Publications by authors named "August B Smit"

249 Publications

Measuring Behavior in the Home Cage: Study Design, Applications, Challenges, and Perspectives.

Front Behav Neurosci 2021 24;15:735387. Epub 2021 Sep 24.

Noldus Information Technology BV, Wageningen, Netherlands.

The reproducibility crisis (or replication crisis) in biomedical research is a particularly existential and under-addressed issue in the field of behavioral neuroscience, where, in spite of efforts to standardize testing and assay protocols, several known and unknown sources of confounding environmental factors add to variance. Human interference is a major contributor to variability both within and across laboratories, as well as novelty-induced anxiety. Attempts to reduce human interference and to measure more "natural" behaviors in subjects has led to the development of automated home-cage monitoring systems. These systems enable prolonged and longitudinal recordings, and provide large continuous measures of spontaneous behavior that can be analyzed across multiple time scales. In this review, a diverse team of neuroscientists and product developers share their experiences using such an automated monitoring system that combines Noldus PhenoTyper home-cages and the video-based tracking software, EthoVision XT, to extract digital biomarkers of motor, emotional, social and cognitive behavior. After presenting our working definition of a "home-cage", we compare home-cage testing with more conventional out-of-cage tests (e.g., the open field) and outline the various advantages of the former, including opportunities for within-subject analyses and assessments of circadian and ultradian activity. Next, we address technical issues pertaining to the acquisition of behavioral data, such as the fine-tuning of the tracking software and the potential for integration with biotelemetry and optogenetics. Finally, we provide guidance on which behavioral measures to emphasize, how to filter, segment, and analyze behavior, and how to use analysis scripts. We summarize how the PhenoTyper has applications to study neuropharmacology as well as animal models of neurodegenerative and neuropsychiatric illness. Looking forward, we examine current challenges and the impact of new developments. Examples include the automated recognition of specific behaviors, unambiguous tracking of individuals in a social context, the development of more animal-centered measures of behavior and ways of dealing with large datasets. Together, we advocate that by embracing standardized home-cage monitoring platforms like the PhenoTyper, we are poised to directly assess issues pertaining to reproducibility, and more importantly, measure features of rodent behavior under more ethologically relevant scenarios.
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http://dx.doi.org/10.3389/fnbeh.2021.735387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8498589PMC
September 2021

Systematic assessment of variability in the proteome of iPSC derivatives.

Stem Cell Res 2021 Aug 20;56:102512. Epub 2021 Aug 20.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, Amsterdam, The Netherlands; Department of Child and Youth Psychiatry, Emma Children's Hospital, Amsterdam Neuroscience, Amsterdam UMC, Amsterdam, The Netherlands. Electronic address:

The use of induced pluripotent stem cells (iPSC) to model human complex diseases is gaining popularity as it allows investigation of human cells that are otherwise sparsely available. However, due to its laborious and cost intensive nature, iPSC research is often plagued by limited sample size and putative large variability between clones, decreasing statistical power for detecting experimental effects. Here, we investigate the source and magnitude of variability in the proteome of parallel differentiated astrocytes using mass spectrometry. We compare three possible sources of variability: inter-donor variability, inter- and intra-clonal variability, at different stages of maturation. We show that the interclonal variability is significantly smaller than the inter-donor variability, and that including more donors has a much larger influence on statistical power than adding more clones per donor. Our results provide insight into the sources of variability at protein level between iPSC samples derived in parallel and will aid in optimizing iPSC studies.
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http://dx.doi.org/10.1016/j.scr.2021.102512DOI Listing
August 2021

The continued need for animals to advance brain research.

Neuron 2021 08;109(15):2374-2379

Radboud University Medical Center, Nijmegen, the Netherlands.

Policymakers aim to move toward animal-free alternatives for scientific research and have introduced very strict regulations for animal research. We argue that, for neuroscience research, until viable and translational alternatives become available and the value of these alternatives has been proven, the use of animals should not be compromised.
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http://dx.doi.org/10.1016/j.neuron.2021.07.015DOI Listing
August 2021

Torpor enhances synaptic strength and restores memory performance in a mouse model of Alzheimer's disease.

Sci Rep 2021 07 29;11(1):15486. Epub 2021 Jul 29.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands.

Hibernation induces neurodegeneration-like changes in the brain, which are completely reversed upon arousal. Hibernation-induced plasticity may therefore be of great relevance for the treatment of neurodegenerative diseases, but remains largely unexplored. Here we show that a single torpor and arousal sequence in mice does not induce dendrite retraction and synapse loss as observed in seasonal hibernators. Instead, it increases hippocampal long-term potentiation and contextual fear memory. This is accompanied by increased levels of key postsynaptic proteins and mitochondrial complex I and IV proteins, indicating mitochondrial reactivation and enhanced synaptic plasticity upon arousal. Interestingly, a single torpor and arousal sequence was also sufficient to restore contextual fear memory in an APP/PS1 mouse model of Alzheimer's disease. Our study demonstrates that torpor in mice evokes an exceptional state of hippocampal plasticity and that naturally occurring plasticity mechanisms during torpor provide an opportunity to identify unique druggable targets for the treatment of cognitive impairment.
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http://dx.doi.org/10.1038/s41598-021-94992-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322095PMC
July 2021

Age-Dependent Hippocampal Proteomics in the APP/PS1 Alzheimer Mouse Model: A Comparative Analysis with Classical SWATH/DIA and directDIA Approaches.

