Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation.

Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn's disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn's disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn's disease when IL-17A or IL-17RA is inhibited.

The role of native cysteine residues in the oligomerization of KCNQ1 channels.

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

Accelerated loss of hypoxia response in zebrafish with familial Alzheimer's disease-like mutation of presenilin 1.

Ageing is the major risk factor for Alzheimer's disease (AD), a condition involving brain hypoxia. The majority of early-onset familial AD (EOfAD) cases involve dominant mutations in the gene PSEN1. PSEN1 null mutations do not cause EOfAD. We exploited putative hypomorphic and EOfAD-like mutations in the zebrafish psen1 gene to explore the effects of age and genotype on brain responses to acute hypoxia. Both mutations accelerate age-dependent changes in hypoxia-sensitive gene expression supporting that ageing is necessary, but insufficient, for AD occurrence. Curiously, the responses to acute hypoxia become inverted in extremely aged fish. This is associated with an apparent inability to upregulate glycolysis. Wild-type PSEN1 allele expression is reduced in post-mortem brains of human EOfAD mutation carriers (and extremely aged fish), possibly contributing to EOfAD pathogenesis. We also observed that age-dependent loss of HIF1 stabilization under hypoxia is a phenomenon conserved across vertebrate classes.

Loss of presenilin 2 age-dependently alters susceptibility to acute seizures and kindling acquisition.

Patients with Alzheimer's disease (AD) experience seizures at higher rates than the general population of that age, suggesting an underexplored role of hyperexcitability in AD. Genetic variants in presenilin (PSEN) 1 and 2 genes lead to autosomal dominant early-onset AD (ADAD); patients with PSEN gene variants also report seizures. Pharmacological control of seizures in AD may be disease-modifying. Preclinical efficacy of FDA-approved antiseizure drugs (ASDs) is well defined in young adult rodents; however, the efficacy of ASDs in aged rodents with chronic seizures is less clear. The mechanism by which ADAD genes lead to AD remains unclear, and even less studied is the pathogenesis of epilepsy in AD. PSEN variants generally all result in a biochemical loss of function (De Strooper, 2007). We herein determined whether well-established models of acute and chronic seizure could be used to explore the relationship between AD genes and seizures through investigating whether loss of normal PSEN2 function age-dependently influenced susceptibility to seizures and/or corneal kindling acquisition. PSEN2 knockout (KO) and age-matched wild-type (WT) mice were screened from 2- to 10-months-old to establish age-dependent focal seizure threshold. Additionally, PSEN2 KO and WT mice aged 2- and 8-months-old underwent corneal kindling such that mice were aged 3- and 9-months old at the beginning of ASD efficacy testing. We then defined the dose-dependent efficacy of mechanistically distinct ASDs on kindled seizures of young versus aged mice to better understand the applicability of corneal kindling to real-world use for geriatric patients. PSEN2 KO mice demonstrated early-life reductions in seizure threshold. However, kindling acquisition was delayed in 2-month-old PSEN2 KO versus WT mice. Young male WT mice took 24.3 ± 1.3 (S.E.M.) stimulations to achieve kindling criterion, whereas age-matched PSEN2 KO male mice took 41.2 ± 1.1 stimulations (p < .0001). The rate of kindling acquisition of 8-month-old mice was no longer different from WT. This study demonstrates that loss of normal PSEN2 function is associated with age-dependent changes in the in vivo susceptibility to acute seizures and kindling. Loss of normal PSEN2 function may be an underexplored molecular contributor to seizures. The use of validated models of chronic seizures in aged rodents may uncover age-related changes in susceptibility to epileptogenesis and/or ASD efficacy in mice with AD-associated genotypes, which may benefit the management of seizures in AD.

Simple Derivatization of RAFT-Synthesized Styrene-Maleic Anhydride Copolymers for Lipid Disk Formulations.

Styrene-maleic acid copolymers have received significant attention because of their ability to interact with lipid bilayers and form styrene-maleic acid copolymer lipid nanoparticles (SMALPs). However, these SMALPs are limited in their chemical diversity, with only phenyl and carboxylic acid functional groups, resulting in limitations because of sensitivity to low pH and high concentrations of divalent metals. To address this limitation, various nucleophiles were reacted with the anhydride unit of well-defined styrene-maleic anhydride copolymers in order to assess the potential for a new lipid disk nanoparticle-forming species. These styrene-maleic anhydride copolymer derivatives (SMADs) can form styrene-maleic acid derivative lipid nanoparticles (SMADLPs) when they interact with lipid molecules. Polymers were synthesized, purified, characterized by Fourier-transform infrared spectroscopy, gel permeation chromatography, and nuclear magnetic resonance and then used to make disk-like SMADLPs, whose sizes were measured by dynamic light scattering (DLS). The SMADs form lipid nanoparticles, observable by DLS and transmission electron microscopy, and were used to reconstitute a spin-labeled transmembrane protein, KCNE1. The polymer method reported here is facile and scalable and results in functional and robust polymers capable of forming lipid nanodisks that are stable against a wide pH range and 100 mM magnesium.

Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy.

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using 31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

Routing of thylakoid lumen proteins by the chloroplast twin arginine transport pathway.

Thylakoids are complex sub-organellar membrane systems whose role in photosynthesis makes them critical to life. Thylakoids require the coordinated expression of both nuclear- and plastid-encoded proteins to allow rapid response to changing environmental conditions. Transport of cytoplasmically synthesized proteins to thylakoids or the thylakoid lumen is complex; the process involves transport across up to three membrane systems with routing through three aqueous compartments. Protein transport in thylakoids is accomplished by conserved ancestral prokaryotic plasma membrane translocases containing novel adaptations for the sub-organellar location. This review focuses on the evolutionarily conserved chloroplast twin arginine transport (cpTat) pathway. An overview is provided of known aspects of the cpTat components, energy requirements, and mechanisms with a focus on recent discoveries. Some of the most exciting new studies have been in determining the structural architecture of the membrane complex involved in forming the point of passage for the precursor and binding features of the translocase components. The cpTat system is of particular interest because it transports folded protein domains using only the proton motive force for energy. The implications for mechanism of translocation by recent studies focusing on interactions between membrane Tat components and with the translocating precursor will be discussed.

Tuning the size of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for biophysical studies.

Characterization of membrane proteins is challenging due to the difficulty in mimicking the native lipid bilayer with properly folded and functional membrane proteins. Recently, styrene-maleic acid (StMA) copolymers have been shown to facilitate the formation of disc-like lipid bilayer mimetics that maintain the structural and dynamic integrity of membrane proteins. Here we report the controlled synthesis and characterization of StMA containing block copolymers. StMA polymers with different compositions and molecular weights were synthesized and characterized by size exclusion chromatography (SEC). These polymers act as macromolecular surfactants for 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol (POPG) lipids, forming disc like structures of the lipids with the polymer wrapping around the hydrophobic lipid edge. A combination of dynamic light scattering (DLS), solid-state nuclear magnetic resonance (SSNMR) spectroscopy, and transmission electron microscopy (TEM) was used to characterize the size of the nanoparticles created using these StMA polymers. At a weight ratio of 1.25:1 StMA to lipid, the nanoparticle size created is 28+1nm for a 2:1 ratio, 10+1nm for a 3:1 StMA ratio and 32+1nm for a 4:1 StMA ratio independent of the molecular weight of the polymer. Due to the polymer acting as a surfactant that forms disc like nanoparticles, we term these StMA based block copolymers "RAFT SMALPs". RAFT SMALPs show promise as a new membrane mimetic with different nanoscale sizes, which can be used for a wide variety of biophysical studies of membrane proteins.

Prevalence and causes of preoperative anaemia in elective major surgery patients.

BACKGROUND: Preoperative anaemia is associated with increased morbidity and mortality in surgical patients. Recent national patient blood management guideline recommended screening surgical patients for anaemia, particularly iron deficiency anaemia, without reference to the prevalence of anaemia or iron deficiency anaemia in this patient population. AIMS: To establish the prevalence and cause of preoperative anaemia in elective major surgery patients. METHODS: Patients attending the anaesthetic pre-admission clinics from 1 July 2013 to 30 June 2014 prior to their major elective surgery in our institution were screened for anaemia and iron deficiency by measuring full blood count, iron studies and C-reactive protein. Patients who were anaemic were either further assessed in the haematology clinic or had their medical records reviewed to ascertain the cause of the anaemia. RESULTS: Of 1494 patients, 208 (13.9%) were anaemic, with a male predominance (70.7%); 57 (27.4%) of them had iron deficiency anaemia. Other common causes of anaemia include underlying malignancy (18.3%), end-stage renal failure (11.5%) and other chronic diseases (7.2%). In 53 patients (25.5%), the cause was unknown. Anaemia was most commonly found in patients scheduled for gastrointestinal surgery. CONCLUSION: Preoperative anaemia affects 13.9% of patients undergoing elective major surgery. The most common causes are iron deficiency and chronic diseases. The cause was unexplained in 25.5% of patients with anaemia. The prevalence of anaemia in different surgical specialties may have implications on the approach to screening, particularly in resource-limited areas.

Probing the interaction of the potassium channel modulating KCNE1 in lipid bilayers via solid-state NMR spectroscopy.

KCNE1 is known to modulate the voltage-gated potassium channel α subunit KCNQ1 to generate slowly activating potassium currents. This potassium channel is essential for the cardiac action potential that mediates a heartbeat as well as the potassium ion homeostasis in the inner ear. Therefore, it is important to know the structure and dynamics of KCNE1 to better understand its modulatory role. Previously, the Sanders group solved the three-dimensional structure of KCNE1 in LMPG micelles, which yielded a better understanding of this KCNQ1/KCNE1 channel activity. However, research in the Lorigan group showed different structural properties of KCNE1 when incorporated into POPC/POPG lipid bilayers as opposed to LMPG micelles. It is hence necessary to study the structure of KCNE1 in a more native-like environment such as multi-lamellar vesicles. In this study, the dynamics of lipid bilayers upon incorporation of the membrane protein KCNE1 were investigated using 31 P solid-state nuclear magnetic resonance (NMR) spectroscopy. Specifically, the protein/lipid interaction was studied at varying molar ratios of protein to lipid content. The static 31 P NMR and T1 relaxation time were investigated. The 31 P NMR powder spectra indicated significant perturbations of KCNE1 on the phospholipid headgroups of multi-lamellar vesicles as shown from the changes in the 31 P spectral line shape and the chemical shift anisotropy line width. 31 P T1 relaxation times were shown to be reversely proportional to the molar ratios of KCNE1 incorporated. The 31 P NMR data clearly indicate that KCNE1 interacts with the membrane. Copyright © 2017 John Wiley & Sons, Ltd.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies.

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy.

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

IL-22 regulates iron availability in vivo through the induction of hepcidin.

Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.

Direct interaction between a precursor mature domain and transport component Tha4 during twin arginine transport of chloroplasts.

Proteins destined for the thylakoid lumen of chloroplasts must cross three membranes en route. The chloroplast twin arginine translocation (cpTat) system facilitates the transport of about one-half of all proteins that cross the thylakoid membrane in chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the protonmotive force generated during photosynthesis for energy. However, key details, such as the structure and composition of the translocation pore, are still unknown. One of the three transmembrane cpTat components, Tha4, is thought to function as the pore by forming an oligomer. Yet, little is known about the topology of Tha4 in thylakoid, and little work has been done to detect precursor-Tha4 interactions, which are expected if Tha4 is the pore. Here, we present evidence of the interaction of the precursor with Tha4 under conditions leading to transport, using cysteine substitutions on the precursor and Tha4 and disulfide bond formation in pea (Pisum sativum). The mature domain of a transport-competent precursor interacts with the amphipathic helix and amino terminus of functional Tha4 under conditions leading to transport. Detergent solubilization of thylakoids post cross linking and blue-native polyacrylamide gel electrophoresis analysis shows that Tha4 is found in a complex containing precursor and Hcf106 (i.e. the cpTat translocase). Affinity precipitation of the cross-linked complex via Tha4 clearly demonstrates that the interaction is with full-length precursor. How these data suggest a role for Tha4 in cpTat transport is discussed.

Dynamic protein-protein interactions of KCNQ1 and KCNE1 measured by EPR line shape analysis.

KCNQ1, also known as Kv7.1, is a voltage gated potassium channel that associates with the KCNE protein family. Mutations in this protein has been found to cause a variety of diseases including Long QT syndrome, a type of cardiac arrhythmia where the QT interval observed on an electrocardiogram is longer than normal. This condition is often aggravated during strenuous exercise and can cause fainting spells or sudden death. KCNE1 is an ancillary protein that interacts with KCNQ1 in the membrane at varying molar ratios. This interaction allows for the flow of potassium ions to be modulated to facilitate repolarization of the heart. The interaction between these two proteins has been studied previously with cysteine crosslinking and electrophysiology. In this study, electron paramagnetic resonance (EPR) spectroscopy line shape analysis in tandem with site directed spin labeling (SDSL) was used to observe changes in side chain dynamics as KCNE1 interacts with KCNQ1. KCNE1 was labeled at different sites that were found to interact with KCNQ1 based on previous literature, along with sites outside of that range as a control. Once labeled KCNE1 was incorporated into vesicles, KCNQ1 (helices S1-S6) was titrated into the vesicles. The line shape differences observed upon addition of KCNQ1 are indicative of an interaction between the two proteins. This method provides a first look at the interactions between KCNE1 and KCNQ1 from a dynamics perspective using the full transmembrane portion of KCNQ1.

The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport.

