An optimized filter trap assay for detecting recombinant authentic tau fibrils.

The development and optimization of the Filter Trap Assay (FTA) for the detection of authentic tau fibrils in vitro mark a pivotal advancement in the realm of tauopathy research, particularly by addressing the limitations of using polyanion-induced tau fibrils, which structurally differ from those isolated from tauopathy patients. Recently it has been shown that truncated tau fragment (297-391), also termed dGAE, can form authentic tau fibrils in the absence of polyanions. This study introduces a refined protocol that reliably detects authentic tau fibrils in a physiologically relevant framework, utilizing nitrocellulose membranes to achieve heightened sensitivity. Our investigation highlights the superior efficacy of sarkosyl, an anionic surfactant traditionally used to prepare protein lysates from brains and cultured neurons, in preserving the aggregated state of tau dGAE fibrils in vitro, underscoring its potential for further exploratory studies. By offering a user-friendly and economically feasible approach, this technique enables a broad range of laboratories to measure the presence of authentic tau fibrils. This methodological enhancement propels our understanding of tauopathies forward and bridges the gap between basic research and advanced structural analyses, enriching the scientific community's methodologies for studying neurodegenerative disorders.

Small Heat Shock Proteins Collaborate with FAIM to Prevent Accumulation of Misfolded Protein Aggregates.

Cells and tissues are continuously subject to environmental insults, such as heat shock and oxidative stress, which cause the accumulation of cytotoxic, aggregated proteins. We previously found that Fas Apoptosis Inhibitory Molecule (FAIM) protects cells from stress-induced cell death by preventing abnormal generation of protein aggregates similar to the effect of small heat shock proteins (HSPs). Protein aggregates are often associated with neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we sought to determine how FAIM protein dynamics change during cellular stress and how FAIM prevents the formation of amyloid-ß aggregates/fibrils, one of the pathological hallmarks of AD. Here, we found that the majority of FAIM protein shifts to the detergent-insoluble fraction in response to cellular stress. A similar shift to the insoluble fraction was also observed in small heat shock protein (sHSP) family molecules, such as HSP27, after stress. We further demonstrate that FAIM is recruited to sHSP-containing complexes after cellular stress induction. These data suggest that FAIM might prevent protein aggregation in concert with sHSPs. In fact, we observed the additional effect of FAIM and HSP27 on the prevention of protein aggregates using an in vitro amyloid-ß aggregation model system. Our work provides new insights into the interrelationships among FAIM, sHSPs, and amyloid-ß aggregation.

Microparticle immunocapture assay for quantitation of protein multimer amount and size.

Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, including target specificity, operative simplicity, and process speed. Here, we report development of a microparticle immunocapture assay that combines the advantages inherent to a monoclonal antibody:protein interaction with highly quantitative flow cytometry analysis. Using established reagents to build our platform, and aggregation-prone amyloid beta 1-42 peptide (Aß42) and alpha-synuclein to demonstrate proof of principle, our results indicate that this assay is a highly adaptable method to measure multimer size and quantity at the same time in a technically streamlined workflow applicable to laboratory and clinical samples.

Fas Apoptosis Inhibitory Molecule Blocks and Dissolves Pathological Amyloid-ß Species.

A number of neurodegenerative diseases are associated with the accumulation of misfolded proteins, including Alzheimer's disease (AD). In AD, misfolded proteins such as tau and amyloid-ß (Aß) form pathological insoluble deposits. It is hypothesized that molecules capable of dissolving such protein aggregates might reverse disease progression and improve the lives of afflicted AD patients. Here we report new functions of the highly conserved mammalian protein, Fas Apoptosis Inhibitory Molecule (FAIM). We found that FAIM-deficient Neuro 2A cells accumulate Aß oligomers/fibrils. We further found that recombinant human FAIM prevents the generation of pathologic Aß oligomers and fibrils in a cell-free system, suggesting that FAIM functions without any additional cellular components. More importantly, recombinant human FAIM disaggregates and solubilizes established Aß fibrils. Our results identify a previously unknown, completely novel candidate for understanding and treating irremediable, irreversible, and unrelenting neurodegenerative diseases.

