MicroRNA-100-5p and microRNA-298-5p released from apoptotic cortical neurons are endogenous Toll-like receptor 7/8 ligands that contribute to neurodegeneration

Background: MicroRNA (miRNA) expression in the brain is altered in neurodegenerative diseases. Recent studies demonstrated that selected miRNAs conventionally regulating gene expression at the post-transcriptional level can act extracellularly as signaling molecules. The identity of miRNA species serving as membrane receptor ligands involved in neuronal apoptosis in the central nervous system (CNS), as well as the miRNAs' sequence and structure required for this mode of action remained largely unresolved. Methods. Using a microarray-based screening approach we analyzed apoptotic cortical neurons of C56BL/6 mice and their supernatant with respect to alterations in miRNA expression/presence. HEK-Blue Toll-like receptor (TLR) 7/8 reporter cells, primary microglia and macrophages derived from human and mouse were employed to test the potential of the identified miRNAs released from apoptotic neurons to serve as signaling molecules for the RNA-sensing receptors. Biophysical and bioinformatical approaches, as well as immunoassays and sequential microscopy were used to analyze the interaction between candidate miRNA and TLR. Immunocytochemical and -histochemical analyses of murine CNS cultures and adult mice intrathecally injected with miRNAs, respectively, were performed to evaluate the impact of miRNA-induced TLR activation on neuronal survival and microglial activation. Results: We identified a specific pattern of miRNAs released from apoptotic cortical neurons that activate TLR7 and/or TLR8, depending on sequence and species. Exposure of microglia and macrophages to certain miRNA classes released from apoptotic neurons resulted in the sequence-specific production of distinct cytokines/chemokines and increased phagocytic activity. Out of those miRNAs miR-100-5p and miR-298-5p, which have consistently been linked to neurodegenerative diseases, entered microglia, located to their endosomes, and directly bound to human TLR8. The miRNA-TLR interaction required novel sequence features, but no specific structure formation of mature miRNA. As a consequence of miR-100-5p- and miR-298-5p-induced TLR activation, cortical neurons underwent cell-autonomous apoptosis. Presence of miR-100-5p and miR-298-5p in cerebrospinal fluid led to neurodegeneration and microglial accumulation in the murine cerebral cortex through TLR7 signaling. Conclusion: Our data demonstrate that specific miRNAs are released from apoptotic cortical neurons, serve as endogenous TLR7/8 ligands, and thereby trigger further neuronal apoptosis in the CNS. Our findings underline the recently discovered role of miRNAs as extracellular signaling molecules, particularly in the context of neurodegeneration

Abnormal brain structure and behavior in MyD88-deficient mice.

While the original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis, the role of its ortholog Toll-like receptors (TLRs), the interleukin 1 receptor (IL-1R) family, and the associated signaling pathways in mammalian brain development and structure is poorly understood. Because the adaptor protein myeloid differentiation primary response protein 88 (MyD88) is essential for downstream signaling of most TLRs and IL-1R, we systematically investigated the effect of MyD88 deficiency on murine brain structure during development and on behavior. In neonatal Myd88 mice, neocortical thickness was reduced, while density of cortical neurons was increased. In contrast, microglia, astrocyte, oligodendrocyte, and proliferating cell numbers were unchanged in these mice compared to wild-type mice. In adult Myd88 mice, neocortical thickness was unaltered, but neuronal density in neocortex and hippocampus was increased. Neuron arborization was less pronounced in adult Myd88 mice compared to wild-type animals. In addition, numbers of microglia and proliferating cells were increased in the neocortex and subventricular zone, respectively, with unaltered astrocyte and oligodendrocyte numbers, and myelinization was enhanced in the adult Myd88 neocortex. These morphologic changes in the brain of adult Myd88 mice were accompanied by specific behavioral traits, such as decreased locomotor activity, increased anxiety-like behavior, but normal day/light activity, satisfactory learning, short- and long-term spatial memory, potential cognitive inflexibility, and increased hanging and locomotor behavior within their home cage. Taken together, MyD88 deficiency results in morphologic and cellular changes in the mouse brain, as well as in altered natural and specific behaviors. Our data indicate a pathophysiological significance of MyD88 for mammalian CNS development, structure, and function.This work was supported by Deutsche Forschungsgemeinschaft (DFG) LE 2420/2-1, SFB-TRR167/B03, NeuroCure Exc 257 (to S.L.), SFB-1315, FOR 3004 (to A.M.K.), and by Charité – Universitätsmedizin Berlin

