A complex synchronization of cell division, exit of cell cycle and differentiation is required for the proper development of the nervous system. The mechanisms of neuronal proliferation and differentiation involve an intricate array of biochemical and morphological changes that require a finely tuned modulation of signaling pathways and lipogenic routes. The particular pattern of expression of SCD5, with the highest levels in embryo and adult brain 
, suggest that a potential role for SCD5 in the mechanisms of proliferation and differentiation of neural cells. Here we report that ectopic expression of SCD5 induces a drastic phenotypical modification in Neuro2a cells, which is characterized by a marked increase in the rate of cell replication and a drastic suppression in the formation of branching neurites, a terminal marker of the neuronal differentiation process.
We hypothesize that the greater rate of cell proliferation of SCD5-expressing cells could have been caused by an acceleration of cell cycle, a notion that is supported by the finding of much greater levels of cyclin D1, a crucial regulator of the G1/S transition in cell cycle progression 
, in these cells. Since exit from cell cycle is a prerequisite for the terminal differentiation of neurons 
, a more active cycle of cell replication could explain the delay, or even failure, of SCD5-expressing neuronal cells in fully developing into differentiated neurons. In this regard, we observed that induction of the differentiation program with retinoic acid markedly halted cell proliferation in both SCD5-expressing and controls cells, however the effect was less marked in the former cell group, indicating a more robust cell growth activity.
In addition, our studies indicate that, although SCD5 may be key factor in defining the biological fate of neuronal cells, the desaturase is itself not targeted by the differentiation program. This notion is supported by our findings that levels of SCD5 protein remained unchanged during the course of differentiation of SH-SY5Y human neuroblastoma cells with retinoic acid, and in similar incubations with retinoic acid in differentiated skin fibroblasts. That is to say, it appears that SCD5 does not lie downstream of the initiation in the pathway, but rather it independently modulates the differentiation pathway.
Modifications in critical biochemical and metabolic features in neuronal cells, such as the changes in acyl-lipid composition and lipid biosynthesis that were detected in Neuro2a cells expressing SCD5, could also contribute to the phenotypical perturbations promoted by this SCD variant. As expected for a Δ9 desaturase, expression of SCD5 increased the MUFA:SFA ratio in cellular lipids, although the enrichment of lipid with MUFA was restricted to MUFA belonging to the n-7 series, chiefly palmitoleic (16
1) and cis-vaccenic (18
1) acids. An intriguing observation in our studies was that the levels of oleic acid remained surprisingly unaltered in the SCD5-expressing Neuro2a cells, suggesting a preference of the desaturase for palmitic acid as substrate. Data from in vitro determinations clearly showed that SCD5 was able to desaturate both palmitic and stearic acid at approximately similar catalytic rates 
. We believe that the particular modifications in fatty acid composition of SCD5-expressing cells could be attributed to cell type-specific fatty acid biosynthetic enzymes, such as differential elongase activity, since a similar SCD5 expression cell model generated in mouse 3T3-L1 preadipocytes significantly elevated the content of oleic acid (data not shown). In any case, the higher MUFA content observed in SCD5-expressing Neuro-2a cells may be functionally associated with their faster cell replication, since it has been established that dividing cells, both normal proliferating cells and cancer cells, have a critical dependence on endogenously synthesized MUFA for sustaining an active mitogenic program 
. Furthermore, in cancer cells in which SCD activity was pharmacologically blocked, addition of n-9 or n-7 MUFA were equally effective in restoring the cell proliferation rate 
, indicating that all MUFA exhibit pro-growth and pro-survival functions.
