Ectodysplasin A (EDA) Signaling: From Skin Appendage to Multiple Diseases.

Ectodysplasin A (EDA) signaling is initially identified as morphogenic signaling regulating the formation of skin appendages including teeth, hair follicles, exocrine glands in mammals, feathers in birds and scales in fish. Gene mutation in EDA signaling causes hypohidrotic ectodermal dysplasia (HED), a congenital hereditary disease with malformation of skin appendages. Interestingly, emerging evidence suggests that EDA and its receptors can modulate the proliferation, apoptosis, differentiation and migration of cancer cells, and thus may regulate tumorigenesis and cancer progression. More recently, as a newly discovered hepatocyte factor, EDA pathway has been demonstrated to be involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and type II diabetes by regulating glucose and lipid metabolism. In this review, we summarize the function of EDA signaling from skin appendage development to multiple other diseases, and discuss the clinical application of recombinant EDA protein as well as other potential targets for disease intervention.

Eda-activated RelB recruits an SWI/SNF (BAF) chromatin-remodeling complex and initiates gene transcription in skin appendage formation.

Ectodysplasin A (Eda) signaling activates NF-κB during skin appendage formation, but how Eda controls specific gene transcription remains unclear. Here, we find that Eda triggers the formation of an NF-κB-associated SWI/SNF (BAF) complex in which p50/RelB recruits a linker protein, Tfg, that interacts with BAF45d in the BAF complex. We further reveal that Tfg is initially induced by Eda-mediated RelB activation and then bridges RelB and BAF for subsequent gene regulation. The BAF component BAF250a is particularly up-regulated in skin appendages, and epidermal knockout of BAF250a impairs skin appendage development, resulting in phenotypes similar to those of Eda-deficient mouse models. Transcription profiling identifies several target genes regulated by Eda, RelB, and BAF. Notably, RelB and the BAF complex are indispensable for transcription of Eda target genes, and both BAF complex and Eda signaling are required to open chromatin of Eda targets. Our studies thus suggest that Eda initiates a signaling cascade and recruits a BAF complex to specific gene loci to facilitate transcription during organogenesis.

ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes.

The APOE ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). ApoE protein aggregation plays a central role in AD pathology, including the accumulation of ß-amyloid (Aß). Lipid-poor ApoE4 protein is prone to aggregate and lipidating ApoE4 protects it from aggregation. The mechanisms regulating ApoE4 aggregation in vivo are surprisingly not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). ABCA1 recycling and degradation is regulated by ADP-ribosylation factor 6 (ARF6). We found that ApoE4 promoted greater expression of ARF6 compared with ApoE3, trapping ABCA1 in late-endosomes and impairing its recycling to the cell membrane. This was associated with lower ABCA1-mediated cholesterol efflux activity, a greater percentage of lipid-free ApoE particles, and lower Aß degradation capacity. Human CSF from APOE ε4/ε4 carriers showed a lower ability to induce ABCA1-mediated cholesterol efflux activity and greater percentage of aggregated ApoE protein compared with CSF from APOE ε3/ε3 carriers. Enhancing ABCA1 activity rescued impaired Aß degradation in ApoE4-treated cells and reduced both ApoE and ABCA1 aggregation in the hippocampus of male ApoE4-targeted replacement mice. Together, our data demonstrate that aggregated and lipid-poor ApoE4 increases ABCA1 aggregation and decreases ABCA1 cell membrane recycling. Enhancing ABCA1 activity to reduce ApoE and ABCA1 aggregation is a potential therapeutic strategy for the prevention of ApoE4 aggregation-driven pathology.SIGNIFICANCE STATEMENT ApoE protein plays a key role in the formation of amyloid plaques, a hallmark of Alzheimer's disease (AD). ApoE4 is more aggregated and hypolipidated compared with ApoE3, but whether enhancing ApoE lipidation in vivo can reverse ApoE aggregation is not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). In this study, we demonstrated that the greater propensity of lipid-poor ApoE4 to aggregate decreased ABCA1 membrane recycling and its ability to lipidate ApoE. Importantly, enhancing ABCA1 activity to lipidate ApoE reduced ApoE and ABCA1 aggregation. This work provides critical insights into the interactions among ABCA1, ApoE lipidation and aggregation, and underscores the promise of stabilizing ABCA1 activity to prevent ApoE-driven aggregation pathology.

