Axonopathy in an α-Synuclein Transgenic Model of Lewy Body Disease Is Associated with Extensive Accumulation of C-Terminal–Truncated α-Synuclein

Abstract

Progressive accumulation of α-synuclein (α-syn) in limbic and striatonigral systems is associated with the neurodegenerative processes in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). The murine Thy-1 (mThy1)-α-syn transgenic (tg) model recapitulates aspects of degenerative processes associated with α-syn accumulation in these disorders. Given that axonal and synaptic pathologies are important features of DLB and PD, we sought to investigate the extent and characteristics of these alterations in mThy1-α-syn tg mice and to determine the contribution of α-syn c-terminally cleaved at amino acid 122 (CT α-syn) to these abnormalities. We generated a novel polyclonal antibody (SYN105) against the c-terminally truncated sequence (amino acids 121 to 123) of α-syn (CT α-syn) and performed immunocytochemical and ultrastructural analyses in mThy1-α-syn tg mice. We found abundant clusters of dystrophic neurites in layers 2 to 3 of the neocortex, the stratum lacunosum, the dentate gyrus, and cornu ammonis 3 of the hippocampus, striatum, thalamus, midbrain, and pons. Dystrophic neurites displayed intense immunoreactivity detected with the SYN105 antibody. Double-labeling studies with antibodies to phosphorylated neurofilaments confirmed the axonal location of full-length and CT α-syn. α-Syn immunoreactive dystrophic neurites contained numerous electrodense laminated structures. These results show that neuritic dystrophy is a prominent pathologic feature of the mThy1-α-syn tg model and suggest that CT α-syn might play an important role in the process of axonal damage in these mice as well as in DLB and PD.

Alpha-synuclein modulates NR2B-containing NMDA receptors and decreases their levels after rotenone exposure

“…To our knowledge, this is one of the first functional descriptions of this specific post-translationally-modified form of α-syn. It has been recently reported that in a transgenic mouse model of Lewy body disease the onset of axonopathy is associated with extensive accumulation of C-terminally cleaved α-syn, thus suggesting that this form of the protein may play a relevant role in the onset of axon damage (Games et al, 2013). Interestingly, presynaptic NMDAR are emerging as crucial mediators of synaptic plasticity along axons during LTP in mature neurons (Bourne et al, 2013;Park et al, 2014).…”

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons

“…To exclude the possibility that the variations in the synapsin-III levels observed in primary neurons were due to non-dopaminergic neurons or glial cells present in the primary cell cultures, we evaluated the expression of synapsin III in a more uniform dopaminergic cell population -dopaminergic differentiated SH-SY5Y neuroblastoma cells (Bellucci et al, 2008) To confirm whether α-syn affected DAT and synapsin III distribution, besides their expression, we used a differentiated neuronal SK-N-SH cell line that stably expressed a green fluorescent protein (GFP)-conjugated form of DAT to overexpress the SYN120 mutant protein (Bellucci et al, 2011a). This form of α-syn can be found in LBs (Prasad et al, 2012), and promotes α-syn aggregation (Crowther et al, 1998), DAT redistribution (Bellucci et al, 2011a) and the induction of axonal and synaptic damage (Games et al, 2013;GarciaReitbock et al, 2010). We found that SYN120-expressing cells showed intracellular α-syn-and synapsin-III-immunopositive inclusions that contained DAT-GFP, whereas, in control SK-N- SH DAT-GFP cells, both synapsin III and DAT localized predominantly at the plasma membrane (supplementary material Fig.…”

Differential effects of immunotherapy with antibodies targeting α-synuclein oligomers and fibrils in a transgenic model of synucleinopathy

“…The line 61 α-syn tg model was selected because these mice develop behavioral motor deficits (Fleming et al, 2004), axonal pathology and accumulation of CT-cleaved α-syn and aggregates in neocortex, limbic system and subcortical regions (Games et al, 2013). In this model, accumulation of α-synin the hippocampus results in Lewy body neurites and degeneration of CA3 neurons similar to DLB (Overk et al, 2014; Rockenstein et al, 2007; Rockenstein et al, 2002).…”

“…Brain sections were incubated overnight at 4 °C with a polyclonal antibody against total α-syn (1:500, affinity-purified rabbit polyclonal, Millipore) (Games et al, 2013; Masliah et al, 2000) in the presence or absence of proteinase K (PK; 10 μg/ml for 15 min) or non-immune IgG controls (background levels), a neuronal marker (NeuN, mouse monoclonal, Millipore 1:2500), an astroglial cell marker (glial fibrillary acidic protein [GFAP], mouse monoclonal, Millipore 1:2500), a microglial cell marker (Iba1; goat polyclonal, Abcam, 1:5000). followed by incubations with secondary antibodies biotinylated (1:100, Vector Laboratories, Inc., Burlin-game, CA), Avidin D-HRP (1:200, ABC Elite, Vector) and detection with 3,3′-diaminobenzidine (Masliah et al, 2011).…”

“…α-Synuclein (α-syn) is a 140 amino acid synaptic protein (Iwai et al, 1995) involved in neurotransmitter release (Liu et al, 2004; Nemani et al, 2010) that accumulates in synaptic terminals (Bellucci et al, 2012; Kramer and Schulz-Schaeffer, 2007; Roy et al, 2007), axons (Dickson et al, 1994; Games et al, 2013), neuronal soma (Spillantini et al, 1997; Takeda et al, 1998), and oligodendrocytes (Papp and Lantos, 1992; Wakabayashi et al, 2000). Under physiological conditions, α-syn is a relatively unstructured monomer (Lashuel et al, 2013; Tsigelny et al, 2007; Tsigelny et al, 2008; Tsigelny et al, 2012) that adopts a β-helical structure when associated with membranes (Ulmer et al, 2005).…”