genes associated with Niemann-Pick disease
of the disease
Niemann–Pick type C (NPC) disease is an early childhood disease exhibiting progressive neurological degeneration. The disease is caused by mutations in either of two genes, NPC1 or NPC2. At the cellular level, the most notable aspect of the disease is a massive accumulation of cholesterol, glycosphingolipids and other lipids in aberrant organelles. The underlying defect appears to be a failure of normal organelle trafficking and a consequent failure of lipid homeostasis (Wang, 2011 and references therein).
Cholesterol is an essential component of cell membranes that influences the permeability and fluidity of the lipid bilayer. Cholesterol also acts as a precursor for steroid hormone biosynthesis and contributes to cell–cell signaling pathways. These critical cellular functions are supported by regulatory mechanisms that maintain normal cholesterol levels and prevent hypercholesterolemia, which is a major risk factor for cardiovascular disease in humans. Cholesterol homeostasis in vertebrates is achieved primarily through de novo synthesis and dietary uptake. Although extensive studies have defined a central role for the sterol regulatory element-binding protein (SREBP) family of transcription factors in controlling cholesterol synthesis, the mechanisms that regulate dietary cholesterol absorption remain more poorly understood. One central component of this pathway is the Niemann-Pick C1-like 1 gene NPC1L1, which encodes a plasma membrane protein that mediates the uptake of dietary cholesterol by the intestine. Mouse mutants for NPC1L1 display significantly reduced levels of cholesterol absorption and are insensitive to treatment with the anti-hypercholesterolemia drug ezetimibe, which acts as a specific NPC1L1 inhibitor. Another major regulator of cholesterol homeostasis is the liver X receptor α (LXRα) nuclear receptor, which binds cholesterol metabolites and regulates the transcription of genes that control cholesterol transport and metabolism, including NPC1L1 (Horner, 2009 and references therein)
About 95% of cases of human NPC disease are caused by mutations in the NPC1 gene. To explore the molecular mechanisms and discover therapeutic treatments for NPC disease, NPC1 mutant models have been generated in yeast, worm, fly and mouse. In Drosophila npc1 mutants, sterol accumulation has been found in many tissues similar to the situation in mammalian NPC mutants. Drosophila npc1 mutants show a first-instar larval lethal phenotype which is due to shortage of the steroid hormone ecdysone and can be rescued by tissue-specific expression of npc1 in the ecdysone producing organ, the ring gland. Moreover, since mutant lethality can be rescued by supplying excess cholesterol or other sterols in the culture medium, a “cholesterol shortage” hypothesis has been proposed to explain NPC disease pathology. Similarly, in NPC1 mutant mice, the neurodegeneration phenotype can also be attributed to a shortage of neurosteroid hormones. Therefore, the cause of NPC disease pathology may be due to a cholesterol trafficking defect and subsequent shortage of steroid hormones (Wang, 2011 and references therein).
Relevant studies of Niemann-Pick disease
Wang, C., Ma, Z., Scott, M.P. and Huang, X. (2011). The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis. Dev Biol 351: 146-155. PubMed ID: 21215267
The report provides insight into how NPC1 is involved in the spermatogenesis process. NPC1 and NPC2 are reported to be involved in sterol trafficking in different model organisms. The first hint that NPC genes are important for male reproduction was the discovery of NPC2, which is highly enriched in epididymal fluid. The Drosophila npc1 model provides the first in vivo link between NPC1 and male infertility. Defective sperm development was later described in a mouse npc1 model, but the underlying mechanism is still not clear. Results from this study in Drosophila add more details about the role of NPC1 in spermatogenesis. First, the function of NPC1 in spermatogenesis is specifically required in the germ cells but not in the somatic cells, suggesting that NPC1 has a cell-autonomous function in this process. Second, NPC1 function is required in the individualization process. Disrupted individualization complexes (ICs), leaky caspase activity and scattered nuclei indicate that cooperative aspects of the individualization process are defective. Third, individualization defects are likely caused by sterol deficiency but are not due to ecdysone shortage. Therefore, npc1 mutant sterility is due to other sterol-involved defects. It is not surprising that npc1 mutants show phenotypes independent of steroid hormone. Previously, npc1 mutants have been reported to display progressive neurodegeneration and this defect cannot be rescued by the ecdysone precursor 7-dC, implying a 7-dC, and probably ecdysone, independent mechanism (Wang, 2011).
Many genes have previously been identified to be involved in the individualization process. However, the functions of most of them are still unclear. So far, actin assembly, apoptosis and membrane trafficking are the three best characterized processes that are known to be important for different aspects of individualization. The individualization complex (IC) is formed by a cohort of 64 actin cones. Therefore, the formation of actin cones, actin dynamics and the integrity of the IC are all essential for individualization. Removal and clearance of excess cytosol is the ultimate goal of individualization. Apoptosis has been shown to be essential for the removal of excess cytosol and this requirement is likely to be conserved in mammals. Lastly, individualization is a rapid membrane-remodeling process. Cog5 and syx5 function in the individualization process by affecting membrane trafficking (Wang, 2011).