Cells 2021 06 24;10(7). Epub 2021 Jun 24.

Center for Neurogenomics and Cognitive Research, Department of Molecular and Cellular Neurobiology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the human population, for which there is currently no cure. The cause of AD is unknown; however, the toxic effects of amyloid-β (Aβ) are believed to play a role in its onset. To investigate this, we examined changes in global protein levels in a hippocampal synaptosome fraction of the Amyloid Precursor Protein swe/Presenelin 1 dE9 (APP/PS1) mouse model of AD at 6 and 12 months of age (moa). Data independent acquisition (DIA), or Sequential Window Acquisition of all THeoretical fragment-ion (SWATH), was used for a quantitative label-free proteomics analysis. We first assessed the usefulness of a recently improved directDIA workflow as an alternative to conventional DIA data analysis using a project-specific spectral library. Subsequently, we applied directDIA to the 6- and 12-moa APP/PS1 datasets and applied the Mass Spectrometry Downstream Analysis Pipeline (MS-DAP) for differential expression analysis and candidate discovery. We observed most regulation at 12-moa, in particular of proteins involved in Aβ homeostasis and microglial-dependent processes, like synaptic pruning and the immune response, such as APOE, CLU and C1QA-C. All proteomics data are available via ProteomeXchange with identifier PXD025777.
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http://dx.doi.org/10.3390/cells10071588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8304546PMC
June 2021

Reduced mGluR5 Activity Modulates Mitochondrial Function.

Cells 2021 06 2;10(6). Epub 2021 Jun 2.

Center for Neurogenomics and Cognitive Research, Department of Molecular and Cellular Neurobiology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 Amsterdam, The Netherlands.

The metabotropic glutamate receptor 5 (mGluR5) is an essential modulator of synaptic plasticity, learning and memory; whereas in pathological conditions, it is an acknowledged therapeutic target that has been implicated in multiple brain disorders. Despite robust pre-clinical data, mGluR5 antagonists failed in several clinical trials, highlighting the need for a better understanding of the mechanisms underlying mGluR5 function. In this study, we dissected the molecular synaptic modulation mediated by mGluR5 using genetic and pharmacological mouse models to chronically and acutely reduce mGluR5 activity. We found that next to dysregulation of synaptic proteins, the major regulation in protein expression in both models concerned specific processes in mitochondria, such as oxidative phosphorylation. Second, we observed morphological alterations in shape and area of specifically postsynaptic mitochondria in mGluR5 KO synapses using electron microscopy. Third, computational and biochemical assays suggested an increase of mitochondrial function in neurons, with increased level of NADP/H and oxidative damage in mGluR5 KO. Altogether, our observations provide diverse lines of evidence of the modulation of synaptic mitochondrial function by mGluR5. This connection suggests a role for mGluR5 as a mediator between synaptic activity and mitochondrial function, a finding which might be relevant for the improvement of the clinical potential of mGluR5.
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http://dx.doi.org/10.3390/cells10061375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228325PMC
June 2021

Longitudinal Assessment of Working Memory Performance in the APPswe/PSEN1dE9 Mouse Model of Alzheimer's Disease Using an Automated Figure-8-Maze.

Front Behav Neurosci 2021 13;15:655449. Epub 2021 May 13.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with a long preclinical and prodromal phase. To enable the study of disease mechanisms, AD has been modeled in many transgenic animal lines and cognitive functioning has been tested using several widely used behavioral tasks. These tasks, however, are not always suited for repeated longitudinal testing and are often associated with acute stress such as animal transfer, handling, novelty, or stress related to the task itself. This makes it challenging to relate cognitive dysfunction in animal models to cognitive decline observed in AD patients. Here, we designed an automated figure-8-maze (F8M) to test mice in a delayed alternation task (DAT) in a longitudinal manner. Mice were rewarded when they entered alternate sides of the maze on subsequent trials. Automation as well as connection of the F8M set-up with a home cage reduces experimenter interference and minimizes acute stress, thus making it suitable for longitudinal testing and facilitating clinical translation. In the present study, we monitored cognitive functioning of 2-month-old APPswe/PSEN1dE9 (APP/PS1) mice over a period of 4 months. The percentage of correct responses in the DAT did not differ between wild-type and transgenic mice from 2 to 6 months of age. However, 6-month-old mice displayed an increase in the number of consecutive incorrect responses. These results demonstrate the feasibility of longitudinal testing using an automated F8M and suggest that APP/PS1 mice are not impaired at delayed spatial alternation until 6 months of age under the current experimental conditions.
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http://dx.doi.org/10.3389/fnbeh.2021.655449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155296PMC
May 2021

Imbalanced post- and extrasynaptic SHANK2A functions during development affect social behavior in SHANK2-mediated neuropsychiatric disorders.