Molecular dynamics of Dkk4 modulates Wnt action and regulates meibomian gland development.

Secreted Dickkopf (Dkk) proteins are major Wnt pathway modulators during organ development. Dkk1 has been widely studied and acts as a general Wnt inhibitor. However, the molecular function of other Dkks remains largely unknown. Here, we show that Dkk4 selectively inhibits a subset of Wnts, but is further inactivated by proteolytic cleavage. Meibomian gland (MG) formation is employed as a model where Dkk4 and its Wnt targets are expressed. Skin-specific expression of Dkk4 arrests MG growth at early germ phase, which is similar to that observed in Eda-ablated Tabby mice. Consistent with transient Dkk4 action, intact Dkk4 inhibits MG extension but the cleaved form progressively increases during MG development with a concomitant upswing in Wnt activity. Furthermore, both Dkk4 and its receptor (and Wnt co-receptor) Lrp6 are direct Eda targets during MG induction. In cell and organotypic cultures, Dkk4 inhibition is eliminated by elevation of Lrp6. Also, Lrp6 upregulation restores MG formation in Tabby mice. Thus, the dynamic state of Dkk4 itself and its interaction with Lrp6 modulates Wnt function during MG development, with a novel limitation of Dkk4 action by proteolytic cleavage.

Involvement of Wnt, Eda and Shh at defined stages of sweat gland development.

To maintain body temperature, sweat glands develop from embryonic ectoderm by a poorly defined mechanism. We demonstrate a temporal cascade of regulation during mouse sweat gland formation. Sweat gland induction failed completely when canonical Wnt signaling was blocked in skin epithelium, and was accompanied by sharp downregulation of downstream Wnt, Eda and Shh pathway genes. The Wnt antagonist Dkk4 appeared to inhibit this induction: Dkk4 was sharply downregulated in ß-catenin-ablated mice, indicating that it is induced by Wnt/ß-catenin; however, its overexpression repressed Wnt target genes and significantly reduced gland numbers. Eda signaling succeeded Wnt. Wnt signaling was still active and nascent sweat gland pre-germs were still seen in Eda-null mice, but the pre-germs failed to develop further and the downstream Shh pathway was not activated. When Wnt and Eda were intact but Shh was ablated, germ induction and subsequent duct formation occurred normally, but the final stage of secretory coil formation failed. Thus, sweat gland development shows a relay of regulatory steps initiated by Wnt/ß-catenin - itself modulated by Dkk4 - with subsequent participation of Eda and Shh pathways.

TaqTth-hpRNA: a novel compact RNA-targeting tool for specific silencing of pathogenic mRNA.

Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. Here, we develop an RNA-cleaving tool, TaqTth-hpRNA, consisting of a small, chimeric TaqTth, and a hairpin RNA guiding probe. With a minimal flanking sequence-motif requirement, in vitro and in vivo studies show TaqTth-hpRNA cleaves RNA efficiently and specifically. In an Alzheimer's disease model, we demonstrate silencing of mutant APPswe mRNA without altering the wild-type APP mRNA. Notably, due to the compact size of TaqTth, we are able to combine with APOE2 overexpression in a single AAV vector, which results in stronger inhibition of pathologies.