Besides modulation of the fatty acid profile of neuronal lipids, we found that SCD5 also is implicated in the control of glucose-mediated lipogenesis. We observed that SCD5 does not appear to globally stimulate lipid biosynthesis from glucose, but to promote a selective increase in the biosynthesis of phosphatidylcholine and cholesterolesters, in parallel to decreases in the production of phosphatidylethanolamine and triacylglycerol. This observation suggests that unlike SCD1, which appears to control the overall rate of lipogenesis in proliferating cells 
, SCD5 activity is functionally linked to more specific biosynthetic routes. Importantly, the alterations in lipid biosynthesis caused by SCD5 expression could also contribute to the drastic change in the rates of cell replication and differentiation observed in SCD5-expressing cells. Phospholipid synthesis, particularly phosphatidylcholine, is critically needed for propelling the progression of cell cycle 
and for avoiding the entry in the apoptotic program 
. Therefore, the activation of the de novo phosphatidylcholine synthesis in cells expressing ectopic SCD5 may provide abundant lipid substrates for the formation of new cell membranes to support their accelerated rate of cell proliferation. The activation of phospholipid biosynthesis, especially phosphatidylcholine though the Kennedy pathway, is also a metabolic requirement for neurite outgrowth and branching 
. We concede that in the present work, for reasons of scope and focus, we did not fully address the potential role of SCD5 activity in phospholipid formation during neuronal differentiation; a potential contribution of the desaturase to the regulation of this lipogenic pathway cannot be ruled out without experimental evidence yet to be established.
The coordination of proliferation and differentiation in neurons not only demands appropriate metabolic conditions but also the action of extrinsic signals, such as EGF, platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), nerve growth factor (NGF), and Wnt proteins, among other cytokines 
. Depending on the phenotypical phase to which the neuronal cell is committed, these factors will trigger selective mechanisms that will activate cell proliferation when expansion of the neuronal population is required, or initiate the program of differentiation 
. The growth factor-mediated activation of tyrosine-kinase receptors, as well as their downstream signaling effectors Akt, ERK and mTOR, are key for the execution of transcriptional mechanisms that leads to cell cycle arrest and morphological differentiation of neurons 
. Activation of EGFR, a paradigmatic receptor that is often associated with the mitogenic response in normal and cancer cells, is particularly critical for the execution of these biological events in neuroblastoma cells 
and in brain astrocytes 
. Therefore, the suppression of neurite outgrowth in SCD5-expressing Neuro-2a cells could then be explained by the marked blockade in the EGF-induced phosphorylation/activation of EGFR and surrogate signals ERK and Akt observed in these cells when the program of differentiation was induced. Data from our group and others demonstrated that SCD1 activity is critical for the functional activation of Akt and ERK 
. Our observation that SCD5 negatively regulates these two signaling proteins upon growth factor stimulation suggests different roles for the human SCD isoforms in the modulation of signaling cascades. However, since our determinations were restricted to a neuronal cell line, the possibility of cell-type specific differences in the participation of SCDs in signaling regulation cannot be ruled out at the moment.
Our results also point out to Wnt proteins as a second signaling mechanism related to neuronal differentiation that is a target for SCD5 activity. Our data showing that SCD5 activity activates the non-canonical Wnt pathways while suppressing the canonical branch of Wnt are in agreement with a previous report of a role for the endogenous biosynthesis of MUFA in the regulation of Wnt signals in mouse skin 
. These authors observed that lack of SCD1 expression led to a suppression in the expression of Lef1, a key transcription factor involved in the regulation of canonical Wnt/β-catenin signaling. Interestingly, ablation of Lef1 downregulates SCD1 expression in mouse skin 
, suggesting a positive activation loop between MUFA and Wnt activation. Since we found that, besides SCD1 
, human skin fibroblasts express significant levels of SCD5, this SCD isoform may also have a role in the regulation of Wnt signaling in human skin tissues.
Perturbations in Wnt signaling pathways caused by ectopic SCD5 activity may be responsible for some of the effects in the processes of proliferation and neurite outgrowth in neuronal cells communicated in this report since a number of studies, albeit conflicting, have demonstrated the critical role of Wnt signaling on neurogenesis and neuronal differentiation. In neural precursor cells and in the developing neocortex of mice, the rate of neuronal differentiation was positively associated to the degree of activation of the canonical Wnt pathway 
, but in neuronal cells canonical Wnt have been also shown to increase neuronal proliferation and to suppress differentiation 
. The discrepancies in the function of Wnt in cell proliferation and differentiation of neuronal cells could also arise from the fact that Wnt may activate or deactivate both events in a time- and stage-specific manner 
. Also importantly, depending on the physiological context of the neuronal cells, a Wnt ligand may elicit either canonical or non-canonical in these cells 
The complexity of the relationship between biological processes like neurogenesis and neuritogenesis and Wnt activity is further highlighted by the fact that posttranslational modifications of Wnt proteins profoundly affect their functional properties. For instance, Wnt-3a is targeted for S-palmitoylation in its Cys77 residue, an event that is critical for signaling activation 
. More recently, Galli and Burrus 
reported that acylation of Wnt1 in specific residues affects both Wnt signaling branches, showing that binding of palmitoleic acid to S224 preferentially signals via the ß-catenin pathway while acylation with palmitic acid (palmitoylation) on C93 primarily impacts the non-canonical, ß-catenin-independent pathway.
