A TREM2-activating antibody with a blood-brain barrier transport vehicle enhances microglial metabolism in Alzheimer's disease models

van Lengerich et al. developed a human TREM2 antibody with a transport vehicle (ATV) that improves brain exposure and biodistribution in mouse models. ATV:TREM2 promotes microglial energetic capacity and metabolism via mitochondrial pathways. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD

Systemic effects of periodontitis treatment in patients with type 2 diabetes: a 12 month, single-centre, investigator-masked, randomised trial

BACKGROUND: Chronic inflammation is believed to be a major mechanism underlying the pathophysiology of type 2 diabetes. Periodontitis is a cause of systemic inflammation. We aimed to assess the effects of periodontal treatment on glycaemic control in people with type 2 diabetes. METHODS: In this 12 month, single-centre, parallel-group, investigator-masked, randomised trial, we recruited patients with type 2 diabetes, moderate-to-severe periodontitis, and at least 15 teeth from four local hospitals and 15 medical or dental practices in the UK. We randomly assigned patients (1:1) using a computer-generated table to receive intensive periodontal treatment (IPT; whole mouth subgingival scaling, surgical periodontal therapy [if the participants showed good oral hygiene practice; otherwise dental cleaning again], and supportive periodontal therapy every 3 months until completion of the study) or control periodontal treatment (CPT; supra-gingival scaling and polishing at the same timepoints as in the IPT group). Treatment allocation included a process of minimisation in terms of diabetes onset, smoking status, sex, and periodontitis severity. Allocation to treatment was concealed in an opaque envelope and revealed to the clinician on the day of first treatment. With the exception of dental staff who performed the treatment and clinical examinations, all study investigators were masked to group allocation. The primary outcome was between-group difference in HbA1c at 12 months in the intention-to-treat population. This study is registered with the ISRCTN registry, number ISRCTN83229304. FINDINGS: Between Oct 1, 2008, and Oct 31, 2012, we randomly assigned 264 patients to IPT (n=133) or CPT (n=131), all of whom were included in the intention-to-treat population. At baseline, mean HbA1c was 8·1% (SD 1·7) in both groups. After 12 months, unadjusted mean HbA1c was 8·3% (SE 0·2) in the CPT group and 7·8% (0·2) in the IPT group; with adjustment for baseline HbA1c, age, sex, ethnicity, smoking status, duration of diabetes, and BMI, HbA1c was 0·6% (95% CI 0·3-0·9; p<0·0001) lower in the IPT group than in the CPT group. At least one adverse event was reported in 30 (23%) of 133 patients in the IPT group and 23 (18%) of 131 patients in the CPT group. Serious adverse events were reported in 11 (8%) patients in the IPT group, including one (1%) death, and 11 (8%) patients in the CPT group, including three (2%) deaths. INTERPRETATION: Compared with CPT, IPT reduced HbA1c in patients with type 2 diabetes and moderate-to-severe periodontitis after 12 months. These results suggest that routine oral health assessment and treatment of periodontitis could be important for effective management of type 2 diabetes. FUNDING: Diabetes UK and UK National Institute for Health Research.Diabetes UK and UK National Institute for Health Researc

Transcriptomic and Genetic Investigations into Lineage Specification of the Neural Crest and Tailbud in Vertebrate Embryo Development

In embryonic development, tissues arise from multipotent progenitor populations and undergo lineage specification and terminal differentiation. Two examples of this are the neural crest (NC) and the multiple progenitor cells of the tailbud. The NC is a multipotent highly migratory cell type that gives rise to numerous tissues and cell types in the adult animal, including pigmentation, cartilage and bone, neurons and glia, and many others. Much is known about the mechanisms driving NC induction and migration, but how lineage specification and migration are coordinated is still not well-understood. To address outstanding questions in NC development, we took a multifaceted approach, employing both traditional functional characterization of a novel Wnt regulator and data-driven single-cell and bulk transcriptomics. Here, I show that Lmo7a regulates both migration and specification of glial and pigment lineages through interactions with focal adhesions (FAs) and canonical Wnt signaling. Lmo7a is expressed in premigratory NC cell, and its loss results in large midline aggregates of NC cells that go on to express markers of pigment and glial lineages. These cells also show abnormally high levels of canonical Wnt signaling. I show that Lmo7a is required for proper formation of FAs, suggesting a novel connection between cell adhesion and lineage specification in the NC through canonical Wnt signaling.I also build a single-cell RNA-seq timeline of cranial NC development to investigate lineage decisions. I identify the precise timing of pigment, skeletal, and neural/glial lineage specification and identify several novel markers of each. Further, using combinatorial analysis of bulk and single-cell RNA-seq data, I computationally model the Wnt signaling dynamics that correspond to lineage specification. I identify a putative Wnt regulatory gene atp6ap2 which encodes the (Pro)renin receptor ((P)RR) protein as a potential regulator of pigment development. Using CRISPR-Cas9 genome editing, I verify a role for ((P)RR) in pigment maturation potentially through canonical Wnt signaling. I also investigate lineage relationships in the tailbud by constructing a comprehensive single-cell transcriptome map of all cells in the tailbud. I uncover evidence for a novel connection between neuromesodermal progenitors and both vascular endothelium and hematopoiesis. I also show that cell cycle arrest is indicative of a notochord fate in midline progenitor cells. In this thesis, I provide novel insights into lineage specification in the NC and tailbud

