Endoplasmic reticulum (ER) stress is recognized as a crucial contributor to the progression of traumatic brain injury (TBI) and represents a potential target for therapeutic intervention. This study aimed to assess the potential of J147, a novel neurotrophic compound, in alleviating ER stress by modulating related signaling pathways, thereby promoting functional recovery in TBI. To this end, adult mice underwent controlled cortical impact (CCI) injury to induce TBI, followed by oral administration of J147 one-hour post-injury, with daily dosing for 3 to 7 days. Multiple behavioral assessments were conducted over 35 days, revealing a significant, dose-dependent improvement in neurofunctional recovery with J147 treatment. The neuropathological analysis demonstrated reduced acute neurodegeneration (observed at three days through FJC staining), enhanced long-term neuron survival (H&E and Nissl staining), and improved neuroplasticity (Golgi staining) at 35 days post-TBI. At the molecular level, TBI-induced AMP-activated protein kinase (AMPK) dephosphorylation, sterol regulatory element binding protein-1 (SREBP-1) activation, and upregulation of ER stress marker proteins, including phosphorylated eukaryotic initiation factor-2α (p-eIF2a), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in perilesional cortex neurons at three days post-injury. Notably, the J147 treatment significantly attenuated AMPK dephosphorylation, SERBP-1 activation, and expression of the ER stress markers. In summary, this study reveals the therapeutic promise of J147 in mitigating secondary brain damage associated with TBI and improving long-term functional recovery by modulating ER stress pathways.

CTCF is a key factor in three-dimensional chromatin folding and transcriptional control that was found to affect cancer cell migration by a mechanism that is still poorly understood. To identify this mechanism, we used mouse melanoma cells with a partial loss of function (pLoF) of CTCF. We found that CTCF pLoF inhibits cell migration rate while leading to an increase in the expression of multiple enzymes in the cholesterol biosynthesis pathway along with an elevation in the cellular cholesterol level. In agreement with the cholesterol change we detected altered membrane dynamics in CTCF pLoF cells as measured by reduced formation of migrasomes, extracellular vesicles formed at the rear side of migrating cells. Inhibition of cholesterol synthesis in CTCF pLoF cells restored the cellular migration rate and migrasome formation, suggesting that CTCF supports cell migration by suppressing cholesterol synthesis. Detailed analysis of the promoter of Hmgcs1, an early enzyme in the cholesterol synthesis pathway, revealed that CTCF prevents formation of a loop between that promoter and another promoter 200 kb away. CTCF also supports PRC2 recruitment to the promoter and deposition of H3K27me3. H3K27me3 at the promoter of Hmgcs1 prevents SREBP2 binding and activation of transcription. By this mechanism, CTCF fine-tunes cholesterol levels to support cell migration. Notably, genome wide association studies suggest a link between CTCF and cholesterol-associated diseases, thus CTCF emerges as a new regulator of cholesterol biosynthesis.

The cGAS-STING innate immunity pathway and the SREBP-activated cholesterol and fatty acid synthesis pathway are abnormally co-regulated in neurodegenerative disease. Activation of STING signaling occurs at the endoplasmic reticulum (ER) membrane with STING anchored by INSIG1 along with SREBP and the sterol-bound SREBP cleavage activating protein (SCAP) when sterols are in abundance. When sterols are low, the INSIG-dependent STING pathway is inactivated and the SREBP-SCAP complex is translocated to the Golgi where SREBP is cleaved and translocated to the nucleus to transactivate genes for cholesterol and fatty acid synthesis. Thus, there is inverse activation of STING vs. SREBP: when innate immunity is active, pathways for cholesterol and fatty acid synthesis are suppressed, and vice versa. The STING pathway is stimulated by foreign viral cytoplasmic nucleic acids interacting with the cyclic GMP-AMP synthase (cGAS) DNA sensor or RIG-I and MDA5 dsRNA sensors, but with neurodegeneration innate immunity is also activated by self-DNAs and double-stranded RNAs that accumulate with neuronal death. Downstream, activated STING recruits TBK1 and stimulates the transactivation of interferon stimulated genes and the autophagy pathway, which are both protective. However, chronic activation of innate immunity contributes to microglia activation, neuroinflammation and autophagy failure leading to neurodegeneration. STING is also a proton channel that when activated stimulates proton exit from STING vesicles leading to cell death. Here we review the salient features of the innate immunity and cholesterol and fatty acid synthesis pathways, observations of abnormal STING and SREBP signaling in neurodegenerative disease, and relevant therapeutic approaches.

Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of the pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL.

OBJECTIVE: Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. In this study, we examined the requirement of SREBP target genes for pancreatic ductal adenocarcinoma (PDAC) tumor growth.
METHODS: Here we constructed a custom CRISPR knockout library containing known SREBP target genes and performed in vitro 2D culture and in vivo orthotopic xenograft CRISPR screens using a patient-derived PDAC cell line. In vitro, we grew cells in medium supplemented with 10% fetal bovine serum (FBS) or 10% lipoprotein-deficient serum (LPDS) to examine differences in gene essentiality in different lipid environments. In vivo, we injected cells into the pancreata of nude mice and collected tumors after 4 weeks.
RESULTS: We identified terpenoid backbone biosynthesis genes as essential for PDAC tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that geranylgeranyl diphosphate synthase 1 (GGPS1) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model.
CONCLUSIONS: These findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.

SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PA→C and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.

As a vital hallmarker of cancer, the metabolic reprogramming has been shown to play a pivotal role in tumour occurrence, metastasis and drug resistance. Amongst a vast variety of signalling molecules and metabolic enzymes involved in the regulation of cancer metabolism, two key transcription factors Nrf1 and Nrf2 are required for redox signal transduction and metabolic homeostasis. However, the regulatory effects of Nrf1 and Nrf2 (both encoded by Nfe2l1 and Nfe2l2, respectively) on the metabolic reprogramming of hepatocellular carcinoma cells have been not well understood to date. Here, we found that the genetic deletion of Nrf1 and Nrf2 from HepG2 cells resulted in distinct metabolic reprogramming. Loss of Nrf1α led to enhanced glycolysis, reduced mitochondrial oxygen consumption, enhanced gluconeogenesis and activation of the pentose phosphate pathway in the hepatocellular carcinoma cells. By striking contrast, loss of Nrf2 attenuated the glycolysis and gluconeogenesis pathways, but with not any significant effects on the pentose phosphate pathway. Moreover, knockout of Nrf1α also caused fat deposition and increased amino acid synthesis and transport, especially serine synthesis, whilst Nrf2 deficiency did not cause fat deposition, but attenuated amino acid synthesis and transport. Further experiments revealed that such distinctive metabolic programming of between Nrf1α-/- and Nrf2-/- resulted from substantial activation of the PI3K-AKT-mTOR signalling pathway upon the loss of Nrf1, leading to increased expression of critical genes for the glucose uptake, glycolysis, the pentose phosphate pathway, and the de novo lipid synthesis, whereas deficiency of Nrf2 resulted in the opposite phenomenon by inhibiting the PI3K-AKT-mTOR pathway. Altogether, these provide a novel insight into the cancer metabolic reprogramming and guide the exploration of a new strategy for targeted cancer therapy.

Progesterone (P4) is a crucial reproductive hormone that acts as a precursor for all other endogenous steroids. P4 modulates transcriptional activity during reproduction by binding to progesterone receptors (PR). However, the physiological role of P4 in the liver is understudied. P4-mediated lipid metabolism in the liver was investigated in this study, as P4 facilitates insulin resistance and influences energy metabolism. While exogenous lipids are mainly obtained from food, the liver synthesizes endogenous triglycerides and cholesterol from a carbohydrate diet. Hepatic de novo lipogenesis (DNL) is primarily determined by acetyl-CoA and its biosynthetic pathways, which involve fatty acid and cholesterol synthesis. While P4 increased the hepatic levels of sterol regulatory element-binding protein 1 C (SREBP-1 C), peroxisome proliferator-activated receptor-gamma (PPARγ), acetyl-CoA carboxylase (ACC), and CD36, co-treatment with the P4 receptor antagonist RU486 blocked these proteins and P4-mediated lipogenesis. RNA sequencing was used to assess the role of P4 in lipogenic events, such as fatty liver and fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism. P4 induced hepatic DNL and lipid anabolism were confirmed in the liver of ovarian resection mice fed a high-fat diet or in pregnant mice. P4 increased lipogenesis directly in mice exposed to P4 and indirectly in fetuses exposed to maternal P4. The lipid balance between lipogenesis and lipolysis determines fat build-up and is linked to lipid metabolism dysfunction, which involves the breakdown and storage of fats for energy and the synthesis of structural and functional lipids. Therefore, P4 may impact the lipid metabolism and reproductive development during gestation.

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap\'s cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap\'s cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.