Synphilin-1 attenuates neuronal degeneration in the A53T alpha-synuclein transgenic mouse model

Abstract

Genetic alterations in alpha-synuclein cause autosomal dominant familial Parkinsonism and may contribute to sporadic Parkinson's disease (PD). Synphilin-1 is an alpha-synuclein-interacting protein, with implications in PD pathogenesis related to protein aggregation. Currently, the in vivo role of synphilin-1 in alpha-synuclein-linked pathogenesis is not fully understood. Using the mouse prion protein promoter, we generated synphilin-1 transgenic mice, which did not display PD-like phenotypes. However, synphilin-1/A53T alpha-synuclein double-transgenic mice survived longer than A53T alpha-synuclein single-transgenic mice. There were attenuated A53T alpha-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration in brains in double-transgenic mice. Overexpression of synphilin-1 decreased caspase-3 activation, increased beclin-1 and LC3 II expression and promoted formation of aggresome-like structures, suggesting that synphilin-1 alters multiple cellular pathways to protect against neuronal degeneration. These studies demonstrate that synphilin-1 can diminish the severity of alpha-synucleinopathy and play a neuroprotective role against A53T alpha-synuclein toxicity in vivo.

Figure 1.

Expression of synphilin-1 increased survival of A53T α-synuclein transgenic mice. (A) Western blot analysis of brain homogenates of non-transgenic mouse (nTg), synphilin-1 transgenic mouse (A6, A4 and A7 lines), rat and human cortex, using a rabbit anti-human synphilin-1 antibody. (B) Western blot analysis of synphilin-1 and A53T-α-synulein expression in double-transgenic mice by anti-synphilin-1 or anti-α-synuclein antibodies. (C) Survival curves for A53T α-synuclein transgenic and double-transgenic mice. Double-transgenic mice displayed a longer life span compared with A53T α-synuclein transgenic mice by Kaplan–Meier survival analysis (log-rank followed by Holm–Sidak method). There were 30 mice (half male and half female) of each genotype. P < 0.05.

Figure 2.

Synphilin-1 attenuated A53T α-synuclein-induced hyperactivity. Novelty-induced activity was measured in cohorts of six to eight mice per group at 4–7 months of age in an open-field test. Horizontal and vertical activities were automatically recorded. The data are presented as the means (beams broken) ± SEM. There was no difference in the novelty-induced activity between mice of various groups at 4 months of age. At 5 and 7 months of age, A53T mice demonstrated significantly increased novelty-induced horizontal and vertical activities compared with mice of other groups (P < 0.001, by statistic analysis with the group by time interaction; P < 0.05 by two-way ANOVA and post hoc Holm–Sidak test). The double-transgenic mice did not display hyperactivity throughout life.

Figure 3.

Abnormal accumulation of α-synuclein was less in double-transgenic mice at the preclinical stage. (A and B) Brain sections from mice at 7 months of age (preclinical stage) were digested with proteinase K for 3 h and then followed by immunostaining using anti-α-synuclein antibodies. Double-transgenic mice displayed less accumulation of abnormal α-synulcein after proteinase K digestion compared with A53T mice at 7 months of age. (A) Representative images of anti-α-synuclein immunostaining of various experimental groups after proteinase K digestion. (B) Graph showing the quantification of α-synuclein accumulation after proteinase K digestion in pons at 7 months of age. (C) Western blot analysis of brainstem homogenates of mice at 7 months of age (preclinical stage) using anti-α-synuclein antibodies. Top, 2% SDS-soluble homogenates; bottom, homogenates were digested with proteinase K followed by western blot analysis using anti-α-synuclein antibodies. (D) Western blot analysis of brain homogenates of mice at 7 months of age (preclinical stage) using anti-beclin-1 antibodies. (E) Graph showing the quantification of (D). P < 0.05 by ANOVA. (F) Western blot analysis of brainstem homogenates (2% SDS-soluble samples) of mice using anti-LC3 and anti-actin antibodies. (G) Graph showing the quantification of (F). *P < 0.05 by ANOVA versus aged-matched non-transgenic control mice or synphilin-1 transgenic mice; ^#P < 0.05 by ANOVA versus aged-matched A53T single-transgenic mice.