Transcriptomic and Genetic Investigations into Lineage Specification of the Neural Crest and Tailbud in Vertebrate Embryo Development

In embryonic development, tissues arise from multipotent progenitor populations and undergo lineage specification and terminal differentiation. Two examples of this are the neural crest (NC) and the multiple progenitor cells of the tailbud. The NC is a multipotent highly migratory cell type that gives rise to numerous tissues and cell types in the adult animal, including pigmentation, cartilage and bone, neurons and glia, and many others. Much is known about the mechanisms driving NC induction and migration, but how lineage specification and migration are coordinated is still not well-understood. To address outstanding questions in NC development, we took a multifaceted approach, employing both traditional functional characterization of a novel Wnt regulator and data-driven single-cell and bulk transcriptomics. Here, I show that Lmo7a regulates both migration and specification of glial and pigment lineages through interactions with focal adhesions (FAs) and canonical Wnt signaling. Lmo7a is expressed in premigratory NC cell, and its loss results in large midline aggregates of NC cells that go on to express markers of pigment and glial lineages. These cells also show abnormally high levels of canonical Wnt signaling. I show that Lmo7a is required for proper formation of FAs, suggesting a novel connection between cell adhesion and lineage specification in the NC through canonical Wnt signaling.I also build a single-cell RNA-seq timeline of cranial NC development to investigate lineage decisions. I identify the precise timing of pigment, skeletal, and neural/glial lineage specification and identify several novel markers of each. Further, using combinatorial analysis of bulk and single-cell RNA-seq data, I computationally model the Wnt signaling dynamics that correspond to lineage specification. I identify a putative Wnt regulatory gene atp6ap2 which encodes the (Pro)renin receptor ((P)RR) protein as a potential regulator of pigment development. Using CRISPR-Cas9 genome editing, I verify a role for ((P)RR) in pigment maturation potentially through canonical Wnt signaling. I also investigate lineage relationships in the tailbud by constructing a comprehensive single-cell transcriptome map of all cells in the tailbud. I uncover evidence for a novel connection between neuromesodermal progenitors and both vascular endothelium and hematopoiesis. I also show that cell cycle arrest is indicative of a notochord fate in midline progenitor cells. In this thesis, I provide novel insights into lineage specification in the NC and tailbud

Single-cell transcriptomic analysis of zebrafish cranial neural crest reveals spatiotemporal regulation of lineage decisions during development.

Neural crest (NC) cells migrate throughout vertebrate embryos to give rise to a huge variety of cell types, but when and where lineages emerge and their regulation remain unclear. We have performed single-cell RNA sequencing (RNA-seq) of cranial NC cells from the first pharyngeal arch in zebrafish over several stages during migration. Computational analysis combining pseudotime and real-time data reveals that these NC cells first adopt a transitional state, becoming specified mid-migration, with the first lineage decisions being skeletal and pigment, followed by neural and glial progenitors. In addition, by computationally integrating these data with RNA-seq data from a transgenic Wnt reporter line, we identify gene cohorts with similar temporal responses to Wnts during migration and show that one, Atp6ap2, is required for melanocyte differentiation. Together, our results show that cranial NC cell lineages arise progressively and uncover a series of spatially restricted cell interactions likely to regulate such cell-fate decisions

The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells

Repeated delivery of chlorhexidine chips for the treatment of peri‐implantitis: A multicenter, randomized, comparative clinical trial

