Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

The functions of HCN channels in neurons depend critically on their subcellular localization, requiring fine-tuned machinery that regulates subcellular channel trafficking. Here we provide evidence that regulatory mechanisms governing axonal HCN channel trafficking involve association of the channels with specific isoforms of the auxiliary subunit TRIP8b. In the medial perforant path, which normally contains HCN1 channels in axon terminals in immature but not in adult rodents, we found axonal HCN1 significantly increased in adult mice lacking TRIP8b (TRIP8b−/−). Interestingly, adult mice harboring a mutation that results in expression of only the two most abundant TRIP8b isoforms (TRIP8b[1b/2]−/−) exhibited an HCN1 expression pattern similar to wildtype mice, suggesting that presence of one or both of these isoforms (TRIP8b(1a), TRIP8b(1a-4)) prevents HCN1 from being transported to medial perforant path axons in adult mice. Concordantly, expression analyses demonstrated a strong increase of expression of both TRIP8b isoforms in rat entorhinal cortex with age. However, when overexpressed in cultured entorhinal neurons of rats, TRIP8b(1a), but not TRIP8b(1a-4), altered substantially the subcellular distribution of HCN1 by promoting somatodendritic and reducing axonal expression of the channels. Taken together, we conclude that TRIP8b isoforms are important regulators of HCN1 trafficking in entorhinal neurons and that the alternatively-spliced isoform TRIP8b(1a) could be responsible for the age-dependent redistribution of HCN channels out of perforant path axon terminals

Differential effects of TRIP8b isoforms on localization of HCN1 in the middle molecular layer (MML) of dentate gyrus.

<p>a–c) Representative low power images of coronal sections of adult wildtype mouse brain show the characteristic distribution of TRIP8b (a) and HCN1 (b) immunoreactivity in hippocampus, with HCN1 and TRIP8b staining most intense in stratum lacunosum-moleculare (SLM) of CA1, and low expression in stratum pyramidale (SP), stratum oriens (SO) and stratum radiatum (SR). In contrast, much lower levels of HCN1 and TRIP8b are expressed in hippocampal dentate gyrus (DG). d–f) Interestingly, in adult TRIP8b^−/− mice the immunoreactivity of HCN1 is increased in the MML of DG (arrows in e), where the granule cells are innervated by axons from medial EC via the perforant path. No changes in HCN1 expression were observed in mossy fibres (empty circles), whereas there is significant reduction of HCN1 staining in the SLM of CA1 (asterisks). g–i) Adult TRIP8b[1b/2]^−/− mice expressing TRIP8b isoforms 1a and 1a-4 show similar HCN1 staining patterns to wildtype mice in all hippocampal subregions. Scale bar: 200 µm. j–l) Higher magnification views of HCN1 immunoreactivity in TRIP8b^−/− DG clearly show the increased HCN1 staining in MML (k) as compared with wildtype (j) and TRIP8b[1b/2]^−/− mice (l). Scale bar: 50 µm. m–n) HCN1 immunofluorescence was measured as a function of distance across the superior blade of the dentate gyrus as indicated in the diagram (m), and demonstrates higher HCN1 expression in MML in TRIP8b^−/− (red circles) as compared to wildtype (black circles) and TRIP8b[1b/2]^−/− (blue circles) mice (n). OML - outer molecular layer, MML - middle molecular layer, IML - inner molecular layer, GCL - granule cell layer, Hil - hilus. Asterisks in (n) denote statistical significance (*p<0.05, **p<0.01, ***p<0.001).</p

Expression analyses of TRIP8b isoforms in entorhinal cortex.

<p>a) Horizontal section showing hippocampus, entorhinal and perirhinal cortex in an adult rat, immunostained (IHC) for TRIP8b. Note: TRIP8b immunoreactivity is strong in areas which also show substantial HCN1 expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032181#pone.0032181-Bender2" target="_blank">[17]</a>: the distal dendritic fields of CA1 and subiculum (Sub) and the outer layers (I–III) of medial entorhinal cortex (medEC, arrows). TRIP8b expression is further visible in deep layer (V) of both the medial and the lateral EC, containing pyramidal cells. In the perirhinal cortex (peCo), this staining is replaced by TRIP8b immunoreactivity in the outermost layers (asterisks), most likely reflecting dendritic positioning of the TRIP8b in the pyramidal cells. b) Higher magnification view of medial EC from a): TRIP8b immunoreactivity is limited to layers I–III and V, but diffuse immunostaining does not permit identification of the TRIP8b-expressing neurons. c) Non-radioactive <i>in situ</i> hybridization (ISH) revealed expression of TRIP8b mRNA specifically in layers II and V, indicating that the stellate cells of layer II and the pyramidal cells of layer V are the major sources of TRIP8b immunoreactivity in medial EC. d–f) Co-labeling of TRIP8b mRNA (d) and reelin protein (e) further showed that many of the TRIP8b mRNA-positive cells in EC layer II co-express reelin (f), identifying them as perforant path projection neurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032181#pone.0032181-Varga1" target="_blank">[25]</a>. Scale bar: 500 µm (a), 200 µm (b, c), 80 µm (d–f). g) Upper panel: Western Blots illustrating expression of TRIP8b and HCN1 in EC tissue of immature (P10) and adult (>P60) rats. As shown previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032181#pone.0032181-Lewis1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032181#pone.0032181-Santoro4" target="_blank">[10]</a>, probing with a pan-TRIP8b antibody revealed two bands, representing predominantly the TRIP8b(1a) and (1a-4) isoforms (∼65 and ∼70 kDa, respectively), which together constitute >80% of cortical TRIP8b. Lower panel: Native TRIP8b in EC (left) run as a control against TRIP8b(1a) (right) and TRIP8b(1a-4) (middle) harvested from HEK293 cells that were single-transfected with those isoforms. h) Quantitative analysis of HCN1 and TRIP8b isoform expression levels in immature vs. adult EC. Data are presented as “% of immature” expression and have been normalized to actin. Expression of both the TRIP8b(1a) (245±41%) and the (1a-4) isoform (200±30%) increased significantly with age (p<0.01 for both), whereas no significant change was detected for HCN1 (140±17%, p>0.05).</p