Mol Psychiatry 2021 May 21. Epub 2021 May 21.

Research Group of the Max Planck Institute for Medical Research at the Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany.

Mutations in SHANK genes play an undisputed role in neuropsychiatric disorders. Until now, research has focused on the postsynaptic function of SHANKs, and prominent postsynaptic alterations in glutamatergic signal transmission have been reported in Shank KO mouse models. Recent studies have also suggested a possible presynaptic function of SHANK proteins, but these remain poorly defined. In this study, we examined how SHANK2 can mediate electrophysiological, molecular, and behavioral effects by conditionally overexpressing either wild-type SHANK2A or the extrasynaptic SHANK2A(R462X) variant. SHANK2A overexpression affected pre- and postsynaptic targets and revealed a reversible, development-dependent autism spectrum disorder-like behavior. SHANK2A also mediated redistribution of Ca-permeable AMPA receptors between apical and basal hippocampal CA1 dendrites, leading to impaired synaptic plasticity in the basal dendrites. Moreover, SHANK2A overexpression reduced social interaction and increased the excitatory noise in the olfactory cortex during odor processing. In contrast, overexpression of the extrasynaptic SHANK2A(R462X) variant did not impair hippocampal synaptic plasticity, but still altered the expression of presynaptic/axonal signaling proteins. We also observed an attention-deficit/hyperactivity-like behavior and improved social interaction along with enhanced signal-to-noise ratio in cortical odor processing. Our results suggest that the disruption of pre- and postsynaptic SHANK2 functions caused by SHANK2 mutations has a strong impact on social behavior. These findings indicate that pre- and postsynaptic SHANK2 actions cooperate for normal neuronal function, and that an imbalance between these functions may lead to different neuropsychiatric disorders.
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http://dx.doi.org/10.1038/s41380-021-01140-yDOI Listing
May 2021

Protein Phosphatase 2B Dual Function Facilitates Synaptic Integrity and Motor Learning.

J Neurosci 2021 Jun 21;41(26):5579-5594. Epub 2021 May 21.

Department of Neuroscience, Erasmus MC, 3015 GE, Rotterdam, The Netherlands.

Protein phosphatase 2B (PP2B) is critical for synaptic plasticity and learning, but the molecular mechanisms involved remain unclear. Here we identified different types of proteins that interact with PP2B, including various structural proteins of the postsynaptic densities (PSDs) of Purkinje cells (PCs) in mice. Deleting PP2B reduced expression of PSD proteins and the relative thickness of PSD at the parallel fiber to PC synapses, whereas reexpression of inactive PP2B partly restored the impaired distribution of nanoclusters of PSD proteins, together indicating a structural role of PP2B. In contrast, lateral mobility of surface glutamate receptors solely depended on PP2B phosphatase activity. Finally, the level of motor learning covaried with both the enzymatic and nonenzymatic functions of PP2B. Thus, PP2B controls synaptic function and learning both through its action as a phosphatase and as a structural protein that facilitates synapse integrity. Phosphatases are generally considered to serve their critical role in learning and memory through their enzymatic operations. Here, we show that protein phosphatase 2B (PP2B) interacts with structural proteins at the synapses of cerebellar Purkinje cells. Differentially manipulating the enzymatic and structural domains of PP2B leads to different phenotypes in cerebellar learning. We propose that PP2B is crucial for cerebellar learning via two complementary actions, an enzymatic and a structural operation.
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http://dx.doi.org/10.1523/JNEUROSCI.1741-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244972PMC
June 2021

NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95.

Nat Commun 2021 05 14;12(1):2849. Epub 2021 May 14.

Interdisciplinary Institute for Neuroscience, CNRS, Univ. Bordeaux, IINS, UMR 5297, Bordeaux, France.

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.
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http://dx.doi.org/10.1038/s41467-021-23133-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121912PMC
May 2021

Auxiliary subunits of the AMPA receptor: The Shisa family of proteins.

Curr Opin Pharmacol 2021 06 21;58:52-61. Epub 2021 Apr 21.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. Electronic address:

AMPA receptors mediate fast synaptic transmission in the CNS and can assemble with several types of auxiliary proteins in a spatio-temporal manner, from newly synthesized AMPA receptor tetramers to mature AMPA receptors in the cell membrane. As such, the interaction of auxiliary subunits with the AMPA receptor plays a major role in the regulation of AMPA receptor biogenesis, trafficking, and biophysical properties. Throughout the years, various 'families' of proteins have been identified and today the approximate full complement of AMPAR auxiliary proteins is known. This review presents the current knowledge on the most prominent AMPA-receptor-interacting auxiliary proteins, highlights recent results regarding the Shisa protein family, and provides a discussion on future research that might contribute to the discovery of novel pharmacological targets of auxiliary subunits.
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http://dx.doi.org/10.1016/j.coph.2021.03.001DOI Listing
June 2021

A Synaptic Framework for the Persistence of Memory Engrams.

