Rhes protein transits from neuron to neuron and facilitates mutant huntingtin spreading in the brain

Abstract

Rhes (RASD2) is a thyroid hormone-induced gene that regulates striatal motor activity and promotes neurodegeneration in Huntington disease (HD) and tauopathy. Rhes moves and transports the HD protein, polyglutamine-expanded huntingtin (mHTT), via tunneling nanotube (TNT)-like membranous protrusions between cultured neurons. However, similar intercellular Rhes transportation in the intact brain was unknown. Here, we report that Rhes induces TNT-like protrusions in the striatal medium spiny neurons (MSNs) and transported between dopamine-1 receptor (D1R)-MSNs and D2R-MSNs of intact striatum and organotypic brain slices. Notably, mHTT is robustly transported within the striatum and from the striatum to the cortical areas in the brain, and Rhes deletion diminishes such transport. Moreover, Rhes moves to the cortical regions following restricted expression in the MSNs of the striatum. Thus, Rhes is a first striatum-enriched protein demonstrated to move and transport mHTT between neurons and brain regions, providing new insights into interneuronal protein transport in the brain.

Fig. 1.. Rhes promotes TNT-like protrusions in MSNs.

(A) AAV Flex Cre-On viral vector design and infection into the D1R^Cre (Drd1a^Cre) mouse primary MSNs. (B and C) Confocal images of D2R^Cre-MSN expressing AAV Cre-On GFP (B) or AAV Cre-On GFP-Rhes (C). Inset b: Blue arrow indicates neurites. Inset c: White open arrow indicates TNT-like protrusions from cell body. Closed arrow indicates TNT-like protrusion from the neurites. Arrowhead indicates GFP-Rhes–positive vesicle-like blub. (D) Organotypic slice culture from D2R^Cre;D1R^tdTomato mice infected with viral particles. (E) Confocal live-slice images and insets of organotypic brain slices transduced with Cre-On AAV viral particles. Yellow and white arrows show neuronal processes (green). The arrowhead shows GFP-Rhes puncta (red). 3D rendering and orthogonal (Ortho, single plane) display show EGFP-Rhes puncta in D1R^tdTomato MSN (arrowhead). (F) Pearson’s coefficient for colocalization (n = 12, D1R^tdTomato neurons from three slices); data are means ± SEM; Student’s t test, ****P < 0.0001. (G) Confocal time-lapse imaging of brain slices. Inset g (top) shows EGFP-Rhes–positive TNT-like protrusions (0 to 30 min) connecting D2R^Cre-MSN (green) to D1R^tdTomato-MSN (red). Inset g (bottom) shows 3D intensity of movement of Rhes (arrowhead) at different time points. (H) 3D intensity from three different brain slice experiments.

Fig. 2.. Rhes moves from D1R-MSN to D2R-MSN in vivo.

(A) AAV-Flex in vivo model. (B) Confocal images of brain sections from D1R^Cre;D2R^EGFP mice injected with Cre-On RFP-Rhes in the striatum. Arrow indicates expression of RFP-Rhes in D1R^Cre(+) neurons. Arrowhead indicates RFP-Rhes expression in D2R^EGFP Cre(−) neurons. Arrowhead in the orthogonal display shows that RFP-Rhes puncta are colocalized with D2R^EGFP neuron. Artistic rendering of inset b is shown. (C) Horizontal reconstruction of confocal images of D1R^Cre;D2R^EGFP mice injected with Cre-On RFP-Rhes in the striatum. At the injected site, inset c1 shows D2R^EGFP neuron (closed arrow), and inset c2 shows D1R^Cre neurons expressing RFP-Rhes (open arrow) and D2R^EGFP neurons with RFP-Rhes (arrowhead). Inset c3 shows RFP-Rhes (arrowhead) in D2R^EGFP neurons, 500 μm away from the injected site, and inset c4 shows RFP-Rhes in the ipsilateral cortical cells. (D) Bar graph shows quantification of the % of indicated neurons from the injected site, and 100 and 500 μm away from the injection in the striatum, ipsilateral cortex [as in (C)], and contralateral striatum (n = 5 per injection, 3 male, 2 female). Data are means ± SEM. **P < 0.01 and ***P < 0.001, two-way ANOVA and Bonferroni post hoc test.

