Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel ß4 subunit explain its role in cell-cell adhesion.

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary ß subunits, designated as ß1/ß1B-ß4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the ß4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the ß4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted ß4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the ß4 cis dimer contributes to the trans homophilic interaction of ß4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of ß4 affects α-ß4 complex formation. These observations provide the structural basis for the parallel dimer formation of ß4 in VGSCs and reveal its mechanism in cell-cell adhesion.

Urinary lumirubin excretion in jaundiced preterm neonates during phototherapy with blue light-emitting diode vs. green fluorescent lamp.

Phototherapy converts lipophilic unconjugated bilirubin to hydrophilic bilirubin photoisomers, such as lumirubin. We comparatively used a blue light-emitting diode (LED) and a green fluorescent lamp (FL) as light sources for phototherapy of hyperbilirubinemic preterm neonates with the aim of examining potential differences in urinary lumirubin excretion between these two wavelengths. Urinary lumirubin levels were measured using a fluorescence assay with blue light exposure in the presence of the unconjugated bilirubin-inducible fluorescent protein UnaG, and denoted as urinary UnaG-bound bilirubin (UUB)/creatinine (Cr) (µg/mg Cr). Preterm neonates born at ≤ 33 weeks gestational age and treated with phototherapy were subjected to this study. The maximum UUB/Cr level during phototherapy per device intensity was compared between neonates treated with the blue LED and the green FL. A total of 61 neonates were examined to determine the maximum UUB/Cr levels. The median of maximum UUB/Cr excretion per light intensity of each device (µg/mg Cr/µW/cm2/nm) was 0.83 for the blue LED and 1.29 for the green FL (p = 0.01). Green light was found to be more effective than blue one for bilirubin excretion via urinary lumirubin excretion. This is the first spectroscopic study to compare the efficacy of phototherapy at different wavelengths using fluorescence assay.

Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

Noninvasive monitoring of bilirubin photoisomer excretion during phototherapy.

Lumirubin is the most prevalently excreted hydrophilic bilirubin photoisomer in phototherapy for neonatal jaundice caused by excess hydrophobic unconjugated bilirubin (ZZ-bilirubin). We developed a simple method to estimate the amount of lumirubin by monitoring the reverse photoisomerization of lumirubin to ZZ-bilirubin. Although lumirubin formation was long considered irreversible, exposure to blue light in the presence of the fluorescent protein UnaG, which binds specifically and tightly to ZZ-bilirubin, enables the reverse photoisomerization of lumirubin. This reaction was first detected using a fluorescence assay of neonatal urine sampled during phototherapy and purified lumirubin. The phenomenon of reverse photoisomerization of lumirubin was validated using liquid chromatography-mass spectrometry, which confirmed that lumirubin is reconverted to ZZ-bilirubin in the presence of UnaG. Analyses of 20 urine samples from 17 neonates revealed a significant correlation (correlation coefficient [r] = 0.978; 95% confidence interval 0.867-0.979; P < .001) between lumirubin and ZZ-bilirubin concentration before and after reverse photoisomerization. In general, the rate of photo-reconversion of lumirubin to ZZ-bilirubin is approximately 40%. In conclusion, we demonstrate here that lumirubin can be photo-reconverted to ZZ-bilirubin via exposure to blue light in the presence of UnaG. Utilizing this approach, urinary lumirubin levels can be estimated using an easy-to-perform fluorescence assay.

Blocking acid-sensing ion channel 1 alleviates Huntington's disease pathology via an ubiquitin-proteasome system-dependent mechanism.

Huntington's disease (HD) is a fatal neurodegenerative disorder. Despite a tremendous effort to develop therapeutic tools in several HD models, there is no effective cure at present. Acidosis has been observed previously in cellular and in in vivo models as well as in the brains of HD patients. Here we challenged HD models with amiloride (Ami) derivative benzamil (Ben), a chemical agent used to rescue acid-sensing ion channel (ASIC)-dependent acidotoxicity, to examine whether chronic acidosis is an important part of the HD pathomechanism and whether these drugs could be used as novel therapeutic agents. Ben markedly reduced the huntingtin-polyglutamine (htt-polyQ) aggregation in an inducible cellular system, and the therapeutic value of Ben was successfully recapitulated in the R6/2 animal model of HD. To reveal the mechanism of action, Ben was found to be able to alleviate the inhibition of the ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. More importantly, we were able to demonstrate that blocking the expression of a specific isoform of ASIC (asic1a), one of the many molecular targets of Ben, led to an enhancement of UPS activity and this blockade also decreased htt-polyQ aggregation in the striatum of R6/2 mice. In conclusion, we believe that chemical compounds that target ASIC1a or pharmacological alleviation of UPS inhibition would be an effective and promising approach to combat HD and other polyQ-related disorders.

Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y.

A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

Genome-wide analyses in neuronal cells reveal that upstream transcription factors regulate lysosomal gene expression.