Expression of HCN1-EGFP and TRIP8b isoforms after single-transfection in immature entorhinal neuron cultures.

<p>Confocal images showing representative neurons in “immature cultures” (P0+4 days <i>in vitro</i>) single-transfected with either HCN1-EGFP (a–c), TRIP8b(1a-4) (d-f) or TRIP8b(1a) (g–i). For the identification of axons, the microtubule-associated protein Tau-1 was co-labeled (b, e, h). Note: Single-transfection resulted in a relatively homogeneous distribution of the overexpressed proteins within neuronal compartments, including the axon (arrows). Scale bar: 20 µm.</p

Expression of endogenous HCN1 and TRIP8b after single-transfection of TRIP8b isoforms in differentiated entorhinal neuron cultures.

<p>a–d) Confocal images showing a representative neuron in “differentiated cultures” (P5+10 days <i>in vitro</i>) that co-expresses endogenous HCN1 (a) and TRIP8b(1a-4) (b). Expression of both proteins is relatively homogenous within the neuron, involving soma, dendrites and the axon (labeled with Tau, arrows; c, d). e–h) Confocal images showing a representative neuron in “differentiated cultures” that co-expresses emdogenous HCN1 (e) and TRIP8b(1a) (f). While expression of both proteins can be observed in all compartments, including the axon (g, h), expression seems to be most intense in the somatodendritic compartment (arrowheads). Scale bar: 20 µm. i–k) Quantitative analyses of the relative subcellular distribution of endogenous HCN1- and TRIP8b-signal in differentiated entorhinal neurons transfected with either TRIP8b(1a) or TRIP8b(1a-4) (n = 25 each). Data from TRIP8b(1a-4)–overexpressing neurons confirm a relatively homogenous distribution of both the HCN1 and TRIP8b throughout the neurons (j, k). In contrast, in neurons transfected with TRIP8b(1a), TRIP8b was preferentially expressed in somatodendritic subdivision 3 (153±12 in 1a- vs. 119±10% in 1a-4-expressing neurons; p = 0.04; k). HCN1 expression in these neurons showed a trend towards enrichment in the somatodendritic compartment, which was, however, not significant (subdivision 1: 63±12 in 1a- vs. 88±12% in 1a-4-expressing neurons; p = 0.14; subdivision 3: 150±14 in 1a- vs. 129±13% in 1a-4-expressing neurons; p = 0.24; j).</p

Ultrastructural localization of HCN1 in middle molecular layer (MML) of dentate gyrus.

<p>a) Light micrographs of silver-intensified immunogold-stained hippocampal slices show the presence of a thin band of HCN1 immunoreactivity in MML in the TRIP8b^−/− (arrows), but not in the wildtype mice. Note also the lack of HCN1 enrichment in stratum lacunosum-moleculare (SLM, asterisks) of CA1 in the TRIP8b^−/−-section, as described by Lewis et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032181#pone.0032181-Lewis2" target="_blank">[12]</a> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032181#pone-0032181-g001" target="_blank">Fig. 1d–f</a>). b, c) Electron micrographs of serial sections showing immunopositive axonal boutons in a TRIP8b^−/− mouse, making both perforated (arrow) and nonperforated synapses with dendritic spines in MML. Abbreviations: at, axon terminal; sp, spine. d) Percentage of axonal boutons, immunopositive for HCN1, making axospinous synapses in MML in wildtype (black) and TRIP8b^−/− mice (white). e) Average number of particles per synaptic bouton in wildtype (black) and TRIP8b^−/− mice (white). Data were obtained from two mice of each genotype, and based on analyses of 25 synaptic boutons from each mouse (100 axonal boutons total) from the MML. Serial ultrathin sections were obtained from slices similar to those shown in (a).</p