Human alpha-synuclein-harboring familial Parkinson's disease-linked Ala-53 --> Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice

Abstract

Mutations in alpha-synuclein (alpha-Syn) cause Parkinson's disease (PD) in a small number of pedigrees with familial PD. Moreover, alpha-Syn accumulates as a major component of Lewy bodies and Lewy neurites, intraneuronal inclusions that are neuropathological hallmarks of PD. To better understand the pathogenic relationship between alterations in the biology of alpha-Syn and PD-associated neurodegeneration, we generated multiple lines of transgenic mice expressing high levels of either wild-type or familial PD-linked Ala-30 --> Pro (A30P) or Ala-53 --> Thr (A53T) human alpha-Syns. The mice expressing the A53T human alpha-Syn, but not wild-type or the A30P variants, develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Pathologically, affected mice exhibit neuronal abnormalities (in perikarya and neurites) including pathological accumulations of alpha-Syn and ubiquitin. Consistent with abnormal neuronal accumulation of alpha-Syn, brain regions with pathology exhibit increases in detergent-insoluble alpha-Syn and alpha-Syn aggregates. Our results demonstrate that the A53T mutant alpha-Syn causes significantly greater in vivo neurotoxicity as compared with other alpha-Syn variants. Further, alpha-Syn-dependent neurodegeneration is associated with abnormal accumulation of detergent-insoluble alpha-Syn.

Figure 1

Expression of Huα-Syn in transgenic mice. (a) Northern blot analysis of total brain RNA from nontransgenic (ntg) littermates, MoPrP–Huα-Syn(WT) transgenic (lines B2-1, B2, and I2-2), MoPrP–Huα-Syn(A53T) transgenic (lines L5, N2-5, H5, and G2-3), and MoPrP–Huα-Syn(A30P) transgenic (lines Q3, O5, T3, O2) mice by using Huα-Syn cDNA probe. The autoradiogram shows high-level expression of Huα-Syn mRNAs in the brains of transgenic mice. Because of sequence homology between Hu- and Moα-Syn, the cRNA probe also detects endogenous Moα-Syn mRNA (Mo). (b and c) Immunoblot analysis of α-Syn polypeptides in MoPrP–Huα-Syn transgenic mice. Total brain protein from nontransgenic and transgenic mice (lanes are the same as described for a) were subjected to immunoblot analysis by using HuSyn-1 rabbit polyclonal Ab (b) or Syn-1 (c), an anti-pan α-Syn mAb.

Figure 2

Cellular localization of Huα-Syn expression in transgenic (tg) mice. (a and b) In situ hybridization analysis of Huα-Syn mRNA expression in brains of Huα-Syn transgenic mice. The antisense (a) and control sense (b) probe to Huα-Syn mRNA shows high-level, widespread expression of Huα-Syn in most brain regions. (c and d) To determine that Huα-Syn mRNA is expressed in the SNpc of transgenic mice, adjacent sections were processed for in situ hybridization (c) and tyrosine hydroxylase immunocytochemistry (d). The arrows outline the location of dopaminergic neurons in the SNpc. (e and f) Immunocytochemical analysis of Huα-Syn polypeptides in MoPrP–Huα-Syn transgenic mice brains. Coronal brain sections from nontransgenic mouse (e) and Huα-Syn(WT) transgenic mouse from I2-2 (f) were immunoreacted with HuSyn-1 antisera. Punctate, neuropil localizations of the Huα-Syn polypeptides are consistent with enrichment of α-Syn at the synaptic terminals. Immunocytochemical analysis of Huα-Syn(A53T) and Huα-Syn(A30P) showed results similar to the WT protein. ctx, cortex; Dent, dentate.

Figure 3

Expression of Huα-Syn(A53T) lead to neurological abnormalities and shortened lifespan of the transgenic mice. (a and b) G2-3(AT) transgenic mice at the middle (a) and late (b) stage of the neurological dysfunction. (c) Survival curves for the Huα-Syn(A53T) transgenic lines. The higher expression of transgene is associated with earlier disease onset and death.

Figure 4

Neuropathology in Huα-Syn transgenic mice. (a–c) Pathological neuronal accumulation of α-Syn in neurons from sick G2-3(A53T) transgenic mice. Abnormal α-Syn accumulations in neurons within deep cerebellar nuclei (a), near reticulo-pontine nuclei (b), and the neocortex (c). (d and f) Increased accumulation of α-Syn in neurons within the a deep cerebellar nuclei of O2(A30P) monitored with HuSyn-1 (a) and Syn-1 (b). Note a more uniform neuronal distribution of α-Syn accumulation. (g–j) Ubiquitin pathology in sick G2-3(A53T) mice. Pathological somal and neuritic accumulation of ubiquitin was prominent in pons (g and h) and cerebellum (i). Occasional cortical neurons (j) also exhibit ubiquitin pathology. (k) Despite α-Syn accumulation in mice from line O2(A30P) (shown in d and f), these mice do not develop ubiquitin pathology. Additional pathology in sick A53T mice includes accumulation of phosphorylated NF-H in deep cerebellar nuclei (l) and thioflavin-S-positive structures (m and Inset). (n) Thioflavin-S-treated nontransgenic mice are shown as negative control for m. (o and p) α-Syn (o) and ubiquitin (p) pathology in the Pons from preclinical H5(A53T) transgenic mice. The asterisks mark examples of perikaryal pathology, and the arrows mark the neuritic pathology.

Figure 5

Astroglial reaction is limited to brain regions showing extensive neuropathology. Sagittal mouse brain sections from a clinically affected G2-3(A53T) transgenic mouse (a, b, and Inset) and an age-matched littermate mouse (c and d) were processed for GFAP immunocytochemistry. The GFAP immunoreaction in the cortex (ctx) and hippocampus (Hippo) of G2-3 transgenic mice (a) shows very little pathology and is almost identical to that of nontransgenic (ntg) littermates (c). However, the midbrain (mid-br) and brainstem areas (b) of affected G2-3 transgenic mice show obvious increases in GFAP-immunostained astroglial cells compared with the nontransgenic littermate (d). cblm, cerebellum.

Figure 6

Accumulation of nonionic detergent-insoluble α-Syn polypeptides in neurologically affected Huα-Syn(AT) transgenic mice. (a and b) Immunoblot analysis, using Syn-1 mAb, of total SDS-soluble brainstem (a) and cortical extracts (b) from Huα-Syn transgenic mice and nontransgenic (nTg) litter mates. In extracts from the transgenic mice, full-length α-Syn at ≈16 kDa (α-Syn16) and a truncated α-Syn at 12 kDa (α-Syn12) are obvious. (c and d) Immunoblot analysis, using Syn-1 mAb, of nonionic detergent-insoluble (P2) fractions from brainstem (c) and cortex (d). (d) Neuropathological changes in the brainstem of clinically affected mice (G2-3-Sick and H5-Sick) are associated with the increased accumulations of full-length α-Syn (α-Syn16), truncated α-Syns (α-Syn12 and α-Syn 10), and high molecular mass α-Syn polypeptides. These biochemical changes of α-Syn were not seen in brainstems of mice without pathology. (d) Consistent with lack of significant pathology in cortex, none of the cortical P2 fractions show abnormal α-Syn polypeptides. The molecular mass standards (tick to the right of the gels) are 9, 13, 20, 35, 50, 60, 80, 111, and 173 kDa.