Our experiments provide evidence that SCD5 modulates not only the activity of Wnt signaling pathways but also the levels of secreted of Wnt ligands, suggesting an additional regulatory mechanism by which the desaturase may control cell proliferation, differentiation, or both. The observation that SCD5 activity enhances the secretion of non-canonical Wnt5a while it reduces the synthesis and, consequently, the secretion of canonical Wnt7b may partly explain the finding of similar changes in the activity in both branches of Wnt signaling in SCD5-expressing cells. The mechanisms by which SCD5 activity controls the production and release of Wnt are currently unknown. Synthesis, intracellular transport and secretion of Wnt are tightly regulated events that also depend on posttranslational modification by fatty acid acylation. Through a battery of elegant experiments, Takada et al. 
demonstrated that Wnt-3a is specifically acylated on Ser209 with palmitoleic acid (16:1 n-7) and that this modification is essential for the intracellular transport and secretion of this Wnt. These authors reported that Ser209 is part of an amino acid sequence that is highly conserved among the members of the Wnt family, suggesting that other Wnt proteins may be modified, structurally and functionally, by palmitoleyl acid (16:1 n-7) acylation. In most acylated proteins, an unsaturated fatty acid could conceivably be replaced by a saturated fatty acid in a residue targeted for acylation, hence in SCD5-expressing cells the abundance of palmitoleic acid may displace palmitic acid from the acylation site in Wnt proteins, potentially modifying their transport, their rate of secretion, or both. However, although it is clear that SCD5 expression alters the homeostatic control of Wnt proteins, at this early stage in the investigation, the questions of whether SCD5-mediated modification of Wnt synthesis and secretion is related to a potential change in their acylation, and whether changing levels of Wnt are responsible for the alterations in the activation of Wnt pathways in SCD5-expressing cells await further experimental confirmation. Finally, given the mounting evidence suggesting a mechanistic association of abnormal Wnt signaling with Alzheimer's and Parkinson's diseases 
, our findings suggest that SCD5 may be a molecular link between signaling and lipogenic pathways mechanisms and these neurodegenerative conditions.
In conclusion, the present study provides the first evidence that human SCD5 activity is implicated in the regulation of critical biological functions in neuronal cells. Our findings imply that, by modulating fatty acid composition, lipogenesis and intracellular signaling, SCD5 controls the rate of replication and differentiation of neuronal cells (). We observed that the constitutive expression of human SCD5 promotes a shift in the fatty acid composition in lipids of neuronal cells, which was characterized by elevated levels of n-7 MUFA with a concomitant reduction in SFA. These modifications in the fatty acid pattern were accompanied by lipogenic alterations, such as the change in the rate of synthesis of phospholipids, which are known to affect cell growth, survival and differentiation. Remarkably, SCD5 expression promotes a profound deregulation of EGF's intracellular signaling mechanisms. We observed that SCD5 expression suppresses the ligand-induced activation of the EGFRAkt/ERK signaling platform. SCD5 activity also reduces the activation of canonical Wnt signaling whereas it stimulates the non-canonical branch of the Wnt pathway. These activity changes could be directly related to the perturbations in the synthesis and secretion of Wnt proteins observed in SCD5-expressing neuronal cells. We also found that SCD5 expression accelerates cell cycle progression while suppressing the program of differentiation, indicating that the fate of neuronal cells is, ultimately, determined by the activity of the desaturase. Lastly, our studies suggest a value for SCD5 as a potential target for clinical interventions in poorly-treated neurological diseases such as brain cancer, Alzheimer and other neurodegenerative conditions.
Hypothetical mechanisms by which SCD5 activity modulates proliferation and differentiation in neuronal cells.