Publisher Correction: Somatic APP gene recombination in Alzheimer's disease and normal neurons.

In this Article, the top label in Fig. 5d should read 'DISH 3/16' instead of 'DISH 3/17'. This error has been corrected online.

Somatic APP gene recombination in Alzheimer's disease and normal neurons.

The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer's disease-related gene APP, which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant 'genomic cDNAs' (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal 'retro-insertion' of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer's disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer's disease that were absent from healthy neurons. Neuronal gene recombination may allow 'recording' of neural activity for selective 'playback' of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain.

Submegabase copy number variations arise during cerebral cortical neurogenesis as revealed by single-cell whole-genome sequencing.

Somatic copy number variations (CNVs) exist in the brain, but their genesis, prevalence, forms, and biological impact remain unclear, even within experimentally tractable animal models. We combined a transposase-based amplification (TbA) methodology for single-cell whole-genome sequencing with a bioinformatic approach for filtering unreliable CNVs (FUnC), developed from machine learning trained on lymphocyte V(D)J recombination. TbA-FUnC offered superior genomic coverage and removed >90% of false-positive CNV calls, allowing extensive examination of submegabase CNVs from over 500 cells throughout the neurogenic period of cerebral cortical development in Mus musculus Thousands of previously undocumented CNVs were identified. Half were less than 1 Mb in size, with deletions 4× more common than amplification events, and were randomly distributed throughout the genome. However, CNV prevalence during embryonic cortical development was nonrandom, peaking at midneurogenesis with levels triple those found at younger ages before falling to intermediate quantities. These data identify pervasive small and large CNVs as early contributors to neural genomic mosaicism, producing genomically diverse cellular building blocks that form the highly organized, mature brain.

The Rac1 inhibitor NSC23766 suppresses CREB signaling by targeting NMDA receptor function.

NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist.

An essential role for inhibitor-2 regulation of protein phosphatase-1 in synaptic scaling.

Protein phosphatase-1 (PP1) activity is important for many calcium-dependent neuronal functions including Hebbian synaptic plasticity and learning and memory. PP1 activity is necessary for the induction of long-term depression, whereas downregulation of PP1 activity is required for the normal induction of long-term potentiation. However, how PP1 is activated is not clear. Moreover, it is not known whether PP1 plays a role in homeostatic synaptic scaling, another form of synaptic plasticity which functions to reset the neuronal firing rate in response to chronic neuronal activity perturbations. In this study, we found that PP1 inhibitor-2 (I-2) is phosphorylated at serine 43 (S43) in rat and mouse cortical neurons in response to bicuculine application. Expression of I-2 phosphorylation-blocking mutant I-2 (S43A) blocked the dephosphorylation of GluA2 at serine 880, AMPA receptor trafficking, and synaptic downscaling induced by bicuculline application. Our data suggest that the phosphorylation of I-2 at S43 appears to be mediated by L-type calcium channels and calcium/calmodulin-dependent myosin light-chain kinase. Our work thus reveals a novel calcium-induced PP1 activation pathway critical for homeostatic synaptic plasticity.

Synaptic activity bidirectionally regulates a novel sequence-specific S-Q phosphoproteome in neurons.

Protein phosphorylation plays a critical role in neuronal transcription, translation, cell viability, and synaptic plasticity. In neurons, phospho-enzymes and specific substrates directly link glutamate release and post-synaptic depolarization to these cellular functions; however, many of these enzymes and their protein substrates remain uncharacterized or unidentified. In this article, we identify a novel, synaptically driven neuronal phosphoproteome characterized by a specific motif of serine/threonine-glutamine ([S/T]-Q, abbreviated as SQ). These SQ-containing substrates are predominantly localized to dendrites, synapses, the soma; and activation of this SQ phosphoproteome by bicuculline application is induced via calcium influx through L-type calcium channels. On the other hand, acute application of NMDA can inactivate this SQ phosphoproteome. We demonstrate that the SQ motif kinase Ataxia-telangiectasia mutated can also localize to dendrites and dendritic spines, in addition to other subcellular compartments, and is activated by bicuculline application. Pharmacology studies indicate that Ataxia-telangiectasia mutated and its sister kinase ataxia telangiectasia mutated and Rad3-related up-regulate these neuronal SQ substrates. Phosphoproteomics identified over 150 SQ-containing substrates whose phosphorylation is bidirectionally regulated by synaptic activity.

Single-Nucleus RNA-seq of Normal-Appearing Brain Regions in Relapsing-Remitting vs. Secondary Progressive Multiple Sclerosis: Implications for the Efficacy of Fingolimod.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease that alters central nervous system (CNS) functions. Relapsing-remitting MS (RRMS) is the most common form, which can transform into secondary-progressive MS (SPMS) that is associated with progressive neurodegeneration. Single-nucleus RNA sequencing (snRNA-seq) of MS lesions identified disease-related transcriptomic alterations; however, their relationship to non-lesioned MS brain regions has not been reported and which could identify prodromal or other disease susceptibility signatures. Here, snRNA-seq was used to generate high-quality RRMS vs. SPMS datasets of 33,197 nuclei from 8 normal-appearing MS brains, which revealed divergent cell type-specific changes. Notably, SPMS brains downregulated astrocytic sphingosine kinases (SPHK1/2) - the enzymes required to phosphorylate and activate the MS drug, fingolimod. This reduction was modeled with astrocyte-specific Sphk1/2 null mice in which fingolimod lost activity, supporting functionality of observed transcriptomic changes. These data provide an initial resource for studies of single cells from non-lesioned RRMS and SPMS brains.