Fig. 3.. Rhes promotes cell-to-cell transport of mHTT in vivo.

(A) Graphical representation of LV vectors and injection. (B) Western blotting of striatal neuronal cells. (C) Confocal image of a brain section of EGFP (green) and wtHTT or mHTT (mCherry, red) at the injection site and 500 μm away from it. Insets c1 to c4 show an enlarged portion. Arrowhead, perinuclear mHTT. Cell nuclei, DAPI (blue). (D to H) Quantification of the intensity of HTT (D), number of HTT-positive cells (E), the total number of HTT/EGFP double-positive cells (F), HTT intensity in the EGFP-positive cells (G), and mHTT intensity 500 μm away from the injection site (H). (I) Confocal images of brain sections from WT and Rhes KO mice showing the expression of EGFP (green) and wtHTT or mHTT (mCherry, red) and cell nuclei stained with DAPI (blue). Insets i1 to i4 show magnified images from the striatum, and insets i1a to i4a and i1b to i4b show magnified images from the cortex. Arrowhead indicates mHTT or wtHTT in the cortex. (J) Quantification of the intensity of HTT in 200-μm² area in the ipsilateral cortex (n = 5 per injection, 3 male, 2 female). Data are means ± SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; not significant, one-way ANOVA and Tukey’s multiple comparisons test.

Fig. 4.. Rhes moves from striatum to cortex.

(A) AAV-Flex and coculture model. (B) Confocal images. Red arrow, CamKII^Cre neurons (Cre+, donor) expressing Cre-On RFP alone or RFP-Rhes. Green arrow, EGFP neurons (Cre−, acceptor). Inset b1, arrowhead, and arrow (orthogonal display), Cre-On RFP-Rhes in the EGFP neurons. (C) Quantification of EGFP neurons positive for Cre-On RFP (n = 29) and RFP-Rhes (n = 41). Data are means ± SEM; Student’s t test, ****P < 0.0001. (D to J) AAV-Flex injection into D1R^Cre [Drd1a^Cre, (D)] or Rgs-9^Cre striatum (G). Coronal brain section of the D1R^Cre mice (E) or Rgs-9Cre (H) injected with Cre-On RFP or RFP-Rhes. DAPI (nuclei, blue). RFP in the striatum (blue arrow). RFP-Rhes in the cortical region (open arrow) and septal regions (closed arrow). Insets e1 to e4 and h1 to h4 show the high-magnification cortex. Insets e3 and e4 (E) and h3 and h4 (H) show perinuclear Rhes in cortical cells (arrowhead). A vertical reconstruction of RFP-Rhes (arrowhead) in the Rgs-9Cre cortex (I). Arrowhead shows RFP-Rhes in the cortex. Quantification of cortical cells in the cortex of D1R^Cre mice [n = 4 per injection, 2 male, 2 female, (F)] or Rgs9^Cre [n = 3 per injection, all male, (J)] positive for RFP or RFP-Rhes. Data are means ± SEM; Student’s t test, **P < 0.01 and ****P < 0.0001.

Fig. 5.. Neuron-to-neuron Rhes transport model.

Our data indicate that Rhes moves between MSNs in the striatum as well as to the cortical areas. Live-cell imaging data from primary neurons and organotypic slice data indicate that TNT-like membranous protrusions are the key routes Rhes contacts the neighboring neurons. Thus, we predict that Rhes transports and facilitates mHTT movements in vivo potentially via the direct physical contact of neurons via membranous protrusions. Both collateral contact between MSNs and corticostriatal contacts of MSN to the cortical projections may occur via TNT-like membranous protrusions.