Parallel with the development of cholinergic hypothesis of geriatric memory dysfunction, cholinergic precursor loading strategy was tried for treating cognitive impairment occurring in Alzheimer's disease. Controlled clinical studies denied clinical usefulness of choline and lecithin phosphatidylcholine , whereas for other phospholipids involved in choline biosynthetic pathways such as cytidine 5'-diphosphocholine CDP-choline or alpha-glyceryl-phosphorylcholine choline alphoscerate a modest improvement of cognitive dysfunction in adult-onset dementia disorders is documented.
These inconsistencies have probably a metabolic explanation. Cholesterol levels were lower in the temporal gyrus of autopsied brains of AD patients compared to control subjects [ ]. Analysis of post squalene cholesterol precursors also provided conflicting results.
Lathosterol was reported to be elevated in the basal ganglia and the pons in AD but the ratio of lathosterol to cholesterol, used as a marker for cholesterol synthesis, was not significantly different between controls and AD patients suggesting that cholesterol synthesis is normal [ ].
More recently a model for cholesterol homeostasis deregulation was proposed based on the measurement of post-squalene cholesterol precursors, cholesterol and oxysterol in brains of individuals with no-cognitive impairment, MCI and AD [ ].
As the disease progresses, massive uptake of cholesterol derived from widespread neuronal degeneration would overwhelm glial efflux pathways resulting in increased brain cholesterol levels and feed-back suppression of de novo cholesterol synthesis. This model could explain the findings in CSF. The authors proposed that reduced expression of DHCR24 also contributes to decreased levels of cholesterol in AD patients and may explain the high levels of desmosterol found in AD in some studies [ , ].
However, in other cases brain levels of desmosterol were reduced in AD [ ]. A further indication that cholesterol synthesis might be inhibited in AD is the finding that neurosteroids, which result from cholesterol metabolism, are reduced in AD temporal cortex as compared to control subjects [ ]. It is important to highlight that changes in levels of cholesterol intermediates in brains of mouse AD models do not parallel changes in human patients.
In the APP transgenic mice carrying the Swedish mutation APP23 , no differences in the levels of lathosterol, desmosterol or cholesterol and were found when compared with wild-type animals [ ]. These differences must be considered when using animal models to study the mevalonate pathway. It is possible that a change in the distribution of cholesterol inside brain cells rather than a change in total cholesterol content may influence AD pathology [ ].
Our findings provide an explanation to the cellular cholesterol overload reported in brains of AD patients [ ]. These studies underscored the relevance of cholesterol sequestration in AD. This is important because a causal relationship between cellular cholesterol sequestration and cell death has been found in Niemann-Pick Type C NPC pathology [ ].
NPC is a disorder characterized by impairment of intracellular cholesterol trafficking and cholesterol sequestration in the endosomal compartment [ ]. Importantly, strategies previously used to reduce cholesterol sequestration in NPC and strategies that reduce cholesterol levels by increasing cholesterol metabolism improved pathological symptoms in mouse models of AD [ , ]. Preclinical and clinical studies have indicated the critical role of cholesterol in AD.
This topic has been reviewed extensively and thoroughly in the past years [ 11 , 12 , 18 , , , - ], thus it is not discussed in this chapter. Brain cholesterol is important in synapse development and maintenance [ 27 , , ]. Synaptic dysfunction is one of the earliest significant events in AD and synapse loss is the strongest anatomical correlate of the degree of clinical impairment [ , ]. Significant decrease in dendritic spine density is present in the hippocampus of patients with AD and in transgenic mouse models of AD [ - ].
Alterations in cholesterol levels, even locally at synapses, may play a role in synapse dysfunction in AD [ ]. Non-sterol isoprenoids pathway The branch of the mevalonate pathway that leads to the production of non-sterol isoprenoids is depicted in Figure 5. The enzymes involved in these steps have been extensively reviewed [ 59 ]. Figure 5. The non-sterol isoprenoid pathway. FPP is the common substrate for synthesis of several end products and for the lipid modification of proteins.
