabotropic glutamate receptor (mGluR) 5 signaling activates ERK1/2. Therefore, we tested whether treatment with the mGluR5 antagonist MPEP normalizes synaptic and learning measures in BTBR mice: MPEP facilitated OLM and decreased synaptic p-ERK1/2 immunolabeling intensity without affecting numbers of p-ERK1/2+ synapses. In contrast, semi-chronic ampakine treatment, which facilitates memory in other models of cognitive impairment, had no effect on OLM in BTBRs. These results suggest that intellectual disabilities associated with different neurodevelopmental disorders on the autism spectrum require distinct therapeutic strategies based on underlying synaptic pathology.

Chemotherapy-related cognitive deficits are a major neurological problem, but the underlying mechanisms are unclear. The death of neural stem/precursor cell (NSC) by cisplatin has been reported as a potential cause, but this requires high doses of chemotherapeutic agents. Cisplatin is frequently used in modern oncology, and it achieves high concentrations in the patient's brain. Here we report that exposure to low concentrations of cisplatin (0.1μM) causes the loss of dendritic spines and synapses within 30min. Longer exposures injured dendritic branches and reduced dendritic complexity. At this low concentration, cisplatin did not affect NSC viability nor provoke apoptosis. However, higher cisplatin levels (1μM) led to the rapid loss of synapses and dendritic disintegration, and neuronal-but not NSC-apoptosis. In-vivo treatment with cisplatin at clinically relevant doses also caused a reduction of dendritic branches and decreased spine density in CA1 and CA3 hippocampal neurons. An acute increase in cell death was measured in the CA1 and CA3 neurons, as well as in the NSC population located in the subgranular zone of the dentate gyrus in the cisplatin treated animals. The density of dendritic spines is related to the degree of neuronal connectivity and function, and pathological changes in spine number or structure have significant consequences for brain function. Therefore, this synapse and dendritic damage might contribute to the cognitive impairment observed after cisplatin treatment.

Neuronal loss is the most common and critical feature of a spectrum of brain traumas and neurodegenerative disorders such as Alzheimer's disease (AD). The capacity to generate new neurons in the central nervous system diminishes early during brain development and is restricted mainly to two brain areas in the mature brain: subventricular zone and subgranular zone. Extensive research on the impact of brain injury on endogenous neurogenesis and cognition has been conducted primarily using young animals, when neurogenesis is most active. However, a critical question remains to elucidate the effect of brain injury on endogenous neurogenesis and cognition in older animals, which is far more relevant for age-related neurodegenerative disorders such as AD. Therefore, we examined the impact of neuronal loss on endogenous neurogenesis in aged animals using CaM/Tet-DTA mice, a transgenic model of hippocampal cell loss. Additionally, we investigated whether the upregulation of adult neurogenesis could mitigate cognitive deficits following substantial hippocampal neuronal loss. Our findings demonstrate that aged CaM/Tet-DTA mice that sustain severe neuronal loss exhibit an upregulation of endogenous neurogenesis. However, despite this significant upregulation, neurogenesis alone is not able to mitigate the cognitive deficits observed. Our studies suggest that the aged brain has the capacity to stimulate neurogenesis post-injury; however, multiple therapeutic approaches, including upregulation of endogenous neurogenesis, will be necessary to recover brain function after severe neurodegeneration.

Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.

In addition to functioning as an activator of fibrinolysis, tissue-type plasminogen activator (tPA) interacts with neurons and regulates multiple aspects of neuronal cell physiology. In this study, we examined the mechanism by which tPA initiates cell signaling in PC12 and N2a neuron-like cells. We demonstrate that enzymatically active and inactive tPA (EI-tPA) activate ERK1/2 in a biphasic manner. Rapid ERK1/2 activation is dependent on LDL receptor-related protein-1 (LRP1). In the second phase, ERK1/2 is activated by tPA independently of LRP1. The length of the LRP1-dependent phase varied inversely with the tPA concentration. Rapid ERK1/2 activation in response to EI-tPA and activated α2-macroglobulin (α2M*) required the NMDA receptor and Trk receptors, which assemble with LRP1 into a single pathway. Assembly of this signaling system may have been facilitated by the bifunctional adapter protein, PSD-95, which associated with LRP1 selectively in cells treated with EI-tPA or α2M*. Myelin-associated glycoprotein binds to LRP1 with high affinity but failed to induce phosphorylation of TrkA or ERK1/2. Instead, myelin-associated glycoprotein recruited p75 neurotrophin receptor (p75NTR) into a complex with LRP1 and activated RhoA. p75NTR was not recruited by other LRP1 ligands, including EI-tPA and α2M*. Lactoferrin functioned as an LRP1 signaling antagonist, inhibiting Trk receptor phosphorylation and ERK1/2 activation in response to EI-tPA. These results demonstrate that LRP1-initiated cell signaling is ligand-dependent. Proteins that activate cell signaling by binding to LRP1 assemble different co-receptor systems. Ligand-specific co-receptor recruitment provides a mechanism by which one receptor, LRP1, may trigger different signaling responses.

