Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor-mediated disease.

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA

SIRT3 is required for liver regeneration but not for the beneficial effect of nicotinamide riboside

Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. In this study, we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require NAD as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here, we show that despite their NAD dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating WT or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Therefore, we provide the first evidence to our knowledge for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy

<div><p>Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. <i>In vitro</i>, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α₉β₁, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.</p></div

EIIIA promotes expression of the vasculature marker VE-cadherin following PHx.

<p>Frozen liver sections taken at day 2 after PHx were stained for VE-cadherin (white). Livers from female and male wild type mice showed increased staining for VE-cadherin <b>(A, C)</b> compared to livers from EIIIA-cFN null mice <b>(B, D)</b>. Scale bar, 50 μm. <b>(E, F)</b> Quantification = minimum to maximum % VE cadherin positive area measurements with line at mean, EIIIA^+/+ (n = 10; 5 female, 5 male); EIIIA^-/- (n = 10; 5 female, 5 male). * p<0.05.</p

Expression of EIIIA-cFN is upregulated early after PHx.

<p><b>(A, B)</b> Wild type mice were euthanized at days 1, 2 and 5 following PHx. mRNA transcript levels for <b>(A)</b> EIIIA-cFN and <b>(B)</b> total fibronectin were measured by qRT-PCR normalized to the expression of <i>tbp</i>. n = 3–4 mice per time point, error bars are mean +/- SEM, *p < 0.05. <b>(C, D)</b> Liver tissue from wild type <b>(C)</b> control mice or <b>(D)</b> mice at day 2 after PHx was stained for EIIIA-cFN (magenta) and with DAPI (blue). Scale bar, 20 μm.</p

Expression of EIIIA-cFN is upregulated early after PHx.

<p><b>(A, B)</b> Wild type mice were euthanized at days 1, 2 and 5 following PHx. mRNA transcript levels for <b>(A)</b> EIIIA-cFN and <b>(B)</b> total fibronectin were measured by qRT-PCR normalized to the expression of <i>tbp</i>. n = 3–4 mice per time point, error bars are mean +/- SEM, *p < 0.05. <b>(C, D)</b> Liver tissue from wild type <b>(C)</b> control mice or <b>(D)</b> mice at day 2 after PHx was stained for EIIIA-cFN (magenta) and with DAPI (blue). Scale bar, 20 μm.</p

EIIIA-cFN null mice have delayed recovery of liver-to-body weight ratios following PHx.

<p>Mice were euthanized at days 2 and 5 following PHx. Liver-to-body weight ratios were measured for EIIIA-cFN null, heterozygous and wild type littermates. <b>(A)</b> Day 2 time point: EIIIA^+/+ n = 11 (6 males, 5 females), EIIIA^+/- n = 10 (5 males, 5 females), EIIIA^-/- n = 11 (6 males, 5 females). <b>(B)</b> Day 5 time point: EIIIA^+/+ n = 8 (4 males, 4 females), EIIIA^+/- n = 8 (4 males, 4 females), EIIIA^-/- n = 8 (4 males, 4 females). Quantification = mean +/- SD, * p<0.05.</p

Liver endothelial cells are more adhesive to cell-deposited matrices containing EIIIA.

<p><b>(A)</b> Mouse liver endothelial cells were allowed to bind to cell-deposited matrices with or without EIIIA-cFN, and with or without blocking antibodies to the integrin α₉ subunit. Graph shows number of cells adhering to either EIIIA⁺ or EIIIA^- matrix, normalized to cells adhering to EIIIA⁺ matrix. Images shown represent 3 independent experiments, mean +/- SEM. <b>(B)</b> Integrin profiling of primary liver endothelial cells. RNA was collected from primary liver endothelial cells at day 8 after isolation. qRT-PCR shows expression of EIIIA-cFN and total FN binding integrins α₄, α₅, α₉, and α_V relative to the housekeeping gene ribosomal protein S12.</p