by
Lori N. Eidson;
George T. Kannarkat;
Christopher J. Barnum;
Jianjun Chang;
Jaegwon Chung;
Chelsea Caspell-Garcia;
Peggy Taylor;
Brit Mollenhauer;
Michael G. Schlossmacher;
Larry Ereshefsky;
Mark Yen;
Catherine Kopil;
Mark Frasier;
Kenneth Marek;
Vicki Hertzberg;
MariadeLourdes Tansey
Background: Efforts to identify fluid biomarkers of Parkinson's disease (PD) have intensified in the last decade. As the role of inflammation in PD pathophysiology becomes increasingly recognized, investigators aim to define inflammatory signatures to help elucidate underlying mechanisms of disease pathogenesis and aid in identification of patients with inflammatory endophenotypes that could benefit from immunomodulatory interventions. However, discordant results in the literature and a lack of information regarding the stability of inflammatory factors over a 24-h period have hampered progress. Methods: Here, we measured inflammatory proteins in serum and CSF of a small cohort of PD (n=12) and age-matched healthy control (HC) subjects (n=6) at 11 time points across 24h to (1) identify potential diurnal variation, (2) reveal differences in PD vs HC, and (3) to correlate with CSF levels of amyloid β (Aβ) and α-synuclein in an effort to generate data-driven hypotheses regarding candidate biomarkers of PD. Results: Despite significant variability in other factors, a repeated measures two-way analysis of variance by time and disease state for each analyte revealed that serum IFNγ, TNF, and neutrophil gelatinase-associated lipocalin (NGAL) were stable across 24h and different between HC and PD. Regression analysis revealed that C-reactive protein (CRP) was the only factor with a strong linear relationship between CSF and serum. PD and HC subjects showed significantly different relationships between CSF Aβ proteins and α-synuclein and specific inflammatory factors, and CSF IFNγ and serum IL-8 positively correlated with clinical measures of PD. Finally, linear discriminant analysis revealed that serum TNF and CSF α-synuclein discriminated between PD and HC with a minimum of 82% sensitivity and 83% specificity. Conclusions: Our findings identify a panel of inflammatory factors in serum and CSF that can be reliably measured, distinguish between PD and HC, and monitor inflammation as disease progresses or in response to interventional therapies. This panel may aid in generating hypotheses and feasible experimental designs towards identifying biomarkers of neurodegenerative disease by focusing on analytes that remain stable regardless of time of sample collection.
by
Vered Padler-Karavani;
Xuezheng Song;
Hai Yu;
Nancy Hurtado-Ziola;
Shengshu Huang;
Saddam Muthana;
Harshal A. Chokhawala;
Jiansong Cheng;
Andrea Verhagen;
Martijn A. Langereis;
Ralf Kleene;
Melitta Schachner;
Raoul J. de Groot;
Yi Lasanajak;
Haruo Matsuda;
Richard Schwab;
Xi Chen;
David Smith;
Richard D. Cummings;
Ajit Varki
Background: Various glycan microarrays are currently widely used, but systematic cross-comparisons are lacking.
Results: We compare and contrast two sialoglycan microarrays using a variety of sialic acid-binding proteins.
Conclusion: Diverse array formats can strengthen the quality of information, but differences between arrays may be observed.
Significance: Glycan arrays with similar glycan structures cannot be simply assumed to give similar results.
Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H2O2). In the cell-attached configuration, H2O2 significantly increased ENaC open probability (Po) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H2O2 are required to elevate intracellular H2O2, probably because catalase, the enzyme that promotes the decomposition of H2O2 to H2O and O2, is highly expressed in A6 cells. The effect of H2O2 on ENaC Po was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H2O2 levels are critical to produce the effect. However, H2O2 did not directly activate ENaC in inside-out patches. The effects of H2O2 on ENaC Po and amiloride-sensitive Na+ current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H2O2 elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P3, these data suggest that H2O2 stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P3.
Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2–3- and α2–6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2–6- and α2–3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-d-glycero-d-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2–6-linked Neu5Ac9Lt or α2–6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.
Background: Epithelial sodium channels (ENaC) are activated by proteolytic cleavage. Several proteases including furin and prostasin cleave ENaC.
Results: Cathepsin B also cleaves and activates ENaC. Cathepsin B cleaves ENaC α but not β or γ subunits.
Conclusion: Cathepsin B is a secreted protease, so it may cleave ENaC at the cell surface.
Significance: Cathepsin B cleavage represents a novel ENaC regulatory mechanism.
We studied the ability of typical unmyelinated cortical axons to conduct action potentials at fever-like temperatures because fever often gives CNS symptoms. We investigated such axons in cerebellar and hippocampal slices from 10 to 25 days old rats at temperatures between 30 and 43°C. By recording with two electrodes along axonal pathways, we confirmed that the axons were able to initiate action potentials, but at temperatures >39°C, the propagation of the action potentials to a more distal recording site was reduced. This temperature-sensitive conduction may be specific for the very thin unmyelinated axons because similar recordings from myelinated CNS axons did not show conduction failures. We found that the conduction fidelity improved with 1 mmol/L TEA in the bath, probably due to block of voltage-sensitive potassium channels responsible for the fast repolarization of action potentials. Furthermore, by recording electrically activated antidromic action potentials from the soma of cerebellar granule cells, we showed that the axons failed less if they were triggered 10–30 msec after another action potential. This was because individual action potentials were followed by a depolarizing after-potential, of constant amplitude and shape, which facilitated conduction of the following action potentials. The temperature-sensitive conduction failures above, but not below, normal body temperature, and the failure-reducing effect of the spike's depolarizing after-potential, are two intrinsic mechanisms in normal gray matter axons that may help us understand how the hyperthermic brain functions.
