Aß Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer's Disease.

We report a highly significant correlation in brain proteome changes between Alzheimers disease (AD) and CRND8 APP695NL/F transgenic mice. However, integrating protein changes observed in the CRND8 mice with co-expression networks derived from human AD, reveals both conserved and divergent module changes. For the most highly conserved module (M42, matrisome) we find many proteins accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A ß ; in plaques and in CAA; further, recombinant MDK and PTN enhance A ß ; aggregation into amyloid. Multiple M42 proteins, annotated as heparan sulfate binding proteins, bind to fibrillar A ß 42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart and islet amyloid polypeptide (IAPP) amyloid in the pancreas. Our findings establish several critical insights. Proteomic changes in modules observed in human AD brains define an A ß ; amyloid responsome that is well conserved from mouse model to human. Further, distinct amyloid structures may serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions. We hypothesize that this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aß scFvs: Applications for gene therapies.

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept in vivo study, we evaluated three different anti-amyloid Aß scFvs (anti-Aß scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aß scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

Modulating innate immune activation states impacts the efficacy of specific Aß immunotherapy.

INTRODUCTION: Passive immunotherapies targeting Aß continue to be evaluated as Alzheimer's disease (AD) therapeutics, but there remains debate over the mechanisms by which these immunotherapies work. Besides the amount of preexisting Aß deposition and the type of deposit (compact or diffuse), there is little data concerning what factors, independent of those intrinsic to the antibody, might influence efficacy. Here we (i) explored how constitutive priming of the underlying innate activation states by Il10 and Il6 might influence passive Aß immunotherapy and (ii) evaluated transcriptomic data generated in the AMP-AD initiative to inform how these two cytokines and their receptors' mRNA levels are altered in human AD and an APP mouse model. METHODS: rAAV2/1 encoding EGFP, Il6 or Il10 were delivered by somatic brain transgenesis to neonatal (P0) TgCRND8 APP mice. Then, at 2 months of age, the mice were treated bi-weekly with a high-affinity anti-Aß1-16 mAb5 monoclonal antibody or control mouse IgG until 6 months of age. rAAV mediated transgene expression, amyloid accumulation, Aß levels and gliosis were assessed. Extensive transcriptomic data was used to evaluate the mRNA expression levels of IL10 and IL6 and their receptors in the postmortem human AD temporal cortex and in the brains of TgCRND8 mice, the later at multiple ages. RESULTS: Priming TgCRND8 mice with Il10 increases Aß loads and blocks efficacy of subsequent mAb5 passive immunotherapy, whereas priming with Il6 priming reduces Aß loads by itself and subsequent Aß immunotherapy shows only a slightly additive effect. Transcriptomic data shows that (i) there are significant increases in the mRNA levels of Il6 and Il10 receptors in the TgCRND8 mouse model and temporal cortex of humans with AD and (ii) there is a great deal of variance in individual mouse brain and the human temporal cortex of these interleukins and their receptors. CONCLUSIONS: The underlying immune activation state can markedly affect the efficacy of passive Aß immunotherapy. These results have important implications for ongoing human AD immunotherapy trials, as they indicate that underlying immune activation states within the brain, which may be highly variable, may influence the ability for passive immunotherapy to alter Aß deposition.

Role of Pnn in alternative splicing of a specific subset of lncRNAs of the corneal epithelium.

PURPOSE: GG-H whole transcriptome array analysis suggested involvement of PININ (PNN) in the alternative splicing of multiple long non-coding RNAs (lncRNAs). To further investigate PNN's role in regulating the alternative splicing of lncRNAs in a corneal epithelial context, we performed detailed analyses for detecting and identifying alternatively spliced lncRNAs. METHODS: Total RNA was isolated from PNN knockdown human corneal epithelial (HCET) cells or Pnn-deficient mouse corneas, and subjected to real-time-PCR (RT-PCR) assays, and the alternatively spliced lncRNAs were counted. Alternatively spliced lncRNAs were detected with in situ hybridization with variant-specific RNA probes on human cornea sections. RESULTS: Our analysis uncovered PNN's impact on the transcript levels of several lncRNAs including Linc00085 and HAS2-AS1. Interestingly, a mouse ortholog of HAS2-AS1, Has2as, clearly exhibited a differential splicing pattern among three major splice variants in the Pnn-deficient mouse cornea. The sequence analyses and quantification of splice variants of candidate lncRNAs, including RP11-295B20.2, RP11-18I14.1, and RP11-322M19.1, demonstrated complex configuration of their splicing changes, with a significant impact of PNN on the process. Knockdown of PNN in HCET cells led to specific changes in the inclusion of multiple cassette exons as well as in the use of alternative splice sites in RP11-322M19.1 and RP11-18I14.1, resulting in considerable net changes in the ratio between the splice variants. Finally, in situ hybridization analyses revealed the presence of RP11-295G20.2 in the nuclei of corneal epithelial cells, but not in the stromal cells of the human cornea, while RP11-322M19.1 was present in epithelial and non-epithelial cells. CONCLUSIONS: The data suggest PNN's role in the alternative splicing of a specific subset of lncRNAs might have a significant impact on the corneal epithelium.