Novel Social Stimulation Ameliorates Memory Deficit in Alzheimer's Disease Model through Activating α-Secretase.

As the most common form of dementia in the world, Alzheimer's disease (AD) is a progressive neurological disorder marked by cognitive and behavioral impairment. According to previous researches, abundant social connections shield against dementia. However, it is still unclear how exactly social interactions benefit cognitive abilities in people with AD and how this process is used to increase their general cognitive performance. In this study, we found that single novel social (SNS) stimulation promoted c-Fos expression and increased the protein levels of mature ADAM10/17 and sAPPα in the ventral hippocampus (vHPC) of wild-type (WT) mice, which are hippocampal dorsal CA2 (dCA2) neuron activity and vHPC NMDAR dependent. Additionally, we discovered that SNS caused similar changes in an AD model, FAD4T mice, and these alterations could be reversed by α-secretase inhibitor. Furthermore, we also found that multiple novel social (MNS) stimulation improved synaptic plasticity and memory impairments in both male and female FAD4T mice, accompanied by α-secretase activation and Aß reduction. These findings provide insight into the process underpinning how social interaction helps AD patients who are experiencing cognitive decline, and we also imply that novel social interaction and activation of the α-secretase may be preventative and therapeutic in the early stages of AD.

Universal, label-free, single-molecule visualization of DNA origami nanodevices across biological samples using origamiFISH.

Structural DNA nanotechnology enables the fabrication of user-defined DNA origami nanostructures (DNs) for biological applications. However, the role of DN design during cellular interactions and subsequent biodistribution remain poorly understood. Current methods for tracking DN fates in situ, including fluorescent-dye labelling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for the single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction probes to achieve 1,000-fold signal amplification. We identify cell-type- and DN shape-specific spatiotemporal distribution patterns within a minute of uptake and at picomolar DN concentrations, 10,000× lower than field standards. We additionally optimize compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo-/vibratome sections, slice cultures and whole-mount organoids. Together, origamiFISH enables the accurate mapping of DN distribution across subcellular and tissue barriers for guiding the development of DN-based therapeutics.

Small-molecule targeting AMPA-mediated excitotoxicity has therapeutic effects in mouse models for multiple sclerosis.

While most research and treatments for multiple sclerosis (MS) focus on autoimmune reactions causing demyelination, it is possible that neurodegeneration precedes the autoimmune response. Hence, glutamate receptor antagonists preventing excitotoxicity showed promise in MS animal models, though blocking glutamate signaling prevents critical neuronal functions. This study reports the discovery of a small molecule that prevents AMPA-mediated excitotoxicity by targeting an allosteric binding site. A machine learning approach was used to screen for small molecules targeting the AMPA receptor GluA2 subunit. The lead candidate has potent effects in restoring neurological function and myelination while reducing the immune response in experimental autoimmune encephalitis and cuprizone MS mouse models without affecting basal neurotransmission or learning and memory. These findings facilitate development of a treatment for MS with a different mechanism of action than current immune modulatory drugs and avoids important off-target effects of glutamate receptor antagonists. This class of MS therapeutics could be useful as an alternative or complementary treatment to existing therapies.

Inhibition of Rac1 in ventral hippocampal excitatory neurons improves social recognition memory and synaptic plasticity.

Rac1 is critically involved in the regulation of the actin cytoskeleton, neuronal structure, synaptic plasticity, and memory. Rac1 overactivation is reported in human patients and animal models of Alzheimer's disease (AD) and contributes to their spatial memory deficits, but whether Rac1 dysregulation is also important in other forms of memory deficits is unknown. In addition, the cell types and synaptic mechanisms involved remain unclear. In this study, we used local injections of AAV virus containing a dominant-negative (DN) Rac1 under the control of CaMKIIα promoter and found that the reduction of Rac1 hyperactivity in ventral hippocampal excitatory neurons improves social recognition memory in APP/PS1 mice. Expression of DN Rac1 also improves long-term potentiation, a key synaptic mechanism for memory formation. Our results suggest that overactivation of Rac1 in hippocampal excitatory neurons contributes to social memory deficits in APP/PS1 mice and that manipulating Rac1 activity may provide a potential therapeutic strategy to treat social deficits in AD.

The GR-FKBP51 interaction modulates fear memory but not spatial or recognition memory.