Inactivation of CREB mediated gene transcription by HDAC8 bound protein phosphatase.

CREB activation via phosphorylation at serine 133 and resulting CREB mediated gene expression is a critical event which can have a significant effect on many cellular processes, including cell survival and plasticity. CREB can be activated by many kinases, for example, it can be phosphorylated by PKA, MAPK, and CaMKIV. The various signaling pathways leading to CREB activation have been extensively studied. On the other hand, CREB is inactivated by PP1 through dephosphorylation at S133 and not much attention has been paid to this aspect of the signaling pathway. It was shown recently that PP1 can be targeted to CREB, for efficient dephosphorylation, through PP1 binding protein HDAC1. In this study, we found that another class-I HDAC family protein, HDAC8, localized in the nucleus of HEK293 cells and also bound to both CREB and PP1. Expression of recombinant HDAC8 results in decreased CREB activation and CREB mediated gene transcription in response to forskolin application. Our study thus elucidated that more than one class-I HDAC family members can regulate the duration of CREB mediated gene transcription.

Genomic mosaicism in the developing and adult brain.

Since the discovery of DNA, the normal developing and functioning brain has been assumed to be composed of cells with identical genomes, which remains the dominant view even today. However, this pervasive assumption is incorrect, as proven by increasing numbers of reports within the last 20 years that have identified multiple forms of somatically produced genomic mosaicism (GM), wherein brain cells-especially neurons-from a single individual show diverse alterations in DNA, distinct from the germline. Critically, these changes alter the actual DNA nucleotide sequences-in contrast to epigenetic mechanisms-and almost certainly contribute to the remarkably diverse phenotypes of single brain cells, including single-cell transcriptomic profiles. Here, we review the history of GM within the normal brain, including its major forms, initiating mechanisms, and possible functions. GM forms include aneuploidies and aneusomies, smaller copy number variations (CNVs), long interspersed nuclear element type 1 (LINE1) repeat elements, and single nucleotide variations (SNVs), as well as DNA content variation (DCV) that reflects all forms of GM with greatest coverage of large, brain cell populations. In addition, technical considerations are examined, along with relationships among GM forms and multiple brain diseases. GM affecting genes and loci within the brain contrast with current neural discovery approaches that rely on sequencing nonbrain DNA (e.g., genome-wide association studies (GWAS)). Increasing knowledge of neural GM has implications for mechanisms of development, diversity, and function, as well as understanding diseases, particularly considering the overwhelming prevalence of sporadic brain diseases that are unlinked to germline mutations. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol, 2018.

Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains.

Previous reports have shown that individual neurons of the brain can display somatic genomic mosaicism of unknown function. In this study, we report altered genomic mosaicism in single, sporadic Alzheimer's disease (AD) neurons characterized by increases in DNA content and amyloid precursor protein (APP) gene copy number. AD cortical nuclei displayed large variability with average DNA content increases of ~8% over non-diseased controls that were unrelated to trisomy 21. Two independent single-cell copy number analyses identified amplifications at the APP locus. The use of single-cell qPCR identified up to 12 copies of APP in sampled neurons. Peptide nucleic acid (PNA) probes targeting APP, combined with super-resolution microscopy detected primarily single fluorescent signals of variable intensity that paralleled single-cell qPCR analyses. These data identify somatic genomic changes in single neurons, affecting known and unknown loci, which are increased in sporadic AD, and further indicate functionality for genomic mosaicism in the CNS.

Molecular mechanisms of homeostatic synaptic downscaling.

Homeostatic synaptic downscaling is a negative feedback response to chronic elevated network activity to reduce the firing rate of neurons. This form of synaptic plasticity decreases the strength of individual synapses to the same proportion, or in a multiplicative manner. Because of this, synaptic downscaling has been hypothesized to counter the potential run-away excitation due to Hebbian type of long term potentiation (LTP), while preserving relative synaptic weight encoded in individual synapses and thus memory information. In this article, we will review the current knowledge on the signaling and molecular mechanisms of synaptic downscaling. Specifically, we focus on three general areas. First the functional roles of several immediate early genes such as Plk2, Homer1a, Arc and Narp are discussed. Secondly, we examine the current knowledge on the regulation of synaptic protein levels by ubiquitination and transcriptional repression in synaptic downscaling. Thirdly, we review the dynamics of signaling molecules such as kinases and phosphatases critical for synaptic downscaling, and their regulation of synaptic scaffolding proteins. Finally we briefly discuss the heterogeneity of homeostatic synaptic downscaling mechanisms. This article is part of the Special Issue entitled 'Homeostatic Synaptic Plasticity'.

Potassium channel Kv2.1 is regulated through protein phosphatase-1 in response to increases in synaptic activity.

The functional stability of neurons in the face of large variations in both activity and efficacy of synaptic connections suggests that neurons possess intrinsic negative feedback mechanisms to balance and tune excitability. While NMDA receptors have been established to play an important role in glutamate receptor-dependent plasticity through protein dephosphorylation, the effects of synaptic activation on intrinsic excitability are less well characterized. We show that increases in synaptic activity result in dephosphorylation of the potassium channel subunit Kv2.1. This dephosphorylation is induced through NMDA receptors and is executed through protein phosphatase-1 (PP1), an enzyme previously established to play a key role in regulating ligand gated ion channels in synaptic plasticity. Dephosphorylation of Kv2.1 by PP1 in response to synaptic activity results in substantial shifts in the inactivation curve of IK, resulting in a reduction in intrinsic excitability, facilitating negative feedback to neuronal excitability.

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5.

The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-D-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.