Front Synaptic Neurosci 2021 24;13:661476. Epub 2021 Mar 24.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

The ability to store and retrieve learned information over prolonged periods of time is an essential and intriguing property of the brain. Insight into the neurobiological mechanisms that underlie memory consolidation is of utmost importance for our understanding of memory persistence and how this is affected in memory disorders. Recent evidence indicates that a given memory is encoded by sparsely distributed neurons that become highly activated during learning, so-called engram cells. Research by us and others confirms the persistent nature of cortical engram cells by showing that these neurons are required for memory expression up to at least 1 month after they were activated during learning. Strengthened synaptic connectivity between engram cells is thought to ensure reactivation of the engram cell network during retrieval. However, given the continuous integration of new information into existing neuronal circuits and the relatively rapid turnover rate of synaptic proteins, it is unclear whether a lasting learning-induced increase in synaptic connectivity is mediated by stable synapses or by continuous dynamic turnover of synapses of the engram cell network. Here, we first discuss evidence for the persistence of engram cells and memory-relevant adaptations in synaptic plasticity, and then propose models of synaptic adaptations and molecular mechanisms that may support memory persistence through the maintenance of enhanced synaptic connectivity within an engram cell network.
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http://dx.doi.org/10.3389/fnsyn.2021.661476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024575PMC
March 2021

Functional brain defects in a mouse model of a chromosomal t(1;11) translocation that disrupts DISC1 and confers increased risk of psychiatric illness.

Transl Psychiatry 2021 02 19;11(1):135. Epub 2021 Feb 19.

Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine at the University of Edinburgh, Edinburgh, UK.

A balanced t(1;11) translocation that directly disrupts DISC1 is linked to schizophrenia and affective disorders. We previously showed that a mutant mouse, named Der1, recapitulates the effect of the translocation upon DISC1 expression. Here, RNAseq analysis of Der1 mouse brain tissue found enrichment for dysregulation of the same genes and molecular pathways as in neuron cultures generated previously from human t(1;11) translocation carriers via the induced pluripotent stem cell route. DISC1 disruption therefore apparently accounts for a substantial proportion of the effects of the t(1;11) translocation. RNAseq and pathway analysis of the mutant mouse predicts multiple Der1-induced alterations converging upon synapse function and plasticity. Synaptosome proteomics confirmed that the Der1 mutation impacts synapse composition, and electrophysiology found reduced AMPA:NMDA ratio in hippocampal neurons, indicating changed excitatory signalling. Moreover, hippocampal parvalbumin-positive interneuron density is increased, suggesting that the Der1 mutation affects inhibitory control of neuronal circuits. These phenotypes predict that neurotransmission is impacted at many levels by DISC1 disruption in human t(1;11) translocation carriers. Notably, genes implicated in schizophrenia, depression and bipolar disorder by large-scale genetic studies are enriched among the Der1-dysregulated genes, just as we previously observed for the t(1;11) translocation carrier-derived neurons. Furthermore, RNAseq analysis predicts that the Der1 mutation primarily targets a subset of cell types, pyramidal neurons and interneurons, previously shown to be vulnerable to the effects of common schizophrenia-associated genetic variants. In conclusion, DISC1 disruption by the t(1;11) translocation may contribute to the psychiatric disorders of translocation carriers through commonly affected pathways and processes in neurotransmission.
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http://dx.doi.org/10.1038/s41398-021-01256-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895946PMC
February 2021

The proteome of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer's disease.

Acta Neuropathol 2021 03 25;141(3):341-358. Epub 2021 Jan 25.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, Amsterdam, The Netherlands.

Granulovacuolar degeneration (GVD) is a common feature in Alzheimer's disease (AD). The occurrence of GVD is closely associated with that of neurofibrillary tangles (NFTs) and GVD is even considered to be a pre-NFT stage in the disease process of AD. Currently, the composition of GVD bodies, the mechanisms associated with GVD and how GVD exactly relates to NFTs is not well understood. By combining immunohistochemistry (IHC) and laser microdissection (LMD) we isolated neurons with GVD and those bearing tangles separately from human post-mortem AD hippocampus (n = 12) using their typical markers casein kinase (CK)1δ and phosphorylated tau (AT8). Control neurons were isolated from cognitively healthy cases (n = 12). 3000 neurons per sample were used for proteome analysis by label free LC-MS/MS. In total 2596 proteins were quantified across samples and a significant change in abundance of 115 proteins in GVD and 197 in tangle bearing neurons was observed compared to control neurons. With IHC the presence of PPIA, TOMM34, HSP70, CHMP1A, TPPP and VXN was confirmed in GVD containing neurons. We found multiple proteins localizing specifically to the GVD bodies, with VXN and TOMM34 being the most prominent new protein markers for GVD bodies. In general, protein groups related to protein folding, proteasomal function, the endolysosomal pathway, microtubule and cytoskeletal related function, RNA processing and glycolysis were found to be changed in GVD neurons. In addition to these protein groups, tangle bearing neurons show a decrease in ribosomal proteins, as well as in various proteins related to protein folding. This study, for the first time, provides a comprehensive human based quantitative assessment of protein abundances in GVD and tangle bearing neurons. In line with previous functional data showing that tau pathology induces GVD, our data support the model that GVD is part of a pre-NFT stage representing a phase in which proteostasis and cellular homeostasis is disrupted. Elucidating the molecular mechanisms and cellular processes affected in GVD and its relation to the presence of tau pathology is highly relevant for the identification of new drug targets for therapy.
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http://dx.doi.org/10.1007/s00401-020-02261-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7882576PMC
March 2021

