IgG Conformer's Binding to Amyloidogenic Aggregates

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs

Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve

Publisher's PDF.Injury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neurodegeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves. IntroductionUniversity of Delaware. Department of Biological Sciences

Distinct pattern of miRNA levels in sciatic nerve in response to injury.

<p>The heatmap with a cluster dendrogram showed significant changes in levels of 63 miRNAs in rat sciatic nerve that received a crush injury and were sacrificed at 1, 4, 7, and 14 days post-injury (DPI), as compared to the sham-operated uninjured control (n = 3/group). <b>A.</b> The color scale shown on the top left denotes the relative expression level of the indicated miRNA across all time points (log₂ scale): red represents an increased change in level and green denotes a decreased level. Clustering analysis was performed using <i>Cluster 3</i>.<i>0</i> with an average linkage and Euclidean distances metric and visualized using Java <i>TreeView</i>. <b>B.</b> Each column represents different time points after injury [1, 4, 7, and 14 days post-injury (DPI)], as compared to the uninjured control (n = 3/group), and rows represents individual miRNAs. Red represents an increased change in level compared to that in the uninjured control and green denotes a decreased level. Insets on the top show three distinct clusters of miRNA changes. Dashed lines indicate miRNA levels in the uninjured control.</p

Ingenuity Pathway Analysis of the differentially expressed up-regulated and down-regulated miRNA predicted targets.

<p>The listed pathways were from the category of “nervous system development and function” and sorted by <i>p</i>-value. Only rows having 3 or more molecules were shown.</p

Validation of mature miRNA sequencing data by real time qPCR assay.

<p>Bar graph showed changes in levels of 8 miRNAs at 1, 4, 7, and 14 days post-injury (DPI), as compared to those of uninjured control. Top panels. miRNAs significantly up-regulated following injury. Bottom panels. miRNAs significantly down-regulated following injury. Error bars indicate standard deviation.</p

Enrichment and detection of small non-coding miRNA from sciatic nerve axoplasm.

<p><b>A.</b> Extended RT-PCR of mRNAs from sciatic nerve axoplasm using either by the conventional whole tissue lysate method or a mechanical squeezing procedure. Note that neither cell body restricted (MAP2, H1F0) nor glial (GFAP, ErbB3) mRNAs were amplified from sciatic nerve cDNAs reverse-transcribed from the squeezed axoplasmic RNAs. <b>B.</b> Size and frequency distribution of regulatory and small non-coding RNAs in uninjured sciatic nerve. <b>C.</b> A size histogram of mapped small RNAs. >40% of small RNAs were mapped to miRNAs, while 30% of small RNA sequences could not be annotated.</p

Seven miRNAs significantly up-regulated and eight miRNAs down-regulated following nerve injury.

<p>Seven miRNAs significantly up-regulated and eight miRNAs down-regulated following nerve injury.</p

Aβ-isolated but not heat-induced Avastin aggregates have enhanced avidity for PFs.

<p>(<b>A</b>) Left panel: SEC chromatograms for 0.3 mg/mL of Aβ-isolated Avastin IgGs, untreated Avastin, and for the antibody diluted into elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound Avastin IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: Antibody binding curves against PFs for unfractionated IVIg and Avastin, and for Aβ-isolated Avastin IgGs. (<b>B</b>) Left panel: SEC chromatograms for ~5 mg/mL of unfractionated Avastin in PBS, pH 7.4, and for IgG conformers contained in supernatant of 71°C heated Avastin monomers (A_400nm 0.5 sup) in PBS, pH 7.4. Right panel: Antibody binding curves against PFs for soluble (A_400nm 0.5 sup) and insoluble (A_400nm 0.5 pellet) IgG conformers of heat-treated Avastin monomers, and for untreated Avastin and IVIg.</p

IgG aggregates are primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated pAb IgGs.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~0.5 mg/mL of Aβ-isolated IVIg IgGs, and for IVIg, untreated, or diluted into column elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound IVIg IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: IgG binding curves against plate-immobilized PFs for untreated IVIg, and for Aβ column-isolated IVIg IgGs that were used unfractionated (Unfrac) or as SEC-isolated monomers (SEC Mon) or aggregates (SEC Aggs). SEC Aggs consisted of a pool of IgG conformers (dimers and HMW species) that eluted before the monomeric antibody. (<b>B</b>) Left panel: SEC chromatograms for ~0.5 mg/mL dye-isolated IVIg IgGs, and for unfractionated IVIg that was untreated or diluted into column elution buffer (PBS containing 1.5 M NaCl, pH 7.4) that was used to elute dye-bound IVIg IgGs. Right panel: IgG binding curves against plate-immobilized PFs for unfractionated and SEC-isolated conformers of dye-isolated IVIg IgGs, and for untreated IVIg.</p

Unfractionated, Aβ- and Cibacron blue-isolated human IgGs binding to plate-immobilized PFs.

<p>¹Mon stands for IgG monomers.</p><p>^2,3SEC-isolated IgG monomers (mon), dimers, and HMW aggregates as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g002" target="_blank">2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">3</a>.</p><p>Each value for EC₅₀ and maximum signal amplitude was determined from the average of two to three sigmoidal fitted antibody binding curves, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g008" target="_blank">8</a>.</p