The glucocorticoid receptor (GR) forms a protein complex with FKBP51 that is increased in post-traumatic stress disorder (PTSD) and by fear conditioned learning. Disrupting the GR-FKBP51 complex with a synthetic peptide can block the storage or retrieval of fear conditioned memories, which could be a novel approach to the alleviate fear associated memory in PTSD. However, a potential unacceptable side effect could be the impairment of other types of memory. Thus, we investigated the effect of disrupting the GR-FKBP51 complex on recognition memory using the novel object and displaced object recognition tasks, spatial memory in the Morris water maze, and on social interaction in Crawley's three-chamber social interaction test. We did not observe adverse effects on these other types of memory and conclude that the GR-FKBP51 interaction remains a promising target for treating psychiatric disorders characterized by unwanted aversive memories such as in PTSD.

The omega-3 hydroxy fatty acid 7(S)-HDHA is a high-affinity PPARα ligand that regulates brain neuronal morphology.

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification-untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7(S)-hydroxy-docosahexaenoic acid [7(S)-HDHA]. Results from mass spectrometric detection of 7(S)-HDHA in mouse and rat brain tissues, time-resolved FRET analyses, and thermal shift assays collectively revealed that 7(S)-HDHA potently activated PPARα with an affinity greater than that of other ligands identified to date. We also found that 7(S)-HDHA activation of PPARα in cultured mouse cortical neurons stimulated neuronal growth and arborization, as well as the expression of genes associated with synaptic plasticity. The findings suggest that this DHA derivative supports and enhances neuronal synaptic capacity in the brain.

Characterizing neurotrophic factor-induced synaptic growth in primary mouse neuronal cultures.

Neurotrophic factors and their signaling cascades play important roles in synaptic growth, which can be investigated in cultured primary neurons to better control the concentrations and timing of neurotrophic factor treatment. Here, we provide a protocol detailing the preparation of cultured primary mouse neurons and the neurotrophic factor treatment. We then describe electrophysiological recording of synaptic transmission, immunocytochemistry of AMPA receptor expression, and imaging analysis of dendritic spines. This platform enables characterization of synaptic growth at functional and morphological levels. For complete details on the use and execution of this profile, please refer to Zhou et al. (2021).

Disruption of PAK3 Signaling in Social Interaction Induced cFos Positive Cells Impairs Social Recognition Memory.

P21-activated kinase 3 (PAK3) gene mutations are linked to several neurodevelopmental disorders, but the underlying mechanisms remain unclear. In this study, we used a tetracycline-inducible system to control the expression of a mutant PAK3 (mPAK3) protein in immediate early gene, namely cFos, positive cells to disrupt PAK signaling, specifically in cells activated by social interaction in transgenic mice. We show that the expression of mPAK3-GFP proteins was in cFos-expressing excitatory and inhibitory neurons in various brain regions, such as the cortex and hippocampus, commonly activated during learning and memory. Basal expression of mPAK3-GFP proteins in cFos-positive cells resulted in social recognition memory deficits in the three-chamber social interaction test, without affecting locomotor activity or other forms of memory. The social memory deficit was rescued by doxycycline to halt the mPAK3-GFP transgene expression. In addition, we show that the expression of mPAK3-GFP proteins in a subset of cFos-positive cells, induced by an antecedent short social interaction, termed social pairing, was sufficient to impair social recognition memory. These results indicate that normal PAK signaling in cFos-positive cells activated during social interaction is critical for social memory.

Rho Signaling in Synaptic Plasticity, Memory, and Brain Disorders.

Memory impairments are associated with many brain disorders such as autism, Alzheimer's disease, and depression. Forming memories involves modifications of synaptic transmission and spine morphology. The Rho family small GTPases are key regulators of synaptic plasticity by affecting various downstream molecules to remodel the actin cytoskeleton. In this paper, we will review recent studies on the roles of Rho proteins in the regulation of hippocampal long-term potentiation (LTP) and long-term depression (LTD), the most extensively studied forms of synaptic plasticity widely regarded as cellular mechanisms for learning and memory. We will also discuss the involvement of Rho signaling in spine morphology, the structural basis of synaptic plasticity and memory formation. Finally, we will review the association between brain disorders and abnormalities of Rho function. It is expected that studying Rho signaling at the synapse will contribute to the understanding of how memory is formed and disrupted in diseases.

NGPF2 triggers synaptic scaling up through ALK-LIMK-cofilin-mediated mechanisms.

