Quantitative Fluorescence Analysis Reveals Dendrite-Specific Thalamocortical Plasticity in L5 Pyramidal Neurons during Learning.

High-throughput anatomic data can stimulate and constrain new hypotheses about how neural circuits change in response to experience. Here, we use fluorescence-based reagents for presynaptic and postsynaptic labeling to monitor changes in thalamocortical synapses onto different compartments of layer 5 (L5) pyramidal (Pyr) neurons in somatosensory (barrel) cortex from mixed-sex mice during whisker-dependent learning (Audette et al., 2019). Using axonal fills and molecular-genetic tags for synapse identification in fixed tissue from Rbp4-Cre transgenic mice, we found that thalamocortical synapses from the higher-order posterior medial thalamic nucleus showed rapid morphologic changes in both presynaptic and postsynaptic structures at the earliest stages of sensory association training. Detected increases in thalamocortical synaptic size were compartment specific, occurring selectively in the proximal dendrites onto L5 Pyr and not at inputs onto their apical tufts in L1. Both axonal and dendritic changes were transient, normalizing back to baseline as animals became expert in the task. Anatomical measurements were corroborated by electrophysiological recordings at different stages of training. Thus, fluorescence-based analysis of input- and target-specific synapses can reveal compartment-specific changes in synapse properties during learning.SIGNIFICANCE STATEMENT Synaptic changes underlie the cellular basis of learning, experience, and neurologic diseases. Neuroanatomical methods to assess synaptic plasticity can provide critical spatial information necessary for building models of neuronal computations during learning and experience but are technically and fiscally intensive. Here, we describe a confocal fluorescence microscopy-based analytical method to assess input, cell type, and dendritic location-specific synaptic plasticity in a sensory learning assay. Our method not only confirms prior electrophysiological measurements but allows us to predict functional strength of synapses in a pathway-specific manner. Our findings also indicate that changes in primary sensory cortices are transient, occurring during early learning. Fluorescence-based synapse identification can be an efficient and easily adopted approach to study synaptic changes in a variety of experimental paradigms.

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons.

Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABAARs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions.

Glutaredoxin 1 Downregulation in the Substantia Nigra Leads to Dopaminergic Degeneration in Mice.

BACKGROUND: Parkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Perturbation of protein thiol redox homeostasis has been shown to play a role in the dysregulation of cell death and cell survival signaling pathways in these neurons. Glutaredoxin 1 (Grx1) is a thiol/disulfide oxidoreductase that catalyzes the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis. OBJECTIVES: We evaluated if the downregulation of Grx1 could lead to dopaminergic degeneration and PD-relevant motor deficits in mice. METHODS: Grx1 was downregulated unilaterally through viral vector-mediated transduction of short hairpin RNA against Grx1 into the SNpc. Behavioral assessment was performed through rotarod and elevated body swing test. Stereological analysis of tyrosine hydroxylase-positive and Nissl-positive neurons was carried out to evaluate neurodegeneration. RESULTS: Downregulation of Grx1 resulted in contralateral bias of elevated body swing and reduced latency to fall off, accelerating rotarod. This was accompanied by a loss of tyrosine hydroxylase-positive neurons in the SNpc and their DA projections in the striatum. Furthermore, there was a loss Nissl-positive neurons in the SNpc, indicating cell death. This was selective to the SNpc neurons because DA neurons in the ventral tegmental area were unaffected akin to that seen in human PD. Furthermore, Grx1 mRNA expression was substantially decreased in the SNpc from PD patients. CONCLUSIONS: Our study indicates that Grx1 is critical for the survival of SNpc DA neurons and that it is downregulated in human PD. © 2020 International Parkinson and Movement Disorder Society.

Glutaredoxin1 Diminishes Amyloid Beta-Mediated Oxidation of F-Actin and Reverses Cognitive Deficits in an Alzheimer's Disease Mouse Model.

Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis through multiple sources are implicated in synaptic pathology observed in the disease. We have previously shown F-actin disassembly in dendritic spines in early AD (34). The actin cytoskeleton can be oxidatively modified resulting in altered F-actin dynamics. Therefore, we investigated whether disruption of redox signaling could contribute to actin network disassembly and downstream effects in the amyloid precursor protein/presenilin-1 double transgenic (APP/PS1) mouse model of AD. Results: Synaptosomal preparations from 1-month-old APP/PS1 mice showed an increase in ROS levels, coupled with a decrease in the reduced form of F-actin and increase in glutathionylated synaptosomal actin. Furthermore, synaptic glutaredoxin 1 (Grx1) and thioredoxin levels were found to be lowered. Overexpressing Grx1 in the brains of these mice not only reversed F-actin loss seen in APP/PS1 mice but also restored memory recall after contextual fear conditioning. F-actin levels and F-actin nanoarchitecture in spines were also stabilized by Grx1 overexpression in APP/PS1 primary cortical neurons, indicating that glutathionylation of F-actin is a critical event in early pathogenesis of AD, which leads to spine loss. Innovation: Loss of thiol/disulfide oxidoreductases in the synapse along with increase in ROS can render F-actin nanoarchitecture susceptible to oxidative modifications in AD. Conclusions: Our findings provide novel evidence that altered redox signaling in the form of S-glutathionylation and reduced Grx1 levels can lead to synaptic dysfunction during AD pathogenesis by directly disrupting the F-actin nanoarchitecture in spines. Increasing Grx1 levels is a potential target for novel disease-modifying therapies for AD.

Progressive Circuit Changes during Learning and Disease.

A critical step toward understanding cognition, learning, and brain dysfunction will be identification of the underlying cellular computations that occur in and across discrete brain areas, as well as how they are progressively altered by experience or disease. These computations will be revealed by targeted analyses of the neurons that perform these calculations, defined not only by their firing properties but also by their molecular identity and how they are wired within the local and broad-scale network of the brain. New studies that take advantage of sophisticated genetic tools for cell-type-specific identification and control are revealing how learning and neurological disorders initiate and successively change the properties of defined neural circuits. Understanding the temporal sequence of adaptive or pathological synaptic changes across multiple synapses within a network will shed light into how small-scale neural circuits contribute to higher cognitive functions during learning and disease.

Rapid Plasticity of Higher-Order Thalamocortical Inputs during Sensory Learning.

Neocortical circuits are sensitive to experience, showing both anatomical and electrophysiological changes in response to altered sensory input. We examined input- and cell-type-specific changes in thalamo- and intracortical pathways during learning using an automated, home-cage sensory association training (SAT) paradigm coupling multi-whisker stimulation to a water reward. We found that the posterior medial nucleus (POm) but not the ventral posterior medial (VPM) nucleus of the thalamus drives increased cortical activity after 24 h of SAT, when behavioral evidence of learning first emerges. Synaptic strengthening within the POm thalamocortical pathway was first observed at thalamic inputs to L5 and was not generated by sensory stimulation alone. Synaptic changes in L2 were delayed relative to L5, requiring 48 h of SAT to drive synaptic plasticity at thalamic and intracortical inputs onto L2 Pyr neurons. These data identify the POm thalamocortical circuit as a site of rapid synaptic plasticity during learning and suggest a temporal sequence to learning-evoked synaptic changes in the sensory cortex.

Aß mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.

Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aß load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression.SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

Thiol Oxidation by Diamide Leads to Dopaminergic Degeneration and Parkinsonism Phenotype in Mice: A Model for Parkinson's Disease.

