Levels of Soluble Interleukin 6 Receptor and Asp358Ala Are Associated With Cognitive Performance and Alzheimer Disease Biomarkers.

BACKGROUND AND OBJECTIVES: Alzheimer disease (AD) is a neurodegenerative disease process manifesting clinically with cognitive impairment and dementia. AD pathology is complex, and in addition to plaques and tangles, neuroinflammation is a consistent feature. Interleukin (IL) 6 is a multifaceted cytokine involved in a plethora of cellular mechanisms including both anti-inflammatory and inflammatory processes. IL6 can signal classically through the membrane-bound receptor or by IL6 trans-signaling forming a complex with the soluble IL6 receptor (sIL6R) and activating membrane-bound glycoprotein 130 on cells not expressing IL6R. IL6 trans-signaling has been demonstrated as the primary mechanism of IL6-mediated events in neurodegenerative processes. In this study, we performed a cross-sectional analysis to investigate whether inheritance of a genetic variation in the IL6R gene and associated elevated sIL6R levels in plasma and CSF were associated with cognitive performance. METHODS: We genotyped the IL6R rs2228145 nonsynonymous variant (Asp358Ala) and assayed IL6 and sIL6R concentrations in paired samples of plasma and CSF obtained from 120 participants with normal cognition, mild cognitive impairment, or probable AD enrolled in the Wake Forest Alzheimer's Disease Research Center's Clinical Core. IL6 rs2228145 genotype and measures of plasma IL6 and sIL6R were assessed for relationships with cognitive status and clinical data, including the Montreal Cognitive Assessment (MoCA), modified Preclinical Alzheimer's Cognitive Composite (mPACC), cognitive domain scores obtained from the Uniform Data Set, and CSF concentrations of phosphoTauT181 (pTau181), ß-amyloid (Aß) Aß40 and Aß42 concentrations. RESULTS: We found that inheritance of the IL6R Ala358 variant and elevated sIL6R levels in plasma and CSF were correlated with lower mPACC, MoCA and memory domain scores, increases in CSF pTau181, and decreases in the CSF Aß42/40 ratio in both unadjusted and covariate-adjusted statistical models. DISCUSSION: These data suggest that IL6 trans-signaling and the inheritance of the IL6R Ala358 variant are related to reduced cognition and greater levels of biomarkers for AD disease pathology. Follow-up prospective studies are necessary, as patients who inherit IL6R Ala358 may be identified as ideally responsive to IL6 receptor-blocking therapies.

Interleukin 6 (IL6) level is a biomarker for functional disease progression within IL6R358Ala variant groups in amyotrophic lateral sclerosis patients.

Interleukin-6 (IL6) expression increases in atrophying muscles and lung tissue during compromised function. Considering ALS patients undergo these same pathological changes, IL6 levels may be relevant for prognostication and treatment. The amount of soluble IL6 receptor, dictated by the IL6R358Ala variant, and local tissue environment in which IL6 signaling occurs is known to influence the ultimate effects of IL6 in multiple diseases. In this longitudinal study, we show that serum IL6 levels negatively correlate both with the patient's functional status as measured by the overall ALSFRS-R and subscores, and with respiratory function as measured by the percent predicted FVC (ppFVC). The correlations are only present in the two-thirds of patients who carry the IL6R358Ala variant that mediates pro-inflammatory transsignaling in the cases of ALSFRS-R limb and respiratory subscores and ppFVC. These results suggest that some observed associations between IL6 and ALS are driven by the subset of patients carrying the IL6R358Ala variant and thus that any IL6-targeted therapeutic approaches may be more advantageous when aimed at this group. Specifically, with relation to respiratory decline, these patients may benefit from closer respiratory follow-up and early initiation of noninvasive ventilation.

Automated Planar Patch-Clamp Recording of P2X Receptors.

