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Neural apoptosis-regulated convertase-1/proprotein convertase subtilisin-kexin like-9 (NARC-1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low-density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT–PCR analyses were performed following retinoic acid (RA)-induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC-1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element-binding protein-2, which normally up-regulates NARC-1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC-1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole-mount in situ hybridization revealed that the pattern of expression of zebrafish NARC-1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC-1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain–midbrain boundaries, leading to embryonic death at ∼ 96 h after fertilization. These data support a novel role for NARC-1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver.
The mammalian proprotein convertases (PCs) constitute a family of nine serine proteinases related to bacterial subtilisin. These include the seven basic amino acid- specific convertases known as PC1/3, PC2, Furin, PC4, PACE4, PC5/6 and PC7 (Seidah and Chretien 1999; Zhou et al. 1999), and two proteinases that cleave at non-basic residues, namely subtilisin kexin isozyme 1/site 1 protease (SKI-1/S1P) (Sakai et al. 1998b; Seidah et al. 1999) and neural apoptosis-regulated convertase-1/proprotein convertase subtilisin-kexin like-9 (NARC-1/PCSK9) (Seidah et al. 2003; Benjannet et al. 2004). These proteinases are implicated in the limited proteolysis of precursors of secretory proteins that participate in several biological functions, such as development, reproduction and immune response, and in numerous pathologies, including neurological disorders, infectious diseases, cancer and dyslipidemias (Decroly et al. 1997; Mbikay et al. 1997; Roebroek et al. 1998; Denis et al. 2000; Benjannet et al. 2001; Lenz et al. 2001; Khatib et al. 2002; Seidah and Prat 2002; Thomas 2002; Abifadel et al. 2003).
Two groups have independently used microarray technology to study mRNAs that are regulated by cholesterol or by the sterol regulatory element-binding proteins (SREBPs), and observed that hepatic rodent NARC-1/PCSK9 expression is up-regulated in SREBP-2 transgenic mice (Horton et al. 2003) and down-regulated by diet-induced excess circulating cholesterol (Maxwell et al. 2003). Recent data from our group demonstrated that NARC-1/PCSK9 mRNA levels are up-regulated in hepatocyte-derived human HepG2 cells and primary hepatocytes by the statins, which are 3-hydroxy-3-methylglutaryl co-enzyme (HMG-CoA) reductase inhibitors, but not by the liver X receptor agonist 22-hydroxycholesterol, nor by the retinoid X receptor agonist 9-cis-retinoic acid (Dubuc et al. 2004). This process can be reversed by mevalonate treatment, in agreement with the implied role of SREBP-2 in the up-regulation of NARC-1/PCSK9 (Dubuc et al. 2004). In contrast to that of NARC-1/PCSK9, the expression of SKI-1/S1P is not sensitive to cholesterol, even though it is a key protease, which, together with the metalloprotease site-2 protease (S2P), activates the formation of the nuclear form of SREBPs (Sakai et al. 1998a; Brown and Goldstein 1999).
During development and in adulthood, NARC-1/PCSK9 is expressed in liver and small intestine, two regenerating organs implicated in cholesterol metabolism. By comparison, NARC-1/PCSK9 is only transiently expressed in cortical cells of the kidney and in specific brain regions where active neurogenesis takes place. These include the telencephalon, rostral extension of the olfactory epithelium and the cerebellum (Seidah et al. 2003). Whether expression in the latter organs is also controlled by cholesterol is unknown. Overexpression of NARC-1/PCSK9 cDNA in primary neurons isolated from telencephalon at embryonic day 12, suggested that this enzyme can enhance neurogenesis of progenitor brain telencephalic cells (Seidah et al. 2003). In order to understand the neural function of NARC-1/PCSK9, we undertook an analysis of its expression in pluripotent mouse P19 embryonal carcinoma cells, which are well suited for studies related to neuroectodermal cell development and maturation. These cells can be efficiently induced to differentiate in culture into neurons and astroglia by combining cell aggregation and brief treatment with all trans-retinoic acid (RA) (Rudnicki et al. 1989; McBurney 1993).
In this study low levels of NARC-1/PCSK9 mRNA were detected in naive P19 cells. Upon neuroectodermal induction of P19 cells by RA, NARC-1/PCSK9 transcripts were induced approximately 7-fold, with a maximum at day 2 of the RA treatment, followed by repression to levels below those of naive P19 cells. These findings suggested that early, transient induction of NARC-1/PCSK9 mRNA may be needed to modulate neurogenesis/gliogenesis, reminiscent of its expression in telencephalon (Seidah et al. 2003). However, in contrast to findings in the liver, NARC-1/PCSK9 mRNA did not seem to be significantly regulated by SREBP-2 in P19 cells and its up-regulation did not affect the protein level of the endogenous LDLR.
We further extended our data into the realm of a whole animal, using the model zebrafish (Danio rerio). As in mouse, zebrafish (zf)-NARC-1/PCSK9 is well expressed in the developing liver and intestine, as well as in cortical neurons and cerebellar granules where continued neurogenesis occurs. Specific knockdown of zf-NARC-1/PCSK9 mRNA in zebrafish embryos using morpholino antisense oligonucleotide technology led to the loss of CNS structures, such as the midbrain and hindbrain, and culminated in lethality at ∼ 3–4 days after fertilization.
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- Materials and methods
Initial studies showed that NARC-1/PCSK9 is transiently expressed during embryonic development in neurogenic centers such as those in the telencephalon and cerebellum, and that it is no longer expressed in mature CNS neurons of rodents (Seidah et al. 2003). We undertook a study of the possible role of this convertase in neuroectodermal differentiation using P19 cells and zebrafish as models. The data revealed that upon RA induction of P19 cells leading to neurons and glial cells, NARC-1/PCSK9 mRNA levels peak at day 2 and fall off thereafter, a profile quite different from that of the other convertases (Fig. 3). This suggests that NARC-1/PCSK9 may have a unique role at the onset of the neuronal/neuroectodermal differentiation process. Interestingly, a minor but statistically significant peak of PC5/6 mRNA at day 2 was also observed, reminiscent of the up-regulation of both NARC-1/PCSK9 and PC5/6 during liver regeneration following partial hepatectomy in rat (Seidah et al. 2003). Thus, it may well be that NARC-1/PCSK9 and PC5/6 have complementary and/or additive roles in the predifferentiation period.
We next attempted to elucidate the mechanism behind the up-regulation of NARC-1/PCSK9 at day 2 in P19 cells. This effect was only observed following RA treatment of either adherent cells or aggregated cells in suspension, but not in untreated controls. Accordingly, we deduced that the aggregation step is not responsible for the observed up-regulation, but rather that RA is the inducing agent. In the nucleus, the RA signal is transduced by binding to a heterodimeric pair of retinoid receptors, retinoic acid receptor/retinoid X receptor (Means and Gudas 1995). Because HepG2 cells, which express both receptors (Denson et al. 2000), do not up-regulate the NARC-1/PCSK9 transcripts when treated with RA (Fig. S2), it is likely that the effect of RA in P19 cells is indirect, possibly involving activation of intervening proteins or transcription factors (Wei et al. 2002), which may then up-regulate NARC-1/PCSK9 mRNA levels. Furthermore, no apparent RA-responsive element could be identified in the proximal ∼ 2 kb of the mouse and human PCSK9 promoters (not shown).
Because NARC-1/PCSK9 transcription is highly up-regulated by SREBP-2 (Horton et al. 2003; Dubuc et al. 2004), we next turned our attention to the effect of RA on SREBP-2 transcription, and found that it was unchanged during the differentiation programme (not shown). However, because it is mostly the nuclear nSREBP-2 protein that is the active ingredient, it is plausible that the activation of the Golgi form of SREBP-2 into its nuclear form nSREBP-2 by the concerted action of SKI-1/S1P and S2P (Brown et al. 2000) may be limiting. Our data revealed that the transcription of SKI-1/S1P was not regulated by RA in P19 cells (Fig. 3). Furthermore, the mRNA levels of the limiting HMG-CoA reductase, which critically depends on nSREBP-2 activity, only increased from days 5–10, indicating that nSREBP-2 is not the key NARC-1/PCSK9 transcription factor at day 2 (Fig. 3). We cannot at the present time eliminate the possibility that the peak observed at day 2 may be due to an enhanced specific stabilization of NARC-1/PCSK9 mRNA, possibly via specific interactions with its 5′ untranslated region, as was the case for insulin, PC1 and PC2 mRNAs at high glucose levels (Schuppin and Rhodes 1996), rather than direct transcriptional activation. However, this mechanism would have to be quite restrictive because mRNA levels of the cholesterogenic genes encoding HMG-CoA reductase, LDLR, SKI-1/S1P and SREBP-2 did not peak at day 2.
The only attributable function of NARC-1/PCSK9 is enhancement of degradation of the LDLR in liver and hepatocyte-derived cell lines (Benjannet et al. 2004; Maxwell and Breslow 2004; Park et al. 2004; Rashid et al. 2005). However, the mechanism behind this effect is not understood, as it does not seem to result from direct cleavage of the LDLR by NARC-1/PCSK9 in an acidic compartment (Attie and Seidah 2005). This suggests that in liver NARC-1/PCSK9 may activate and/or change the trafficking of another protein(s) that is responsible for the degradation of the LDLR. Because the protein level of the LDLR in P19 cells treated with RA did not change from days 0–4 (Fig. 4b), even though NARC-1/PCSK9 was up-regulated at day 2, this indicates that the cellular conditions in P19 cells may not be appropriate for LDLR-enhanced degradation by NARC-1/PCSK9. This led us to conclude that, similar to findings in Chinese hamster ovary cells (Park et al. 2004), the NARC-1/PCSK9-induced enhanced degradation of the LDLR is not efficient in P19 cells, and that NARC-1/PCSK9 may have other function(s) in these cells. Indeed, comparative ISH of NARC-1/PCSK9 and LDLR in consecutive sections of a whole mouse on the first day after birth (P1) revealed both distinct and overlapping expression patterns (Fig. 4c). Thus, both mRNAs were co-localized in liver, intestine and kidney, whereas the high level of expression of NARC-1/PCSK9 in cerebellum did not coincide with that of the LDLR, which is generally not abundant in the CNS. Therefore, NARC-1/PCSK9 may have new functions in the nervous system, possibly related to neurogenesis, among others (Seidah et al. 2003). This may involve the NARC-1/PCSK9-enhanced degradation of other receptor types or proteins during development of the cerebellum and telencephalon.
We decided to study such function(s) within the realm of a whole animal such as the zebrafish, which represents a well studied experimental model as it develops externally with visual clarity. These qualities, together with antisense-based translation inhibition techniques, allow visual phenotypic assessment of targeted gene knockdown. Mining of zebrafish genomic databases allowed us to put together a complete primary structure of zf-NARC-1/PCSK9 (Fig. S3). The deduced sequence predicts a 667-amino acid proprotein with a 22-amino acid signal peptide (amino acids 1–22), and a 121-amino acid prosegment (amino acids 23–143), resulting in a 525-amino acid mature protein. The zymogen activation site predicted at SSIFAQ143SIPWN is very similar to that found in human and mouse, with the only conservative variation of zebrafish Ile140 to Val in mammals. Notably, we also deduced the possible presence of an extra N-glycosylation site within the prosegment of zf-NARC-1/PCSK9 (Asn75) that is not found in mammalian orthologs. Finally, the C-terminal Cys/His-rich domain of zf-NARC-1/PCSK9 is similar to that found in the human and mouse sequences, with the presence of two CysCys-X6-Cys motifs.
We used MOs selectively to inhibit zf-NARC-1/PCSK9 translation (Fig. 6), to create morphant phenotypes and to document the resulting visible morphological defects. Based on murine NARC-1/PCSK9 gene expression patterns (Seidah et al. 2003), it was predicted that defects would be associated with neural and/or hepatic/intestinal/renal organogenesis. In the zebrafish, neurogenesis begins postgastrulation (approximately 10 hpf) with the formation of the neural plate, a conserved vertebrate structure. The neural plate then undergoes anterior–posterior patterning during segmentation, creating visible regionalized structures by 24 hpf (Schier et al. 1996). Hepatic cell fate specification from the presumptive anterior endoderm begins at approximately 16–18 hpf before primitive gut tube formation (Korzh et al. 2001). However, the liver only acquires a histologically distinct architecture at 34 hpf (Wallace and Pack 2003). At 24 hpf, embryos injected with MO1 displayed defective neurogenesis (Fig. 6d) as compared with wild type (WT) (Fig. 6c). This included absence of tectum (red arrow), midbrain–hindbrain boundary (arrow head), as well as decreased hindbrain architecture (black arrow and asterisk) (Figs 6c and d). Comparison of WT (Fig. 6e) and MO1-injected (Fig. 6f) embryos at 48 hpf indicated the presence of further abnormalities in neural development. The observed overall neural degeneration was followed by regionalization at 48 hpf into several chambers of unknown identity (Fig. 6f). The knockdown of zf-NARC-1/PCSK9 expression resulted in an ∼ 60% decreased in protein levels, but not complete loss of protein (Fig. 6a). The specific phenotypes observed suggest that in zebrafish neurogenesis is highly sensitive to altered NARC-1/PCSK9 expression. This is reminiscent of the effects observed in human NARC-1/PCSK9 heterozygote missense mutations (50% loss of function), resulting in familial hypocholesterolemia (Cohen et al. 2005; Kotowski et al. 2006). It remains unclear at this time whether hepatic specification or liver organogenesis is affected in the zf-NARC-1/PCSK9 morphants. Further investigation by marker gene in situ and histological analysis is required.
The refractile (as opposed to transparent) appearance of neural tissue in morphants is a hallmark of cell death, particularly associated with neural degeneration, and was used initially to identify neuron survival mutants in large-scale zebrafish chemical mutagenesis screens (Furutani-Seiki et al. 1996). Although not visibly evident, apoptosis is an essential component of normal neurogenesis, whereby neuronal pathways form via axonal extension in a ‘first-come-first-served’ basis, requiring redundant projections to undergo programmed cell death (Lossi and Merighi 2003). The close association between differentiation and apoptosis during neurogenesis may confer increased sensitivity to cellular alterations, particularly those affecting the cell cycle and metabolism (Furutani-Seiki et al. 1996). Interestingly, the known autosomal dominant natural human NARC-1/PCSK9 mutations resulting in hypercholesterolemia place this gene within such a metabolic context (Abifadel et al. 2003; Benjannet et al. 2004; Timms et al. 2004; Attie and Seidah 2005).
While this work was in progress, the complete knockout of NARC-1/PCSK9 in mouse was reported (Rashid et al. 2005). The latter work confirmed earlier studies, showing that NARC-1/PCSK9 enhances degradation of the LDLR; its loss would therefore result in higher hepatic LDLR levels (Benjannet et al. 2004; Maxwell and Breslow 2004; Park et al. 2004). Nevertheless, it was astonishing to discover that in mouse the loss of NARC-1/PCSK9 expression does not result in a lethal phenotype (Rashid et al. 2005), as is the case in zebrafish (this work).
The association of NARC-1/PCSK9 with LDLR degradation is insufficient to explain the zebrafish morphant defects because LDLR–/– mice are viable (Ishibashi et al. 1993), suggesting that this PC embodies biological activities beyond LDLR homeostasis, as also suggested by its expression in brain areas lacking the LDLR (Fig. 4c). Nevertheless, differences do exist between the mouse and zebrafish, including the absence of placenta and external embryonic growth in the fish. In this context, a recent study on Veph showed that the knockdown of this transiently expressed CNS protein in neurogenesis centers results in a lethal phenotype in zebrafish but not in Veph–/– mice (Muto et al. 2004). Death in developing zebrafish lacking Veph transcripts seems to follow defects in the midbrain–hindbrain boundary and otic vesicle formation. A similar phenotype was observed upon knockdown of zf-NARC-1/PCSK9, with defective neurogenesis including loss of the tectum and midbrain–hindbrain boundary, as well as disorganized hindbrain architecture (Figs 6c and d). It remains to be seen whether NARC-1/PCSK9 has different functions in mammals and fish. Alternatively, the CNS of mammals may have developed mechanisms to compensate for the lack of NARC-1/PCSK9 that are absent in teleosts. Detailed mapping of the functional domains of NARC-1/PCSK9 in zebrafish and mouse may lead to a better understanding of the various functions of this fascinating convertase.