Prognostic Value of Tumour-Infiltrating Lymphocytes in an Unselected Cohort of Breast Cancer Patients

Tumour-infiltrating lymphocytes (TILs) are considered to have prognostic and predictive value for patients with early breast cancer. We examined 1166 breast cancer patients from a prospective, multicentre cohort (Prognostic Assessment in Routine Application (PiA), n = 1270, NCT 01592825) following recommendations from the International TILs Working Group. TIL quantification was performed using predefined groups and as a continuous variable in 10% increments. The primary objective was the distribution of TILs in different breast cancer types. The second objective was the association with the recurrence-free interval (RFI) and overall survival (OS). Stromal infiltration with more than 60% TILs appeared in 2% of hormone receptor (HR)-positive and HER2-negative tumours, in 9.8% of HER2-positive tumours (any HR) and 19.4% of triple-negative breast cancers (TNBCs). Each 10% increment was associated with an improvement in the prognosis in HER2-positive samples (RFI, hazard ratio 0.773, 95% CI 0.587–1.017; OS, hazard ratio 0.700, 95% CI 0.523–0.937). When defining exploratory cut-offs for TILs, the use of a 30% threshold for the HR-positive and HER2-negative group, a 20% threshold for the HER2 group and a 60% threshold for the TNBC group appeared to be the most suitable. TILs bore prognostic value, especially in HER2-positive breast cancer. For clinical use, additional research on the components of immune infiltration might be reasonable

Comparison of the K78 chromosome to representative chromosomes within Borrelia.

<p>^aSequence identities and indel contents calculated with stretcher (EMBOSS package [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref044" target="_blank">44</a>])</p><p>^bSum of two unconnected non-overlapping contigs (436,767 + 466,749 bp)</p><p>^cUnfinished assembly (5 contigs, of which the shortest with 1774 bp length has been left out of the comparative analysis)</p><p>*Approximate values due to incompleteness of the chromosome assemblies.</p><p>Comparison of the K78 chromosome to representative chromosomes within Borrelia.</p

Chromosomal region of 5S-23S rRNA and 16S rRNA for the <i>B</i>. <i>afzelii</i> strains K78, ACA-1, PKo, Tom3107, HLJ01 and for <i>B</i>. <i>burgdorferi</i> B31.

<p>The rRNAs (marked red), are presented with transcription from right to left as located on the chromosome, and are composed of two copies of 16S rRNA, separated by tRNA-Ala. A tRNA-Ile (transcribed left to right) precedes the tandem repeats of the 23S-5S cassette. In many cases one of the 16S copies has undergone degeneration. In the case of ACA-1 the two contigs constituting the chromosome are separated at the position where the second 23S rRNA copy is expected (vertical red line), meaning the presence or absence of the second copy of 23S rRNA could not be determined due to the lower sequencing quality in this region. There is a high sequence homology among the four <i>B</i>. <i>afzelii</i> strains (except for the second copy of 23S rRNA of ACA-1) in contrast to the sequences in <i>B</i>. <i>burgdorferi</i> B31 rRNAs. The similarity score plots of the Mauve alignments use the backbone color scheme [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref043" target="_blank">43</a>] which shows overall similarity in a mauve color or clustering blocks among cluster members in the same color.</p

Comparison of the replicons found in <i>Borrelia afzelii</i> K78 to the published sequences of <i>B</i>. <i>afzelii</i> strains ACA-1, PKo and <i>B</i>. <i>burgdorferi</i> strain B31.

<p>^a Accession numbers (GenBank, RefSeq) are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.s010" target="_blank">S3 Table</a>.</p><p>^b Another cp9 plasmid has been described for B31 which is named cp9–2 (renaming the listed to cp9–1) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref065" target="_blank">65</a>]</p><p>^c The attribution to code “Q” which is the naming for cp32–10 has been made via the presence of the respective plasmid partitioning protein type of the paralogous family 32 (PFam32). The linear plasmid lp56 in B31 is longer and contains parts analog to the cp32–10 type plasmids therefore this plasmid has been proposed to be attributed to code “Q” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref046" target="_blank">46</a>]. Linear plasmids lp32–10, as seen in PKo and ACA-1, carry a PFam32 gene similar to cp32–10 and therefore also get the code “Q” in spite of carrying different gene content.</p><p>^d There is data from an earlier PKo genome project available, with a chromosome length of 905.4 kbp, GenBank CP000395) with an apparent insert of two genes (BAPKO_0393, BAPKO_0395) and a full definition of the 3’-terminal <i>arcB</i> gene (truncated in the listed chromosome).</p><p>^e Two more plasmids, cp32–2, which has identical PFam32 and PFam49 genes as cp32–7, and cp32–5 have been described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref066" target="_blank">66</a>] but have not yet been sequenced in full length.</p><p>Comparison of the replicons found in <i>Borrelia afzelii</i> K78 to the published sequences of <i>B</i>. <i>afzelii</i> strains ACA-1, PKo and <i>B</i>. <i>burgdorferi</i> strain B31.</p

