Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by expansion of CAG repeats in the Huntingtin (HTT) gene.
Aim
There is human evidence of increased complement biosynthesis and activation in HD, with increased immunoreactivity for C1q, C4, C3 and C3b observed in striatal neurons, astrocyte, and microglia cells. However, the role of the classical pathway in HD pathogenesis is unclear.
Methods
Using the R6/2 mouse model of HD with ~ 120 CAG expansion, we investigated alteration of classical complement cascade in plasma and cerebral spinal fluid (CSF), we evaluated correlation of classical complement C1q levels with the neurodegenerative biomarker Neurofilament Light Chain (NFL) levels and assessed efficacy of anti C1q mAb therapy in preventing neurodegeneration and ameliorating disease progression.
Results
We found evidence of perturbations in the complement pathway, such as significant increase in plasma levels of C1q, C1s, C4, C3 and C3d during disease progression. Positive correlation of plasma C1q with CSF NFL levels was observed, suggesting a role of the classical complement cascade in neurodegeneration and disease progression. To test this hypothesis, we pharmacologically inhibited the classical pathway via systemic administration of anti-C1q mAb (ANX-M1, Annexon Biosciences). We observed that reduction of free C1q by anti C1q mAb therapy resulted in normalized complement pathway activity, reduced levels of CSF NFL, improved motor behavior measured with the open field assay and improved R6/2 mouse survival.
Conclusions
Altogether this study suggests that inhibiting C1q restores synaptic and neuronal health in mice with expanded CAG repeats in the HTT gene and C1q is a potential pharmacological target in HD. A Phase 2 study of ANX005 anti-C1q therapy in HD patients (clinical trials.gov NCT04514367) is ongoing.
ABSTRACT Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 ( t ransformation e ffector s ite 2 [TES2]/C-terminal activation region 2 [CTAR2]) activates NF-κB, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. By using a false discovery rate (FDR) of <0.001 after correction for multiple hypotheses, LMP1 TES2 caused >2-fold changes in 1,916 mRNAs; 1,479 RNAs were upregulated and 437 were downregulated. In contrast to tumor necrosis factor alpha (TNF-α) stimulation, which transiently upregulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IκBα and A20. LMP1 TES2-regulated RNAs encode many NF-κB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene set enrichment analyses found LMP1 TES2-upregulated genes to be significantly enriched for pathways in cancer, B- and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IκBα superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-κB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.
ABSTRACT Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jκ/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jκ, did not change EBNA3C association with RBP-Jκ or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jκ, decreased c- myc expression, and caused G 0 /G 1 growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c- myc -induced apoptosis. In assays of EBNA2- and RBP-Jκ-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jκ binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c- myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jκ binding domain. The dependence of EBNA3A effects on the core RBP-Jκ interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jκ association strongly implicates RBP-Jκ in c- myc promoter regulation.
