Distinct calcium sources regulate temporal profiles of NMDAR and mGluR-mediated protein synthesis.

Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity-mediated protein synthesis, another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases-increased translation inhibition, followed by a decrease in translation inhibition, and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition, which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs, RyRs, and SOCE. Furthermore, we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally, we signify the relevance of our findings in the context of Alzheimer's disease. Using neurons derived from human fAD iPSCs and transgenic AD mice, we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis, and its implications in neurodegeneration.

Association between ApoE ε4 genotype and attentional function in non-demented, middle-aged, and older adults from rural India.

Objectives: Several genetic factors have been associated with cognitive decline in aging. Apolipoprotein E (ApoE) ε4 has been widely studied in the risk for pathological cognitive decline, including dementia. However, the association between ApoE ε4 and cognitive functioning in the healthy aging Indian population has been understudied, and the results are ambiguous. Materials and Methods: This study aims to examine the role of the ApoE genotype with attentional function in aging adults (≥45 years) in a rural Indian population. Cross-sectional (baseline) data (n = 2100) was utilized from an ongoing longitudinal cohort study on aging (Srinivaspura Aging, Neurosenescence, and Cognition study). Participants hailed from villages of Srinivaspura in Karnataka, southern India. Participants were categorized based on ApoE-ε4 status into three categories: No ε4, heterozygous ε4, and homozygous ε4. Attentional function was assessed using the auditory and visual attention subtests from a computerized neurocognitive test battery. Linear regression was performed adjusting for age, gender, and education. Results: In model 1 (unadjusted), we did not find an association between ApoE and attention function. In the partially adjusted model 2 (adjusting for age), ApoE ε4 with age was significantly associated with the attention function. Further, with increasing age, there was a decline in attention among homozygous ε4 individuals. Model 3 (model 2 + gender) found that ApoE ε4, age, and gender explained a significant variance in attention function. In addition, with increasing age, males had poor attention in the homozygous as compared to heterozygous group. Model 4 (model 3+ education) explained a significant variance in attention and also revealed that with increasing age, attention declined in the illiterate and low literacy groups in both homozygous and heterozygous groups among both genders. Conclusion: Although ApoE ε4 alone was not associated, it interacted with age, gender, and education to affect attention function in this rural Indian population. Longitudinal cognitive monitoring will yield insights into understanding whether the ApoE ε4 genotype influences the rate of cognitive decline in this rural, aging population.

Akt activation ameliorates deficits in hippocampal-dependent memory and activity-dependent synaptic protein synthesis in an Alzheimer's disease mouse model.

Protein kinase-B (Akt) and the mechanistic target of rapamycin (mTOR) signaling pathways are implicated in Alzheimer's disease (AD) pathology. Akt/mTOR signaling pathways, activated by external inputs, enable new protein synthesis at the synapse and synaptic plasticity. The molecular mechanisms impeding new protein synthesis at the synapse in AD pathogenesis remain elusive. Here, we aimed to understand the molecular mechanisms prior to the manifestation of histopathological hallmarks by characterizing Akt1/mTOR signaling cascades and new protein synthesis in the hippocampus of WT and amyloid precursor protein/presenilin-1 (APP/PS1) male mice. Intriguingly, compared to those in WT mice, we found significant decreases in pAkt1, pGSK3ß, pmTOR, pS6 ribosomal protein, and p4E-BP1 levels in both post nuclear supernatant and synaptosomes isolated from the hippocampus of one-month-old (presymptomatic) APP/PS1 mice. In synaptoneurosomes prepared from the hippocampus of presymptomatic APP/PS1 mice, activity-dependent protein synthesis at the synapse was impaired and this deficit was sustained in young adults. In hippocampal neurons from C57BL/6 mice, downregulation of Akt1 precluded synaptic activity-dependent protein synthesis at the dendrites but not in the soma. In three-month-old APP/PS1 mice, Akt activator (SC79) administration restored deficits in memory recall and activity-dependent synaptic protein synthesis. C57BL/6 mice administered with an Akt inhibitor (MK2206) resulted in memory recall deficits compared to those treated with vehicle. We conclude that dysregulation of Akt1/mTOR and its downstream signaling molecules in the hippocampus contribute to memory recall deficits and loss of activity-dependent synaptic protein synthesis. In AD mice, however, Akt activation ameliorates deficits in memory recall and activity-dependent synaptic protein synthesis.

