ALBERT

Description: TDP-43 aggregation in the cytoplasm or nucleus is a key feature of the pathology of amyotrophic lateral sclerosis and frontotemporal dementia and is observed in numerous other neurodegenerative diseases, including Alzheimer's disease. Despite this fact, the inciting events leading to TDP-43 aggregation remain unclear. We observed that endogenous TDP-43 undergoes reversible aggregation in the nucleus after the heat shock and that this behavior is mediated by the C-terminal prion domain. Substitution of the prion domain from TIA-1 or an authentic yeast prion domain from RNQ1 into TDP-43 can completely recapitulate heat shock-induced aggregation. TDP-43 is constitutively bound to members of the Hsp40/Hsp70 family, and we found that heat shock-induced TDP-43 aggregation is mediated by the availability of these chaperones interacting with the inherently disordered C-terminal prion domain. Finally, we observed that the aggregation of TDP-43 during heat shock led to decreased binding to hnRNPA1, and a change in TDP-43 RNA-binding partners suggesting that TDP-43 aggregation alters its function in response to misfolded protein stress. These findings indicate that TDP-43 shares properties with physiologic prions from yeast, in that self-aggregation is mediated by a Q/N-rich disordered domain, is modulated by chaperone proteins and leads to altered function of the protein. Furthermore, they indicate that TDP-43 aggregation is regulated by chaperone availability, explaining the recurrent observation of TDP-43 aggregates in degenerative diseases of both the brain and muscle where protein homeostasis is disrupted.

Description: Increasing attention has focused on the significance of RNA in sperm, in light of its contribution to the birth and long-term health of a child, role in sperm function and diagnostic potential. As the composition of sperm RNA is in flux, assigning specific roles to individual RNAs presents a significant challenge. For the first time RNA-seq was used to characterize the population of coding and non-coding transcripts in human sperm. Examining RNA representation as a function of multiple methods of library preparation revealed unique features indicative of very specific and stage-dependent maturation and regulation of sperm RNA, illuminating their various transitional roles. Correlation of sperm transcript abundance with epigenetic marks suggested roles for these elements in the pre- and post-fertilization genome. Several classes of non-coding RNAs including lncRNAs, CARs, pri-miRNAs, novel elements and mRNAs have been identified which, based on factors including relative abundance, integrity in sperm, available knockout data of embryonic effect and presence or absence in the unfertilized human oocyte, are likely to be essential male factors critical to early post-fertilization development. The diverse and unique attributes of sperm transcripts that were revealed provides the first detailed analysis of the biology and anticipated clinical significance of spermatozoal RNAs.