Which process during individualization are sterols required for? Though NPC1 proteins are highly conserved in sequence and npc mutants in different animal models have sterol trafficking defects, global mutant phenotypes differ greatly. An evolutionally conserved mechanism of NPC pathology has emerged from studies on NPC1 mutants in the worm, fly and mouse. Shortage of available sterols, which in turn leads to shortage of cholesterol-derived steroid hormones, appears to be the source of defects in NPC models. Are the individualization defects observed in Drosophila npc1 mutants caused by the same mechanism? Consistent with the sterol trafficking defect observed in the npc1 testis, adding 7-dC rescues male sterility in npc1 mutants, indicating that the sterility is likely due to sterol shortage. However, it was found that although EcRA is expressed in spermatocytes, neither EcR (for ecdysone signaling) nor dib (for ecdysone biosynthesis) is required in the individualization process. These results indicate that the steroid hormone ecdysone is not required (Wang, 2011).
There are two possibilities for the requirement of sterols in individualization. First, instead of ecdysone, another unidentified steroid hormone is required for individualization. If this is true, since npc1 is required in the germ cell, this steroid must act in a paracrine or autocrine manner to affect individualization. Alternatively, sterols may be intrinsically important for individualization. The individualization process requires membrane bending and remodeling, as well as a large amount of membrane addition. Sterols are key membrane components and sterol content can affect membrane bending rigidity/curvature and fluidity. It is possible that abnormal sterol distribution and sterol deficiency in npc1 mutants may lead to impaired membrane trafficking or membrane-remodeling. It has been shown earlier that sterol-rich structures are present in the leading edge of the IC and that, in mutants of the sterol binding protein OSBP, sterol-rich structures are absent and the individualization process is defective. Adding cholesterol or 7-dC can rescue male infertility in osbp mutants, indicating that sterility is caused by the sterol deficiency. Interestingly, the OSBP-rich or sterol-rich puncta are present in npc1 mutants, suggesting that npc1 may act independent or downstream of Osbp in individualization. Moreover, a temperature-sensitive feature of the individualization defects of npc1 mutants was observed, implying a membrane defects in npc1 mutants (Wang, 2011).
In summary, this study reports the male sterile phenotype of the npc1 mutant fly in detail and successfully connects the NPC1 protein with the spermatogenesis process. It was found that while rescuing both lethality and male infertility, 7-dC usually yields better rescuing results than cholesterol. Exploiting the difference between 7-dC and cholesterol in rescue efficiency may be a useful therapeutic strategy for the treatment of NPC disease. New drugs or treatments that improve the efficiency of delivery and usage of sterols and steroid hormones may have better therapeutic effects (Wang, 2011).
Bujold, M., Gopalakrishnan, A., Nally, E. and King-Jones, K. (2010). Nuclear receptor DHR96 acts as a sentinel for low cholesterol concentrations in Drosophila melanogaster. Mol Cell Biol 30: 793-805. PubMed ID: 19933845
It appears likely that insufficient cholesterol absorption in DHR96 mutants merely contributes to the failure to survive on low cholesterol, because only a 20% reduction of total cholesterol and cholesteryl ester levels was found in mutant animals compared to controls. With this in mind, it is interesting that genes with roles in reducing cellular cholesterol concentrations are substantially overexpressed in DHR96 mutants, suggesting that cellular cholesterol efflux is increased in mutant animals compared to the wild type. The ACAT and ABCA1 genes fall into this category, since ACAT reduces the pool of free cellular cholesterol through an esterification reaction that adds a fatty acid to the molecule, while ABCA1 encodes an ATP transporter that is involved in the active removal of cholesterol by pumping it across the cell membrane. Wild-type ACAT and ABCA1 are downregulated in response to declining cholesterol concentrations, which is in agreement with the idea that under conditions of dangerously low cholesterol levels, genes that increase cholesterol efflux must be turned off. On lipid-depleted medium, ACAT and ABCA1 mRNA levels are substantially higher in DHR96 mutants than in controls, suggesting that mutant cells actively reduce cellular cholesterol concentrations under conditions of low dietary cholesterol. At the same time, cholesterol uptake is reduced as well, thus aggravating the situation (Bujold, 2010).