Sex difference in evolution of cognitive decline: studies on mouse model and the Dominantly Inherited Alzheimer Network cohort.

Women carry a higher burden of Alzheimer's disease (AD) compared to men, which is not accounted entirely by differences in lifespan. To identify the mechanisms underlying this effect, we investigated sex-specific differences in the progression of familial AD in humans and in APPswe/PS1ΔE9 mice. Activity dependent protein translation and associative learning and memory deficits were examined in APPswe/PS1ΔE9 mice and wild-type mice. As a human comparator group, progression of cognitive dysfunction was assessed in mutation carriers and non-carriers from DIAN (Dominantly Inherited Alzheimer Network) cohort. Female APPswe/PS1ΔE9 mice did not show recall deficits after contextual fear conditioning until 8 months of age. Further, activity dependent protein translation and Akt1-mTOR signaling at the synapse were impaired in male but not in female mice until 8 months of age. Ovariectomized APPswe/PS1ΔE9 mice displayed recall deficits at 4 months of age and these were sustained until 8 months of age. Moreover, activity dependent protein translation was also impaired in 4 months old ovariectomized APPswe/PS1ΔE9 mice compared with sham female APPswe/PS1ΔE9 mice. Progression of memory impairment differed between men and women in the DIAN cohort as analyzed using linear mixed effects model, wherein men showed steeper cognitive decline irrespective of the age of entry in the study, while women showed significantly greater performance and slower decline in immediate recall (LOGIMEM) and delayed recall (MEMUNITS) than men. However, when the performance of men and women in several cognitive tasks (such as Wechsler's logical memory) are compared with the estimated year from expected symptom onset (EYO) we found no significant differences between men and women. We conclude that in familial AD patients and mouse models, females are protected, and the onset of disease is delayed as long as estrogen levels are intact.

Genes critical for development and differentiation of dopaminergic neurons are downregulated in Parkinson's disease.

We performed transcriptome analysis using RNA sequencing on substantia nigra pars compacta (SNpc) from mice after acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment and from Parkinson's disease (PD) patients. Acute and chronic exposure to MPTP resulted in decreased expression of genes involved in sodium channel regulation. However, upregulation of pro-inflammatory pathways was seen after single dose but not after chronic MPTP treatment. Dopamine biosynthesis and synaptic vesicle recycling pathways were downregulated in PD patients and after chronic MPTP treatment in mice. Genes essential for midbrain development and determination of dopaminergic phenotype such as, LMX1B, FOXA1, RSPO2, KLHL1, EBF3, PITX3, RGS4, ALDH1A1, RET, FOXA2, EN1, DLK1, GFRA1, LMX1A, NR4A2, GAP43, SNCA, PBX1, and GRB10 were downregulated in human PD and overexpression of GFP tagged LMX1B rescued MPP+ induced death in SH-SY5Y neurons. Downregulation of gene ensemble involved in development and differentiation of dopaminergic neurons indicate their potential involvement in pathogenesis and progression of human PD.

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.

Isoform-specific hyperactivation of calpain-2 occurs presymptomatically at the synapse in Alzheimer's disease mice and correlates with memory deficits in human subjects.

