Abstract
Background
Tauopathies, such as frontotemporal dementia (FTD) and Alzheimer’s dimentia (AD), are neurodegenerative diseases characterized by the pathological aggregation of paired helical ...filaments (PHFs) or neurofibrillary tangles (NFTs) within neurons and glia, leading to cell death
1
. PHFs and NFTs are formed by aggregation of hyperphosphorylated tau
1
. Mutations in the microtubule‐associated protein tau (MAPT) gene result in tauopathies. Moreover, FTD is a common clinical syndrome of 4 repeat (4R) tauopathies, as defined by aggregation of tau protein isoforms with four microtubule binding domains
2
. Here, we aimed to develop and characterize a physiologically relevant and robust in vitro FTD model, to aid the future development of FTD disease therapeutics.
Method
Using CRISPR‐Cas9 gene editing technology, familial mutations MAPT P301S and N279K underlying AD/FTD
3
were engineered into an iPSC line that carries the opti‐ox
TM
technology and can rapidly be reprogrammed into glutamatergic neurons
4
. By means of immunocytochemistry we characterized b‐amyloid oligomer treated neurons, and neurons with the MAPT mutations to assess tau hyperphosphorylation.
Result
The CRISPR‐edited MAPT neurons can mature and show a healthy morphology. b‐amyloid oligomers (dose dependently) induce tau hyperphosphorylation in wild type glutamatergic neurons. Total tau expression is reduced in MAPT P301S homozygous and N279K heterozygous cells when compared to wild type cells. The MAPT P301S cells also show hyperphosphorylation for p‐tau 217, p‐tau202/205, and p‐tau404, and the N279K cells show hyperphosphorylation for p‐tau202/205, and p‐tau404.
Conclusion
The elevated p‐tau to total tau ratio measured by the immunocytochemistry assay indicates the potential of at least two cell lines as possible disease models to aid future research into developing AD/FTD disease therapeutics.
References:
1. Silva MC, eLife, 2019
2. Seward ME, et al, 2013. Journal Cell Sci 126(5):1278‐1286
3. Hutton M, et al, 1998, Nature 393:702‐705
4. PawlowskiM, et al, 2017. Stem cell reports 8(4), 803‐812
Abstract
Background
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. Many ALS cases are associated with nuclear‐to‐cytosolic mislocalization and ...phosphorylation of the transactivation response DNA binding protein (TDP)‐43
1
. This is caused by several mutations such as M337V in the TAR DNA binding protein (TARDBP) gene
1
. In addition, proteasomal degradation is often affected, resulting in aggregation of TDP‐43
2
. TDP‐43 is involved in many mRNA processes, and its depletion results in mis‐splicing of the neuronal growth associated factor, stathmin 2 (STMN‐2)
3
. This mis‐splicing contributes to axonal degeneration. To model the ALS phenotypes in vitro, proteasomal inhibition is often used to trigger TDP‐43‐associated phenotypes
2
.
Methods
In this report we assessed if homozygous or heterozygous CRISPR‐edited TPD‐43 M337V mutations in induced pluripotent stem cell (iPSC)‐derived glutamatergic neurons, generated by bit.bio using the opti‐ox technology
4‐5
, alone, or in combination with an MG‐132 proteasomal inhibition trigger, would result in TDP‐43 mislocalization and/or STMN‐2 splicing, and could therefore be used as an ALS‐relevant disease model. This was assessed in highly mature (DIV21), and less mature (DIV7) neurons.
Results
Immunocytochemistry, high‐content imaging, and automated image analysis were used for visualization, localization, and quantification of proteins of interest in individual cells. Here, increase in TDP‐43 mislocalization, phosphorylation (and aggregation) was observed upon acute proteasomal inhibition in all cell lines compared to their respective untreated sample, with more extreme phenotypes on DIV7. More interestingly, the homozygous M337V cell line D5 showed showed TDP‐43 associated phenotypes in untreated conditions at DIV7. Digital droplet PCR was used to evaluate mRNA expression, using absolute quantification of nucleic acid molecules. This showed decrease of full length STMN‐2 and the generation of truncated STMN‐2 upon MG‐132 treatment in most cell lines. Additionally, preliminary functional data generated using Multi Electrode Arrays (MEA) showed that all neuronal lines demonstrated formation of synchronous activity at late stage of maturation, suggesting these neurons are electrophysiologically active and amenable to functional studies.
Conclusion
These results suggest that wildtype and mutant clones show TDP‐43 dysregulation and mis‐splicing of STMN‐2 upon acute proteasomal inhibition, thus establishing an ALS relevant phenotype that can be used for in vitro drug discovery.
