<i>skn-1</i> is required for interneuron sensory integration and foraging behavior in <i>Caenorhabditis elegans</i>

<div><p>Nrf2/<i>skn-1</i>, a transcription factor known to mediate adaptive responses of cells to stress, also regulates energy metabolism in response to changes in nutrient availability. The ability to locate food sources depends upon chemosensation. Here we show that Nrf2/<i>skn-1</i> is expressed in olfactory interneurons, and is required for proper integration of multiple food-related sensory cues in <i>Caenorhabditis elegans</i>. Compared to wild type worms, <i>skn-1</i> mutants fail to perceive that food density is limiting, and display altered chemo- and thermotactic responses. These behavioral deficits are associated with aberrant AIY interneuron morphology and migration in <i>skn-1</i> mutants. Both <i>skn-1</i>-dependent AIY autonomous and non-autonomous mechanisms regulate the neural circuitry underlying multisensory integration of environmental cues related to energy acquisition.</p></div

<i>skn-1</i> is expressed in AIY neurons and regulates their functions.

<p>(A) Schematic of the neuronal circuits of <i>C</i>. <i>elegans</i> chemoattractive and thermotactic behaviors. Green triangles represent sensory neurons and blue hexagons command interneurons. Arrows indicate direct interactions, and their thickness is proportional to the frequency of synaptic contacts between the neurons (Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176798#pone.0176798.ref036" target="_blank">36</a>]). T, thermophilic; C, cryophilic.(Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176798#pone.0176798.ref041" target="_blank">41</a>]). (B) Population thermotaxis experiments were performed using a radial thermal gradient. After 60 minutes the position of the worms was scored and the percentages of worms in the colder (Zone 1) and warmer (Zone 2) regions determined. N2 animals moved toward the region of the plate closer to their previous cultivation temperature (23°C), while both <i>skn-1</i> null strains <i>zu135</i> and <i>zu67</i> showed a cryophilic phenotype similar to <i>ttx-3</i> (AIY) null worms. (C) Representative 3D reconstruction of confocal sections through the head region of double transgenic <i>wglS342;otlS133</i> worms is shown. The <i>skn-1</i>:<i>EGFP</i> reporter (green), containing the entire <i>skn-1</i> gene, is highly expressed in neurons in the head ganglia, and colocalizes with the AIY-specific marker <i>ttx3</i>:RFP (Red). Arrows point at AIY. Scale bar 10 μm. (D) Relative mRNA levels of the AIY cell fate specification homeobox genes <i>ttx-3</i> and <i>ceh-23</i>, and their targets <i>ser-2a</i>, <i>ser-2b</i>, <i>sra-11</i>, <i>kal-1</i> and <i>hen-1</i> in N2 and <i>skn-1</i> null worms. (E) Representative 3D reconstructions showing the expression of the transcriptional reporter <i>ser2</i>:<i>prom1</i>:<i>EGFP</i> in the head region of N2 and <i>skn-1(zu135)</i> null worms. Data are mean and S.E.M. of 3–5 experiments. *p<0.05; **p<0.01; ***p<0.001 versus N2 (Student’s <i>t</i>-test).</p

<i>skn-1</i> regulates AIY neuron morphology and food-seeking behavior by cell-autonomous and non-autonomous mechanisms.

<p>(A) Schematic depiction, representative images, and quantification of the neuroanatomical defects observed in <i>skn-1(zu135)</i> worms. Wild type morphology is shown in black, while aberrant branches from the axon or cell body are depicted in red. Also shown are examples of premature stops, and axon misrouting. (B) Schematic depiction and representative images of the AIY position relative to the pharynx grinder in N2 (black line) and <i>skn-1(zu135)</i> worms (red line). a, distance between grinder and the AIY axon fasciculation at the nerve ring; b, distance between the grinder and AIY cell body. Quantification of the AIY migratory defects in the indicated worm strains. AIY-specific expression of <i>skn-1b</i> has no effect on migration (B), and food-leaving behavior (C) (OP50 = 0.033x). Pan-neuronal expression of <i>skn-1b</i> significantly decreases AIY migratory defects (B) and partially rescues the food-leaving behavior (D). Data are mean and S.E.M. of 3–7 experiments. ***p<0.001 versus N2; ###p<0.001 versus <i>skn-1</i> (zu135) (Student’s <i>t</i>-test).</p

Nrf2 is highly expressed in olfactory bulb interneurons.

<p>(A) Representative immunoblots and quantification of Nrf2 protein levels in adult murine brain (n = 4). Values were normalized by GAPDH and expressed as mean percentage (and S.E.M.) compared to olfactory bulb (OB). Cx, cortex; Hp, hippocampus; Cb, cerebellum; Me, medulla. (B) Schematic showing the rostral migratory stream (RMS), the route followed by neuroblasts originating in the sub ventricular zone (SVZ) to reach the olfactory bulb (OB). Immunohistochemistry showing the expression of Nrf2 in the RMS at low magnification (left) and high magnification (right). The boxed area indicates the regions shown in the immunostaining. (C) Immunohistochemistry showing the distribution of Nrf2 in the various regions of the olfactory bulb (upper panel). GCL: granule cell layer; MCL: mitral cell layer; EPL: external plexiform layer; GL: glomerular layer. The higher magnification panels show Nrf2 subcellular localization in mitral cells (MCL) and granule cells (GCL).</p

Iron chelation decreases BDNF expression in human neuroblastoma cells.