Functional classification of the <i>B</i>. <i>afzelii</i> K78 annotated genome, describing a total of 1,309 proteins.

<p>The best-hits per category from rpsblast against COG with a cutoff of E-value 0.01 are counted. Proteins with the best-hit falling into more than one category are counted as hit in each category which results in the addition of 51 hits, resulting in a total of 758 hits to defined COGs.</p><p>Functional classification of the <i>B</i>. <i>afzelii</i> K78 annotated genome, describing a total of 1,309 proteins.</p

Alignment of <i>B</i>. <i>afzelii</i> K78 <i>ospC</i> sequence against the sequences of <i>B</i>. <i>afzelii</i> strains from public databases.

<p>A non-redundant set of partial <i>ospC</i> sequences according to BAFK78_B0019 bp 97–583, comprising 59 <i>B</i>. <i>afzelii</i> strains and the sequence of <i>B</i>. <i>burgdorferi</i> B31 as external root reference were included in the analyses. A: Maximum likelihood tree representation, re-rooted with <i>B</i>. <i>burgdorferi</i> B31 as outgroup. Clusters containing strains attributed to human infectivity are boxed, of which the previously identified groups were labelled A1–A8. The strains compared in this study are highlighted in blue. B: A recombination network representation is shown for the sequences in an unrooted distance phylogram. The pairwise homoplasy index test for the <i>B</i>. <i>afzelii</i> sequences (p = 7.8x10^-15) indicates significance for the presence of recombination events. The strains compared in this study are highlighted by a yellow background.</p

Species-specific variation of the intergenic variable region in the circular plasmid cp26.

<p>The variable sequence segment in the cp26 plasmids of K78 is compared with 25 <i>Borrelia</i> strains. A maximum likelihood tree rooted with a <i>B</i>. <i>bissettii</i> sequence as outgroup, shows the relationship for the intergenic part, which in <i>B</i>. <i>afzelii</i> K78 is situated between BAFK78_A0014 and BAFK78_A0015 (bacterial extracellular solute-binding protein), and which shows a species-specific length. Insertions and deletions within this region have been analyzed with the program Mauve, and the compositional analysis for 16 of the 26 sequences (underlined in the tree view) is shown with related segments marked by color and/or boxes together with a similarity score diagram for each sequence. Blue bars denote segments which, in some strains, have been annotated as short hypothetical proteins (the number of assigned proteins in this region is indicated in parentheses in the tree view).</p

Comparison of the sequence types of <i>B</i>. <i>afzelii</i> strains according to multi locus sequence typing (MLST), and <i>ospA</i> and <i>ospC</i> typing.

<p>MLST typing, according to the system described by Margos <i>et al</i>.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref054" target="_blank">54</a>] comprising 592 defined profiles, assigned K78 to sequence type ST335 which is identical to the Italian strains 0600839I and 05001891I in the Borrelia MLST database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref055" target="_blank">55</a>]. No match for ACA-1 and Tom3107 was found in the MLST data base with their respective sequence profiles. Column ospA lists the ospA sequence type from the MLSA database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref056" target="_blank">56</a>] and in parentheses the OspA serotypes. The nearest hit for Tom3107 is ospA sequence type-3 with 1 bp mismatch: ospC classification follows the scheme of Seinost <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref057" target="_blank">57</a>] and Lagal <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120548#pone.0120548.ref058" target="_blank">58</a>]. Tom3107 do not fall into any invasive group. For strain HLJ01 only the chromosome sequence is available.</p><p>Comparison of the sequence types of <i>B</i>. <i>afzelii</i> strains according to multi locus sequence typing (MLST), and <i>ospA</i> and <i>ospC</i> typing.</p