AIMS: This study investigates the role of thiol homeostasis disruption in Parkinson's disease (PD) pathogenesis using a novel animal model. A single unilateral administration of the thiol oxidant, diamide (1.45 µmol) into substantia nigra (SN) of mice leads to locomotor deficits and degeneration of dopaminergic (DA) neurons in SN pars compacta (SNpc). RESULTS: Diamide-injected mice showed hemiparkinsonian behavior, measured as spontaneous contralateral body rotations, poor grip strength, and impaired locomotion on a rotarod. We observed a significant loss of DA neurons in ipsilateral but not contralateral SNpc and their striatal fibers. This was accompanied by increased Fluoro-Jade C-positive cells and a loss of NeuN-positive neurons, indicative of neurodegeneration. Importantly, diamide injection led to α-synuclein aggregation in ipsilateral SNpc, a hallmark of PD pathology not often seen in animal models of PD. On investigating putative mechanism(s) involved, we observed a loss of glutathione, which is essential for maintaining protein thiol homeostasis (PTH). Concomitantly, the redox-sensitive ASK1-p38 mitogen-activated protein kinase (MAPK) death signaling pathway was activated in the ipsilateral but not contralateral ventral midbrain through dissociation of ASK1-Trx1 complex. In Neuro-2a cells, diamide activated ASK1-p38 cascade through Trx1 oxidation, leading to cell death, which was abolished by ASK1 knockdown. INNOVATION: Since diamide selectively disrupts PTH, DA neurons appear to be vulnerable to such perturbations and even a single insult with a thiol oxidant can result in long-lasting degeneration. CONCLUSION: Identification of the role of PTH dysregulation in neurodegeneration, especially in early PD, not only facilitates an understanding of novel regulatory features of molecular signaling cascades but also may aid in developing disease-modifying strategies for PD. Antioxid. Redox Signal. 25, 252-267.

Regulation of DJ-1 by Glutaredoxin 1 in Vivo: Implications for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx(-/-) mice we provide show that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homologue in a C. elegans in vivo model of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.

MPTP activates ASK1-p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model.

Activation of apoptosis signal-regulating kinase 1 (ASK1)-p38 MAPK death signaling cascade is implicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream p38 MAPK in a time-dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thioredoxin (Trx1), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase µ 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein covariation network analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mediated TNF signaling through intrathecal administration of TNF-neutralizing antibody prevented Trx1 oxidation and downstream ASK1-p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1-p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states.

DJ-1 loss by glutaredoxin but not glutathione depletion triggers Daxx translocation and cell death.

Environmental and genetic causes are implicated in the etiopathogenesis of Parkinson's disease (PD), a neurodegenerative movement disorder. DJ-1, a putative gene recessively linked to early onset PD, functions as an antioxidant, transcriptional co-activator, and molecular chaperone. We examined DJ-1 status following global perturbation of protein thiol homeostasis by depleting cellular antioxidant glutathione or downregulating glutaredoxin 1, a thiol disulfide oxidoreductase, wherein both paradigms generate oxidative stress. While these perturbations did not affect expression of DJ-1 mRNA, downregulation of glutaredoxin 1 but not glutathione depletion caused loss of DJ-1 protein, translocation of Daxx (a death-associated protein) from nucleus, and cell death. Overexpression of wild-type DJ-1, but not the cysteine mutants, prevented Daxx translocation and cytotoxicity. Protease inhibitors prevented constitutive DJ-1 loss. Residual DJ-1 was present in reduced state, indicating that DJ-1 when oxidized was degraded through proteolysis. Thus, loss of DJ-1 occurring through its oxidative modification and subsequent proteolysis mediated through dysregulation of thiol disulfide oxidoreductase may contribute to pathogenesis of sporadic PD, thus providing a link between environmental challenges and constitutive levels of this vital protein.

Complex I assay in mitochondrial preparations from CNS.

Mammalian NADH:ubiquinone oxidoreductase (complex I) is made up of at least 46 subunits and is one of the largest enzyme complexes known. It catalyzes the first step of the respiratory electron transport chain through the oxidation of NADH, providing two electrons for the reduction of ubiquinone to ubiquinol, thus propelling protons across the inner membrane of the mitochondria, which subsequently drive ATP synthesis. Dysfunction of complex I has been implicated in various neurodegenerative disorders, and it is probably the most vulnerable component of the electron transport chain to inhibition by reactive oxygen species. We describe a simple spectrophotometric method for estimating the activity of complex I from mitochondria isolated from regions of the central nervous system of mice.