FAIM Opposes Aggregation of Mutant SOD1 That Typifies Some Forms of Familial Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative illness that is unremittingly fatal and for which no effective treatment exists. All forms of ALS are characterized by protein aggregation. In familial forms of ALS, specific and heritable aggregation-prone proteins have been identified, such as mutant superoxide dismutase (SOD1). It has been suggested that a factor capable of preventing mutant SOD1 protein aggregation and/or disassembling mutant SOD1 protein aggregates would ameliorate SOD1-associated forms of familial ALS. Here we identify Fas Apoptosis Inhibitory Molecule (FAIM), a highly evolutionarily conserved 20 kDa protein, as an agent with this activity. We show FAIM counteracts intracellular accumulation of mutant SOD1 protein aggregates, which is increased in the absence of FAIM, as determined by pulse-shape analysis and filter trap assays. In a cell-free system, FAIM inhibits aggregation of mutant SOD1, and further disassembles and solubilizes established mutant SOD1 protein aggregates, as determined by thioflavin T (ThT), filter trap, and sedimentation assays. In sum, we report here a previously unknown activity of FAIM that opposes ALS disease-related protein aggregation and promotes proteostasis of an aggregation-prone ALS protein.

FAIM Is a Non-redundant Defender of Cellular Viability in the Face of Heat and Oxidative Stress and Interferes With Accumulation of Stress-Induced Protein Aggregates.

A key element of cellular homeostasis lies in the way in which misfolded and dysfunctional proteins are handled. Cellular pathways that include proteasomal destruction and autophagic disposal are components of normal proteostasis. Here we report a novel molecule that plays a non-redundant role in maintaining homeostasis, Fas Apoptosis Inhibitory Molecule (FAIM). FAIM is highly conserved throughout evolution and bears no homology to any other protein. We found that FAIM counteracts heat and oxidative stress-induced loss of cell viability. FAIM is recruited to ubiquitinated proteins induced by cellular stress and the levels of stress-induced protein aggregates are much greater in FAIM-deficient cell lines. Primary fibroblasts from FAIM-deficient mice showed the same proteostasis deficits as cell lines. Administration of a mediator of oxidative stress to FAIM-deficient animals induced more ubiquitinated protein aggregates and more organ damage as compared to wild type mice. These results identify a completely new actor that protects cells against stress-induced loss of viability by preventing protein aggregation.

CD25 + B-1a Cells Express Aicda.

B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID), a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO) mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC) B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM) progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

A novel mechanism of B cell-mediated immune suppression through CD73 expression and adenosine production.

Immune suppression by regulatory T cells and regulatory B cells is a critical mechanism to limit excess inflammation and autoimmunity. IL-10 is considered the major mediator of B cell-induced immune suppression. We report a novel mechanism for immune suppression through adenosine generation by B cells. We identified a novel population of B cells that expresses CD73 as well as CD39, two ectoenzymes that together catalyze the extracellular dephosphorylation of adenine nucleotides to adenosine. Whereas CD39 expression is common among B cells, CD73 expression is not. Approximately 30-50% of B-1 cells (B220(+)CD23(-)) and IL-10-producing B (B10) cells (B220(+)CD5(+)CD1d(hi)) are CD73(hi), depending on mouse strain, whereas few conventional B-2 cells (B220(+)CD23(+)AA4.1(-)) express CD73. In keeping with expression of both CD73 and CD39, we found that CD73(+) B cells produce adenosine in the presence of substrate, whereas B-2 cells do not. CD73(-/-) mice were more susceptible to dextran sulfate sodium salt (DSS)-induced colitis than wild type (WT) mice were, and transfer of CD73(+) B cells ameliorated the severity of colitis, suggesting that B cell CD73/CD39/adenosine can modulate DSS-induced colitis. IL-10 production by B cells is not affected by CD73 deficiency. Interestingly, adenosine generation by IL-10(-/-) B cells is impaired because of reduced expression of CD73, indicating an unexpected connection between IL-10 and adenosine and suggesting caution in interpreting the results of studies with IL-10(-/-) cells. Our findings demonstrate a novel regulatory role of B cells on colitis through adenosine generation in an IL-10-independent manner.

Human B-1 cells take the stage.

B-1 cells play critical roles in defending against microbial invasion and in housekeeping removal of cellular debris. B-1 cells secrete natural antibody and manifest functions that influence T cell expansion and differentiation and in these and other ways differ from conventional B-2 cells. B-1 cells were originally studied in mice where they are easily distinguished from B-2 cells, but their identity in the human system remained poorly defined for many years. Recently, functional criteria for human B-1 cells were established on the basis of murine findings, and reverse engineering resulted in identification of the phenotypic profile, CD20(+)CD27(+)CD43(+)CD70(-), for B-1 cells found in both umbilical cord blood and adult peripheral blood. Human B-1 cells may contribute to multiple disease states through production of autoantibody and stimulation/modulation of T cell activity. Human B-1 cells could be a rich source of antibodies useful in treating diseases present in elderly populations where natural antibody protection may have eroded. Manipulation of human B-1 cell numbers and/or activity may be a new avenue for altering T cell function and treating immune dyscrasias.

