Participant-derived cell line transcriptomic analyses and mouse studies reveal a role for ZNF335 in plasma cholesterol statin response.

Background: Statins lower circulating low-density lipoprotein cholesterol (LDLC) levels and reduce cardiovascular disease risk. Though highly efficacious in general, there is considerable inter-individual variation in statin efficacy that remains largely unexplained. Methods: To identify novel genes that may modulate statin-induced LDLC lowering, we used RNA-sequencing data from 426 control- and 2 µM simvastatin-treated lymphoblastoid cell lines (LCLs) derived from European and African American ancestry participants of the Cholesterol and Pharmacogenetics (CAP) 40 mg/day 6-week simvastatin clinical trial (ClinicalTrials.gov Identifier: NCT00451828). We correlated statin-induced changes in LCL gene expression with plasma LDLC statin response in the corresponding CAP participants. For the most correlated gene identified (ZNF335), we followed up in vivo by comparing plasma cholesterol levels, lipoprotein profiles, and lipid statin response between wild-type mice and carriers of a hypomorphic (partial loss of function) missense mutation in Zfp335 (the mouse homolog of ZNF335). Results: The statin-induced expression changes of 147 human LCL genes were significantly correlated to the plasma LDLC statin responses of the corresponding CAP participants in vivo (FDR=5%). The two genes with the strongest correlations were zinc finger protein 335 (ZNF335 aka NIF-1, rho=0.237, FDR-adj p=0.0085) and CCR4-NOT transcription complex subunit 3 (CNOT3, rho=0.233, FDR-adj p=0.0085). Chow-fed mice carrying a hypomorphic missense (R1092W; aka bloto) mutation in Zfp335 had significantly lower non-HDL cholesterol levels than wild type C57BL/6J mice in a sex combined model (p=0.04). Furthermore, male (but not female) mice carrying the Zfp335R1092W allele had significantly lower total and HDL cholesterol levels than wild-type mice. In a separate experiment, wild-type mice fed a control diet for 4 weeks and a matched simvastatin diet for an additional 4 weeks had significant statin-induced reductions in non-HDLC (-43±18% and -23±19% for males and females, respectively). Wild-type male (but not female) mice experienced significant reductions in plasma LDL particle concentrations, while male mice carrying Zfp335R1092W allele(s) exhibited a significantly blunted LDL statin response. Conclusions: Our in vitro and in vivo studies identified ZNF335 as a novel modulator of plasma cholesterol levels and statin response, suggesting that variation in ZNF335 activity could contribute to inter-individual differences in statin clinical efficacy.

Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.

Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome.

Smith Lemli Opitz syndrome (SLOS) is an inherited malformation and mental retardation metabolic disorder with no cure. Mutations in the last enzyme of the cholesterol biosynthetic pathway, 7-dehydrocholesterol reductase (DHCR7), lead to cholesterol insufficiency and accumulation of its dehyrdocholesterol precursors, and contribute to its pathogenesis. The central nervous system (CNS) constitutes a major pathophysiological component of this disorder and remains unamenable to dietary cholesterol therapy due to the impenetrability of the blood brain barrier (BBB). The goal of this study was to restore sterol homeostasis in the CNS. To bypass the BBB, gene therapy using an adeno-associated virus (AAV-8) vector carrying a functional copy of the DHCR7 gene was administered by intrathecal (IT) injection directly into the cerebrospinal fluid of newborn mice. Two months post-treatment, vector DNA and DHCR7 expression was observed in the brain and a corresponding improvement of sterol levels seen in the brain and spinal cord. Interestingly, sterol levels in the peripheral nervous system also showed a similar improvement. This study shows that IT gene therapy can have a positive biochemical effect on sterol homeostasis in the central and peripheral nervous systems in a SLOS animal model. A single dose delivered three days after birth had a sustained effect into adulthood, eight weeks post-treatment. These observations pave the way for further studies to understand the effect of biochemical improvement of sterol levels on neuronal function, to provide a greater understanding of neuronal cholesterol homeostasis, and to develop potential therapies.