Description: Abstract 2054 Poster Board II-31 The serine proteinases, human neutrophil elastase (HNE) and proteinase 3 (PR3) are degradative enzymes stored in cytoplasmic azurophilic granules of neutrophils. Both proteins are aberrantly expressed in human myeloid leukemias. Over-expression of PR3 is important in maintaining a leukemia phenotype, while HNE may suppress hematopoietic progenitors. PR3 and HNE contain a conserved nonameric HLA-A*02 restricted T-cell epitope called PR1. The presence of PR1-specific CTL has been correlated with molecular remission in CML. A PR1 nonameric peptide vaccine has been evaluated clinically, with promising results. A limitation of the PR1 peptide vaccine is its restriction to patients with the HLA-A*02 allele. A vaccine comprising a larger portion of the PR3 and/or HNE protein and capable of inducing broader T-cell responses restricted by a wider range of HLA alleles is a potential alternative. We have generated numerous recombinant vaccinia viruses (rVV) expressing forms of PR3 and HNE and have evaluated CTL responses in an HLA-A*02 transgenic mouse model (Tg-A2): (1) rVV-EGFP-PR3, contains a gene encoding EGFP fused to human PR3 lacking the N-terminal signal peptide, while retaining both pro-dipeptide and C-terminal propeptide sequences; (2) EGFP fused to C-terminal truncated PR3 (rVV-EGFP-PR3-T) deleted of PR2 CTL epitope, but maintaining PR1 and; (3) rVV-EGFP-HNE. Tg-A2 were immunized with rVV-EGFP-PR3, and 2 weeks later immune splenocytes were expanded by in vitrostimulation (IVS) with syngeneic naïve mouse splenocytes loaded with an overlapping PR3 peptide library. Intracellular cytokine (ICC) assays using a PR3 peptide library or PR1 9-mer epitope peptide demonstrated a robust PR3-specific CD8+ T-cell response, surprisingly without a detectable response to the PR1 peptide. However, PR2, a decamer, is immunodominant in mice following this immunization. This result was unexpected, since the PR2 epitope was identified by computer prediction as a potential HLA-A*02-restricted epitope within PR3 but, unlike the HLA-A*02-restricted 9-mer epitope PR1, has not been extensively studied as a T-cell epitope in humans. We tested whether the immunodominant PR2-specific response in rVV-EGFP-PR3 immunized mice is also found when a naturally-processed PR3 protein is used as the stimulating antigen, expressed from the cell line K562-A2-PR3, a derivative of K562-A2 endogenously expressing human PR3. Splenocytes from mice immunized with rVV-EGFP-PR3 were expanded by co-culture with irradiated K562-A2-PR3 cells, then tested in ICC assays using PR1 and PR2 CTL epitope peptides. We observed a robust response to PR2 but not PR1. While the PR1 epitope is absolutely conserved between human and murine versions of PR3 and HNE, the PR2 epitope is unique to human PR3, diverging from murine PR3 by 3 amino acids, and by 5 amino acid from human and murine HNE. We speculated that Tg-A2 mice may be tolerant to PR1 due to the presence of this epitope within murine PR3 and HNE, which may account for dominance of PR2-specific responses. To investigate the possibility of inducing PR1-specific CTL in Tg-A2, mice were immunized with rVV-EGFP-HNE, and we detected PR1-specific responses by IVS followed by ICC with the PR1 peptide. These results indicate that tolerance to PR1 can likely be broken by immunization with rVV expressing HNE protein that contains the PR1 epitope. These observations can be explained by either the PR2〉PR1 competition hypothesis, or by different amino acid sequences flanking the PR1 epitope in PR3 and HNE proteins affecting processing of the protein to generate the PR1 peptide. To test this question, we used rVV-PR3-T to immunize Tg-A2 mice, then conducted IVS followed by ICC with the PR1 peptide to detect PR1-specific CTL. Results showed that immunization with rVV expressing truncated PR3 reliably induced PR1-specific CTL, unlike immunization with rVV expressing the full-length PR3 antigen. We concluded that PR2 epitope prevented the recognition of PR1. Therefore in Tg-A2 mice, vaccination with poxvirus vaccines expressing PR3 will induce an HLA-A2-restricted CTL response almost entirely focused on PR2, whereas immunization with rVV-HNE will induce PR1-specific CTL. If this were replicated in humans, the majority of PR1-specific CTL detected in CML patients could be derived from HNE rather than PR3 antigen presentation, and that PR2-specific CTL derived from PR3 should be equally frequent as PR1 and likely contribute to clinical GVL and tumor regression. Disclosures: No relevant conflicts of interest to declare.

Description: It has been suggested that the BCR-ABL gene of chronic myeloid leukemia (CML) is not uniformly expressed in Philadelphia (Ph)-positive cells, and that BCR-ABL gene expression precludes transcription of the normal BCR or ABL genes. Therefore, we have analyzed granulocyte-macrophage colony-forming unit (CFU-GM) colonies derived from peripheral blood of 11 CML patients by cytogenetic and by reverse transcriptase-polymerase chain reaction (PCR) amplification of BCR-ABL, ABL-BCR, BCR, and ABL. All CFU-GM colonies with analyzable metaphases were found to contain a Ph chromosome. In 2 patients, the initial PCR screening failed to detect BCR-ABL transcripts in 2 of 11 and 1 of 7 Ph-positive colonies. However, when amplification for BCR-ABL was repeated in quintuplicate, all but 1 colony from a single patient showed one or more positive results. Amplifications of the four genes in each colony showed that BCR-ABL, ABL-BCR, and the normal BCR and ABL were simultaneously expressed in the majority of CFU-GM colonies. Replicate PCR tests for BCR and for ABL in colonies initially scored as negative also uncovered previously undetected positive amplifications. We conclude that BCR-ABL expression does not suppress transcription from the normal BCR and ABL genes, and that Ph-positive, BCR-ABL-negative colonies derived from peripheral blood CFU-GM are rare or nonexistent.