Estelle R. Bennett

The membrane topology of GAT-1, a (Na+ + Cl-)-coupled gamma-aminobutyric acid transporter from rat brain.

The membrane topology of GAT-1, a sodium- and chloride-coupled γ-aminobutyric acid transporter from rat brain, has been probed using N-glycosylation scanning mutagenesis. Overall, the results support the theoretical 12-transmembrane segment model. This model (based on hydropathy analysis) was originally proposed for GAT-1 and adopted for all other members of the sodium- and chloride-dependent neurotransmitter transporter superfamily. However, our data indicate that the loop connecting putative transmembrane domains 2 and 3, which was predicted to be located intracellularly, can be glycosylated in vivo. Furthermore, studies with permeant and impermeant methanesulfonate reagents suggest that cysteine 74, located in the hydrophilic loop connecting transmembrane domains 1 and 2, is intracellular rather than extracellular. We present a model in which the topology deviates from the theoretical one in the amino-terminal third of the transporter. It also contains 12 transmembrane segments, but the highly conserved domain 1 does not form a conventional transmembrane α-helix.

The Butyrylcholinesterase K Variant Confers Structurally Derived Risks for Alzheimer Pathology

The K variant of butyrylcholinesterase (BChE-K, 20% incidence) is a long debated risk factor for Alzheimer disease (AD). The A539T substitution in BChE-K is located at the C terminus, which is essential both for BChE tetramerization and for its capacity to attenuate β-amyloid (Aβ) fibril formation. Here, we report that BChE-K is inherently unstable as compared with the “usual” BChE (BChE-U), resulting in reduced hydrolytic activity and predicting prolonged acetylcholine maintenance and protection from AD. A synthetic peptide derived from the C terminus of BChE-K (BSP-K), which displayed impaired intermolecular interactions, was less potent in suppressing Aβ oligomerization than its BSP-U counterpart. Correspondingly, highly purified recombinant human rBChE-U monomers suppressed β-amyloid fibril formation less effectively than dimers, which also protected cultured neuroblastoma cells from Aβ neurotoxicity. Dual activity structurally derived changes due to the A539T substitution can thus account for both neuroprotective characteristics caused by sustained acetylcholine levels and elevated AD risk due to inefficient interference with amyloidogenic processes.

Competing targets of microRNA-608 affect anxiety and hypertension

MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA–target interaction can simultaneously affect multiple other miRNA–target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3′-untranslated region (3′UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.

Activation of the alternative NFκB pathway improves disease symptoms in a model of Sjogren's syndrome.

The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogrens syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9−/− mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-κB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogrens syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-κB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9−/− mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogrens disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-κB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-κB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogrens syndrome and may be applicable to additional autoimmune syndromes.

AChE and RACK1 promote the anti-inflammatory properties of fluoxetine.

Selective serotonin reuptake inhibitors (SSRIs) show anti-inflammatory effects, suggesting a possible interaction with both Toll-like-receptor 4 (TLR4) responses and cholinergic signaling through as yet unclear molecular mechanism(s). Our results of structural modeling support the concept that the antidepressant fluoxetine physically interacts with the TLR4–myeloid differentiation factor-2 complex at the same site as bacterial lipopolysaccharide (LPS). We also demonstrate reduced LPS-induced pro-inflammatory interleukin-6 and tumor necrosis factor alpha in human peripheral blood mononuclear cells preincubated with fluoxetine. Furthermore, we show that fluoxetine intercepts the LPS-induced decreases in intracellular acetylcholinesterase (AChE-S) and that AChE-S interacts with the nuclear factor kappa B (NFκB)-activating intracellular receptor for activated C kinase 1 (RACK1). This interaction may prevent NFκB activation by residual RACK1 and its interacting protein kinase PKCβII. Our findings attribute the anti-inflammatory properties of SSRI to surface membrane interference with leukocyte TLR4 activation accompanied by intracellular limitation of pathogen-inducible changes in AChE-S, RACK1, and PKCβII.

Butyrylcholinesterase interactions with amylin may protect pancreatic cells in metabolic syndrome.

The metabolic syndrome (MetS) is a risk factor for type 2 diabetes mellitus (T2DM). However, the mechanisms underlying the transition from MetS to T2DM are unknown. Our goal was to study the potential contribution of butyrylcholinesterase (BChE) to this process. We first determined the hydrolytic activity of BChE in serum from MetS, T2DM and healthy individuals. The ‘Kalow’ variant of BChE (BChE‐K), which has been proposed to be a risk factor for T2DM, was genotyped in the last two groups. Our results show that in MetS patients serum BChE activity is elevated compared to T2DM patients and healthy controls (P < 0.001). The BChE‐K genotype showed similar prevalence in T2DM and healthy individuals, excluding this genotype as a risk factor for T2DM. However, the activity differences remained unexplained. Previous results from our laboratory have shown BChE to attenuate the formation of β‐amyloid fibrils, and protect cultured neurons from their cytotoxicity. Therefore, we next studied the in vitro interactions between recombinant human butyrylcholinesterase and amylin by surface plasmon resonance, Thioflavine T fluorescence assay and cross‐linking, and used cultured pancreatic β cells to test protection by BChE from amylin cytotoxicity. We demonstrate that BChE interacts with amylin through its core domain and efficiently attenuates both amylin fibril and oligomer formation. Furthermore, application of BChE to cultured β cells protects them from amylin cytotoxicity. Taken together, our results suggest that MetS‐associated BChE increases could protect pancreatic β‐cells in vivo by decreasing the formation of toxic amylin oligomers.

miRNA-132 induces hepatic steatosis and hyperlipidaemia by synergistic multitarget suppression

Objective Both non-alcoholic fatty liver disease (NAFLD) and the multitarget complexity of microRNA (miR) suppression have recently raised much interest, but the in vivo impact and context-dependence of hepatic miR-target interactions are incompletely understood. Assessing the relative in vivo contributions of specific targets to miR-mediated phenotypes is pivotal for investigating metabolic processes. Design We quantified fatty liver parameters and the levels of miR-132 and its targets in novel transgenic mice overexpressing miR-132, in liver tissues from patients with NAFLD, and in diverse mouse models of hepatic steatosis. We tested the causal nature of miR-132 excess in these phenotypes by injecting diet-induced obese mice with antisense oligonucleotide suppressors of miR-132 or its target genes, and measured changes in metabolic parameters and transcripts. Results Transgenic mice overexpressing miR-132 showed a severe fatty liver phenotype and increased body weight, serum low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and liver triglycerides, accompanied by decreases in validated miR-132 targets and increases in lipogenesis and lipid accumulation-related transcripts. Likewise, liver samples from both patients with NAFLD and mouse models of hepatic steatosis or non-alcoholic steatohepatitis (NASH) displayed dramatic increases in miR-132 and varying decreases in miR-132 targets compared with controls. Furthermore, injecting diet-induced obese mice with anti-miR-132 oligonucleotides, but not suppressing its individual targets, reversed the hepatic miR-132 excess and hyperlipidemic phenotype. Conclusions Our findings identify miR-132 as a key regulator of hepatic lipid homeostasis, functioning in a context-dependent fashion via suppression of multiple targets and with cumulative synergistic effects. This indicates reduction of miR-132 levels as a possible treatment of hepatic steatosis.

Quantitative analysis of influenza M2 channel blockers.

The influenza M2 H(+) channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed K(s) measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system.

TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis

See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.

Alzheimer's brains show inter-related changes in RNA and lipid metabolism

Alzheimers disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with ADs cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3′-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in ADs lipid processing, alternative splicing and 3′-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications.