Maarit Hölttä-Vuori

A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD

The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.

BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms.

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon‐24 (BODIPY‐cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol‐storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY‐cholesterol did not disturb zebrafish development and was targeted to sterol‐enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time‐lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.

Zebrafish: gaining popularity in lipid research

Zebrafish are an increasingly popular vertebrate model organism in which to study biological phenomena. It has been widely used, especially in developmental biology and neurobiology, and many aspects of its development and physiology are similar to those of mammals. The popularity of zebrafish relies on its relatively low cost, rapid development and ease of genetic manipulation. Moreover, the optical transparency of the developing fish together with novel imaging techniques enable the direct visualization of complex phenomena at the level of the entire organism. This potential is now also being increasingly appreciated by the lipid research community. In the present review we summarize basic information on the lipid composition and distribution in zebrafish tissues, including lipoprotein metabolism, intestinal lipid absorption, the yolk lipids and their mobilization, as well as lipids in the nervous system. We also discuss studies in which zebrafish have been employed for the visualization of whole-body lipid distribution and trafficking. Finally, recent advances in using zebrafish as a model for lipid-related diseases, including atherosclerosis, obesity, diabetes and hepatic steatosis are highlighted. As the insights into zebrafish lipid metabolism increase, it is likely that zebrafish as a model organism will become an increasingly powerful tool in lipid research.

PNPLA3 mediates hepatocyte triacylglycerol remodeling

The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3I148M) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3WT) and PNPLA3I148M on HuH7 cell lipidome after [13C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3I148M induced a net accumulation of unlabeled TAGs, but not newly synthesized total [13C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3WT and PNPLA3I148M induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3WT associated in PCA with newly synthesized [13C]TAGs, particularly 52:1 and 50:1, while PNPLA3I148M associated with similar preexisting TAGs. PNPLA3WT overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3I148M. PNPLA3I148M localized more extensively to lipid droplets (LDs) than PNPLA3WT, suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3I148M.

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein‐2 (LAMP‐2) and LAMP‐1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP‐1 and LAMP‐2 (LAMP−/−), low‐density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild‐type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP‐2 deficient liver and that overexpression of LAMP‐2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP‐2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP−/− cells was diminished by overexpression of any of the three isoforms of LAMP‐2, but not by LAMP‐1. The LAMP‐2 luminal domain, the membrane‐proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP‐2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone‐mediated autophagy function of LAMP‐2.

Seipin regulates ER–lipid droplet contacts and cargo delivery

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.

The CCHCR1 (HCR) gene is relevant for skin steroidogenesis and downregulated in cultured psoriatic keratinocytes.

The HCR gene, officially called Coiled-Coil α-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.

Alleviation of seipinopathy-related ER stress by triglyceride storage

Mutations affecting the N-glycosylation site in Berardinelli-Seip lipodystrophy (BSCL)-associated gene BSCL2/seipin lead to a dominantly inherited spastic paraplegia termed seipinopathy. While the loss of function of seipin leads to severe congenital lipodystrophy, the effects of seipin N-glycosylation mutations on lipid balance in the nervous system are unknown. In this study, we show that expression of seipin N-glycosylation mutant N88S led to decreased triglyceride (TG) content in astrocytoma and motor neuron cell lines. This was corrected by supplementation with exogenous oleic acid. Upon oleic acid loading, seipin N88S protein was relocated from the endoplasmic reticulum (ER) to the surface of lipid droplets and this was paralleled by alleviation of ER stress induced by the mutant protein. This effect was not limited to seipin N88S, as oleic acid loading also reduced tunicamycin-induced ER stress in motor neuron cells. Furthermore, both seipin N88S and tunicamycin-induced ER stress were decreased by inhibiting lipolysis, suggesting that lipid droplets protected neuronal cells from ER stress. In developing zebrafish larvae, seipin N88S expression led to TG imbalance and reduced spontaneous free swimming. Importantly, supplementation with exogenous oleic acid reduced ER stress in the zebrafish head and increased fish motility. We propose that the decreased TG content contributes to the pathology induced by seipin N88S, and that rescuing TG levels may provide a novel therapeutic strategy in seipinopathy.

Endosomal Actin Remodeling by Coronin-1A Controls Lipoprotein Uptake and Degradation in Macrophages

Rationale: The actin cytoskeleton has been implicated in the processing of atherogenic lipoproteins in macrophages. However, the functional role of actin and the regulatory proteins involved are unknown. Objective: Coronin-1A (Coro1A) was identified as a differentially expressed transcript in wild-type versus Niemann-Pick type C1 deficient macrophages exposed to acetylated low-density lipoproteins (AcLDL). We investigated whether Coro1A plays a role in the uptake or processing of modified lipoproteins in macrophages and if this is related to its actin regulatory functions. Methods and Results: In wild-type primary macrophages, filamentous actin transiently decorated AcLDL containing endosomes that also recruited Coro1A. This dynamic association of F-actin with endosomes was disturbed in Coro1A deficient macrophages. In Coro1A knockout macrophages the uptake of AcLDL was increased, rate of AcLDL delivery to lysosomes enhanced, and lipoprotein-derived cholesteryl ester hydrolysis accelerated. Overexpression of wild-type Coro1A normalized AcLDL uptake in Coro1A knockout macrophages while a Coro1A actin binding mutant did not. Furthermore, the effects of macrophage Coro1A silencing on endosomal actin association and AcLDL delivery to lysosomes resembled those of cofilin silencing. Conclusions: Coro1A controls actin association with endocytic organelles, thereby negatively regulating endo-lysosomal delivery, degradation of modified lipoproteins and cholesterol deposition in macrophages.

Elevated Levels of StAR-Related Lipid Transfer Protein 3 Alter Cholesterol Balance and Adhesiveness of Breast Cancer Cells: Potential Mechanisms Contributing to Progression of HER2-Positive Breast Cancers

The STARD3 gene belongs to the minimal amplicon in HER2-positive breast cancers and encodes a cholesterol-binding membrane protein. To study how elevated StAR-related lipid transfer protein 3 (StARD3) expression affects breast cancer cells, we generated MCF-7 cells stably overexpressing StARD3-green fluorescent protein. We found that StARD3-overexpressing cells exhibited nonadherent morphological features, had increased Src levels, and had altered cholesterol balance, as evidenced by elevated mRNA levels of the cholesterol biosynthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and increased plasma membrane cholesterol content. On removal of serum and insulin from the culture medium, the morphological characteristics of the StARD3-overexpressing cells changed, the cells became adherent, and they developed enlarged focal adhesions. Under these conditions, the StARD3-overexpressing cells maintained elevated Src and plasma membrane cholesterol content and showed increased phosphorylation of focal adhesion kinase. In two Finnish nationwide patient cohorts, approximately 10% (212/2220) breast cancers exhibited high StARD3 protein levels, which was strongly associated with HER2 amplification; several factors related to poor disease outcome and poor breast cancer-specific survival. In addition, high StARD3 levels in breast cancers were associated with elevated 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA levels and anti-Src-Tyr416 immunoreactivity. These results provide evidence that StARD3 overexpression results in increased cholesterol biosynthesis and Src kinase activity in breast cancer cells and suggest that elevated StARD3 expression may contribute to breast cancer aggressiveness by increasing membrane cholesterol and enhancing oncogenic signaling.