The enzymes responsible for synthesis of FPP and its non-sterol derivatives are prenyl-transferases that catalyze consecutive condensations of IPPs with primer substrates to form linear backbones for all isoprenoid compounds [ ].
In mouse brain cytosol, FPPS and GGPPS activities were higher than those in the corresponding fractions from the liver, perhaps reflecting a higher demand for protein prenylation in the brain [ ].
GGPPS activity was lowest in the cerebellum [ ]. FPPS is the target of nitrogen-containing bisphosphonate N-BP inhibitors, drugs used extensively to treat bone diseases [ ]. A few bisphosphonate selective inhibitors for GGPPS have been reported but a clinically proven inhibitor of GGPPS has not yet been identified, limiting the validation of this enzyme as a therapeutic target [ ]. Dolichol phosphate is a lipid carrier embedded in the ER membrane, essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation [ , ].
Dolichol is present, as a family with different chain lengths, in the hippocampus and spinal cord in a relatively low concentration compared to other areas of the brain [ ].
Dolichol increases in brain and in peripheral organs during aging [ ] and is associated with increased HMGCR activity [ ]. The use of dolichol level as a marker for aging has been proposed [ ]. In humans, the main ubiquinone is ubiquinone 10, or CoQ10, with 10 isoprene units. Ubiquinone performs major functions as an electron carrier in the electron transfers of the respiratory chain, and as an antioxidant component in cell membranes and as a key component in the maintenance of the redox homeostasis of the cell [ - ].
FOH and GGOH can also be formed by degradation of isoprenylated proteins in reactions catalyzed by prenylcysteine lyases, enzymes highly expressed in the brain [ ]. Contrary to the existence of a salvage pathway that uses FOH and GGOH for protein prenylation, it was demonstrated that overexpressing phosphatases that convert FPP and GGPP to FOH and GGOH in mammalian cells, decreases rather than increases protein isoprenylation as evaluated by a decreased of Rho protein level in cell membranes and results in defects in cell growth and cytoskeletal organization that are associated with dysregulation of Rho family GTPases [ ].
Whether any of these mechanisms take place in the brain is uncertain. FOH has been shown to modulate the activity of the farnesoid X receptor FXR , a member of the nuclear hormone receptor superfamily [ ]. Non-sterol isoprenoids and protein prenylation FPP and GGPP are substrates for protein farnesylation and geranylgeranylation collectively called isoprenylation.
In the human genome, there are approximately hypothetical prenylated proteins [ ]. Among them heterotrimeric G protein subunits, nuclear lamins and small GTPases have been confirmed to be prenylated [ ]. Small GTPases represent the largest group of prenylated proteins.
When X is a methionine or serine, as in Ras proteins, then the protein is farnesylated by FTase. However, when X is a leucine residue, as in Rho proteins e. GGTase II catalyzes prenylation of Rab proteins, which contain at their C-termini either one or, more frequently, two cysteine residues, both of which are modified by geranylgeranyl groups [ , ].
The covalent attachment of the lipophilic isoprenyl group s enables prenylated proteins to anchor to cell membranes, which is an essential requirement for biological function [ ]. The localization of small GTPases in distinct subcellular sites defines which signaling pathways they activate, thus defining their participation in disease. As an example, some singly prenylated Rabs are mistargeted and dysfunctional [ ]. Inhibiting the membrane localization of small GTPases is a therapeutic strategy in cancer [ ].
In addition, isoprenoid moieties are essential in the protein-protein interaction functions of prenylated proteins since they work as molecular handles that bind to hydrophobic grooves on the surface of soluble protein factors; these factors remove the prenylated protein from membranes in a regulated manner [ ].
There is evidence that unprenylated versions of some proteins may also have physiological functional effects [ , ] or may interfere with the activity of the isoprenylated proteins during disease [ , ]. The requirement of prenylation for membrane association has also been recently challenged [ ]. The interest in understanding the regulation of isoprenoid production and protein prenylation in the brain has increased considerably in the past few years due to the importance of protein prenylation in several cellular processes such as cell growth, cytoskeletal organization and remodeling, and vesicle trafficking; and to the fact that some of the beneficial effects of statins in neurodegenerative diseases have been attributed to changes of protein prenylation [ , - ].