P2X receptors are a structurally and functionally distinctive family of ligand-gated ion channels that play important roles in mediating extracellular adenosine 5'-triphosphate (ATP) signaling in diverse physiological and pathophysiological processes. For several decades, the "manual" patch-clamp technique was regarded as the gold standard assay for investigating ion channel properties. More recently, breakthroughs in the development of automated patch-clamp technologies are enabling the study of ion channels, with much greater throughput capacities. These automated platforms, of which there are many, generate consistent, reliable, high-fidelity data. This chapter demonstrates the versatility of one of these technologies for ligand-gated ion channels, with a particular emphasis on protocols that address some of the issues of receptor desensitization that are commonly associated with P2X receptor-mediated currents.

IL6 receptor358Ala variant and trans-signaling are disease modifiers in amyotrophic lateral sclerosis.

OBJECTIVE: To test the hypothesis that patients with amyotrophic lateral sclerosis (ALS) inheriting the common interleukin 6 receptor (IL6R) coding variant (Asp358Ala, rs2228145, C allele) have associated increases in interleukin 6 (IL6) and IL6R levels in serum and CSF and faster disease progression than noncarriers. METHODS: An observational, case-control study of paired serum and CSF of 47 patients with ALS, 46 healthy, and 23 neurologic disease controls from the Northeastern ALS Consortium Biofluid Repository (cohort 1) was performed to determine serum levels of IL6, sIL6R, and soluble glycoprotein 130 and compared across groups and IL6R genotype. Clinical data regarding disease progression from a separate cohort of 35 patients with ALS from the Wake Forest ALS Center (cohort 2) were used to determine change in ALSFRS-R scores by genotype. RESULTS: Patients with ALS had increased CSF IL6 levels compared with healthy (p < 0.001) and neurologic (p = 0.021) controls. Patients with ALS also had increased serum IL6 compared with healthy (p = 0.040) but not neurologic controls. Additive allelic increases in serum IL6R were observed in all groups (average increase of 52% with the presence of the IL6R C allele; p < 0.001). However, only subjects with ALS had significantly increased CSF sIL6R levels compared with controls (p < 0.001). When compared across genotypes, only patients with ALS inheriting the IL6R C allele exhibit increased CSF IL6. ALSFRS-R scores decreased more in patients with ALS with the IL6R C allele than in those without (p = 0.019). CONCLUSIONS: Theses results suggest that for individuals inheriting the IL6R C allele, the cytokine exerts a disease- and location-specific role in ALS. Follow-up, prospective studies are necessary, as this subgroup of patients may be identified as ideally responsive to IL6 receptor-blocking therapies.

Extracellular heat shock proteins in neurodegenerative diseases: New perspectives.

One pathological hallmark of neurodegenerative diseases and CNS trauma is accumulation of insoluble, hydrophobic molecules and protein aggregations found both within and outside cells. These may be the consequences of an inadequate or overburdened cellular response to stresses resulting from potentially toxic changes in extra- and intracellular environments. The upregulated expression of heat shock proteins (HSPs) is one example of a highly conserved cellular response to both internal and external stress. Intracellularly these proteins act as chaperones, playing vital roles in the folding of nascent polypeptides, the translocation of proteins between subcellular locations, and the disaggregation of misfolded or aggregated proteins in an attempt to maintain cellular proteostasis during both homeostatic and stressful conditions. While the predominant study of the HSPs has focused on their intracellular chaperone functions, it remains unclear if all neuronal populations can mount a complete stress response. Alternately, it is now well established that some members of this family of proteins can be secreted by nearby, non-neuronal cells to act in the extracellular environment. This review addresses the current literature detailing the use of exogenous and extracellular HSPs in the treatment of cellular and animal models of neurodegenerative disease. These findings offer a new measure of therapeutic potential to the HSPs, but obstacles must be overcome before they can be efficiently used in a clinical setting.

Purinergic receptors P2RX4 and P2RX7 in familial multiple sclerosis.

Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study, we set out to evaluate whether rare coding variants of major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T [p.T205M], P2RX7 rs201921967:A>G [p.N361S], and P2RX4 rs765866317:G>A [p.G135S]) segregating with disease in a multi-incident family with six family members diagnosed with MS (logarithm of odds = 3.07). Functional analysis of this haplotype in HEK293 cells revealed impaired P2X7 surface expression (P < 0.01), resulting in over 95% inhibition of adenosine triphosphate (ATP)-induced pore function (P < 0.001) and a marked reduction in phagocytic ability (P < 0.05). In addition, transfected cells showed 40% increased peak ATP-induced inward current (P < 0.01), and a greater Ca2+ response to the P2X4 135S variant compared with wild type (P < 0.0001). Our study nominates rare genetic variants in P2RX4 and P2RX7 as major genetic contributors to disease, further supporting a role for these purinergic receptors in MS and the disruption of transmembrane cation channels leading to impairment of phagocytosis as the pathological mechanisms of disease.

The applications of the novel polymeric fluoropyrimidine F10 in cancer treatment: current evidence.

F10 is a novel polymeric fluoropyrimidine drug candidate with strong anticancer activity in multiple preclinical models. F10 has strong potential for impacting cancer treatment because it displays high cytotoxicity toward proliferating malignant cells with minimal systemic toxicities thus providing an improved therapeutic window relative to traditional fluoropyrimidine drugs, such as 5-fluorouracil. F10 has a unique mechanism that involves dual targeting of thymidylate synthase and Top1. In this review, the authors provide an overview of the studies that revealed the novel aspects of F10's cytotoxic mechanism and summarize results obtained in preclinical models of acute myeloid leukemia, acute lymphocytic leukemia, glioblastoma and prostate cancer that demonstrate the strong potential of F10 to improve treatment outcomes.

Selective anti-tumor activity of the novel fluoropyrimidine polymer F10 towards G48a orthotopic GBM tumors.

F10 is a novel anti-tumor agent with minimal systemic toxicity in vivo and which displays strong cytotoxicity towards glioblastoma (GBM) cells in vitro. Here we investigate the cytotoxicity of F10 towards GBM cells and evaluate the anti-tumor activity of locally-administered F10 towards an orthotopic xenograft model of GBM. The effects of F10 on thymidylate synthase (TS) inhibition and Topoisomerase 1 (Top1) cleavage complex formation were evaluated using TS activity assays and in vivo complex of enzyme bioassays. Cytotoxicity of F10 towards normal brain was evaluated using cortices from embryonic (day 18) mice. F10 displays minimal penetrance of the blood-brain barrier and was delivered by intra-cerebral (i.c.) administration and prospective anti-tumor response towards luciferase-expressing G48a human GBM tumors in nude mice was evaluated using IVIS imaging. Histological examination of tumor and normal brain tissue was used to assess the selectivity of anti-tumor activity. F10 is cytotoxic towards G48a, SNB-19, and U-251 MG GBM cells through dual targeting of TS and Top1. F10 is not toxic to murine primary neuronal cultures. F10 is well-tolerated upon i.c. administration and induces significant regression of G48a tumors that is dose-dependent. Histological analysis from F10-treated mice revealed tumors were essentially completely eradicated in F10-treated mice while vehicle-treated mice displayed substantial infiltration into normal tissue. F10 displays strong efficacy for GBM treatment with minimal toxicity upon i.c. administration establishing F10 as a promising drug-candidate for treating GBM in human patients.

A single-dose resveratrol treatment in a mouse model of amyotrophic lateral sclerosis.

The underlying causes of denervation of the neuromuscular junction and eventual motor neuron death in amyotrophic lateral sclerosis (ALS) have not been resolved. The superoxide dismutase 1 (SOD1)(G93A) mutant mouse is a frequently used animal model of ALS. We hypothesized that resveratrol (RSV), a polyphenolic molecule that enhances mammalian NAD(+)-dependent SIRT1 deacetylases and may increase life span, would improve motor function and survival in the SOD1 mouse model via modulation of p53 acetylation. Data were collected for mean survival times, neuromuscular performance on the ROTOR-ROD™ (San Diego Instruments, San Diego, CA, USA), body weight, and p53 acetylation. Mean survival times were not statistically different (P=.23) between control and experimental (RSV-fed) groups (mean +/- SD, control [n=11] 138 +/- 6 days vs. experimental [n=10] 135 +/- 8 days). Performance was not significantly different between groups at time points corresponding to 50%, 80%, and 90% mean life span (P=.46), nor did RSV treatment attenuate body weight loss. Thus although manipulation of SIRT1 deacetylase activity has effects at the protein level in healthy aging organisms, we conclude that RSV treatment does not lead to functional improvement or increased longevity in a mouse model of ALS. We speculate that RSV-mediated modulation of p53 acetylation is either incapable of increasing or insufficient to increase motor performance and longevity in this model of ALS.

