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. 2017 Nov 15;7(1):15637.
doi: 10.1038/s41598-017-15778-8.

Differential overexpression of SERPINA3 in human prion diseases

S Vanni  1 , F Moda  2 , M Zattoni  1 , E Bistaffa  1 , E De Cecco  1 , M Rossi  1 , G Giaccone  2 , F Tagliavini  2 , S Haïk  3 , J P Deslys  4 , G Zanusso  5 , J W Ironside  6 , I Ferrer  7 , G G Kovacs  8 , G Legname  9
Affiliations

Differential overexpression of SERPINA3 in human prion diseases

S Vanni et al. Sci Rep. .

Abstract

Prion diseases are fatal neurodegenerative disorders with sporadic, genetic or acquired etiologies. The molecular alterations leading to the onset and the spreading of these diseases are still unknown. In a previous work we identified a five-gene signature able to distinguish intracranially BSE-infected macaques from healthy ones, with SERPINA3 showing the most prominent dysregulation. We analyzed 128 suitable frontal cortex samples, from prion-affected patients (variant Creutzfeldt-Jakob disease (vCJD) n = 20, iatrogenic CJD (iCJD) n = 11, sporadic CJD (sCJD) n = 23, familial CJD (gCJD) n = 17, fatal familial insomnia (FFI) n = 9, Gerstmann-Sträussler-Scheinker syndrome (GSS)) n = 4), patients with Alzheimer disease (AD, n = 14) and age-matched controls (n = 30). Real Time-quantitative PCR was performed for SERPINA3 transcript, and ACTB, RPL19, GAPDH and B2M were used as reference genes. We report SERPINA3 to be strongly up-regulated in the brain of all human prion diseases, with only a mild up-regulation in AD. We show that this striking up-regulation, both at the mRNA and at the protein level, is present in all types of human prion diseases analyzed, although to a different extent for each specific disorder. Our data suggest that SERPINA3 may be involved in the pathogenesis and the progression of prion diseases, representing a valid tool for distinguishing different forms of these disorders in humans.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
RT-qPCR analysis for SERPINA3 mRNA expression in brain normalized against GAPDH. gCJD (n = 17) *****p < 0.000000005, FFI (n = 9) **p < 0.0001, vCJD (n = 20) ***p < 0.000001, iCJD (n = 11) ****p < 0.00000005, sCJD (n = 23) ******p < 0.0000000005, AD (n = 14) *p < 0.005). Fold Change (FC) values are presented as average with CI.
Figure 2
Figure 2
RT-qPCR analysis for GFAP mRNA expression in brain normalized against GAPDH. gCJD (n = 17) ****p ≤ 0.000001, FFI (n = 9) *p < 0.05, vCJD (n = 20) **p < 0.001, iCJD (n = 11) ***p < 0.0005, sCJD (n = 23) ***p < 0.0005. Fold Change (FC) values are presented as average with CI.
Figure 3
Figure 3
RT-qPCR analysis for PRNP mRNA expression in brain normalized against GAPDH. iCJD (n = 11) **p < 0.0005, sCJD (n = 23) *p < 0.05. Fold Change (FC) values are presented as average with CI.
Figure 4
Figure 4
Western blotting analysis of brain samples from CJD, AD patients and healthy controls. The expression levels of SERPINA3 and PrPSc in brain samples of representative cases from each group were assessed by means of Western blotting, on two different gels run in parallel within the same experiment. β-actin was used as proteins loading control and to normalize the expression level of SERPINA3 for densitometric analysis. SERPINA3 and β-actin were developed on the same membrane, sequentially. SERPINA3 image has been cropped to improve clarity. Rec lane refers to recombinant SERPINA3 used as control. Molecular weight is represented on the left (kDa). Original full-length images are provided in Supplementary Material.
Figure 5
Figure 5
RT-qPCR analysis for serpina3n in the brain of RML-infected CD1 mice (n = 4 for each time point) *p < 0.05, **p < 0,005, ***p < 0.0005, ****p < 0.0001. Dashed column vs Gapdh, plain gray vs Tubb3, dotted column vs Actb. Fold changes are presented averaged with CI.
Figure 6
Figure 6
Western blotting analysis for serpina3n in the brain of RML-infected CD1 mice and related age-matched controls. The expression levels of serpina3n and PrPSc were assessed by means of Western blotting, on two different gels run in parallel within the same experiment. β-actin was used as proteins loading control and to normalize the expression level of serpina3n for densitometric analysis. serpina3n and β-actin were developed on the same membrane, sequentially. serpina3n image has been cropped to improve clarity. Molecular weights are shown on the left. Original full-length images are provided in Supplementary Material.
Figure 7
Figure 7
Neuropathological analysis. Immunohistochemical analysis revealed strong SERPINA3 immunoreaction within residual neurons and reactive glial cells in the frontal cortex of CJD patients [variant (E,F), sporadic (D) and iatrogenic (C)] while it was scanty and limited to isolated neurons in control subjects (A). In AD patients, anti-SERPINA3 decorated some cortical neurons and the amyloid of senile plaques (B).
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