Twin arginine transport (Tat) systems transport folded proteins using proton-motive force as sole energy source. The thylakoid Tat system comprises three membrane components. A complex composed of cpTatC and Hcf106 is the twin arginine signal peptide receptor. Signal peptide binding triggers assembly of Tha4 for the translocation step. Tha4 is thought to serve as the protein-conducting element, and the topology it adopts during transport produces the transmembrane passageway. We analyzed Tha4 topology and conformation in actively transporting translocases and compared that with Tha4 in nontransporting membranes. Using cysteine accessibility labeling techniques and diagnostic protease protection assays, we confirm an overall N(OUT)-C(IN) topology for Tha4 that is maintained under transport conditions. Significantly, the amphipathic helix (APH) and C-tail exhibited substantial changes in accessibility when actively engaged in protein transport. Compared with resting state, cysteines within the APH became less accessible to stromally applied modifying reagent. The APH proximal C-tail, although still accessible to Cys-directed reagents, was much less accessible to protease. We attribute these changes in accessibility to indicate the Tha4 conformation that is adopted in the translocase primed for translocation. We propose that in the primed translocase, the APH partitions more extensively and uniformly into the membrane interface and the C-tails pack closer together in a mesh-like network. Implications for the mode by which the substrate protein crosses the bilayer are discussed.

Arabidopsis ETHE1 encodes a sulfur dioxygenase that is essential for embryo and endosperm development.

Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants. Arabidopsis ETHE1 is localized in the mitochondrion and exhibits sulfur dioxygenase activity. Seeds homozygous for a DNA insertion in ETHE1 exhibit alterations in endosperm development that are accompanied by a delay in embryo development followed by embryo arrest by early heart stage. Strong ETHE1 labeling was observed in the peripheral and chalazal endosperm of wild-type seeds prior to cellularization. Therefore, ETHE1 appears to play an essential role in regulating sulfide levels in seeds.

Chronic seizures induce sex-specific cognitive deficits with loss of presenilin 2 function.

Patients with early-onset Alzheimer's disease (EOAD) are at elevated risk for seizures, including patients with presenilin 2 (PSEN2) variants. Like people with epilepsy, uncontrolled seizures may worsen cognitive function in AD. While the relationship between seizures and amyloid beta accumulation has been more thoroughly investigated, the role of other drivers of seizure susceptibility in EOAD remain relatively understudied. We therefore sought to define the impact of loss of normal PSEN2 function and chronic seizures on cognitive function in the aged brain. Male and female PSEN2 KO and age- and sex-matched wild-type (WT) mice were sham or corneal kindled beginning at 6-months-old. Kindled and sham-kindled mice were then challenged up to 6 weeks later in a battery of cognitive tests: non-habituated open field (OF), T-maze spontaneous alternation (TM), and Barnes maze (BM), followed by immunohistochemistry for markers of neuroinflammation and neuroplasticity. PSEN2 KO mice required significantly more stimulations to kindle (males: p < 0.02; females: p < 0.02) versus WT. Across a range of behavioral tests, the cognitive performance of kindled female PSEN2 KO mice was most significantly impaired versus age-matched WT females. Male BM performance was generally worsened by seizures (p = 0.038), but loss of PSEN2 function did not itself worsen cognitive performance. Conversely, kindled PSEN2 KO females made the most BM errors (p = 0.007). Chronic seizures also significantly altered expression of hippocampal neuroinflammation and neuroplasticity markers in a sex-specific manner. Chronic seizures may thus significantly worsen hippocampus-dependent cognitive deficits in aged female, but not male, PSEN2 KO mice. Our work suggests that untreated focal seizures may worsen cognitive burden with loss of normal PSEN2 function in a sex-related manner.

Human microglia show unique transcriptional changes in Alzheimer's disease.

Microglia, the innate immune cells of the brain, influence Alzheimer's disease (AD) progression and are potential therapeutic targets. However, microglia exhibit diverse functions, the regulation of which is not fully understood, complicating therapeutics development. To better define the transcriptomic phenotypes and gene regulatory networks associated with AD, we enriched for microglia nuclei from 12 AD and 10 control human dorsolateral prefrontal cortices (7 males and 15 females, all aged >60 years) before single-nucleus RNA sequencing. Here we describe both established and previously unrecognized microglial molecular phenotypes, the inferred gene networks driving observed transcriptomic change, and apply trajectory analysis to reveal the putative relationships between microglial phenotypes. We identify microglial phenotypes more prevalent in AD cases compared with controls. Further, we describe the heterogeneity in microglia subclusters expressing homeostatic markers. Our study demonstrates that deep profiling of microglia in human AD brain can provide insight into microglial transcriptional changes associated with AD.