Edaravone Dexborneol mitigates pathology in animal and cell culture models of Alzheimer's disease by inhibiting neuroinflammation and neuronal necroptosis.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent neurodegenerative disease with limited disease-modifying treatments. Drug repositioning strategy has now emerged as a promising approach for anti-AD drug discovery. Using 5×FAD mice and Aß-treated neurons in culture, we tested the efficacy of Y-2, a compounded drug containing the antioxidant Edaravone (Eda), a pyrazolone and (+)-Borneol, an anti-inflammatory diterpenoid from cinnamon, approved for use in amyotrophic lateral sclerosis patients. RESULTS: We examined effects of Y-2 versus Eda alone by i.p. administered in 8-week-old 5×FAD mice (females) for 4 months by comparing cognitive function, Aß pathologies, neuronal necroptosis and neuroinflammation. Using primary neurons and astrocytes, as well as neuronal and astrocytic cell lines, we elucidated the molecular mechanisms of Y-2 by examining neuronal injury, astrocyte-mediated inflammation and necroptosis. Here, we find that Y-2 improves cognitive function in AD mice. Histopathological data show that Y-2, better than Eda alone, markedly ameliorates Aß pathologies including Aß burden, astrogliosis/microgliosis, and Tau phosphorylation. In addition, Y-2 reduces Aß-induced neuronal injury including neurite damage, mitochondrial impairment, reactive oxygen species production and NAD+ depletion. Notably, Y-2 inhibits astrocyte-mediated neuroinflammation and attenuates TNF-α-triggered neuronal necroptosis in cell cultures and AD mice. RNA-seq further demonstrates that Y-2, compared to Eda, indeed upregulates anti-inflammation pathways in astrocytes. CONCLUSIONS: Our findings infer that Y-2, better than Eda alone, mitigates AD pathology and may provide a potential drug candidate for AD treatment.

EDA ligand triggers plasma membrane trafficking of its receptor EDAR via PKA activation and SNAP23-containing complexes.

BACKGROUND: Ectodysplasin-A (EDA), a skin-specific TNF ligand, interacts with its membrane receptor EDAR to trigger EDA signaling in skin appendage formation. Gene mutations in EDA signaling cause Anhidrotic/Hypohidrotic Ectodermal Dysplasia (A/HED), which affects the formation of skin appendages including hair, teeth, and several exocrine glands. RESULTS: We report that EDA triggers the translocation of its receptor EDAR from a cytosolic compartment into the plasma membrane. We use protein affinity purification to show that upon EDA stimulation EDAR associates with SNAP23-STX6-VAMP1/2/3 vesicle trafficking complexes. We find that EDA-dependent PKA activation is critical for the association. Notably, either of two HED-linked EDAR mutations, T346M and R420W, prevents EDA-induced EDAR translocation; and both EDA-induced PKA activation and SNAP23 are required for Meibomian gland (MG) growth in a skin appendage model. CONCLUSIONS: Overall, in a novel regulatory mechanism, EDA increases plasma membrane translocation of its own receptor EDAR, augmenting EDA-EDAR signaling in skin appendage formation. Our findings also provide PKA and SNAP23 as potential targets for the intervention of HED.

Semaphorin-3A guides radial migration of cortical neurons during development.

Postmitotic neurons in the developing cortex migrate along radial glial fibers to their proper location in the cortical plate and form the layered structure. Here we report that the radial migration of rat layer II/III cortical neurons requires guidance by the extracellular diffusible factor Semaphorin-3A (Sema3A). This factor is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is highly expressed in migrating neurons. Downregulation or conditional knockout of NP1 in newborn cortical neurons impedes their radial migration by disrupting their radial orientation during migration without altering their cell fate. Studies in cultured cortical slices further show that the endogenous gradient of Sema3A is required for the proper migration of newborn neurons. In addition, transwell chemotaxis assays show that isolated newborn neurons are attracted by Sema3A. Thus, Sema3A may function as a chemoattractive guidance signal for the radial migration of newborn cortical neurons toward upper layers.

Salidroside reduces neuropathology in Alzheimer's disease models by targeting NRF2/SIRT3 pathway.

BACKGROUND: Neurite dystrophy is a pathologic hallmark of Alzheimer's disease (AD). However, drug discovery targeting neurite protection in AD remains largely unexplored. METHODS: Aß-induced neurite and mitochondrial damage assays were used to evaluate Aß toxicity and the neuroprotective efficacy of a natural compound salidroside (SAL). The 5×FAD transgenic mouse model of AD was used to study the neuroprotective function of SAL. To verify the direct target of SAL, we used surface plasmon resonance and cellular thermal shift assays to analyze the drug-protein interaction. RESULTS: SAL ameliorates Aß-mediated neurite damage in cell culture. We further reveal that SAL represses mitochondrial damage in neurites by promoting mitophagy and maintaining mitochondrial homeostasis, dependent on an NAD-dependent deacetylase SIRT3. In AD mice, SAL protects neurite morphology, mitigates Aß pathology, and improves cognitive function, which are all SIRT3-dependent. Notably, SAL directly binds to transcription factor NRF2, inhibits its degradation by blocking its interaction with KEAP1 ubiquitin ligase, and then advances NRF2-mediated SIRT3 transcription. CONCLUSIONS: Overall, we demonstrate that SAL, a potential anti-aging drug candidate, attenuates AD pathology by targeting NRF2/SIRT3 pathway for mitochondrial and neurite protection. Drug discovery strategies focusing on SAL may thus provide promising therapeutics for AD.