UNLABELLED: The upstream transcription factors (USFs) USF1 and USF2 are ubiquitously expressed transcription factors that are characterized by a conserved basic helix-loop-helix/leucine zipper DNA-binding domain. They form homo- or heterodimers, and recognize E-box motifs to modulate gene expression. They are known to regulate diverse cellular functions, including the cell cycle, immune responses and glucose/lipid metabolism, but their roles in neuronal cells remain to be clarified. Here, we performed chromatin immunoprecipitation of USF1 from mouse brain cortex. Subsequent promoter array analysis (ChIP-chip) indicated that USF1 exclusively bound to the CACGTG E-box motifs in the proximal promoter regions. Importantly, functional annotation of the USF1-binding targets revealed an enrichment of genes related to lysosomal functions. Gene expression array analysis using a neuronal cell line subsequently revealed that knockdown of USFs de-regulated lysosomal gene expression. Altered expression was validated by quantitative RT-PCR, supporting the conclusion that USFs regulate lysosomal gene expression. Furthermore, USF knockdown slightly increased LysoTracker Red staining, implying a role for USFs in modulating lysosomal homeostasis. Together, our comprehensive genome-scale analyses identified lysosomal genes as targets of USFs in neuronal cells, suggesting a potential additional pathway of lysosomal regulation. DATABASE: The data for the gene expression array and ChIP-chip have been submitted to the Gene Expression Omnibus (GEO) under accession numbers GSE76615 and GSE76616, respectively.

Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS.

The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons, and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression.

Structure-based site-directed photo-crosslinking analyses of multimeric cell-adhesive interactions of voltage-gated sodium channel ß subunits.

The ß1, ß2, and ß4 subunits of voltage-gated sodium channels reportedly function as cell adhesion molecules. The present crystallographic analysis of the ß4 extracellular domain revealed an antiparallel arrangement of the ß4 molecules in the crystal lattice. The interface between the two antiparallel ß4 molecules is asymmetric, and results in a multimeric assembly. Structure-based mutagenesis and site-directed photo-crosslinking analyses of the ß4-mediated cell-cell adhesion revealed that the interface between the antiparallel ß4 molecules corresponds to that in the trans homophilic interaction for the multimeric assembly of ß4 in cell-cell adhesion. This trans interaction mode is also employed in the ß1-mediated cell-cell adhesion. Moreover, the ß1 gene mutations associated with generalized epilepsy with febrile seizures plus (GEFS+) impaired the ß1-mediated cell-cell adhesion, which should underlie the GEFS+ pathogenesis. Thus, the structural basis for the ß-subunit-mediated cell-cell adhesion has been established.

NF-Y inactivation causes atypical neurodegeneration characterized by ubiquitin and p62 accumulation and endoplasmic reticulum disorganization.

Nuclear transcription factor-Y (NF-Y), a key regulator of cell-cycle progression, often loses its activity during differentiation into nonproliferative cells. In contrast, NF-Y is still active in mature, differentiated neurons, although its neuronal significance remains obscure. Here we show that conditional deletion of the subunit NF-YA in postmitotic mouse neurons induces progressive neurodegeneration with distinctive ubiquitin/p62 pathology; these proteins are not incorporated into filamentous inclusion but co-accumulated with insoluble membrane proteins broadly on endoplasmic reticulum (ER). The degeneration also accompanies drastic ER disorganization, that is, an aberrant increase in ribosome-free ER in the perinuclear region, without inducing ER stress response. We further perform chromatin immunoprecipitation and identify several NF-Y physiological targets including Grp94 potentially involved in ER disorganization. We propose that NF-Y is involved in a unique regulation mechanism of ER organization in mature neurons and its disruption causes previously undescribed novel neuropathology accompanying abnormal ubiquitin/p62 accumulation.

Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

Loss of aPKCλ in differentiated neurons disrupts the polarity complex but does not induce obvious neuronal loss or disorientation in mouse brains.

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6ß, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6ß and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.

Crystal structure of an active form of BACE1, an enzyme responsible for amyloid beta protein production.

BACE1 (beta-secretase) is a transmembrane aspartic protease that cleaves the beta-amyloid precursor protein and generates the amyloid beta peptide (Abeta). BACE1 cycles between the cell surface and the endosomal system many times and becomes activated interconvertibly during its cellular trafficking, leading to the production of Abeta. Here we report the crystal structure of the catalytically active form of BACE1. The active form has novel structural features involving the conformation of the flap and subsites that promote substrate binding. The functionally essential residues and water molecules are well defined and play a key role in the iterative activation of BACE1. We further describe the crystal structure of the dehydrated form of BACE1, showing that BACE1 activity is dependent on the dynamics of a catalytically required Asp-bound water molecule, which directly affects its catalytic properties. These findings provide insight into a novel regulation of BACE1 activity and elucidate how BACE1 modulates its activity during cellular trafficking.