Inhibition of apoE proteolysis in presence of glia conditioned medium.

<p>A) rE3 and rE4 proteins were incubated with conditioned media obtained from primary cortical neurons (DIV22) for indicated times. Full length apoE and its proteolytic products were analyzed by western immunoblotting. Longer exposure of same blot is shown in the right panel. B) Comparison of time dependent changes in full length apoE4 levels upon incubation in cortical (CTX) and hippocampal (HPC, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093120#pone-0093120-g001" target="_blank">fig. 1A</a>) conditioned medium. C) Ratio of apoE fragments lesser than 15 kDa to initial apoE levels was quantified using ImageJ. D) Conditioned medium obtained from primary rat hippocampal (HPC), cortical (CTX) and glia cells were mixed with equal volumes of PBS or each other as indicated. 2 ug rE4 was incubated with these conditioned medium for 6 h at 37°C, followed by western immunoblot analysis of apoE. Full length apoE levels were quantified as indicated.</p

Extracellular Proteolysis of Apolipoprotein E (apoE) by Secreted Serine Neuronal Protease

<div><p>Under normal conditions, brain apolipoprotein E (apoE) is secreted and lipidated by astrocytes, then taken up by neurons via receptor mediated endocytosis. Free apoE is either degraded in intraneuronal lysosomal compartments or released. Here we identified a novel way by which apoE undergoes proteolysis in the extracellular space via a secreted neuronal protease. We show that apoE is cleaved in neuronal conditioned media by a secreted serine protease. This apoE cleavage was inhibited by PMSF and α1-antichymotrypsin, but not neuroserpin-1 or inhibitors of thrombin and cathepsin G, supporting its identity as a chymotrypsin like protease. In addition, apoE incubation with purified chymotrypsin produced a similar pattern of apoE fragments. Analysis of apoE fragments by mass spectrometry showed cleavages occuring at the C-terminal side of apoE tryptophan residues, further supporting our identification of cleavage by chymotrypsin like protease. Hippocampal neurons were more efficient in mediating this apoE cleavage than cortical neurons. Proteolysis of apoE4 generated higher levels of low molecular weight fragments compared to apoE3. Primary glial cultures released an inhibitor of this proteolytic activity. Together, these studies reveal novel mechanism by which apoE can be regulated and therefore could be useful in designing apoE directed AD therapeutic approaches.</p></div

Detection of chymotrypsin cleavage sites in apoE3 using mass spectrometry.

<p>A) Representation of full length apoE3 protein sequence and observed proteolytic sites in ∼25, 20 and 17 kDa fragments generated upon incubation of rE3 in hippocampal conditioned medium. Two common cleavages sites at the carboxy side of W-20 and W-39 are indicated by red lines. Additional cleavage site in the ∼25 kDa apoE fragments at W-210 is indicated by arrow. Additional 6 cleavage sites in ∼20 kDa fragments are indicated by black lines, whereas additional two cleavage sites at W-26 and E-171 in ∼17 kDa fragments are marked by arrows with solid heads. Amino acid position is indicated by numbers. B) Annotated tandem mass spectra of identified peptides generated upon cleavage by chymotrypsin like protease at W-20 (<i>i</i>), W-26 (<i>ii</i>) W-39 (<i>iii</i>) and W-210 (<i>iv</i>).</p

Characterization of chymotrypsin like protease activity and its inhibitor in neuronal and glia conditioned medium respectively.