There is now compelling evidence that members of the family of small heat shock proteins (HSP) can be secreted by a variety of different types of cells. Secretion of small HSP may at times represent ...altruistic delivery of supporting and stabilizing factors from one cell to another. A probably more general effect of extracellular small HSP, however, is exerted by their ability to activate macrophages and macrophage-like cells. When doing so, small HSP induce an immune-regulatory state of activation, stimulating macrophages to suppress inflammation. For this reason, small HSP deserve consideration as broadly applicable therapeutic agents for inflammatory disorders.
In one particular case, however, adaptive immune responses to the small HSP itself may subvert the protective quality of the innate immune response it triggers. This situation only applies to alpha B-crystallin, and is unique for humans as well. In this special case, local concentrations of alpha B-crystallin determine the balance between protective innate responses and destructive adaptive responses, the latter of which are held responsible for the development of multiple sclerosis lesions.
This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.
Activated microglia and macrophages play a key role in driving demyelination during multiple sclerosis (MS), but the factors responsible for their activation remain poorly understood. Here, we ...present evidence for a dual-trigger role of IFN-γ and alpha B-crystallin (HSPB5) in this context. In MS-affected brain tissue, accumulation of the molecular chaperone HSPB5 by stressed oligodendrocytes is a frequent event. We have shown before that this triggers a TLR2-mediated protective response in surrounding microglia, the molecular signature of which is widespread in normal-appearing brain tissue during MS. Here, we show that IFN-γ, which can be released by infiltrated T cells, changes the protective response of microglia and macrophages to HSPB5 into a robust pro-inflammatory classical response. Exposure of cultured microglia and macrophages to IFN-γ abrogated subsequent IL-10 induction by HSPB5, and strongly promoted HSPB5-triggered release of TNF-α, IL-6, IL-12, IL-1β and reactive oxygen and nitrogen species. In addition, high levels of CXCL9, CXCL10, CXL11, several guanylate-binding proteins and the ubiquitin-like protein FAT10 were induced by combined activation with IFN-γ and HSPB5. As immunohistochemical markers for microglia and macrophages exposed to both IFN-γ and HSPB5, these latter factors were found to be selectively expressed in inflammatory infiltrates in areas of demyelination during MS. In contrast, they were absent from activated microglia in normal-appearing brain tissue. Together, our data suggest that inflammatory demyelination during MS is selectively associated with IFN-γ-induced re-programming of an otherwise protective response of microglia and macrophages to the endogenous TLR2 agonist HSPB5.
Pentraxin‐3 (PTX3), an acute‐phase protein released during inflammation, aids phagocytic clearance of pathogens and apoptotic cells, and plays diverse immunoregulatory roles in tissue injury. In ...neuroinflammatory diseases, like MS, resident microglia could become activated by endogenous agonists for Toll like receptors (TLRs). Previously we showed a strong TLR2‐mediated induction of PTX3 in cultured human microglia and macrophages by HspB5, which accumulates in glia during MS. Given the anti‐inflammatory effects of HspB5, we examined the contribution of PTX3 to these effects in MS and its animal model EAE. Our data indicate that TLR engagement effectively induces PTX3 expression in human microglia, and that such expression is readily detectable in MS lesions. Enhanced PTX3 expression is prominently expressed in microglia in preactive MS lesions, and in microglia/macrophages engaged in myelin phagocytosis in actively demyelinating lesions. Yet, we did not detect PTX3 in cerebrospinal fluid of MS patients. PTX3 expression is also elevated in spinal cords during chronic relapsing EAE in Biozzi ABH mice, but the EAE severity and time course in PTX3‐deficient mice did not differ from WT mice. Moreover, systemic PTX3 administration did not alter the disease onset or severity. Our findings reveal local functions of PTX3 during neuroinflammation in facilitating myelin phagocytosis, but do not point to a role for PTX3 in controlling the development of autoimmune neuroinflammation.
In neuroinflammatory diseases, like multiple sclerosis, resident brain microglia become activated by endogenous agonists for Toll‐like receptors (TLRs). These induce secretion of large amounts of pentraxin‐3 (PTX3), an antibody‐like protein. In this study, we found that PTX3 likely fulfils only local functions and does not inhibit main pathways in experimental autoimmune neuroinflammation (EAE).