Synaptic scaling is an extensively studied form of homeostatic plasticity critically involved in various brain functions. Although it is accepted that synaptic scaling is expressed through the postsynaptic accumulation of AMPA receptors (AMPARs), the induction mechanism remains elusive. In this study, we show that TTX treatment induces rapid but transient release of the neurite growth-promoting factor 2 (NGPF2), and this release is necessary and sufficient for TTX-induced scaling up. In addition, we show that inhibition of the anaplastic lymphoma kinase (ALK)-LIMK-cofilin signaling pathway blocks TTX- and NGPF2-induced synaptic scaling up. Furthermore, we show that TTX-induced release of NGPF2 is protein synthesis dependent and requires fragile X mental retardation protein 1 (FMRP1). These results indicate that activity blockade induces NGPF2 synthesis and release to trigger synaptic scaling up through LIMK-cofilin-dependent actin reorganization, spine enlargement, and stabilization of AMPARs at the synapse.

LIM-Kinases in Synaptic Plasticity, Memory, and Brain Diseases.

Learning and memory require structural and functional modifications of synaptic connections, and synaptic deficits are believed to underlie many brain disorders. The LIM-domain-containing protein kinases (LIMK1 and LIMK2) are key regulators of the actin cytoskeleton by affecting the actin-binding protein, cofilin. In addition, LIMK1 is implicated in the regulation of gene expression by interacting with the cAMP-response element-binding protein. Accumulating evidence indicates that LIMKs are critically involved in brain function and dysfunction. In this paper, we will review studies on the roles and underlying mechanisms of LIMKs in the regulation of long-term potentiation (LTP) and depression (LTD), the most extensively studied forms of long-lasting synaptic plasticity widely regarded as cellular mechanisms underlying learning and memory. We will also discuss the involvement of LIMKs in the regulation of the dendritic spine, the structural basis of synaptic plasticity, and memory formation. Finally, we will discuss recent progress on investigations of LIMKs in neurological and mental disorders, including Alzheimer's, Parkinson's, Williams-Beuren syndrome, schizophrenia, and autism spectrum disorders.

Overexpression of LIMK1 in hippocampal excitatory neurons improves synaptic plasticity and social recognition memory in APP/PS1 mice.

Accumulating evidence indicates that the actin regulator cofilin is overactivated in Alzheimer's Disease (AD), but whether this abnormality contributes to synaptic and cognitive impairments in AD is unclear. In addition, the brain region and cell types involved remain unknown. In this study, we specifically manipulate LIMK1, the key protein kinase that phosphorylates and inactivates cofilin, in the hippocampus of APP/PS1 transgenic mice. Using local injections of the AAV virus containing LIMK1 under the control of the CaMKIIα promoter, we show that expression of LIMK1 in hippocampal excitatory neurons increases cofilin phosphorylation (i.e., decreases cofilin activity), rescues impairments in long-term potentiation, and improves social memory in APP/PS1 mice. Our results suggest that deficits in LIMK1/cofilin signaling in the hippocampal excitatory neurons contribute to AD pathology and that manipulations of LIMK1/cofilin activity provide a potential therapeutic strategy to treat AD.

Genetic manipulations of AMPA glutamate receptors in hippocampal synaptic plasticity.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are the principal mediators of fast excitatory synaptic transmission and they are required for various forms of synaptic plasticity, including long-term potentiation (LTP) and depression (LTD), which are key mechanisms of learning and memory. AMPARs are tetrameric complexes assembled from four subunits (GluA1-4), however, the lack of subunit-specific pharmacological tools has made the assessment of individual subunits difficult. The application of genetic techniques, particularly gene targeting, allows for precise manipulation and dissection of each subunit in the regulation of neuronal function and behaviour. In this review, we summarize studies using various mouse models with genetically altered AMPARs and focus on their roles in basal synaptic transmission, LTP, and LTD at the hippocampal CA1 synapse. These studies provide strong evidence that there are multiple forms of LTP and LTD at this synapse which can be induced by various induction protocols, and they are differentially regulated by different AMPAR subunits and domains. We conclude that it is necessary to delineate the mechanism of each of these forms of plasticity and their contribution to memory and brain disorders.

Disruption of the autism-related gene Pak1 causes stereocilia disorganization, hair cell loss, and deafness in mice.

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases (PAKs) are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout (Pak1-/-) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell (HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of ßII-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1-/- mice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.

Transcriptome Regulation by Oncogenic ALK Pathway in Mammalian Cortical Development Revealed by Single-Cell RNA Sequencing.