The fact that treatment with 1% cholesterol largely phenocopies the genome-wide effects of the DHR96 mutation strongly suggests that DHR96 functions at the top of a gene network controlling the systemic response to varying levels of dietary sterols. It has been shown earlier that DHR96 can bind cholesterol in vivo, suggesting that this nuclear receptor is a cellular sensor for varying sterol levels. This would suggest that cholesterol or a very similar metabolite acts as a direct ligand for DHR96; however, a key question is whether such a ligand would act as an agonist or an antagonist. The “constitutive androstane receptor” (CAR), which is one of the three vertebrate nuclear receptor orthologs of DHR96, displays the unusual feature that it acts as a constitutively active transcription factor in the absence of a ligand. Androstane metabolites, however, act as inverse agonists and deactivate murine CAR upon ligand binding. Prior to this finding, all nuclear receptors were believed to be activated by ligand binding; however, it remains unclear how widespread this mode of nuclear receptor inactivation is (Bujold, 2010).
Observations of this study are best explained by an inverse agonist mechanism similar to what has been described for CAR. Given the fact that DHR96 is only required for survival when the animal is feeding on a low-cholesterol diet, it follows that cholesterol metabolites that might act as ligands for DHR96 are scarce under these conditions. This suggests that DHR96 is active in the absence of a ligand. Conversely, the DHR96 gene is not required for survival when cholesterol concentrations are sufficiently high, suggesting that the DHR96 protein is inactive when potential ligands are abundant, again favoring the view of an inverse agonist mechanism. Perhaps the strongest argument for a mechanism through an inverse agonist derives from the fact that it provides the simplest explanation for the fact that a high-cholesterol diet is able to phenocopy the DHR96 mutation. In accordance with the finding that DHR96 binds to cholesterol, this model predicts that a high-cholesterol diet would result in a widespread deactivation of DHR96 receptor molecules, essentially turning off DHR96 activity. Consequently, one would expect that the molecular consequences of deactivated DHR96 protein (via a high-cholesterol treatment) or removing functional protein altogether (via a null mutation) are very similar indeed. The observation that DHR96 mRNA levels decline in response to increasing cholesterol levels is also compatible with the idea of inverse agonism. Since DHR96 mRNA is possibly regulated by its own protein, one would predict that increasing cholesterol levels reduce DHR96 activity, which in turn results in reduced transcription of the DHR96 gene itself (Bujold, 2010).
Nutrigenomics is a powerful strategy for identifying genes that act in nutrient-dependent pathways, and this study represents a first step toward the identification of genes with hitherto unknown roles in cholesterol homeostasis. It was shown that the expression of four Niemann-Pick genes—NPC1b and NPC2c, -d, and -e—is strongly dependent on the concentration of dietary cholesterol. Other identified genes with predicted roles in sterol biology are ACAT, ABCA1, Lip3, and Cyp12d1. Cholesterol-responsive genes with no known links to cholesterol homeostasis were also found. For instance, CG5932, encoding a gastric lipase; FANCL, encoding a ubiquitin E3 ligase; and CG31148, encoding a glucosylceramidase, are all downregulated in response to increasing cholesterol concentrations. An earlier study shows that DHR96 mutants are resistant to diet-induced obesity, which is at least in part due to the role of DHR96 in regulating CG5932, confirming that this nuclear receptor also has important roles in controlling lipid metabolism. In addition, a study of mice demonstrates that the “Idol” ubiquitin E3 ligase is transcriptionally induced by LXR and triggers proteolytic degradation of the LDL receptor via ubiquitination, thereby downregulating cellular cholesterol uptake. Future work may provide insight into whether FANCL has comparable roles in regulating cholesterol homeostasis in Drosophila (Bujold, 2010).
Shi, X.Z., Zhong, X. and Yu, X.Q. (2012). Drosophila melanogaster NPC2 proteins bind bacterial cell wall components and may function in immune signal pathways. Insect Biochem Mol Biol 42: 545-556. PubMed ID: 22580186
Horner, M.A., Pardee, K., Liu, S., King-Jones, K., Lajoie, G., Edwards, A., Krause, H.M. and Thummel, C.S. (2009). The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis. Genes Dev 23:2711-2716. PubMed ID: 19952106
Gazi, M., Shyamala, B.V. and Bhat, K.M. (2009). A neurodegenerative disease affecting synaptic connections in Drosophila mutant for the tumor suppressor morphogen Patched. Dev Biol 334: 311-323. PubMed ID: 19635474
This study shows that loss of function for ptc causes a neurodegenerative disease that has similarity to the NPC disease in vertebrates. This involvement of Ptc in a neurodegenerative disease defines a novel role for Ptc. The similarity to the NPC disease is consistent with the fact that NPC1 and Ptc share significant sequence homology. It was also shown that the loss of function for Ptc results in a reduction in the levels of cholesterol esters in the brain. Loss of Ptc function also progressively affects the number of Syt-positive synaptic connections in such structures as the dendritic-rich calyx of the brain. The fact that levels of cholesterol esters, and the number of connections in the mutant brain can be restored by feeding cholesterol, and that the disease can be suppressed by feeding cholesterol argues a primary role for cholesterol esters and synaptic connections in the progression of the disease. These results are also supported by the EM data that the number of connections are reduced in the mutant brain, which can be restored by feeding cholesterol. These results also reveal a role for cholesterol esters in forming and or maintaining synaptic connections, either directly or indirectly. Moreover, it was also shown that inclusions per se are not the problem since wild type flies can have the inclusion but that does not cause the disease. These could be novel results which shed new insight into the basic science of neurodegenerative diseases in general. The study also demonstrates new tools to analyze the adult brain that should generally be applicable in adult brain development and functional studies (Gazi, 2009).