Abstract As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects ...are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal/phagosomal CD14 and Toll-like receptors 1 and 2. Humans, however, possess natural antibodies against HSPB5 that block receptor binding. To protect it from these antibodies, we encapsulated HSPB5 in porous PLGA microparticles. We document here size, morphology, protein loading and release characteristics of such microparticles. Apart from effectively protecting HSPB5 from neutralization, PLGA microparticles also strongly promoted macrophage targeting of HSPB via phagocytosis. As a result, HSPB5 in porous PLGA microparticles was more than 100-fold more effective in activating macrophages than free soluble protein. Yet, the immune-regulatory nature of the macrophage response, as documented here by microarray transcript profiling, remained the same. In mice developing cigarette smoke-induced COPD, HSPB5-loaded PLGA microparticles were selectively taken up by alveolar macrophages upon intratracheal administration, and significantly suppressed lung infiltration by lymphocytes and neutrophils. In contrast, 30-fold higher doses of free soluble HSPB5 remained ineffective. Our data indicate that porous HSPB5-PLGA microparticles hold considerable promise as an anti-inflammatory biomaterial for humans.
ABSTRACTMicroglial nodules are frequently observed in the normal-appearing white matter of multiple sclerosis (MS) patients. Previously, we have shown that these clusters, which we call “preactive MS ...lesions,” are closely associated with stressed oligodendrocytes and myelin sheaths that contain markedly elevated levels of the small stress protein alpha-B-crystallin (HspB5). Here, we show that microglia in these lesions express the recently identified receptors for HspB5, that is, CD14, Toll-like receptor family 1 and 2 (TLR1 and TLR2), and several molecular markers of the microglial response to HspB5. These markers were identified by genome-wide transcript profiling of 12 primary human microglial cultures at 2 time points after exposure to HspB5. These data indicate that HspB5 activates production by microglia of an array of chemokines, immune-regulatory mediators, and a striking number of antiviral genes that are generally inducible by type I interferons. Together, our data suggest that preactive MS lesions are at least in part driven by HspB5 derived from stressed oligodendrocytes and may reflect a local attempt to restore tissue homeostasis.
Activated microglia and macrophages play a key role in driving demyelination during multiple sclerosis (MS), but the factors responsible for their activation remain poorly understood. Here, we ...present evidence for a dual-trigger role of IFN-γ and alpha B-crystallin (HSPB5) in this context. In MS-affected brain tissue, accumulation of the molecular chaperone HSPB5 by stressed oligodendrocytes is a frequent event. We have shown before that this triggers a TLR2-mediated protective response in surrounding microglia, the molecular signature of which is widespread in normal-appearing brain tissue during MS. Here, we show that IFN-γ, which can be released by infiltrated T cells, changes the protective response of microglia and macrophages to HSPB5 into a robust pro-inflammatory classical response. Exposure of cultured microglia and macrophages to IFN-γ abrogated subsequent IL-10 induction by HSPB5, and strongly promoted HSPB5-triggered release of TNF-alpha, IL-6, IL-12, IL-1beta and reactive oxygen and nitrogen species. In addition, high levels of CXCL9, CXCL10, CXL11, several guanylate-binding proteins and the ubiquitin-like protein FAT10 were induced by combined activation with IFN-γ and HSPB5. As immunohistochemical markers for microglia and macrophages exposed to both IFN-γ and HSPB5, these latter factors were found to be selectively expressed in inflammatory infiltrates in areas of demyelination during MS. In contrast, they were absent from activated microglia in normal-appearing brain tissue. Together, our data suggest that inflammatory demyelination during MS is selectively associated with IFN-γ-induced re-programming of an otherwise protective response of microglia and macrophages to the endogenous TLR2 agonist HSPB5.PUBLICATION ABSTRACT
CEP‐1347 is a potent inhibitor of the mixed lineage kinases (MLKs), a distinct family of mitogen‐activated protein kinase kinase kinases (MAPKKK). It blocks the activation of the c‐Jun/JNK apoptotic ...pathway in neurons exposed to various stressors and attenuates neurodegeneration in animal models of Parkinson's disease (PD). Microglial activation may involve kinase pathways controlled by MLKs and might contribute to the pathology of neurodegenerative diseases. Therefore, the possibility that CEP‐1347 modulates the microglial inflammatory response tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and monocyte chemotactic protein‐1 (MCP‐1) was explored. Indeed, the MLK inhibitor CEP‐1347 reduced cytokine production in primary cultures of human and murine microglia, and in monocyte/macrophage‐derived cell lines, stimulated with various endotoxins or the plaque forming peptide Aβ1–40. Moreover, CEP‐1347 inhibited brain TNF production induced by intracerebroventricular injection of lipopolysaccharide in mice. As expected from a MLK inhibitor, CEP‐1347 acted upstream of p38 and c‐Jun activation in microglia by dampening the activity of both pathways. These data imply MLKs as important, yet unrecognized, modulators of microglial inflammation, and demonstrate a novel anti‐inflammatory potential of CEP‐1347.
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