BET bromodomain inhibitors PFI-1 and JQ1 are identified in an epigenetic compound screen to enhance C9ORF72 gene expression and shown to ameliorate C9ORF72-associated pathological and behavioral abnormalities in a C9ALS/FTD model

Abstract

Background An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), referred to as C9ALS/FTD. No cure or effective treatment exist for C9ALS/FTD. Three major molecular mechanisms have emerged to explain C9ALS/FTD disease mechanisms: (1) C9ORF72 loss-of-function through haploinsufficiency, (2) dipeptide repeat (DPR) proteins mediated toxicity by the translation of the repeat RNAs, and more controversial, (3) RNA-mediated toxicity by bidirectional transcription of the repeats that form intranuclear RNA foci. Recent studies indicate a double-hit pathogenic mechanism in C9ALS/FTD, where reduced C9ORF72 protein levels lead to impaired clearance of toxic DPRs. Here we explored whether pharmacological compounds can revert these pathological hallmarks in vitro and cognitive impairment in a C9ALS/FTD mouse model (C9BAC). We specifically focused our study on small molecule inhibitors targeting chromatin-regulating proteins (epidrugs) with the goal of increasing C9ORF72 gene expression and reduce toxic DPRs. Results We generated luciferase reporter cell lines containing 10 (control) or ≥ 90 (mutant) G4C2 HRE located between exon 1a and 1b of the human C9ORF72 gene. In a screen of 14 different epidrugs targeting bromodomains, chromodomains and histone-modifying enzymes, we found that several bromodomain and extra-terminal domain (BET) inhibitors (BETi), including PFI-1 and JQ1, increased luciferase reporter activity. Using primary cortical cultures from C9BAC mice, we further found that PFI-1 treatment increased the expression of V1-V3 transcripts of the human mutant C9ORF72 gene, reduced poly(GP)-DPR inclusions but enhanced intranuclear RNA foci. We also tested whether JQ1, an BETi previously shown to reach the mouse brain by intraperitoneal (i.p.) injection, can revert behavioral abnormalities in C9BAC mice. Interestingly, it was found that JQ1 administration (daily i.p. administration for 7 days) rescued hippocampal-dependent cognitive deficits in C9BAC mice. Conclusions Our findings place BET bromodomain inhibitors as a potential therapy for C9ALS/FTD by ameliorating C9ORF72-associated pathological and behavioral abnormalities. Our finding that PFI-1 increases accumulation of intranuclear RNA foci is in agreement with recent data in flies suggesting that nuclear RNA foci can be neuroprotective by sequestering repeat transcripts that result in toxic DPRs.

Bromodomain and Extra-Terminal Proteins in Brain Physiology and Pathology: BET-ing on Epigenetic Regulation

“…Furthermore, BET proteins have also been associated with cognitive dysfunction and neuropsychiatric diseases, such as SUD, FTD, and schizophrenia [ 16 , 153 , 192 ].…”

“…Supporting data were recently obtained by Quezada and collaborators, showing that the BET inhibitor PFI-1 induced V1-V3 transcripts of the mutant C9ORF72 gene and facilitated the formation of nuclear RNA foci with a consistent reduction in DPR inclusions in cell models of C9ALS/FTD. In addition, BET blockade suppressed the hippocampal-dependent cognitive deficits in a C9BAC mouse model of C9ALS/FTD [ 153 ]. Hence, BET inhibitors could be considered as a valuable therapeutic approach for ALS and FTD, even though further studies are essential to better identify the effective dosage, as well as the long-term efficacy in the pathology progression.…”

Recapitulating and reversing human brain ribosomopathy defects via the maladaptive integrated stress response

“…Therefore, we used SNORD118 -mutant human iPSC-derived NPCs and performed cell-based small-molecule screen for reversing proliferation defects in mutant NPCs. On the basis of published studies, we selected those chemical compounds that potentially affect ribosome biogenesis and nucleoli functions, including 2BAct ( 38 ), MCL-186 ( 39 ), bromosporine ( 40 ), nicotinamide ( 41 ), tideglusib ( 42 , 43 ), PFI-1 ( 44 ), YM201636 ( 45 ), cilostazol ( 46 ), leucine ( 47 ), and prostetin ( 48 ). Our initial EdU assay–based cell proliferation screen identified 2BAct, MCL-186, and cilostazol, which significantly increased the proliferation of SNORD118 *5C>G -mutant NPCs (Fig.…”

Epigenetic Changes and Chromatin Reorganization in Brain Function: Lessons from Fear Memory Ensemble and Alzheimer’s Disease

“…These screens have been carried out in experimental models of frontotemporal dementia (FTD) (i.e., expressing hexanucleotide repeat expansion in the C9ORF72 gene), the second most common dementia after AD [ 125 ] that exhibit alterations in the repressive marks H3K9me3, H3K9me27 and DNA methylation in neurons and astrocytes [ 126 , 127 , 128 , 129 ]. In some of these studies, treatment with a specific class of epidrugs (i.e., JQ1 and PFI-1, both members of the bromodomain and extra-terminal domain (BET) inhibitor family) was shown to restore gene transcription in mouse and human iPSC-derived neurons, and moreover, to ameliorate cognitive deficits in FTD mice [ 130 , 131 ]. However, in a comparable JQ1 treatment it was shown that this BET inhibitor can inhibit non-spatial learning in wild-type mice [ 132 ], indicating that additional studies that precisely determine the genomic regions affected by JQ1 treatment are required.…”