Synaptic plasticity within the spinal cord has great potential to facilitate recovery of function after spinal cord injury (SCI). Spinal plasticity can be induced in an activity-dependent manner even without input from the brain after complete SCI. A mechanistic basis for these effects is provided by research demonstrating that spinal synapses have many of the same plasticity mechanisms that are known to underlie learning and memory in the brain. In addition, the lumbar spinal cord can sustain several forms of learning and memory, including limb-position training. However, not all spinal plasticity promotes recovery of function. Central sensitization of nociceptive (pain) pathways in the spinal cord may emerge in response to various noxious inputs, demonstrating that plasticity within the spinal cord may contribute to maladaptive pain states. In this review we discuss interactions between adaptive and maladaptive forms of activity-dependent plasticity in the spinal cord below the level of SCI. The literature demonstrates that activity-dependent plasticity within the spinal cord must be carefully tuned to promote adaptive spinal training. Prior work from our group has shown that stimulation that is delivered in a limb position-dependent manner or on a fixed interval can induce adaptive plasticity that promotes future spinal cord learning and reduces nociceptive hyper-reactivity. On the other hand, stimulation that is delivered in an unsynchronized fashion, such as randomized electrical stimulation or peripheral skin injuries, can generate maladaptive spinal plasticity that undermines future spinal cord learning, reduces recovery of locomotor function, and promotes nociceptive hyper-reactivity after SCI. We review these basic phenomena, how these findings relate to the broader spinal plasticity literature, discuss the cellular and molecular mechanisms, and finally discuss implications of these and other findings for improved rehabilitative therapies after SCI.
Uncontrollable nociceptive stimulation adversely affects recovery in spinally contused rats. Spinal cord injury (SCI) results in altered microRNA (miRNA) expression both at, and distal to the lesion site. We hypothesized that uncontrollable nociception further influences SCIsensitive miRNAs and associated gene targets, potentially explaining the progression of maladaptive plasticity. Our data validated previously described sensitivity of miRNAs to SCI alone. Moreover, following SCI, intermittent noxious stimulation decreased expression of miR124 in dorsal spinal cord 24 h after stimulation and increased expression of miR1292 in dorsal, and miR1 in ventral spinal cord at 7 days. We also found that brain-derived neurotrophic factor (BDNF) mRNA expression was significantly down-regulated 1 day after SCI alone, and significantly more so, after SCI followed by tailshock. Insulin-like growth factor-1 (IGF-1) mRNA expression was significantly increased at both 1 and 7 days postSCI, and significantly more so, 7 days post-SCI with shock. MiR1 expression was positively and significantly correlated with IGF-1, but not BDNF mRNA expression. Further, stepwise linear regression analysis indicated that a significant proportion of the changes in BDNF and IGF-1 mRNA expression were explained by variance in two groups of miRNAs, implying co-regulation. Collectively, these data show that uncontrollable nociception which activates sensorimotor circuits distal to the injury site, influences SCI-miRNAs and target mRNAs within the lesion site. SCI-sensitive miRNAs may well mediate adverse consequences of uncontrolled sensorimotor activation on functional recovery. However, their sensitivity to distal sensory input also implicates these miRNAs as candidate targets for the management of SCI and neuropathic pain.
The trunk plays a pivotal role in limbed locomotion. Yet, little is known about how the brain stem controls trunk activity during walking. In this study, we assessed the spatiotemporal activity patterns of axial and hindlimb motoneurons (MNs) during drug-induced fictive locomotor-like activity (LLA) in an isolated brain stem-spinal cord preparation of the neonatal mouse. We also evaluated the extent to which these activity patterns are affected by removal of brain stem. Recordings were made in the segments T7, L2, and L5 using calcium imaging from individual axial MNs in the medial motor column (MMC) and hindlimb MNs in lateral motor column (LMC). The MN activities were analyzed during both the rhythmic and the tonic components of LLA, the tonic component being used as a readout of generalized increase in excitability in spinal locomotor networks. The most salient effect of brain stem removal was an increase in locomotor rhythm frequency and a concomitant reduction in burst durations in both MMC and LMC MNs. The lack of effect on the tonic component of LLA indicated specificity of action during the rhythmic component. Cooling-induced silencing of the brain stem reproduced the increase in rhythm frequency and accompanying decrease in burst durations in L2 MMC and LMC, suggesting a dependency on brain stem neuron activity. The work supports the idea that the brain stem locomotor circuits are operational already at birth and further suggests an important role in modulating trunk activity. The brain stem may influence the axial and hindlimb spinal locomotor rhythm generating circuits by extending their range of operation. This may represent a critical step of locomotor development when learning how to walk in different conditions and environments is a major endeavor.