A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain.

Nat Commun 2021 01 19;12(1):463. Epub 2021 Jan 19.

Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, New York, NY, USA.

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.
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http://dx.doi.org/10.1038/s41467-020-20343-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815907PMC
January 2021

Recent Developments in Data Independent Acquisition (DIA) Mass Spectrometry: Application of Quantitative Analysis of the Brain Proteome.

Front Mol Neurosci 2020 23;13:564446. Epub 2020 Dec 23.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Mass spectrometry is the driving force behind current brain proteome analysis. In a typical proteomics approach, a protein isolate is digested into tryptic peptides and then analyzed by liquid chromatography-mass spectrometry. The recent advancements in data independent acquisition (DIA) mass spectrometry provide higher sensitivity and protein coverage than the classic data dependent acquisition. DIA cycles through a pre-defined set of peptide precursor isolation windows stepping through 400-1,200 m/z across the whole liquid chromatography gradient. All peptides within an isolation window are fragmented simultaneously and detected by tandem mass spectrometry. Peptides are identified by matching the ion peaks in a mass spectrum to a spectral library that contains information of the peptide fragment ions' pattern and its chromatography elution time. Currently, there are several reports on DIA in brain research, in particular the quantitative analysis of cellular and synaptic proteomes to reveal the spatial and/or temporal changes of proteins that underlie neuronal plasticity and disease mechanisms. Protocols in DIA are continuously improving in both acquisition and data analysis. The depth of analysis is currently approaching proteome-wide coverage, while maintaining high reproducibility in a stable and standardisable MS environment. DIA can be positioned as the method of choice for routine proteome analysis in basic brain research and clinical applications.
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http://dx.doi.org/10.3389/fnmol.2020.564446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793698PMC
December 2020

Cerebrospinal fluid total tau levels indicate aberrant neuronal plasticity in Alzheimer's disease.

medRxiv 2020 Nov 3. Epub 2020 Nov 3.

Alzheimer's disease (AD) is characterised by abnormal amyloid beta and tau processing. Previous studies reported that cerebrospinal fluid (CSF) total tau (t-tau) levels vary between patients. Here we show that CSF t-tau variability is associated with distinct impairments in neuronal plasticity mediated by gene repression factors SUZ12 and REST. AD individuals with abnormal t-tau levels have increased CSF concentrations of plasticity proteins regulated by SUZ12 and REST. AD individuals with normal t-tau, on the contrary, have decreased concentrations of these plasticity proteins and increased concentrations in proteins associated with blood-brain and blood CSF-barrier dysfunction. Genomic analyses suggested that t-tau levels in part depend on genes involved in gene expression. The distinct plasticity abnormalities in AD as signaled by t-tau urge the need for personalised treatment.
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http://dx.doi.org/10.1101/2020.10.29.20211920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654872PMC
November 2020

Somatic TARDBP variants as a cause of semantic dementia.

Brain 2020 12;143(12):3827-3841

Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands.

The aetiology of late-onset neurodegenerative diseases is largely unknown. Here we investigated whether de novo somatic variants for semantic dementia can be detected, thereby arguing for a more general role of somatic variants in neurodegenerative disease. Semantic dementia is characterized by a non-familial occurrence, early onset (<65 years), focal temporal atrophy and TDP-43 pathology. To test whether somatic variants in neural progenitor cells during brain development might lead to semantic dementia, we compared deep exome sequencing data of DNA derived from brain and blood of 16 semantic dementia cases. Somatic variants observed in brain tissue and absent in blood were validated using amplicon sequencing and digital PCR. We identified two variants in exon one of the TARDBP gene (L41F and R42H) at low level (1-3%) in cortical regions and in dentate gyrus in two semantic dementia brains, respectively. The pathogenicity of both variants is supported by demonstrating impaired splicing regulation of TDP-43 and by altered subcellular localization of the mutant TDP-43 protein. These findings indicate that somatic variants may cause semantic dementia as a non-hereditary neurodegenerative disease, which might be exemplary for other late-onset neurodegenerative disorders.
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http://dx.doi.org/10.1093/brain/awaa317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805802PMC
December 2020

Genetic Variation in CNS Myelination and Functional Brain Connectivity in Recombinant Inbred Mice.