Precise regulation of embryonic neurodevelopment is crucial for proper structural organization and functioning of the adult brain. The key molecular machinery orchestrating this process remains unclear. Anaplastic lymphoma kinase (ALK) is an oncogenic receptor-type protein tyrosine kinase that is specifically and transiently expressed in developing nervous system. However, its role in the mammalian brain development is unknown. We found that transient embryonic ALK inactivation caused long-lasting abnormalities in the adult mouse brain, including impaired neuronal connectivity and cognition, along with delayed neuronal migration and decreased neuronal proliferation during neurodevelopment. scRNA-seq on human cerebral organoids revealed a delayed transition of cell-type composition. Molecular characterization identified a group of differentially expressed genes (DEGs) that were temporally regulated by ALK at distinct developmental stages. In addition to oncogenes, many DEGs found by scRNA-seq are associated with neurological or neuropsychiatric disorders. Our study demonstrates a pivotal role of oncogenic ALK pathway in neurodevelopment and characterized cell-type-specific transcriptome regulated by ALK for better understanding mammalian cortical development.

PQBP1 promotes translational elongation and regulates hippocampal mGluR-LTD by suppressing eEF2 phosphorylation.

Eukaryotic elongation factor 2 (eEF2) mediates translocation of peptidyl-tRNA from the ribosomal A site to the P site to promote translational elongation. Its phosphorylation on Thr56 by its single known kinase eEF2K inactivates it and inhibits translational elongation. Extensive studies have revealed that different signal cascades modulate eEF2K activity, but whether additional factors regulate phosphorylation of eEF2 remains unclear. Here, we find that the X chromosome-linked intellectual disability protein polyglutamine-binding protein 1 (PQBP1) specifically binds to non-phosphorylated eEF2 and suppresses eEF2K-mediated phosphorylation at Thr56. Loss of PQBP1 significantly reduces general protein synthesis by suppressing translational elongation. Moreover, we show that PQBP1 regulates hippocampal metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) and mGluR-LTD-associated behaviors by suppressing eEF2K-mediated phosphorylation. Our results identify PQBP1 as a novel regulator in translational elongation and mGluR-LTD, and this newly revealed regulator in the eEF2K/eEF2 pathway is also an excellent therapeutic target for various disease conditions, such as neural diseases, virus infection, and cancer.

Norwogonin attenuates hypoxia-induced oxidative stress and apoptosis in PC12 cells.

BACKGROUND: Norwogonin is a natural flavone with three phenolic hydroxyl groups in skeletal structure and has excellent antioxidant activity. However, the neuroprotective effect of norwogonin remains unclear. Here, we investigated the protective capacity of norwogonin against oxidative damage elicited by hypoxia in PC12 cells. METHODS: The cell viability and apoptosis were examined by MTT assay and Annexin V-FITC/PI staining, respectively. Reactive oxygen species (ROS) content was measured using DCFH-DA assay. Lactate dehydrogenase (LDH), malondialdehyde (MDA) and antioxidant enzyme levels were determined using commercial kits. The expression of related genes and proteins was measured by real-time quantitative PCR and Western blotting, respectively. RESULTS: We found that norwogonin alleviated hypoxia-induced injury in PC12 cells by increasing the cell viability, reducing LDH release, and ameliorating the changes of cell morphology. Norwogonin also acted as an antioxidant by scavenging ROS, reducing MDA production, maintaining the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and decreasing the expression levels of HIF-1α and VEGF. In addition, norwogonin prevented cell apoptosis via inhibiting the expression levels of caspase-3, cytochrome c and Bax, while increasing the expression levels of Bcl-2 and the ratio of Bcl-2/Bax. CONCLUSIONS: Norwogonin attenuates hypoxia-induced injury in PC12 cells by quenching ROS, maintaining the activities of antioxidant enzymes, and inhibiting mitochondrial apoptosis pathway.

The Role of ADF/Cofilin in Synaptic Physiology and Alzheimer's Disease.

Actin-depolymerization factor (ADF)/cofilin, a family of actin-binding proteins, are critical for the regulation of actin reorganization in response to various signals. Accumulating evidence indicates that ADF/cofilin also play important roles in neuronal structure and function, including long-term potentiation and depression. These are the most extensively studied forms of long-lasting synaptic plasticity and are widely regarded as cellular mechanisms underlying learning and memory. ADF/cofilin regulate synaptic function through their effects on dendritic spines and the trafficking of glutamate receptors, the principal mediator of excitatory synaptic transmission in vertebrates. Regulation of ADF/cofilin involves various signaling pathways converging on LIM domain kinases and slingshot phosphatases, which phosphorylate/inactivate and dephosphorylate/activate ADF/cofilin, respectively. Actin-depolymerization factor/cofilin activity is also regulated by other actin-binding proteins, activity-dependent subcellular distribution and protein translation. Abnormalities in ADF/cofilin have been associated with several neurodegenerative disorders such as Alzheimer's disease. Therefore, investigating the roles of ADF/cofilin in the brain is not only important for understanding the fundamental processes governing neuronal structure and function, but also may provide potential therapeutic strategies to treat brain disorders.