Short-term stimulation of calcium-permeable transient receptor potential canonical 5-containing channels by oxidized phospholipids.

OBJECTIVE: To determine whether calcium-permeable channels are targets for the oxidized phospholipids: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC). METHODS AND RESULTS: Oxidized phospholipids are key factors in inflammation and associated diseases, including atherosclerosis; however, the initial reception mechanisms for cellular responses to the factors are poorly understood. Low micromolar concentrations of PGPC and POVPC evoked increases in intracellular calcium in human embryonic kidney 293 cells that overexpressed human transient receptor potential canonical 5 (TRPC5) but not human TRP melastatin (TRPM) 2 or 3. The results of electrophysiological experiments confirmed stimulation of TRPC5. To investigate relevance to endogenous channels, we studied proliferating vascular smooth muscle cells from patients undergoing coronary artery bypass surgery. PGPC and POVPC elicited calcium entry that was inhibited by anti-TRPC5 or anti-TRPC1 antibodies or dominant-negative mutant TRPC5. Calcium release did not occur. The effect was functionally relevant because it enhanced cell migration. The actions of PGPC and POVPC depended on G(i/o) proteins but not on previously identified G protein-coupled receptors for oxidized phospholipids. CONCLUSIONS: Stimulation of calcium-permeable TRPC5-containing channels may be an early event in cellular responses to oxidized phospholipids that couples to cell migration and requires an unidentified G protein-coupled receptor.

Interactions, functions, and independence of plasma membrane STIM1 and TRPC1 in vascular smooth muscle cells.

Stromal interaction molecule 1 (STIM1) is a predicted single membrane-spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane-spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation.

Inhibitory interaction between P2X4 and GABA(C) rho1 receptors.

Reciprocal functional inhibition between P2X and GABA(A/C) receptors represents a novel mechanism fine-tuning neuronal excitability. However, the participating receptors and underlying mechanisms are not fully understood. P2X(4) receptor is widely found in neurons that express GABA(C) rho1 receptor. Thus, we co-expressed P2X(4) and rho1 receptors in HEK293 cells and, using patch-clamp recording, examined whether they have mutual functional inhibition. Currents evoked by simultaneous application of ATP and GABA (I(ATP+GABA)) were significantly smaller compared to the addition of I(ATP) and I(GABA). Furthermore, I(ATP) were strongly suppressed during rho1 receptor activation. Similarly, I(GABA) were greatly attenuated during P2X(4) receptor activation. Such mutual inhibition was absent in cells only expressing P2X(4) or rho1 receptor. Taken together, these functional data support negative cross-talk between P2X(4) and rho1 receptors.

Phosphorylation of c-Jun in avian and mammalian motoneurons in vivo during programmed cell death: an early reversible event in the apoptotic cascade.

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo. We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.

Transcription of the M1 muscarinic receptor gene in neurons and neuronal progenitors of the embryonic rat forebrain.

Development of the nervous system is accompanied by expansion and differentiation of the neuronal progenitors within the embryonic neuroepithelium. Although the role of growth factors in this process is well documented, there is increasing evidence for a role of neurotransmitters. Acetylcholine is known to exert many actions on developing neural cells, but its potential role in neurogenesis is unclear. Here, we show that the M1 muscarinic acetylcholine receptor is expressed in the neuroepithelium of the rat forebrain, where it is found on both nestin+ progenitor cells and TuJ1+ newly differentiated neurons. Furthermore, transcription is governed, at least in part, by regulatory cis elements that are also responsible for driving transcription in neuroblastoma cells. This represents the first demonstration of M1 receptors on neuronal progenitor cells and supports the notion that M1 muscarinic receptors may play a role in development of the nervous system prior to the onset of synaptogenesis and their subsequent role in neurotransmission.

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus.

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.