Lipocalin-2 protects the brain during inflammatory conditions.

Sepsis is a prevalent health issue that can lead to central nervous system (CNS) inflammation with long-term behavioral and cognitive alterations. Using unbiased proteomic profiling of over 100 different cytokines, we found that Lipocalin-2 (LCN2) was the most substantially elevated protein in the CNS after peripheral administration of lipopolysaccharide (LPS). To determine whether the high level of LCN2 in the CNS is protective or deleterious, we challenged Lcn2-/- mice with peripheral LPS and determined effects on behavior and neuroinflammation. At a time corresponding to peak LCN2 induction in wild-type (WT) mice injected with LPS, Lcn2-/- mice challenged with LPS had exacerbated levels of pro-inflammatory cytokines and exhibited significantly worsened behavioral phenotypes. To determine the extent of global inflammatory changes dependent upon LCN2, we performed an RNAseq transcriptomic analysis. Compared with WT mice injected with LPS, Lcn2-/- mice injected with LPS had unique transcriptional profiles and significantly elevated levels of multiple pro-inflammatory molecules. Several LCN2-dependent pathways were revealed with this analysis including, cytokine and chemokine signaling, nucleotide-binding oligomerization domain-like receptor signaling and Janus kinase-signal transducer and activator of transcription signaling. These findings demonstrate that LCN2 serves as a potent protective factor in the CNS in response to systemic inflammation and may be a potential candidate for limiting sepsis-related CNS sequelae.

Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice.

Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinson's disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia.

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Exercise training-induced modification of the gut microbiota persists after microbiota colonization and attenuates the response to chemically-induced colitis in gnotobiotic mice.

Exercise reduces the risk of inflammatory disease by modulating a variety of tissue and cell types, including those within the gastrointestinal tract. Recent data indicates that exercise can also alter the gut microbiota, but little is known as to whether these changes affect host function. Here, we use a germ-free (GF) animal model to test whether exercise-induced modifications in the gut microbiota can directly affect host responses to microbiota colonization and chemically-induced colitis. Donor mice (n = 19) received access to a running wheel (n = 10) or remained without access (n = 9) for a period of six weeks. After euthanasia, cecal contents were pooled by activity treatment and transplanted into two separate cohorts of GF mice. Two experiments were then conducted. First, mice were euthanized five weeks after the microbiota transplant and tissues were collected for analysis. A second cohort of GF mice were colonized by donor microbiotas for four weeks before dextran-sodium-sulfate was administered to induce acute colitis, after which mice were euthanized for tissue analysis. We observed that microbial transplants from donor (exercised or control) mice led to differences in microbiota ß-diversity, metabolite profiles, colon inflammation, and body mass in recipient mice five weeks after colonization. We also demonstrate that colonization of mice with a gut microbiota from exercise-trained mice led to an attenuated response to chemical colitis, evidenced by reduced colon shortening, attenuated mucus depletion and augmented expression of cytokines involved in tissue regeneration. Exercise-induced modifications in the gut microbiota can mediate host-microbial interactions with potentially beneficial outcomes for the host.

5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor.

The present study examines the interaction of the neurotransmitter 5-hydroxytryptamine (5-HT) with muscle-type nicotinic acetylcholine receptors. 5-HT inhibits the initial rate of [125I]alpha-bungarotoxin binding to Torpedo acetylcholine receptor membranes (IC(50)=8.5+/-0.32 mM) and [3H]5-HT can be photoincorporated into acetylcholine receptor subunits, with labeling of the alpha-subunit inhibitable by both agonists and competitive antagonists. Within the agonist-binding domain, [3H]5-HT photoincorporates into alphaTyr(190), alphaCys(192) and alphaCys(193). Functional studies using the human clonal cell line TE671/RD, show that 5-HT is a weak inhibitor (IC(50)=1.55+/-0.25 mM) of acetylcholine receptor activity. In this regard, agonist-response profiles in the absence and presence of 5-HT indicate a noncompetitive mode of inhibition. In addition, 5-HT displaces high affinity [3H]thienylcyclohexylpiperidine binding to the desensitized Torpedo acetylcholine receptor channel (IC(50)=1.61+/-0.07 mM). Collectively, these results indicate that 5-HT interacts weakly with the agonist recognition site and inhibits receptor function noncompetitively by binding to the acetylcholine receptor channel.

