Skip to main content
. 2012 Mar 7;32(10):3306–3320. doi: 10.1523/JNEUROSCI.5367-11.2012

Figure 4.

Figure 4.

Accumulation and aggregation of αS in the ER. A, Schematic representation of the protocol used for subcellular fractionation. B, Various subcellular fractions, as shown in (A), from presymptomatic A53TαS mice were analyzed for organelle markers: calnexin (CLN) for ER, synaptotagmin (ST) for synaptic vesicles, syntaxin 6 (STX) for Golgi, cathepsin D (CTS D) for lysosomes, VDAC for mitochondria, and p38 for cytosol. Parallel analysis for αS and βS shows ER-associated αS but not βS. C, αS, but not βS, accumulates within the microsome fraction. ER/M (P100) and other intermediate fractions from nTg mice, human pons, and SH-SY5Y cell lines stably transfected with αS or βS were analyzed for αS/βS and organelle markers. In all cases, αS, but not βS is associated with ER/M fraction. Note that even with the overexpression of βS in the SH-SY5Y cell lines, βS does not associate with ER/M fractions (P100). D, ER-associated αS monomers in nTg, A53TαS Tg, and WTαS Tg mice are protected from proteolysis by PK, indicating that αS is in the ER lumen. In the presence of 1% TX-100, both αS and the ER-resident grp78 are completely proteolyzed by PK. Identical results were obtained with A30PαS mice. E, Accumulation of αS aggregates with ER-enriched microsomes fractions. One microgram of pure organelle fractions were also evaluated using organelle-specific markers: calnexin (CLN) for ER, synaptotagmin (ST) for synaptic vesicles, syntaxin 6 (STX) for Golgi, NeuN for nuclei (N), cytochrome c (CytoC) for mitochondria (M), βS for cytosol (C). Total SDS-soluble (T) and initial ER/M (P100) fractions are also shown. The gradient-enriched ER fraction is free from other organelle markers but is enriched for ER markers. The ER fraction also shows significant enrichments in αS monomer and the presence of SDS-stable high molecular weight αS aggregates (arrowheads). Quantitative data show that αS monomer is significantly enriched in the ER fractions, compared with M and N. All organelles were fractionated from SpCs of end-stage A53TαS Tg mice. Values are mean ± SEM (n = 3–4). *p < 0.05, **p < 0.01, one-way ANOVA. F, Microsomes from end-stage A53TαS Tg mice show accumulation of pS129 αS and oligomeric αS (arrowheads). Equal amounts of microsomes obtained from SpC of nTg and A53TαS Tg mice were immunoblotted for pS129 αS and total αS. Asymptomatic, (PreS); end stage, (Sick). G, Membrane floatation analysis of microsomes and detergent-insoluble αS. Microsome-associated αS from end-stage A53TαS Tg mice (Sick) and age-matched A30PαS Tg mice shows that αS floats with membranes. Lanes are input microsomes (P100), membrane fractions (M), and free (F) fractions from the floatation gradient. The detergent-insoluble αS aggregates (P-Tx) do not float with the M fractions. Arrowheads are high molecular weight aggregates of αS.