scholarly journals TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology

2021 ◽  
Author(s):  
Wenhui Qiao ◽  
Yixing Chen ◽  
Yuka Martens ◽  
Chia-chen Liu ◽  
Joshua Knight ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Mouse Model ◽  
Proteolytic Processing ◽  
Synaptic Function ◽  
Amyloid Burden ◽  
Wild Type ◽  
Amyloid Pathology ◽  
Soluble Trem2 ◽  
Aβ Clearance

The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimer's disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site, leading to enhanced shedding. Here, we generated a novel Trem2 H157Y knock-in mouse model to investigate how this H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates Aβ clearance and reduces amyloid burden and microgliosis. Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

scholarly journals ­TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology

2021 ◽  
Author(s):  
Wenhui Qiao ◽  
Yixing Chen ◽  
Yuka A. Martens ◽  
Chia-Chen Liu ◽  
Joshua A. Knight ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Proteolytic Processing ◽  
Synaptic Function ◽  
Hek293 Cells ◽  
Immunofluorescent Staining ◽  
Amyloid Pathology ◽  
Biochemical Assays ◽  
Soluble Trem2

Abstract Background: The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimer’s disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site and was shown to enhance shedding and impair phagocytosis in vitro by ectopic expression of TREM2-H157Y in HEK293 cells. However, the physiological and AD-related outcomes of TREM2 H157Y mutation in vivo remain unknown.Methods: We generated a novel Trem2 H157Y knock-in mouse model through CRISPR-Cas9 technology and investigated how Trem2 H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology by conducting biochemical assays, immunofluorescent staining, hippocampal electrophysiology, and in vivo micro-dialysis in awake, free-moving animals.Results: Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates Aβ clearance and reduces amyloid burden and microgliosis. Conclusion: Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

scholarly journals Loss of Non-Apoptotic Role of Caspase-3 in the PINK1 Mouse Model of Parkinson’s Disease

2019 ◽  
Vol 20 (14) ◽  
pp. 3407 ◽  
Author(s):  
Paola Imbriani ◽  
Annalisa Tassone ◽  
Maria Meringolo ◽  
Giulia Ponterio ◽  
Graziella Madeo ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Synaptic Plasticity ◽  
Mouse Model ◽  
Caspase 3 ◽  
Cysteine Proteases ◽  
Synaptic Function ◽  
Adult Brain ◽  
Striatal Slices

Caspases are a family of conserved cysteine proteases that play key roles in multiple cellular processes, including programmed cell death and inflammation. Recent evidence shows that caspases are also involved in crucial non-apoptotic functions, such as dendrite development, axon pruning, and synaptic plasticity mechanisms underlying learning and memory processes. The activated form of caspase-3, which is known to trigger widespread damage and degeneration, can also modulate synaptic function in the adult brain. Thus, in the present study, we tested the hypothesis that caspase-3 modulates synaptic plasticity at corticostriatal synapses in the phosphatase and tensin homolog (PTEN) induced kinase 1 (PINK1) mouse model of Parkinson’s disease (PD). Loss of PINK1 has been previously associated with an impairment of corticostriatal long-term depression (LTD), rescued by amphetamine-induced dopamine release. Here, we show that caspase-3 activity, measured after LTD induction, is significantly decreased in the PINK1 knockout model compared with wild-type mice. Accordingly, pretreatment of striatal slices with the caspase-3 activator α-(Trichloromethyl)-4-pyridineethanol (PETCM) rescues a physiological LTD in PINK1 knockout mice. Furthermore, the inhibition of caspase-3 prevents the amphetamine-induced rescue of LTD in the same model. Our data support a hormesis-based double role of caspase-3; when massively activated, it induces apoptosis, while at lower level of activation, it modulates physiological phenomena, like the expression of corticostriatal LTD. Exploring the non-apoptotic activation of caspase-3 may contribute to clarify the mechanisms involved in synaptic failure in PD, as well as in view of new potential pharmacological targets.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

scholarly journals Increased levels of Stress-inducible phosphoprotein-1 accelerates amyloid-β deposition in a mouse model of Alzheimer’s disease

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Rachel E. Lackie ◽  
Jose Marques-Lopes ◽  
Valeriy G. Ostapchenko ◽  
Sarah Good ◽  
Wing-Yiu Choy ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Model ◽  
Protein Quality ◽  
Amyloid Β ◽  
Amyloid Burden ◽  
C Elegans ◽  
Model Of Alzheimer’S Disease