A CD25⁻ positive population of activated B1 cells expresses LIFR and responds to LIF.

B1 B cells defend against infectious microorganisms by spontaneous secretion of broadly reactive "natural" immunoglobulin that appears in the absence of immunization. Among many distinguishing characteristics, B1 B cells display evidence of activation that includes phosphorylated STAT3. In order to identify the origin of pSTAT3 we examined interleukin-2 receptor (IL-2R) expression on B1 cells. We found that some (about 1/5) B1a cells express the IL-2R α chain, CD25. Although lacking CD122 and unresponsive to IL-2, B1a cells marked by CD25 express increased levels of activated signaling intermediates, interruption of which results in diminished CD25. Further, CD25⁺ B1a cells contain most of the pSTAT3 found in the B1a population as a whole. Moreover, CD25⁺ B1a cells express leukemia inhibitory factor receptor (LIFR), and respond to LIF by upregulating pSTAT3. Together, these results define a new subset of B1a cells that is marked by activation-dependent CD25 expression, expresses substantial amounts of activated STAT3, and contains a functional LIFR.

Fas apoptosis inhibitory molecule expression in B cells is regulated through IRF4 in a feed-forward mechanism.

Fas apoptosis inhibitory molecule (FAIM) was originally cloned as an inhibitor of Fas-mediated apoptosis in B cells that has been reported to affect multiple cell types. Recently, we found that FAIM enhances CD40L-mediated signal transduction, including induction of IFN regulatory factor (IRF)4, in vitro and augments plasma cell production in vivo. These results have keyed interest in the regulation of FAIM expression, about which little is known. Here, we show that Faim is regulated by IRF4. The Faim promoter contains three IRF binding sites, any two of which promote Faim expression. Faim promoter activity is lost following mutation of all three IRF binding sites, whereas activity of the full promoter is enhanced by concurrent expression of IRF4. In stimulated primary B cells, IRF4 expression precedes FAIM expression, IRF4 binds directly to the Faim promoter, and loss of IRF4 results in the failure of stimulated Faim up-regulation. Finally, FAIM is preferentially expressed in germinal center B cells. Taken together, these results indicate that FAIM expression is regulated through IRF4 and that this most likely occurs as part of germinal center formation. Because FAIM enhances CD40-induced IRF4 expression in B cells, these results suggest that induction of FAIM initiates a positive reinforcing (i.e., feed-forward) system in which IRF4 expression is both enhanced by FAIM and promotes FAIM expression.

Fas apoptosis inhibitory molecule enhances CD40 signaling in B cells and augments the plasma cell compartment.

Fas apoptosis inhibitory molecule (FAIM) was cloned as a mediator of Fas resistance that is highly evolutionarily conserved but contains no known effector motifs. In this study, we report entirely new functions of FAIM that regulate B cell signaling and differentiation. FAIM acts to specifically enhance CD40 signaling for NF-kappaB activation, IRF-4 expression, and BCL-6 down-regulation in vitro, but has no effect on its own or in conjunction with LPS or anti-Ig stimulation. In keeping with its effects on IRF-4 and BCL-6, FAIM overexpression augments the plasma cell compartment in vivo. These results indicate that FAIM is a new player on the field of B cell differentiation and acts as a force multiplier for a series of events that begins with CD40 engagement and ends with plasma cell differentiation.

Alteration of enzymatic properties of cell-surface antigen CD38 by agonistic anti-CD38 antibodies that prolong B cell survival and induce activation.