Recent exome sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several human intellectual disabilities and cognitive disorders. However, it is currently unknown how mutations in BAF complexes result in impaired cognitive function. Postmitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Mice harboring selective genetic manipulations of BAF53b have severe defects in long-term memory and long-lasting forms of hippocampal synaptic plasticity. We rescued memory impairments in BAF53b mutant mice by reintroducing BAF53b in the adult hippocampus, which suggests a role for BAF53b beyond neuronal development. The defects in BAF53b mutant mice appeared to derive from alterations in gene expression that produce abnormal postsynaptic components, such as spine structure and function, and ultimately lead to deficits in synaptic plasticity. Our results provide new insight into the role of dominant mutations in subunits of BAF complexes in human intellectual and cognitive disorders.

Synapses are highly dynamic structures that mediate cell-cell communication in the central nervous system. Their molecular composition is altered in an activity-dependent fashion, which modulates the efficacy of subsequent synaptic transmission events. Whereas activity-dependent trafficking of individual key synaptic proteins into and out of the synapse has been characterized previously, global activity-dependent changes in the synaptic proteome have not been studied. To test the feasibility of carrying out an unbiased large-scale approach, we investigated alterations in the molecular composition of synaptic spines following mass stimulation of the central nervous system induced by pilocarpine. We observed widespread changes in relative synaptic abundances encompassing essentially all proteins, supporting the view that the molecular composition of the postsynaptic density is tightly regulated. In most cases, we observed that members of gene families displayed coordinate regulation even when they were not known to physically interact. Analysis of correlated synaptic localization revealed a tightly co-regulated cluster of proteins, consisting of mainly glutamate receptors and their adaptors. This cluster constitutes a functional core of the postsynaptic machinery, and changes in its size affect synaptic strength and synaptic size. Our data show that the unbiased investigation of activity-dependent signaling of the postsynaptic density proteome can offer valuable new information on synaptic plasticity.

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., (© 2024. The Author(s).)

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 β1-catenin, insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-β), glycogen synthase kinase-3β (GSK-3β), 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 β1-catenin, IGF-1, PSD-95, and TGF-β genes decreased, whereas NR2B and GSK-3β expression increased. Treatment with CM restores the expression of PSD-95 and GSK-3β 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., (© 2023 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.

Postsynaptic density protein 95 (PSD-95) is essential for synaptic maturation and plasticity. Although its synaptic regulation has been widely studied, the control of PSD-95 cellular expression is not understood. We found that Psd-95 was controlled post-transcriptionally during neural development. Psd-95 was transcribed early in mouse embryonic brain, but most of its product transcripts were degraded. The polypyrimidine tract binding proteins PTBP1 and PTBP2 repressed Psd-95 (also known as Dlg4) exon 18 splicing, leading to premature translation termination and nonsense-mediated mRNA decay. The loss of first PTBP1 and then of PTBP2 during embryonic development allowed splicing of exon 18 and expression of PSD-95 late in neuronal maturation. Re-expression of PTBP1 or PTBP2 in differentiated neurons inhibited PSD-95 expression and impaired the development of glutamatergic synapses. Thus, expression of PSD-95 during early neural development is controlled at the RNA level by two PTB proteins whose sequential downregulation is necessary for synapse maturation.

An increasing number of individuals in our population are surviving to over 90 years and a subset is at risk for developing dementia. However, senile plaque and neurofibrillary tangle pathology do not consistently differentiate individuals with and without dementia. Synaptic protein loss is a feature of aging and dementia and may dissociate 90+ individuals with and without dementia. Synaptophysin (SYN), postsynaptic density 95 (PSD-95) and growth-associated protein 43 (GAP-43) were studied in the frontal cortex of an autopsy series of 32 prospectively followed individuals (92-105 years) with a range of cognitive function. SYN protein levels were decreased in individuals with dementia and increased in those with clinical signs of cognitive impairment insufficient for a diagnosis of dementia. SYN but neither PSD-95 nor GAP-43 protein levels were significantly correlated with mini-mental status examination (MMSE) scores. Frontal cortex SYN protein levels may protect neuronal function in oldest-old individuals and reflect compensatory responses that may be involved with maintaining cognition.