Figure 4.

Synphilin-1 promoted aggresome-like inclusion formation in double-transgenic mice at end-stage disease. (A) Sagittal mouse brain sections were subjected to hematoxylin and eosin (HE) staining, immunostaining with DAB detection using anti-ubiquitin (B) and anti-synphilin-1 (C) antibodies, proteinase K digestion followed by anti-α-synuclein staining (D), double-immunostaining using anti-α-synuclein and anti-synphilin-1 antibodies (E). (A) Eosinophilic cytoplasmic inclusions (arrow) in the pons in sick double-transgenic mice by HE staining. (B) Ubiquitin pathology in sick double-transgenic mice. Pathological somal and neuritic (arrow) accumulation of ubiquitin was prominent in pons. (C) Synphilin-1 was highly expressed in neurons in pons in sick double-transgenic mice. Arrow showing the synphilin-1-positive inclusions. (D) Brain sections from double transgenic mice over digested with proteinase for 3 h and then followed by immunostaining using anti-α-synuclein antibodies. The representative image showing the abnormal accumlation of α-synuclein in pons in sick double-transgenic mice (11 months). (E) Inclusions in double-transgenic mice labeled using anti-α-synuclein and anti-synphilin-1antibodies. Red, α-synuclein; green or synphilin-1 staining; DAPI staining for nuclei. (F) Aggresome-like inclusions in double-transgenic mice. Red, α-synuclein (left) or synphilin-1 (right) staining; green, γ-tublin staining; DAPI staining for nuclei. (G) Graph showing the quantification of neurons in brainstem with inclusions that were positively stained with both anti-α-synuclein and anti-γ-tublin antibodies, which resembled aggresome-like structures. P < 0.05 by Student's t-test.

Figure 5.

Synphilin-1 attenuated A53T α-synuclein-induced axonal degeneration in brainstem. HE and silver staining was used to visualize axonal degeneration in brainstem of various mice from each experimental group. (A) Representative images of various brainstems with silver staining in sagittal sections that were matched for level. The yellow-gold color is the typical background seen with FD NeuroSilver staining. Axonal degeneration in the brainstem of age-matched non-transgenic littermates is inconspicuous. The brainstem in A53T transgenic mice shows prominent axonal degeneration (black fibers), including axonal swellings (arrow). The inside rectangle shows a typical axonal swelling by HE staining. The axonal degeneration in double-transgenic mice was dramatically decreased compared with A53T transgenic mice. (B) Graph showing the quantification of silver degeneration in brainstem at 9 months of age. Values are mean ± SEM. *P < 0.05, versus non-transgenic mice; ^#P< 0.05, versus A53T α-synuclein mice by ANOVA.

Figure 6.

Synphilin-1 reduced A53T α-synuclein-induced astroglial activation in the brainstem. Sagittal mouse brain sections from 9-month-old mice were subjected to GFAP immunostaining. (A and B) Graph showing the quantification of GFAP-positive cells in the brainstem retrorubal field and facial motor nucleus at 9 months of age. Values represent mean ± SEM. *P < 0.05, versus non-transgenic mice; ^#P < 0.05, versus A53T α-synuclein mice by ANOVA. (C) Representative images of brainstem immunostaining for GFAP in sagittal sections that were matched for level.

Figure 7.

Synphilin-1 reduced A53T α-synuclein-induced caspase-3 activation. (A and B) Graphs showing the quantification of the number of positive motor neurons for anti-cleaved caspase-3 immunostaining in the brainstem of various mice at 7 months of age. Values are mean ± SEM. *P < 0.05, versus non-transgenic mice, ^#P < 0.05, versus A53T α-synuclein transgenic mice by ANOVA. (C) Representative images of immunostaining using anti-cleaved caspase-3 antibodies.