Non-sterol isoprenoids and protein prenyltransferases have emerged as attractive therapeutic targets for several diseases [ , ] but we still need a deeper understanding of their roles in the brain in order to determine their value for treating neurodegeneration in general, and AD in particular.
Until recently, protein prenylation was considered to function constitutively. However, there is evidence that signaling cascades activated by druggable surface receptors affect prenylation of specific small GTPases by posttranslational modifications e. Prenyltransferases are expressed in the brain, which contains the highest activity of GGTase I [ ]. GGTase I plays important roles in synapse formation, where it is activated through acetylcholine receptor clustering at the postsynaptic membrane [ ].
There is a growing body of evidence indicating that inhibition of protein prenyltransferases and inhibition of the mevalonate pathway to an extent that reduces the levels of FPP and GGPP, alter many mechanisms critical for normal brain function. When analyzing studies in which statins are used it is important to consider that different statins differ in terms of their potency, stability and ability to cross the BBB [ , , ]. Studies on the effect of statins or protein prenyl transferase inhibitors on neurite dendrites or axons extension and branching provided conflicting results depending on the type of neurons, the class of statin used and the duration of the treatments.
Statins decreased neurite initiation but increased neurite branching in neuroblastoma cells [ ]. The field of AD research will benefit from a deeper understanding of the roles of non-sterol isoprenoids and protein prenylation in axon regrowth. Under certain experimental conditions statins affect survival of neurons and neuron-like cells, acting through the decrease of non-sterol isoprenoids and protein prenylation.
Lovastatin but not pravastatin induced apoptosis of rat brain neuroblasts and caused a significant reduction of the membrane pool of Ras and RhoA proteins, suggesting an impairment of protein prenylation as the result of reduced isoprenoid production [ ]. Similarly, we found no effect of pravastatin on survival of sympathetic and cortical neurons at concentrations that significantly reduced cholesterol synthesis [ , , ].
Under these conditions, however, pravastatin did not affect protein prenylation [ ]. Statins induced stellation, followed by apoptosis in cerebellar astrocytes and cell death of cerebellar neurons [ ]. These latter effects were independent of reduced cholesterol synthesis but were prevented by GGPP. The authors speculated that astroglia cells might provide neuroprotective signals, perhaps GGPP, against the damaging effects that result from downregulation the mevalonate pathway.
This idea of communication between glia and neurons through intermediates of the mevalonate pathway is further discussed in Section 4. Results were analysed as in Fig. Full size image The mevalonate pathway is important for the biosynthesis of terpenes and steroids such as cholesterol 5 , 6. Therefore, we performed a metabolomic analysis to determine the effect of ATV on intracellular metabolite composition during primitive streak formation in EBs. Control and ATV-treated EBs were collected on days 4 and 5, and metabolites were identified followed by unsupervised clustering and heat map visualisation Fig.
ATV-treated EBs had a strikingly different metabolomic profile compared to control EBs; ATV specifically downregulated 46 metabolites and upregulated 37 metabolites on days 4 and 5. We performed principal component analysis to graphically visualise the relationship between ATV-treated and vehicle-treated EB populations and days of differentiation Fig.
The first principal component distinguished ATV-treated EBs from controls, while the second principal component characterised the differences in differentiation between days 4 and 5.
Interestingly, the free fatty acids FAs palmitic acid , stearic acid and oleic acid were increased following ATV treatment, whereas intermediates of FA biosynthesis from acetyl-CoA, namely palmitoylcarnitine , acylcarnitine and acylcarnitine were decreased Fig.
These observations support the idea that a statin-dependent increase in free FA and FAS activity would promote neurogenesis. On the other hand, statins markedly decreased amino acids and dipeptides such as threonine, arginine and Gly-Asp Supplementary Table 3 , which might contribute to the inhibition of the primitive streak formation. Surprisingly, given the major role of statins in inhibiting cholesterol production, the intracellular cholesterol level in ATV-treated EBs was actually higher than in control EBs Fig.