<p>A) DIV 18 hippocampal conditioned medium was incubated in presence or absence of PMSF (1 ug/ml) or 0.25 ug α1-ACT for 2 h at 37°C, followed by casein zymography. B) 0.5 ug α-chymotrypsin (chym) was incubated alone or together with 0.25 ug α1-ACT in PBS for 2 h at 37°C. Similarly, hippocampal (HPC), cortical (CTX) and glia (Glia) conditioned media were incubated alone or together with 0.25 ug α1-ACT for 2 h at 37°C. Samples were later analyzed by casein zymography. C) HPC, CTX and Glia conditioned medium was mixed with PBS or each other in 50∶50 ratio and incubated for 2 h at 37°C and was analyzed by casein zymograpgy. D) Glia conditioned medium was mixed with HPC conditioned medium or PBS as indicated in 90∶10 ratio and incubated 2 h at 37°C followed by casein zymograpgy. Hippocampal conditioned medium alone or pre-incubated with α1-ACT was loaded as control. E) Decreasing amounts of α-chymotrypsin (chym) was incubated with conditioned glia medium as indicated for 2 h at 37°C and was analyzed by casein zymograpgy.</p

Inhibition of apoE degradation by PMSF and protease inhibitor mix.

<p>A) rE3 and rE4 proteins were incubated with DIV20 hippocampal medium for 4 hours in the absence (ctrl) or presence of 10 ug/ml PMSF or 1× complete protease inhibitor mix (PI). ApoE and its proteolytic fragments were later analyzed by immunoblotting. B, C) Full length apoE levels (B) and ratio of apoE fragments greater than 15 kDa to full length apoE (C) were measured using ImageJ. D) rE4 was incubated with indicated concentrations of α-chymotrypsin (chym) for 4 hours and generated proteolytic fragments of apoE were analyzed by western immunoblotting. A longer exposure to highlight smaller apoE fragments in shown in the right panel.</p

Thrombin and cathepsin G do not contribute to apoE proteolysis in conditioned neuronal medium.

<p>A) Rec apoE4 (2 ug) was incubated together with purified human thrombin (5 U) in presence and absence of direct thrombin inhibitor argotroban (AGTB) at indicated concentrations for 16 h at 37°C. Since AGTB was dissolved in DMSO, DMSO control was included. AGTB was very potent in inhibiting thrombin mediated degradation of apoE. B) Rec apoE4 (2 ug) was incubated together with purified cathepsin G (cath G) at indicated concentrations in absence and presence of 100 nM cathepsin G inhibitor (CGI) for 16 h. ApoE proteolysis was analyzed by immunoblotting. C) Rec apoE3 and apoE4 (2 ug) were incubated with conditioned hippocampal medium for 16 h at 37°C in presence and absence of thrombin inhibitor, AGTB and cathepsin G inhibitor, CGI at indicated concentrations. ApoE was analyzed by western immunoblotting. Full length apoE levels were quantified as above.</p

<i>Ex-vivo</i> degradation of rec apoE in conditioned medium obtained from rat primary hippocampal neuron cultures.

<p>A–C) Conditioned medium was obtained from DIV 22 rat primary hippocampal neurons, and rec apoE3 (rE3) and apoE4 (rE4) (2 ug) were incubated with conditioned medium for indicated times at 37°C. Degradation of apoE at each time point was analyzed by western immunoblotting (A). Longer exposure of same blot is shown on the right panel. The levels of apoE fragments greater than 15 kDa (B) and lesser than 15 kDa (C) compared to apoE at t = 0 were determined using ImageJ. D–H) Commercially obtained rE3 and rE4 was incubated with hippocampal conditioned medium for indicated time periods followed by analysis using western immunoblotting (D). Longer exposure of same blot is shown on the right panel. Levels of full length apoE (E), total apoE fragments (F) and ratio of apoE fragments greater than 15 kDa (G) and lesser than 15 kDa (H) to full length apoE levels were quantified using ImageJ.</p

α1-ACT impairs apoE proteolysis.

<p>A) rE4 was incubated with hippocampal conditioned medium for 6 hours in absence (ctrl) or presence of either α1-antichymotrypsin (α1-ACT) or neuroserpin-1 (NSP1) at 1 ug/ml. Degradation of apoE was analyzed by western immunoblotting. A longer exposure is in the lower panel. B–D) Levels of full length apoE (B), ratio of apoE fragments greater than 15 kDa (C) and lesser than 15 kDa (D) to full length apoE levels were quantified using ImageJ.</p