BackgroundPeri‐implantitis is a challenging condition to manage and is frequently treated using non‐surgical debridement. The local delivery of antimicrobial agents has demonstrated benefit in mild to moderate cases of peri‐implantitis. This study compared the safety and efficacy of chlorhexidine gluconate 2.5 mg chip (CHX chips) as an adjunctive treatment to subgingival debridement in patients afflicted with peri‐implantitis.MethodsA multicenter, randomized, single‐blind, two‐arm, parallel Phase‐3 study was conducted. Peri‐implantitis patients with implant pocket depths (IPD) of 5‐8 mm underwent subgingival implant surface debridement followed by repeated bi‐weekly supragingival plaque removal and chlorhexidine chips application (ChxC group) for 12 weeks, or similar therapy but without application of ChxC (control group). All patients were followed for 24 weeks. Plaque and gingival indices were measured at every visit whereas IPD, recession, and bleeding on probing were assessed at 8, 12, 16, 24 week.ResultsA total of 290 patients were included: 146 in the ChxC group and 144 in the control. At 24 weeks, a significant reduction in IPD (P = 0.01) was measured in the ChxC group (1.76 ± 1.13 mm) compared with the control group (1.54 ± 1.13 mm). IPD reduction of ≥2 mm was found in 59% and 47.2% of the implants in the ChxC and control groups, respectively (P = 0.03). Changes in gingival recession (0.29 ± 0.68 mm versus 0.15 ± 0.55 mm, P = 0.015) and relative attachment gain (1.47 ± 1.32 mm and 1.39 ± 1.27 mm, P = 0.0017) were significantly larger in the ChxC group. Patients in the ChxC group that were < 65 years exhibited significantly better responses (P < 0.02); likewise, non‐smokers had similarly better response (P < 0.02). Both protocols were well tolerated, and no severe treatment‐related adverse events were recorded throughout the study.ConclusionsPatients with peri‐implantitis that were treated with an intensive treatment protocol of bi‐weekly supragingival plaque removal and local application of chlorhexidine chips had greater mean IPD reduction and greater percentile of sites with IPD reduction of ≥2 mm as compared with bi‐weekly supra‐gingival plaque removal.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/166183/1/jper10672.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166183/2/jper10672_am.pd

CLR-1/RPTP acts in the UNC-6/Netrin and UNC-40/DCC pathway to direct SPR.

<p>In this model, limiting amounts of UNC-6/Netrin secreted from AVA interneurons bind UNC-40/DCC expressed in PHB neurons. CLR-1/RPTP expressed in AVA neurons acts genetically downstream of UNC-6/Netrin in the SPR pathway, and its extracellular domain is required for full SPR function, suggesting that it may interact with UNC-6/Netrin, UNC-40/DCC, or another unidentified ligand or receptor. The requirement of CLR-1/RPTP’s phosphatase domain for rescue indicates that phosphatase activity is also required for its function in SPR.</p

CLR-1/RPTP acts in postsynaptic neurons, and is localized to the synaptic region.

<p>(A) Schematics and micrographs of normal PHB-AVA NLG-1 GRASP fluorescence in wild-type and <i>clr-1/RPTP(e1745)</i> mutant animals expressing a transgene that drives expression of the <i>clr-1</i> cDNA in AVA neurons (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP</i>), and reduced PHB-AVA NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutant animals expressing either a construct that drives expression of the <i>clr-1/RPTP</i> cDNA in PHB neurons (_<i>p</i><i>PHB</i>::<i>clr-1/RPTP</i>), a transgene that drives expression of the <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted in AVA neurons (_<i>p</i><i>AVA</i>::<i>clr-1/RPTPΔxcd)</i>, or a transgene that drives expression of the <i>clr-1/RPTP</i> cDNA with a mutation that inactivates the phosphatase domain (_<i>p</i><i>AVA</i>::<i>clr-1/RPTPpd</i>). (B) Quantification of NLG-1 GRASP fluorescence. Expression of <i>clr-1/RPTP</i> in AVAs, but not PHBs restores NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutants (n>75). Expression of the <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted or with a mutation in the active site of the phosphatase domain does not fully restore NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutants (n>100). Two or more lines were examined with each transgene, and combined in the graph above. Values for each individual transgenic line are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s006" target="_blank">S2 Table</a>. NS, not significant, ***<i>P</i><0.001, *<i>P</i><0.05, U-test. Comparison to <i>clr-1/RPTP</i> indicated over individual bars. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>. (C) Expression of <i>clr-1/RPTP</i> in AVAs, but not PHBs, rescues the behavioral defect in <i>clr-1/RPTP(e1745)</i> mutants (n>75). Expression of <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted or with a mutation in the active site of the phosphatase domain does not fully rescue the behavioral defect in <i>clr-1/RPTP(e1745)</i> mutants (n≥60). NS, not significant, ***<i>P</i><0.001, t-test. Comparison to <i>clr-1/RPTP</i> indicated over individual bars. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. Error bars are SEM. (D) Schematic and micrograph of an animal expressing the <i>clr-1/RPTP</i> cDNA linked to <i>YFP</i> in AVA (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP</i>::<i>YFP</i>). (E) Schematic and micrograph of an animal expressing the <i>clr-1/RPTP</i> cDNA linked to <i>mCherry</i> in AVA (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP</i>::<i>mCherry</i>) and PHB-AVA NLG-1 GRASP, and overlay in a <i>clr-1/RPTP</i> mutant animal. (D-E), CLR-1 localization is brightest in the preanal ganglion (yellow box), and the majority of animals show localization in the anterior half of this region, where PHB-AVA synapses usually form (green fluorescence).</p