Cholesterol homeostasis is maintained by a balance of de novo synthesis and incorporation Thus, the observed elevation in intracellular cholesterol might be due to enhanced expression of the low-density lipoprotein receptor Supplementary Table 4 , which takes up extracellular cholesterol and is induced by statins Together, our results indicate that the inhibition of statin-mediated primitive streak formation is not caused by a decrease in intracellular cholesterol but is associated with other metabolic anomalies.
Figure 3: Metabolomic analysis of statin-treated mouse EBs. Principal components 1 and 2 account for Full size image To identify the signalling pathways underlying statin-induced inhibition of primitive streak formation, we analysed the role of cholesterol, geranylgeranyl diphosphate and farnesyl diphosphate, which are all downstream of the mevalonate pathway Supplementary Figure 3a.
In contrast, a squalene synthetase inhibitor zaragozic acid , which blocks the biosynthesis of cholesterol, and a geranylgeranyltransferase inhibitor GGTI did not suppress cardiomyogenesis Fig. Moreover, FTI inhibited primitive streak formation and induced Sox2 expression ectodermal differentiation in a dose-dependent manner Supplementary Figure 3f.
Farnesyltransferase is an enzyme transferring farnesyl diphosphate to the cysteines at the C-terminus of proteins. Importantly, farnesol rescued T expression in ATV-treated EBs, confirming the involvement of farnesyl diphosphate in primitive streak formation Fig. These data indicate that the inhibition of farnesyl diphosphate inhibits primitive streak formation, similar to statin treatment.
Figure 4: Identification of the effector pathway downstream of mevalonate. Results are representative of three cultures. Nuclei were visualised by Hoechst staining. EBs were collected at the indicated times and analysed as in Fig. Full size image Farnesylation is a protein modification in which a farnesyl group is covalently attached to cysteine residues within carboxyl-terminal CaaX motifs To identify the farnesylated proteins involved in EB primitive streak formation, we used a comprehensive tagging-via-substrate TAS approach 13 , Supplementary Figure 4a.
These spots corresponded to several small G proteins, including the well-known farnesyl substrates H-, N- and K-Ras and Cdc42, all of which have crucial roles in early embryogenesis 15 , 16 , In the present study, we focused on the 42 kDa spot appearing specifically in EBs white arrowhead in Fig. To examine the subcellular localisation of lamin B1 in mouse ES cells, we introduced Myc-tagged wild type WT lamin B1 or a mutant lamin B1C-S in which the cysteine residue of CaaX is changed to serine so it can no longer be farnesylated WT farnesylated lamin B1 localised to the nuclear membrane, whereas non-farnesylated mutant lamin B1 accumulated in the nucleoplasm Fig.
These data confirmed that lamin B1 is tethered to the nuclear membrane by C-terminal farnesylation as previously shown To determine the biological function of lamin B1 in primitive streak formation, we used haploid genetics to generate lamin B1-deficient KO and lamin B1-revertant REV clones Supplementary Figure 5a and b Lamin B1 REV ES cells are positive isogenic controls directly derived from the lamin B1 KO cell line by inducing Cre expression and thereby reactivating expression of lamin B1, which excludes any effect of genetic background.
We used real-time PCR to examine marker genes of primitive streak formation. These results indicate that Lamin B1 has a crucial role in the induction of primitive streak genes. Importantly, the U. Food and Drug Administration has classified ATV to pregnancy category X, meaning that the risks involved in use of the drug in pregnant women clearly outweigh potential benefits.Choices contain precursors for the Kandutsch-Russel pathway e. A horrifying-via-substrate biosynthesis revealed that made lamin B1 and suddenly G proteins were farnesylated in embryoid bands and important for acetylcholine travel gene expression. The Kandutsch—Russell interruption includes lathosterol and 7-dehydrocholesterol 7-DHC as columns; while the Bloch pathway, uses desmosterol as an unforgivable reviewed in [ ].