Abstract Molecular chaperones and co-chaperones, which are part of the protein quality control machinery, have been shown to regulate distinct aspects of Alzheimer’s Disease (AD) pathology in multiple ways. Notably, the co-chaperone STI1, which presents increased levels in AD, can protect mammalian neurons from amyloid-β toxicity in vitro and reduced STI1 levels worsen Aβ toxicity in C. elegans. However, whether increased STI1 levels can protect neurons in vivo remains unknown. We determined that overexpression of STI1 and/or Hsp90 protected C. elegans expressing Aβ(3–42) against Aβ-mediated paralysis. Mammalian neurons were also protected by elevated levels of endogenous STI1 in vitro, and this effect was mainly due to extracellular STI1. Surprisingly, in the 5xFAD mouse model of AD, by overexpressing STI1, we find increased amyloid burden, which amplifies neurotoxicity and worsens spatial memory deficits in these mutants. Increased levels of STI1 disturbed the expression of Aβ-regulating enzymes (BACE1 and MMP-2), suggesting potential mechanisms by which amyloid burden is increased in mice. Notably, we observed that STI1 accumulates in dense-core AD plaques in both 5xFAD mice and human brain tissue. Our findings suggest that elevated levels of STI1 contribute to Aβ accumulation, and that STI1 is deposited in AD plaques in mice and humans. We conclude that despite the protective effects of STI1 in C. elegans and in mammalian cultured neurons, in vivo, the predominant effect of elevated STI1 is deleterious in AD.

scholarly journals Efficacy of BB-83698, a Novel Peptide Deformylase Inhibitor, in a Mouse Model of Pneumococcal Pneumonia

2004 ◽  
Vol 48 (1) ◽  
pp. 80-85 ◽  
Author(s):  
E. Azoulay-Dupuis ◽  
J. Mohler ◽  
J. P. Bédos
Keyword(s):  
Mouse Model ◽  
Lung Tissue ◽  
Resistant Strain ◽  
Peptide Deformylase ◽  
In Vitro Activity ◽  
Wild Type ◽  
Virulent Strains ◽  
Resistant Strains

ABSTRACT The efficacy of BB-83698, a novel potent peptide deformylase inhibitor, was evaluated in a mouse model of acute pneumonia. The Streptococcus pneumoniae isolates tested included four virulent strains (one penicillin-susceptible wild-type strain, one macrolide-resistant strain, and two quinolone-resistant mutants [a mutant carrying mutations in ParC and GyrA and an efflux mutant] isogenic to the wild type) and two poorly virulent penicillin-resistant strains. Pneumonia was induced by intratracheal inoculation of 105 CFU (virulent strains) into immunocompetent mice or 107 CFU (less virulent strains) into leukopenic mice. Animals received three or six subcutaneous injections of antibiotics at 12- or 24-h intervals, with antibiotic treatment initiated at 3, 6, 12, or 18 h postinfection (p.i.). BB-83698 showed potent in vitro activity against all strains (MICs, 0.06 to 0.25 μg/ml). In the in vivo model, all control animals died within 2 to 5 days of infection. BB-83698 (80 mg/kg of body weight twice daily or 160 mg/kg once daily) protected 70 to 100% of the animals, as measured 10 days p.i., regardless of the preexisting resistance mechanisms. In contrast, the survival rates for animals treated with the comparator antibiotics were 30% for animals treated with erythromycin (100 mg/kg) and infected with the macrolide-resistant strain, 34% for animals treated with amoxicillin (200 mg/kg every 8 h) and infected with the penicillin-resistant strain, and 0 and 78% for animals treated with ciprofloxacin (250 mg/kg) and infected with the ParC and GyrA mutant and the efflux mutant, respectively. At 80 mg/kg, BB-83698 generated a peak concentration in lung tissue of 61.9 μg/ml within 1 h and areas under the concentration-times curves of 57.4 and 229.4 μg · h/ml for plasma and lung tissue, respectively. The emergence of S. pneumoniae isolates with reduced susceptibilities to BB-83698 was not observed following treatment with a suboptimal dosing regimen. In conclusion, the potent in vitro activity of BB-83698 against S. pneumoniae, including resistant strains, translates into good in vivo efficacy in a mouse pneumonia model.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

scholarly journals Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence of Neisseria meningitidis

2006 ◽  
Vol 74 (2) ◽  
pp. 1360-1367 ◽  
Author(s):  
Laura Plant ◽  
Johanna Sundqvist ◽  
Susu Zughaier ◽  
Lena Lövkvist ◽  
David S. Stephens ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Neisseria Meningitidis ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Inner Core ◽  
Wild Type ◽  
Proinflammatory Response

ABSTRACT Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

scholarly journals Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation

2006 ◽  
Vol 74 (12) ◽  
pp. 6907-6919 ◽  
Author(s):  
Andrea Hamilton ◽  
Carl Robinson ◽  
Iain C. Sutcliffe ◽  
Josh Slater ◽  
Duncan J. Maskell ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mutant Strain ◽  
Natural Host ◽  
Wild Type ◽  
Animal Suffering ◽  
Organ Cultures ◽  
Mucus Production ◽  
Streptococcus Equi

ABSTRACT Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM 138 - 213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt 190 - 685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt 190 - 685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt 190 - 685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM 138 - 213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt 190 - 685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

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