Calpain hyperactivation is implicated in late-stages of neurodegenerative diseases including Alzheimer's disease (AD). However, calpains are also critical for synaptic function and plasticity, and hence memory formation and learning. Since synaptic deficits appear early in AD pathogenesis prior to appearance of overt disease symptoms, we examined if localized dysregulation of calpain-1 and/or 2 contributes to early synaptic dysfunction in AD. Increased activity of synaptosomal calpain-2, but not calpain-1 was observed in presymptomatic 1 month old APPswe/PS1ΔE9 mice (a mouse model of AD) which have no evident pathological or behavioural hallmarks of AD and persisted up to 10 months of age. However, total cellular levels of calpain-2 remained unaffected. Moreover, synaptosomal calpain-2 was hyperactivated in frontal neocortical tissue samples of post-mortem brains of AD-dementia subjects and correlated significantly with decline in tests for cognitive and memory functions, and increase in levels of ß-amyloid deposits in brain. We conclude that isoform-specific hyperactivation of calpain-2, but not calpain-1 occurs at the synapse early in the pathogenesis of AD potentially contributing to the deregulation of synaptic signaling in AD. Our findings would be important in paving the way for potential therapeutic strategies for amelioration of cognitive deficits observed in ageing-related dementia disorders like AD.

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.

Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of ß-adrenergic receptor signaling.

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and ß-arrestin2, thereby regulating ß-adrenergic receptor (ßAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects ßAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac ß1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable ßAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued ß1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of ßAR signaling.

Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease.

AIMS: Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by ß-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. RESULTS: Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APPSwe/PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9-10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. INNOVATION: Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. CONCLUSIONS: We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269-1280.

Phosphorylation of the deubiquitinase USP20 by protein kinase A regulates post-endocytic trafficking of ß2 adrenergic receptors to autophagosomes during physiological stress.

Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated ß2 adrenergic receptors (ß2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated ß2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a ß2AR-dependent manner. Upon ß2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated ß2AR complex, and facilitates trafficking of the ubiquitinated ß2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the ß2AR to autophagosomes while promoting plasma membrane recycling of internalized ß2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis.

Arrestins and protein ubiquitination.

The adaptor proteins, ß-arrestins 1 and 2, were originally identified as inhibitors of G protein signaling at the seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors or GPCRs). Subsequent studies have established ß-arrestins as critical multifunctional 7TMR adaptors that mediate receptor trafficking and activate G protein-independent signaling pathways. 7TMR activation leads not only to the recruitment of arrestin proteins upon phosphorylation by GPCR kinases but also to ß-arrestin ubiquitination. This posttranslational modification of ß-arrestin is appended by specific E3 ubiquitin ligases and reversed by deubiquitinases, which are also recruited in a receptor- and agonist-specific manner. ß-Arrestin ubiquitination allows it to form protein complexes with activated 7TMRs, endocytic proteins such as clathrin, and phosphorylated ERK1/2. ß-Arrestin ubiquitination is dependent on its activated conformation and likely regulates timing and subcellular localization of various protein interactions during receptor trafficking and signaling. ß-Arrestins also serve as adaptors that escort E3 ubiquitin ligases to mediate ubiquitination of a wide list of substrate proteins including 7TMRs and provide an added layer of regulation for defining substrate specificity in the cellular ubiquitination pathway.

The p75NTR intracellular domain generated by neurotrophin-induced receptor cleavage potentiates Trk signaling.

The p75 neurotrophin receptor (p75NTR) potentiates Trk signaling, but the underlying mechanisms remain uncertain. Here, we examine the relationship between p75NTR cleavage and Trk signaling. We found that, in PC12 cells, nerve growth factor (NGF) induces rapid and robust alpha-secretase- and gamma-secretase-dependent cleavage of p75NTR, releasing the resulting intracellular domain into the cytosol. Brain-derived neurotrophic factor similarly induces p75NTR cleavage in primary cerebellar granule neurons. p75NTR cleavage occurs by means of Trk-dependent activation of MEK-Erk signaling and induction of alpha-secretase activity, and is independent of ligand binding to p75NTR. Neurons and PC12 cells lacking p75NTR display defects in neurotrophin-dependent Akt activation. Normal Akt activation is rescued using full-length p75NTR or the p75 intracellular domain, but not cleavage-resistant p75NTR. We then demonstrate that NGF-dependent growth arrest of PC12 cells requires p75NTR cleavage and generation of the intracellular domain. We conclude that generation of the soluble p75NTR intracellular domain by Trk-induced cleavage plays a fundamental role in Trk-dependent signaling events.