FOH and GGOH can also be formed by degradation of isoprenylated proteins in reactions catalyzed by prenylcysteine lyases, enzymes highly expressed in the brain [ ]. Whether any of these mechanisms take place in the brain is uncertain. Furthermore, supplementation of phosphatidylinositol 4,5-bisphosphate, extracellular perfusion of phospholipase C inhibitor or a protein kinase C PKC inhibitor had no effect on the inhibitory activity of simvastatin on IKACh.
The product of HMGCR, mevalonic acid, is phosphorylated sequentially to 5-phosphomevalonate by the enzyme mevalonate kinase MK and to 5-pyrophosphomevalonate by phosphomevalonate kinase PMK. In mouse brain cytosol, FPPS and GGPPS activities were higher than those in the corresponding fractions from the liver, perhaps reflecting a higher demand for protein prenylation in the brain [ ]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
FOH and GGOH can also be formed by degradation of isoprenylated proteins in reactions catalyzed by prenylcysteine lyases, enzymes highly expressed in the brain [ ]. Disruption of the DHCR24 gene results in accumulation of desmosterol and is characterized by multiple congenital anomalies in humans and mice [ , , ]. In the present study, we screened a library of 1, well-characterised drugs to evaluate their effect on cell fate and potentially identify new mechanisms involved in early embryogenesis Supplementary Figure 1a. In their recent study they found that carriers of a specific variant of HMGCR display a protective effect that resembled in size and gender to what has been reported for APOE2 in humans [ ]. Patch pipettes were made from glass capillaries with a diameter of 1.
Moreover, neuron-specific ablation of selenoprotein expression causes a neurodevelopmental and neurodegenerative phenotype affecting the cerebral cortex and hippocampus [ ]; and impaired expression of selenoproteins in the brain triggers striatal neuronal loss leading to coordination defects in mice [ ]. Alterations in cholesterol levels, even locally at synapses, may play a role in synapse dysfunction in AD [ ]. There is only limited information of changes in the mevalonate pathway enzymes and lipid intermediates in AD brains, although certain exceptions exist.
Following fragmentation, cRNA 0. Disturbances in either of these two pathways may result in replacement of cholesterol with its precursors in the brain, which causes serious disorders of the nervous system [ , ]. The time required for depletion of the non-sterol isoprenoids pools may also be tissue-or cell-specific. All full blots are shown in Supplementary Figure 6.
Contrary to the existence of a salvage pathway that uses FOH and GGOH for protein prenylation, it was demonstrated that overexpressing phosphatases that convert FPP and GGPP to FOH and GGOH in mammalian cells, decreases rather than increases protein isoprenylation as evaluated by a decreased of Rho protein level in cell membranes and results in defects in cell growth and cytoskeletal organization that are associated with dysregulation of Rho family GTPases [ ].
FOH has been shown to modulate the activity of the farnesoid X receptor FXR , a member of the nuclear hormone receptor superfamily [ ]. For each experiment, small aliquots of the HMG-CoA reductase inhibitor stock solutions were added to normal Tyrode's solution. SREBP-2 belongs to a family of membrane-bound transcription factors that regulate cholesterol and fatty acid homeostasis. These properties can partially explain ionic mechanisms of the anti-arrhythmic effect of statins. However, current evidence indicates that DHCR24 has functions that go beyond those expected from its enzymatic activity in the mevalonate pathway. These data confirmed that lamin B1 is tethered to the nuclear membrane by C-terminal farnesylation as previously shown
According to the Sarr et al.
Recently, it was revealed that chromatin-Lamin B1 interactions are tightly linked to gene repression during ES cell lineage commitment This idea of communication between glia and neurons through intermediates of the mevalonate pathway is further discussed in Section 4. Importantly, DHCR24 mediates the protective effects of estrogens in cultured human neuroblasts since estrogen and selective estrogen receptor modulators SERMs stimulate the expression of DHCR24 in human neuroblast long-term cell cultures [ , ]. Full size image To identify the signalling pathways underlying statin-induced inhibition of primitive streak formation, we analysed the role of cholesterol, geranylgeranyl diphosphate and farnesyl diphosphate, which are all downstream of the mevalonate pathway Supplementary Figure 3a. The metabolic cues regulating primitive streak formation remain largely unknown.