TNF-α-dependent neuronal necroptosis regulated in Alzheimer's disease by coordination of RIPK1-p62 complex with autophagic UVRAG.

Background: Neuronal death is a major hallmark of Alzheimer's disease (AD). Necroptosis, as a programmed necrotic process, is activated in AD. However, what signals and factors initiate necroptosis in AD is largely unknown. Methods: We examined the expression levels of critical molecules in necroptotic signaling pathway by immunohistochemistry (IHC) staining and immunoblotting using brain tissues from AD patients and AD mouse models of APP/PS1 and 5×FAD. We performed brain stereotaxic injection with recombinant TNF-α, anti-TNFR1 neutralizing antibody or AAV-mediated gene expression and knockdown in APP/PS1 mice. For in vitro studies, we used TNF-α combined with zVAD-fmk and Smac mimetic to establish neuronal necroptosis models and utilized pharmacological or molecular biological approaches to study the signaling pathways. Results: We find that activated neuronal necroptosis is dependent on upstream TNF-α/TNFR1 signaling in both neuronal cell cultures and AD mouse models. Upon TNF-α stimulation, accumulated p62 recruits RIPK1 and induces its self-oligomerization, and activates downstream RIPK1/RIPK3/MLKL cascade, leading to neuronal necroptosis. Ectopic accumulation of p62 is caused by impaired autophagy flux, which is mediated by UVRAG downregulation during the TNF-α-promoted necroptosis. Notably, UVRAG overexpression inhibits neuronal necroptosis in cell and mouse models of AD. Conclusions: We identify a finely controlled regulation of neuronal necroptosis in AD by coordinated TNF-α signaling, RIPK1/3 activity and autophagy machinery. Strategies that could fine-tune necroptosis and autophagy may bring in promising therapeutics for AD.

The effect of C-terminal fragment of JNK2 on the stability of p53 and cell proliferation.

The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramatically inhibited 24 h later. However, the expression of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.

Top3ß is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.

Topoisomerases are crucial for solving DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3ß (Top3ß) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein that is deficient in fragile X syndrome and is known to regulate the translation of mRNAs that are important for neuronal function, abnormalities of which are linked to autism. Notably, the FMRP-Top3ß interaction is abolished by a disease-associated mutation of FMRP, suggesting that Top3ß may contribute to the pathogenesis of mental disorders. Top3ß binds multiple mRNAs encoded by genes with neuronal functions linked to schizophrenia and autism. Expression of one such gene, that encoding protein tyrosine kinase 2 (ptk2, also known as focal adhesion kinase or FAK), is reduced in the neuromuscular junctions of Top3ß mutant flies. Synapse formation is defective in Top3ß mutant flies and mice, as well as in FMRP mutant flies and mice. Our findings suggest that Top3ß acts as an RNA topoisomerase and works with FMRP to promote the expression of mRNAs that are crucial for neurodevelopment and mental health.

[The effects of cysteines on the function of human glutathion S-transferase pi(GSTp) under cell oxidative stress].

Site-directed mutagenesis was used to generate three cysteine mutants of GSTp, C(47/101), C(14/47/101) and C(14/47/101/169). GSTp, C(47/101), C(14/47/101) and C(14/47/101/169) were transfected into 293 cells separately and GST activity was determined by using CDNB as substrate. Data showed that each cysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant negative effect. The expression vectors of wide type GSTp and its cysteine mutants were co-transfected with c-Jun, NF-kappaB, or p21 luciferase reporting vector, into 293 cells separately, luciferase activity showed that C(14/47/101) and C(14/47/101/169) can dramatically activate c-Jun and p21 transcriptional activity. Each cysteine mutant can increase endogenous p21 level, and also increased mortality rate of 293 cells when exposed to H2O2. These results suggest that cysteine residues of GSTp play an important role in protecting cells against oxitative stress.