<p><b>A.</b> SH-SY5Y cells were treated with either vehicle or 100 µM deferoxamine (DFO) for 24 hours. RT-PCR measures of BDNF transcripts show a decrease in total BDNF transcripts (tBDNF, variants 1–14, 16–18 (*p<0.01, n = 4–5). <b>B.</b> Iron chelation did not cause changes in cell survival as assessed by trypan blue exclusion 24 hours post-treatment (n = 5). <b>C.</b> SH-SY5Y cells were transfected with luciferase reporters for the promoter regions of BDNF I–IV and were then treated with 100 µM deferoxamine for 24 hours. No significant differences were observed between control and treated cells (n = 3–4).</p

CpKO mice have decreased concentrations of iron in the cerebral cortex and striatum.

<p>Atomic absorption measures of cortical and striatal biopsy punches show significant decreases in iron concentrations in CpKO compared to wild type mice in both the cerebral cortex ( n = 18; *p<0.02) and the striatum (n = 7; *p<0.03).</p

CpKO mice have increased cerebral infarct volume after focal ischemic stroke.

<p><b>A.</b> Representative images of TTC-stained brain sections in mice subjected to 45 min MCAO with a 72 hours post-stroke survival. <b>B.</b> Calculation of percent infarct volume show a significant increase in the cortex and hemisphere of the CpKO mice compared to WT mice. (*p<0.002; n = 8 WT and 6 KO mice).</p

The expression of BDNF is reduced in the cerebral cortex and striatum of ceruloplasmin-deficient mice.

<p><b>A.</b> RT-PCR measures of the 4 BDNF transcripts show a decrease in BDNF transcripts 1, 2 and 4 in the cortex of the CpKO mouse (*p<0.04, n = 5). <b>B.</b> RT-PCR measures of the 4 BDNF transcripts show a decrease in BDNF transcripts 1, 2, 3 and 4 in the striatum of the CpKO mouse (*p<0.05, n = 5). <b>C.</b> Examples of immunoblots of BDNF protein levels in the cortex and striatum (top panel) and densitometry results showing significant decreases BDNF protein levels (normalized to actin level) using Image J software. (*p<0.03; n = 4 WT and 3 CpKO mice).</p

<i>Bhlhe40</i> KO mice have increased neuronal excitability.

<p>(A) <i>Bhlhe40</i> KO mice have a 14.4% decrease in IPSC amplitude (<i>Bhlhe40</i> KO: 85.6 ± 12.2, WT: 100 ± 11.6); n = 4 mice for each <i>Bhlhe40</i> KO (5 cells) and WT (6 cells). (B) Left: <i>Bhlhe40</i> KO mice have a 40% increase in mEPSC amplitude, relative to WT levels (<i>Bhlhe40</i> KO: 18.99 ± 0.69 pA, WT: 13.6 ± 0.88 pA). Right: Example traces of mEPSCs from WT and <i>Bhlhe40</i> KO slices. Error bars are standard error of the mean; unpaired t-tests were used for both mEPSC and IPSC amplitude comparisons <i>Bhlhe40</i> KO hippocampal slices compared to WT slices; * = p<0.05.</p

Mice lacking the transcriptional regulator Bhlhe40 have enhanced neuronal excitability and impaired synaptic plasticity in the hippocampus

<div><p>Bhlhe40 is a transcription factor that is highly expressed in the hippocampus; however, its role in neuronal function is not well understood. Here, we used <i>Bhlhe40</i> null mice on a congenic C57Bl6/J background (<i>Bhlhe40</i> KO) to investigate the impact of Bhlhe40 on neuronal excitability and synaptic plasticity in the hippocampus. <i>Bhlhe40</i> KO CA1 neurons had increased miniature excitatory post-synaptic current amplitude and decreased inhibitory post-synaptic current amplitude, indicating CA1 neuronal hyperexcitability. Increased CA1 neuronal excitability was not associated with increased seizure severity as <i>Bhlhe40</i> KO relative to +/+ (WT) control mice injected with the convulsant kainic acid. However, significant reductions in long term potentiation and long term depression at CA1 synapses were observed in <i>Bhlhe40</i> KO mice, indicating impaired hippocampal synaptic plasticity. Behavioral testing for spatial learning and memory on the Morris Water Maze (MWM) revealed that while <i>Bhlhe40</i> KO mice performed similarly to WT controls initially, when the hidden platform was moved to the opposite quadrant <i>Bhlhe40</i> KO mice showed impairments in relearning, consistent with decreased hippocampal synaptic plasticity. To investigate possible mechanisms for increased neuronal excitability and decreased synaptic plasticity, a whole genome mRNA expression profile of <i>Bhlhe40</i> KO hippocampus was performed followed by a chromatin immunoprecipitation sequencing (ChIP-Seq) screen of the validated candidate genes for Bhlhe40 protein-DNA interactions consistent with transcriptional regulation. Of the validated genes identified from mRNA expression analysis, insulin degrading enzyme (<i>Ide</i>) had the most significantly altered expression in hippocampus and was significantly downregulated on the RNA and protein levels; although Bhlhe40 did not occupy the <i>Ide</i> gene by ChIP-Seq. Together, these findings support a role for Bhlhe40 in regulating neuronal excitability and synaptic plasticity in the hippocampus and that indirect regulation of <i>Ide</i> transcription may be involved in these phenotypes.</p></div