Leukocyte cell-surface antigen CD38 is a single-transmembrane protein. CD38 ligation by anti-CD38 antibodies triggers the growth or apoptosis of immune cells. Although the extracellular domain of CD38 has multifunctional catalytic activities including NAD(+) glycohydrolase and cyclase, the CD38-mediated cell survival or death appears to be independent of its catalytic activity. It is proposed that a conformational change of CD38 triggers the signalling. The conformational change of CD38 could influence its catalytic activity. However, the agonistic anti-CD38 antibody that alters the catalytic activity of CD38 has not been reported so far. In the present study, we demonstrated that two agonistic anti-mouse CD38 mAbs (CS/2 and clone 90) change the catalytic activities of CD38. CS/2 was clearly more potent than clone 90 in prolonging B cell survival and activation. CS/2 inhibited the NAD(+) glycohydrolase activity of both the isolated extracellular domain of CD38 (FLAG-CD38) and cell-surface CD38. Kinetic analysis suggested a non-competitive inhibition. On the other hand, clone 90 stimulated the NAD(+) glycohydrolase activity of FLAG-CD38 and had little effect on the NAD(+) glycohydrolase activity of cell-surface CD38. CS/2 and clone 90 had no effect on the cyclase activity of FLAG-CD38 and inhibited the cyclase activity of cell-surface CD38. Accordingly, these agonistic antibodies probably induce the conformational changes of CD38 that are evident in the distinct alterations of the catalytic site. The antibodies will be useful tools to analyze the conformational change of CD38 in the process of triggering B cell survival and the activation signal.

B-1 cells express transgelin 2: unexpected lymphocyte expression of a smooth muscle protein identified by proteomic analysis of peritoneal B-1 cells.

B-1 cells constitute a unique B cell subset that differs phenotypically, biochemically, and functionally from the predominant population of conventional B-2 cells. Functional differences include constitutive secretion of natural immunoglobulin and failure of BCR signaling to initiate proliferation. The origin of these differences remains uncertain. We hypothesized that unbiased analysis of differences in protein expression between highly pure populations of B-1 and B-2 cells might provide information not readily available through other means. To pursue this, we undertook 2D gel analysis of B-1 and B-2 cells combined with mass spectrometry. We identified the smooth muscle protein, transgelin 2, in peritoneal (but not splenic) B-1 cells and did not find it in splenic B-2 cells; these results were confirmed by Western blot analysis, which showed a more than 60-fold difference in transgelin 2 expression between peritoneal B-1 and splenic B-2 cells. In contrast, levels of transgelin 2 RNA differed to a much lesser extent (3-fold) in the two B cell populations, so transgelin 2 is an example of a molecule whose subset-specific expression is more readily detected by proteomic than transcriptomic analyses. Finally, transgelin 2 protein expression was induced in splenic B-2 cells; thus, transgelin 2 joins a number of other inducible molecules that are constitutively expressed by peritoneal B-1 but not splenic B-2 cells. Although the role of transgelin 2 in B-1 cell function remains uncertain, identification of this molecule demonstrates the value of examining protein expression in this B cell subset.

NF-kappaB is required for CD38-mediated induction of C(gamma)1 germline transcripts in murine B lymphocytes.

Ligation of CD38 on murine B cells with agonistic anti-CD38 mAb induces B cell proliferation, expression of germline gamma1 transcripts and enhances IL-5 receptor expression. This leads to Ig class switch recombination from the micro to gamma1 heavy chain gene, and high levels of IgM and lgG1 production, particularly in response to anti-CD38 and IL-5 co-stimulation. Although some of the post-receptor signaling events initiated by CD38 ligation have been characterized, signaling pathways involved in CD38-mediated germline gamma1 transcript expression in B cells are poorly understood. Here we show that CD38 ligation of murine splenic B cells activates members of the NF-kappaB/Rel family of proteins including c-Rel, p65 and p50. The activation patterns and kinetics of NF-kappaB-like proteins in CD38-stimulated B cells differ somewhat from those seen in CD40-stimulated B cells. Activation of NF-kappaB-like proteins by CD38 ligation is not observed in splenic B cells from Bruton's tyrosine kinase (Btk)-deficient (Btk(-/-)) mice, with inhibitors of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase also suppressing NF-kappaB activation in CD38-activated B cells. We infer from these results that activation of Btk, PI-3 kinase and PKC play, at least in part, important roles in the induction of NF-kappaB in CD38-stimulated murine B cells. Consistent with a role for NF-kappaB/Rel signaling in CD38-mediated germline gamma1 transcript expression, p50(-/-) B cells show significant impairment of germline gamma1 transcript expression in response to CD38 ligation, whereas the CD40-induced response was not altered. In contrast, c-Rel(-/-) B cells show a severe impairment of germline gamma1 transcript expression in response to CD38 or CD40 ligation. These results indicate an essential role for NF-kappaB proteins in the induction of germline gamma1 transcripts by CD38-ligated murine B cells giving rise to IL-5-induced IgG1 production.