Statins are ineffective at reducing neuroinflammation or prolonging survival in scrapie-infected mice

Abstract

Neuroinflammation is a prominent component of several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease, Parkinson's disease, tauopathies, amyotrophic lateral sclerosis and prion diseases. In such conditions, the ability to decrease neuroinflammation by drug therapy may influence disease progression. Statins have been used to treat hyperlipidemia as well as reduce neuroinflammation and oxidative stress in various tissues. In previous studies, treatment of scrapie-infected mice with the type 1 statins, simvastatin or pravastatin, showed a small beneficial effect on survival time. In the current study, to increase the effectiveness of statin therapy, we treated infected mice with atorvastatin, a type 2 statin that has improved pharmacokinetics over many type 1 statins. Treatments with either simvastatin or pravastatin were tested for comparison. We evaluated scrapie-infected mice for protease-resistant PrP (PrPres) accumulation, gliosis, neuroinflammation and time until advanced clinical disease requiring euthanasia. All three statin treatments reduced total serum cholesterol ≥40 % in mice. However, gliosis and PrPres deposition were similar in statin-treated and untreated infected mice. Time to euthanasia due to advanced clinical signs was not changed in statin-treated mice relative to untreated mice, a finding at odds with previous reports. Expression of 84 inflammatory genes involved in neuroinflammation was also quantitated. Seven genes were reduced by pravastatin, and one gene was reduced by atorvastatin. In contrast, simvastatin therapy did not reduce any of the tested genes, but did slightly increase the expression of Ccl2 and Cxcl13. Our studies indicate that none of the three statins tested were effective in reducing scrapie-induced neuroinflammation or neuropathogenesis.

Fig. 1.

Mouse serum cholesterol levels with simvastatin (SMV), pravastatin (PRV), or atorvastatin (ATV) treatment. Total serum cholesterol was measured in statin-treated and untreated mice after 150 days of consuming statin-supplemented or control chow. There was a ≥40 % reduction in the average cholesterol in all three statins tested relative to control, indicating an effect on the mevalonate pathway. Each dot represents a single mouse. Error bars indicate one standard deviation. *** P value ≤0.001 by one-way ANOVA relative to untreated.

Fig. 2.

Euthanasia plots and immunoblot detection of PrPres in 22L-infected mice treated with simvastatin, pravastatin, or atorvastatin. The time until euthanasia for untreated control scrapie-infected C57BL/10 mice was compared to that for mice treated with simvastatin (a), pravastatin (b), or atorvastatin (c). Mice were inoculated intracerebrally with scrapie strain 22L, and the x-axis shows the day post-inoculation (p.i.) when animals were euthanized due to advanced signs of clinical scrapie (see the Methods section for details). Curves were statistically analysed using the Mantel–Cox log rank. The number of mice per group (n) is indicated in each graph. (d) Brain protein samples from representative end-stage 22L-infected C57BL/10 mice treated with simvastatin, pravastatin, or atorvastatin, or untreated, were separated by SDS-PAGE. All samples were treated with proteinase K prior to separation as described in the Methods section. Each lane was loaded with 0.36 mg whole-brain equivalents and probed with anti-PrP antibody D13. The 21 and 31 kiloDalton protein standards are indicated to the left of the immunoblot.

Fig. 3.

Neuropathology and immunohistochemical assessment of gliosis and PrP deposition in cortex brain sections from 22L-infected mice treated with simvastatin, pravastatin, or atorvastatin. (a) Brain sections from 22L-infected mice treated with simvastatin, pravastatin, atorvastatin, or untreated, were probed with antibodies specific for GFAP, IBA-1, or PrP. A representative H and E stained section shows that similar amounts of spongiform change (arrow heads) were observed in all scrapie-infected mice. Sections from the brain of untreated uninfected mice were similarly probed as a negative control. Representative images of the cerebral cortex are shown for all at the same scale as indicated. (b) Sagittal sections from brains of 22L-infected mice treated with statins or untreated as indicated were scanned to quantitatively assess the percentage of positive pixels after probing with antibodies specific for GFAP, IBA-1, or PrP. Uninfected mice were included as a negative control. The background represents several brain sections from 22L-infected mice that were analysed with the primary antibody omitted. Each character represents an independent sagittal section from a single mouse. Bars indicate the mean, and error bars indicate one standard deviation. GFAP, IBA-1 and PrP were not decreased in the statin-treated infected mice versus the untreated infected mice, indicating that statin treatment was not highly effective at reducing the presence of activated microglia or astroglia, or in reducing the amount of PrPSc observed. All 22L-infected mice, regardless of treatment, showed significantly higher staining for GFAP, IBA-1 and PrP relative to the uninfected controls. Data were analysed by one-way ANOVA. ***P≤0.0001.