Cells 2020 09 18;9(9). Epub 2020 Sep 18.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands.

Myelination greatly increases the speed of action potential propagation of neurons, thereby enhancing the efficacy of inter-neuronal communication and hence, potentially, optimizing the brain's signal processing capability. The impact of genetic variation on the extent of axonal myelination and its consequences for brain functioning remain to be determined. Here we investigated this question using a genetic reference panel (GRP) of mouse BXD recombinant inbred (RI) strains, which partly model genetic diversity as observed in human populations, and which show substantial genetic differences in a variety of behaviors, including learning, memory and anxiety. We found coherent differences in the expression of myelin genes in brain tissue of RI strains of the BXD panel, with the largest differences in the hippocampus. The parental C57BL/6J (C57) and DBA/2J (DBA) strains were on opposite ends of the expression spectrum, with C57 showing higher myelin transcript expression compared with DBA. Our experiments showed accompanying differences between C57 and DBA in myelin protein composition, total myelin content, and white matter conduction velocity. Finally, the hippocampal myelin gene expression of the BXD strains correlated significantly with behavioral traits involving anxiety and/or activity. Taken together, our data indicate that genetic variation in myelin gene expression translates to differences observed in myelination, axonal conduction speed, and possibly in anxiety/activity related behaviors.
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http://dx.doi.org/10.3390/cells9092119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564997PMC
September 2020

Hyperexcitable Parvalbumin Interneurons Render Hippocampal Circuitry Vulnerable to Amyloid Beta.

iScience 2020 Jul 14;23(7):101271. Epub 2020 Jun 14.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands. Electronic address:

Parvalbumin (PV) interneuron dysfunction is associated with various brain disorders, including Alzheimer disease (AD). Here, we asked whether early PV neuron hyperexcitability primes the hippocampus for amyloid beta-induced functional impairment. We show that prolonged chemogenetic activation of PV neurons induces long-term hyperexcitability of these cells, disrupts synaptic transmission, and causes spatial memory deficits on the short-term. On the long-term, pyramidal cells also become hyperexcitable, and synaptic transmission and spatial memory are restored. However, under these conditions of increased excitability of both PV and pyramidal cells, a single low-dose injection of amyloid beta directly into the hippocampus significantly impairs PV neuron function, increases pyramidal neuron excitability, and reduces synaptic transmission, resulting in significant spatial memory deficits. Taken together, our data show that an initial hyperexcitable state of PV neurons renders hippocampal function vulnerable to amyloid beta and may contribute to an increased risk for developing AD.
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http://dx.doi.org/10.1016/j.isci.2020.101271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327841PMC
July 2020

A persistent alcohol cue memory trace drives relapse to alcohol seeking after prolonged abstinence.

Sci Adv 2020 May 6;6(19):eaax7060. Epub 2020 May 6.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 HV, Netherlands.

Alcohol use disorder is characterized by a high risk of relapse during periods of abstinence. Relapse is often triggered by retrieval of persistent alcohol memories upon exposure to alcohol-associated environmental cues, but little is known about the neuronal circuitry that supports the long-term storage of alcohol cue associations. We found that a small ensemble of neurons in the medial prefrontal cortex (mPFC) of mice was activated during cue-paired alcohol self-administration (SA) and that selective suppression of these neurons 1 month later attenuated cue-induced relapse to alcohol seeking. Inhibition of alcohol seeking was specific to these neurons as suppression of a non-alcohol-related or sucrose SA-activated mPFC ensemble did not affect relapse behavior. Hence, the mPFC neuronal ensemble activated during cue-paired alcohol consumption functions as a lasting memory trace that mediates cue-evoked relapse long after cessation of alcohol intake, thereby providing a potential target for treatment of alcohol relapse vulnerability.
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http://dx.doi.org/10.1126/sciadv.aax7060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202866PMC
May 2020

APOE ε4 genotype-dependent cerebrospinal fluid proteomic signatures in Alzheimer's disease.

Alzheimers Res Ther 2020 05 27;12(1):65. Epub 2020 May 27.

Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Amsterdam UMC, Vrije Universiteit Amsterdam, PO Box 7057, 1007 MB, Amsterdam, The Netherlands.

Background: Aggregation of amyloid β into plaques in the brain is one of the earliest pathological events in Alzheimer's disease (AD). The exact pathophysiology leading to dementia is still uncertain, but the apolipoprotein E (APOE) ε4 genotype plays a major role. We aimed to identify the molecular pathways associated with amyloid β aggregation using cerebrospinal fluid (CSF) proteomics and to study the potential modifying effects of APOE ε4 genotype.

