Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model - PubMed (original) (raw)

Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model

Shalesh Kaushal et al. Exp Biol Med (Maywood). 2010 Jun.

Abstract

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in one in 2000 Americans. The Z mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes and chronic liver injury. Autophagy is one of several proteolytic mechanisms activated to cope with this hepatocellular protein burden, and is likely important in disposal of the unique polymerized conformation of the a1AT mutant Z protein, which is thought to be especially injurious to the cell. Recent data indicate that rapamycin may more efficiently upregulate autophagy when given in weekly dose pulses, as compared with a daily regimen. Therefore, we evaluated the effect of rapamycin on PiZ mice, a well-characterized model which recapitulates human a1AT liver disease. Daily dosing had no effect on autophagy, on accumulation of a1AT mutant Z protein or on liver injury. Weekly dosing of rapamycin did increase autophagic activity, as shown by increased numbers of autophagic vacuoles. This was associated with reduction in the intrahepatic accumulation of a1AT mutant Z protein in the polymerized conformation. Markers of hepatocellular injury, including cleavage of caspase 12 and hepatic fibrosis, were also decreased. In conclusion, this is the first report of a successful in vivo method for reduction of intrahepatic a1AT mutant Z polymerized protein. Application of this finding may be therapeutic in patients with a1AT deficiency by reducing the intracellular burden of the polymerized, mutant Z protein and by reducing the progression of liver injury.

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Figures

Figure 1. Lack of effect of daily dosing rapamycin on autophagy and a1AT protein in the liver in PiZ mice

Panel a, percent area of hepatocellular cytoplasm occupied by autophagic vacuoles, determined as described by morphometric counting by a single blinded examiner, in PiZ mice treated with daily dosing rapamycin versus vehicle control PiZ mice (p>0.4 by ANOVA). Bars +/− S.D. Panel b, representative examples of SDS-PAGE followed by immunoblot for a1AT from PiZ mouse liver treated with daily dose rapamycin (rapa) or vehicle control (con) of total hepatic protein lysate (T) or after separation of a1AT monomers (M) from polymers (P). Actin loading controls below, as shown. Note, the polymers are denatured to monomers before loading and therefore run at the monomeric 52kDa of the intracellular form of a1AT protein. Also, actin is only recovered in the M fraction. Molecular weight markers, Mrx103 as shown.

Figure 1. Lack of effect of daily dosing rapamycin on autophagy and a1AT protein in the liver in PiZ mice

Figure 2. Effect of weekly pulse dosing rapamycin on autophagy and a1AT protein in the liver in PiZ mice

Panel a, percent area of hepatocellular cytoplasm occupied by autophagic vacuoles in PiZ mice treated with weekly dosing rapamycin versus vehicle control PiZ mice (p<0.5 by ANOVA). Bars +/− S.D. Panel b, representative example of SDS-PAGE followed by immunoblot for LC3 from a WT mouse liver fasted 12 hours (fast) as a positive control, PiZ mouse liver treated with weekly dose rapamycin (rapa) or vehicle control (con) of total hepatic protein lysate. LC3-I and LC3-II reactive bands are shown. Actin loading controls as shown, below, and molecular weight markers, Mrx103 as shown. Panel c, representative examples of SDS-PAGE followed by immunoblot for a1AT from PiZ mouse liver treated with weekly dose rapamycin (rapa) or vehicle control (con) of total hepatic protein lysate (T) or after separation of a1AT monomers (M) from polymers (P). Note, the polymers are denatured to monomers before loading and therefore run at the monomeric 52kDa of the intracellular form of a1AT protein. Molecular weight markers, Mrx103 as shown. Panel d, quantification by electronic gel scanning of the means of the weekly treated mice and controls, as from panel b. Left panel is total (Total) intrahepatic a1AT mutant Z protein, right panel is monomer (M) and polymer (P) separation. Comparisons as noted, #; p<0.03, *; p<0.02, ^; p<0.02. Panel e, mean serum a1AT protein levels at sacrifice in PiZ mice treated with weekly rapamycin compared to controls. Bars +/− S.D. Comparison p>0.4.

Figure 2. Effect of weekly pulse dosing rapamycin on autophagy and a1AT protein in the liver in PiZ mice

Figure 3. Effect of weekly pulse dosing rapamycin on the distribution of a1AT protein in the liver in PiZ mice

Panel a, a1AT immunohistochemical staining of globule devoid areas of PiZ mouse liver from weekly rapamycin treated (left panel) and controls (right panel). Dark areas indicate increased a1AT immunoreactivity. Panel b, photomicrograph of PiZ mouse liver treated with weekly dose rapamycin (left panel) versus control (right panel) stained with Periodic Acid-Schiff and digestion to highlight globules (dark hepatocellular inclusions shown on light background) of a1AT mutant Z protein.

Figure 3. Effect of weekly pulse dosing rapamycin on the distribution of a1AT protein in the liver in PiZ mice

Figure 4. Effect of weekly pulse dosing rapamycin on liver injury in PiZ mice

Panel a, inflammation graded 1+ to 4+ by blinded examiner and shown as means in weekly rapamycin treated (left panel) versus controls (right panel). Bars +/− S.D. Comparison p>0.3. Panel b, example of SDS-PAGE followed by immunoblot for caspase 12 (left panel) and means of densitometric quantification of replicates (right panel) of PiZ mice treated with weekly rapamycin versus controls. Arrow indicates 37–39kDa cleaved (activated) caspase 12. Bars +/− S.D. Molecular weight markers, Mrx103 as shown. Comparison * p<0.04. Panel c, quantification of mean percent BrdU labeled hepatocellular nuclei from male PiZ mice treated with weekly rapamycin compared to controls. Bars +/− S.D. Comparison * p<0.03. Panel d, hepatic fibrosis as shown by red fibers in representative photomicrographs stained by Sirius red in WT mice (left panel), weekly PiZ rapa treated (middle panel) compared to control (right panel) PiZ mice.

Figure 4. Effect of weekly pulse dosing rapamycin on liver injury in PiZ mice

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Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model - PubMed (original) (raw)