Noncompetitive functional inhibition at diverse, human nicotinic acetylcholine receptor subtypes by bupropion, phencyclidine, and ibogaine.

Nicotinic acetylcholine receptors (nAChR) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes the acute functional effects of bupropion, phencyclidine, and ibogaine on two human nAChR subtypes. Function of muscle-type nAChR (alpha1 beta gamma delta) in TE671/RD cells or of ganglionic nAChR (alpha3 beta4 alpha5+/-beta2) in SH-SY5Y neuroblastoma cells was measured with 86Rb+ efflux assays. Functional blockade of human muscle-type and ganglionic nAChR is produced by each of the drugs in the low to intermediate micromolar range. Functional blockade is insurmountable by increasing agonist concentrations in TE671/RD and SH-SY5Y cells for each of these drugs, suggesting noncompetitive inhibition of nAChR function. Based on these findings, we hypothesize that nAChR are targets of diverse substances of abuse and agents used in antiaddiction/smoking cessation strategies. We also hypothesize that nAChR play heretofore underappreciated roles in depression and as targets for clinically useful antidepressants.

Antidepressants noncompetitively inhibit nicotinic acetylcholine receptor function.

Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.

Differences in the Abeta40/Abeta42 ratio associated with cerebrospinal fluid lipoproteins as a function of apolipoprotein E genotype.

The epsilon4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD). ApoE, which is important for lipid metabolism, is also a major constituent of cerebrospinal fluid (CSF) lipoproteins (LPs). Although ApoE in the CSF is derived from the central nervous system, the relation between LP metabolism in plasma and CSF is not clear. Soluble amyloid-beta (Abeta) protein may normally be associated with CSF LPs. It is converted in AD to a fibrillar form in brain parenchyma. ApoE and CSF LPs may regulate this process. The purpose of this study was to characterize CSF LPs from healthy, cognitively normal, fasted, elderly individuals at different risk for AD based on ApoE genotype. Lipid composition of CSF LPs did not differ with ApoE genotype. Interestingly, plasma and CSF high-density lipoprotein (HDL) cholesterol and apolipoprotein AI (ApoAI) levels were correlated. Importantly, as assessed by size-exclusion chromatography, Abeta in CSF coeluted in fractions containing LPs and was influenced by ApoE genotype: E4-positive subjects displayed significant elevations in Abeta40/Abeta42 ratios. These results suggest that plasma ApoAI/HDL levels can influence CSF ApoAI/HDL levels and that interactions between Abeta and central nervous system LPs may reflect changes in brain Abeta metabolism before the onset of clinical disease.

Neurotoxicity of channel mutations in heterologously expressed alpha7-nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) composed of chick alpha7 subunits mutated to threonine at amino acid valine-251 in the putative channel-lining M2 domain were expressed heterologously in several neuron-like and non-neuronal mammalian cell lines. Expression of mutant alpha7-nAChR is toxic to neuron-like cells of the human neuroblastoma cell lines SH-SY5Y and IMR-32, but not to several other cell types. Growth in the presence of the alpha7-nAChR antagonist methyllycaconitine (MLA) protects against neurotoxicity, as does gradual downregulation of functional, mutant alpha7-nAChR in surviving transfected SH-SY5Y cells. Relative to wild-type alpha7-nAChR, functional alpha7-nAChR mutants show a higher affinity for agonists, slower rates of desensitization, and sensitivity to dihydro-beta-erythroidine (DHbetaE) as an agonist, but they retain sensitivity to MLA as a competitive antagonist. These findings demonstrate that expression of hyperfunctional, mutant forms of Ca2+-permeable alpha7-nAChR is toxic to neuron-like cells.

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.