Methods: We tested 243 proteins and protein fragments in CSF comparing 193 subjects with AD across the cognitive spectrum (65% APOE ε4 carriers, average age 75 ± 7 years) against 60 controls with normal CSF amyloid β, normal cognition, and no APOE ε4 allele (average age 75 ± 6 years).

Results: One hundred twenty-nine proteins (53%) were associated with aggregated amyloid β. APOE ε4 carriers with AD showed altered concentrations of proteins involved in the complement pathway and glycolysis when cognition was normal and lower concentrations of proteins involved in synapse structure and function when cognitive impairment was moderately severe. APOE ε4 non-carriers with AD showed lower expression of proteins involved in synapse structure and function when cognition was normal and lower concentrations of proteins that were associated with complement and other inflammatory processes when cognitive impairment was mild. Repeating analyses for 114 proteins that were available in an independent EMIF-AD MBD dataset (n = 275) showed that 80% of the proteins showed group differences in a similar direction, but overall, 28% effects reached statistical significance (ranging between 6 and 87% depending on the disease stage and genotype), suggesting variable reproducibility.

Conclusions: These results imply that AD pathophysiology depends on APOE genotype and that treatment for AD may need to be tailored according to APOE genotype and severity of the cognitive impairment.
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http://dx.doi.org/10.1186/s13195-020-00628-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254647PMC
May 2020

Splice-dependent trans-synaptic PTPδ-IL1RAPL1 interaction regulates synapse formation and non-REM sleep.

EMBO J 2020 06 29;39(11):e104150. Epub 2020 Apr 29.

Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon, Korea.

Alternative splicing regulates trans-synaptic adhesions and synapse development, but supporting in vivo evidence is limited. PTPδ, a receptor tyrosine phosphatase adhering to multiple synaptic adhesion molecules, is associated with various neuropsychiatric disorders; however, its in vivo functions remain unclear. Here, we show that PTPδ is mainly present at excitatory presynaptic sites by endogenous PTPδ tagging. Global PTPδ deletion in mice leads to input-specific decreases in excitatory synapse development and strength. This involves tyrosine dephosphorylation and synaptic loss of IL1RAPL1, a postsynaptic partner of PTPδ requiring the PTPδ-meA splice insert for binding. Importantly, PTPδ-mutant mice lacking the PTPδ-meA insert, and thus lacking the PTPδ interaction with IL1RAPL1 but not other postsynaptic partners, recapitulate biochemical and synaptic phenotypes of global PTPδ-mutant mice. Behaviorally, both global and meA-specific PTPδ-mutant mice display abnormal sleep behavior and non-REM rhythms. Therefore, alternative splicing in PTPδ regulates excitatory synapse development and sleep by modulating a specific trans-synaptic adhesion.
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http://dx.doi.org/10.15252/embj.2019104150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265247PMC
June 2020

AMPAR Auxiliary Protein SHISA6 Facilitates Purkinje Cell Synaptic Excitability and Procedural Memory Formation.

Cell Rep 2020 04;31(2):107515

Department of Neuroscience, Erasmus MC, 3000 DR Rotterdam, the Netherlands; Netherlands Institute for Neuroscience, 1105 CA Amsterdam, the Netherlands. Electronic address:

The majority of excitatory postsynaptic currents in the brain are gated through AMPA-type glutamate receptors, the kinetics and trafficking of which can be modulated by auxiliary proteins. It remains to be elucidated whether and how auxiliary proteins can modulate synaptic function to contribute to procedural memory formation. In this study, we report that the AMPA-type glutamate receptor (AMPAR) auxiliary protein SHISA6 (CKAMP52) is expressed in cerebellar Purkinje cells, where it co-localizes with GluA2-containing AMPARs. The absence of SHISA6 in Purkinje cells results in severe impairments in the adaptation of the vestibulo-ocular reflex and eyeblink conditioning. The physiological abnormalities include decreased presence of AMPARs in synaptosomes, impaired excitatory transmission, increased deactivation of AMPA receptors, and reduced induction of long-term potentiation at Purkinje cell synapses. Our data indicate that Purkinje cells require SHISA6-dependent modification of AMPAR function in order to facilitate cerebellar, procedural memory formation.
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http://dx.doi.org/10.1016/j.celrep.2020.03.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175376PMC
April 2020

Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits.

Cell 2020 03;180(6):1178-1197.e20

Department of Fundamental Neurosciences, University of Lausanne, Lausanne 1005, Switzerland; University of Rome Tor Vergata, Department of Biomedicine and Prevention, Rome 00133, Italy. Electronic address:

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.
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http://dx.doi.org/10.1016/j.cell.2020.02.044DOI Listing
March 2020

Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry.

Proteomics 2020 02 6;20(3-4):e1900403. Epub 2020 Feb 6.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.

The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1β-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1β-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.
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http://dx.doi.org/10.1002/pmic.201900403DOI Listing
February 2020

Deficiency Impacts Hippocampal CA1 Neuronal Excitability, Dendritic Architecture, and Affects Spatial Learning.

Front Cell Neurosci 2019 25;13:465. Epub 2019 Oct 25.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit, Amsterdam, Netherlands.

G-protein-coupled receptor 158 () is highly expressed in striatum, hippocampus and prefrontal cortex. It gained attention as it was implicated in physiological responses to stress and depression. Recently, has been shown to act as a pathway-specific synaptic organizer in the hippocampus, required for proper mossy fiber-CA3 neurocircuitry establishment, structure, and function. Although rodent expression is highest in CA3, considerable expression occurs in CA1 especially after the first postnatal month. Here, we combined hippocampal-dependent behavioral paradigms with subsequent electrophysiological and morphological analyses from the same group of mice to assess the effects of deficiency on CA1 physiology and function. We demonstrate deficits in spatial memory acquisition and retrieval in the Morris water maze paradigm, along with deficits in the acquisition of extinction memory in the passive avoidance test in KO mice. Electrophysiological recordings from CA1 pyramidal neurons revealed normal basal excitatory and inhibitory synaptic transmission, however, Schaffer collateral stimulation yielded dramatically reduced post-synaptic currents. Interestingly, intrinsic excitability of CA1 pyramidals was found increased, potentially acting as a compensatory mechanism to the reductions in Schaffer collateral-mediated drive. Both and , neurons deficient for or with lowered levels of exhibited robust reductions in dendritic architecture and complexity, i.e., reduced length, surface, bifurcations, and branching. This effect was localized in the apical but not basal dendrites of adult CA1 pyramidals, indicative of compartment-specific alterations. A significant positive correlation between spatial memory acquisition and extent of complexity of CA1 pyramidals was found. Taken together, we provide first evidence of significant disruptions in hippocampal CA1 neuronal dendritic architecture and physiology, driven by deficiency. Importantly, the hippocampal neuronal morphology deficits appear to support the impairments in spatial memory acquisition observed in KO mice.
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http://dx.doi.org/10.3389/fncel.2019.00465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843000PMC
October 2019

The Effects of Sindbis Viral Vectors on Neuronal Function.

Front Cell Neurosci 2019 8;13:362. Epub 2019 Aug 8.

Laboratory for Neuroregeneration, The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, Netherlands.

Viral vectors are attractive tools to express genes in neurons. Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. However, to which extent Sindbis by itself may affect neurons is not fully understood. We assessed effects of neuronal transduction with a Sindbis viral vector on the transcriptome and proteome in organotypic hippocampal slice cultures, and analyzed the electrophysiological properties of individual CA1 neurons, at 24 h and 72 h after viral vector injection. Whereas Sindbis caused substantial gene expression alterations, changes at the protein level were less pronounced. Alterations in transcriptome and proteome were predominantly limited to proteins involved in mediating anti-viral innate immune responses. Sindbis transduction did not affect the intrinsic electrophysiological properties of individual neurons: the membrane potential and neuronal excitability were similar between transduced and non-transduced CA1 neurons up to 72 h after Sindbis injection. Synaptic currents also remained unchanged upon Sindbis transduction, unless slices were massively infected for 72 h. We conclude that Sindbis viral vectors at low transduction rates are suitable for studying short-term effects of a protein of interest on electrophysiological properties of neurons, but not for studies on the regulation of gene expression.
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http://dx.doi.org/10.3389/fncel.2019.00362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694438PMC
August 2019

Early restoration of parvalbumin interneuron activity prevents memory loss and network hyperexcitability in a mouse model of Alzheimer's disease.

Mol Psychiatry 2020 12 20;25(12):3380-3398. Epub 2019 Aug 20.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, Amsterdam, the Netherlands.

Neuronal network dysfunction is increasingly recognized as an early symptom in Alzheimer's disease (AD) and may provide new entry points for diagnosis and intervention. Here, we show that amyloid-beta-induced hyperexcitability of hippocampal inhibitory parvalbumin (PV) interneurons importantly contributes to neuronal network dysfunction and memory impairment in APP/PS1 mice, a mouse model of increased amyloidosis. We demonstrate that hippocampal PV interneurons become hyperexcitable at ~16 weeks of age, when no changes are observed yet in the intrinsic properties of pyramidal cells. This hyperexcitable state of PV interneurons coincides with increased inhibitory transmission onto hippocampal pyramidal neurons and deficits in spatial learning and memory. We show that treatment aimed at preventing PV interneurons from becoming hyperexcitable is sufficient to restore PV interneuron properties to wild-type levels, reduce inhibitory input onto pyramidal cells, and rescue memory deficits in APP/PS1 mice. Importantly, we demonstrate that early intervention aimed at restoring PV interneuron activity has long-term beneficial effects on memory and hippocampal network activity, and reduces amyloid plaque deposition, a hallmark of AD pathology. Taken together, these findings suggest that early treatment of PV interneuron hyperactivity might be clinically relevant in preventing memory decline and delaying AD progression.
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http://dx.doi.org/10.1038/s41380-019-0483-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714697PMC
December 2020
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