Genome scan for loci involved in nonsyndromic cleft lip with or without cleft palate in families from West Bengal, India.

In order to identify genes or regions involved in nonsyndromic cleft lip with or without cleft palate (CL/P) in families from India, we analyzed 38 multiplex families (DNA from 272 individuals, 82 affected with CL/P, 190 unaffected) for 285 genome-wide markers (average spacing 12.6 cM), including markers in six candidate loci or regions on chromosomes 2, 4, 6, 14, 17, and 19 that have been implicated in other studies of CL/P. LOD scores (two-point and multipoint), and model-free association (TDT) and linkage (NPL) statistics, were calculated between each of the markers and a hypothetical CL/P susceptibility locus. The most statistically significant two-point linkage results were with markers on chromosome 7 (LOD = 1.89 with D7S435, 7p15, 47 cM), chromosome 5 (LOD = 1.76 with D5S407, 5q11, 65 cM), chromosome 15 (LOD = 1.55 with D15S652, 15q26, 90 cM), and chromosome 20 (LOD = 1.46 with STS155130, 20q13, 54 cM). The most significant multipoint linkage result was on chromosome 5q, again near D5S407 (HLOD = 1.40). Regions on chromosomes 1p, 1q, 7q, 12q, 16q, 18q, and Xp also had a LOD or HLOD > or = 1.0. Of seven candidate markers and regions with previous positive reports in the literature (TGFA, MSX1, D4S175, F13A1, TGFB3, D17S250, and APOC2), none had a significant linkage result, but one (the APOC2 region) had a significant association result and three others (TGFA, MSX1, F13A1) had suggestive results. The results are consistent with the involvement of multiple loci in CL/P expression in this West Bengal population, which concurs with results found in other CL/P study populations.

Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.

BACKGROUND: Cleft lip or palate (or the two in combination) is a common birth defect that results from a mixture of genetic and environmental factors. We searched for a specific genetic factor contributing to this complex trait by examining large numbers of affected patients and families and evaluating a specific candidate gene. METHODS: We identified the gene that encodes interferon regulatory factor 6 (IRF6) as a candidate gene on the basis of its involvement in an autosomal dominant form of cleft lip and palate, Van der Woude's syndrome. A single-nucleotide polymorphism in this gene results in either a valine or an isoleucine at amino acid position 274 (V274I). We carried out transmission-disequilibrium testing for V274I in 8003 individual subjects in 1968 families derived from 10 populations with ancestry in Asia, Europe, and South America, haplotype and linkage analyses, and case-control analyses, and determined the risk of cleft lip or palate that is associated with genetic variation in IRF6. RESULTS: Strong evidence of overtransmission of the valine (V) allele was found in the entire population data set (P<10(-9)); moreover, the results for some individual populations from South America and Asia were highly significant. Variation at IRF6 was responsible for 12 percent of the genetic contribution to cleft lip or palate and tripled the risk of recurrence in families that had already had one affected child. CONCLUSIONS: DNA-sequence variants associated with IRF6 are major contributors to cleft lip, with or without cleft palate. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling.

Meta-analysis of 13 genome scans reveals multiple cleft lip/palate genes with novel loci on 9q21 and 2q32-35.

Isolated or nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex etiology. A 10-cM genome scan of 388 extended multiplex families with CL/P from seven diverse populations (2,551 genotyped individuals) revealed CL/P genes in six chromosomal regions, including a novel region at 9q21 (heterogeneity LOD score [HLOD]=6.6). In addition, meta-analyses with the addition of results from 186 more families (six populations; 1,033 genotyped individuals) showed genomewide significance for 10 more regions, including another novel region at 2q32-35 (P=.0004). These are the first genomewide significant linkage results ever reported for CL/P, and they represent an unprecedented demonstration of the power of linkage analysis to detect multiple genes simultaneously for a complex disorder.