There are two NPC1 genes in Drosophila, NPC1a and NPC1b. The Ptc protein shares significant homology to the NPC1 proteins in Drosophila or to the vertebrate NPC1 protein. Two previous studies show that null mutants for NPC1a die as first instar larvae, but these mutant larvae can be rescued to adulthood by feeding them cholesterol. Also, that while the brain in these rescued mutants are normal, the malphigian tubule (the kidney equivalent in flies) has the highest sterol accumulation and contains large multi-lamellar inclusions. These inclusions are strikingly similar to what was found in the brain of ptc mutants in this study. Moreover, in NPC disease, it is thought that unesterified cholesterol accumulates in the E/L system. It was found that there is a reduction in the pool of esterified cholesterol. These results suggest that the cholesterol metabolism is affected in both NPC disease and ptc mutants (Gazi, 2009).
These results further suggest that NPC1 genes in flies may play a smaller role in the brain. This possibility is consistent with another study which reports that null mutants for dNPC1a gene mimic human NPC patients with progressive motor defects and reduced life span; the brains of these mutants reportedly contain higher levels of cholesterol and multi-lamellar inclusions. What is the relationship between ptc and dNPC1? It was observed that ptc mutant individuals that are also heterozygous for NPC1a (ptc/ptc; NPC1a/+) do not show an enhancement of the disease. While it was not examined if the double mutants between ptc and dNPC1 mutants show an enhanced neurodegenerative phenotype, it may be that one needs to eliminate also both NPC1 genes (dNPC1a and dNPC1b) in order to observe a strong brain phenotype. Nonetheless, it seems likely that the ptc-neurodegenerative disease in flies is closest to the NPC-disease in vertebrates. In vertebrates, since loss of function for ptc die as embryos, it is not known if Ptc has any role in preventing a neurodegenerative disease; however, at least in Drosophila, Ptc may function in the same pathway as the NPC1 proteins. Additional work is needed to fully determine the relationship between dNPC1a and dNPC1b as well as between dNPC1 genes and ptc (Gazi, 2009).
The role of Hh-Ptc-Smo signaling in development and disease has been examined in many different studies over the last several years. This study reveals that Ptc has a role in preventing a neurodegenerative disease. This disease is not due to a strange allele of ptc but due to the loss of function for ptc. The disease was observed in several different allelic combinations of ptc, including combinations of known loss of function alleles and deficiencies (which also rules out a background-mediated locomotor defect). The phenotype can also be rescued by a ptc transgene. Ptc is widely expressed in the adult brain, and at the same time, the expression of Hh is restricted to mostly olfactory lobes, suggesting that Ptc plays a repressive role in the development of the disease. This is also consistent with the result that a gain of function for Hh causes the same disease as the loss of function for ptc. Moreover, the fact that inducing Hh in the brain at any point in the adult life will cause the disease indicates that the disease in the loss of function for ptc is not due to some developmental abnormality. Results with Hs-hh also indicate that Ptc functions indirectly via repressing Smo. This conclusion is consistent with the result that reducing the dosage of smo alleviates the disease (Gazi, 2009).
It is thought that in NPC disease, formation of inclusions in neurons causes neuronal death, which results in a disease-state. However, no direct evidence exists to link the inclusions to disease-state. A previous study suggests that in the case of Huntington’s disease, formation of inclusion bodies is a cellular response to decrease the mutant protein and prevent the poisoning of other neurons. Therefore, formation of such inclusions is a good thing for neurons in slowing down the disease. It was shown that the inclusions in the brain of ptc mutants are due to cholesterol: feeding a large amount of cholesterol to wild type flies causes formation of inclusions in the brain. This also indicates that the presence of inclusions in the brain of ptc mutants is indeed due to an aberrant metabolism or accumulation of cholesterol in neurons. Quantifying the number of inclusions in the entire brain in EM experiments was found to be a difficult proposition for several reasons. First is the fact that there are no serial section EM maps of the fly brain and it is nearly impossible to pinpoint the precise location within the brain to be able to compare between brains of different genotypes, conditions and age. Second, the inclusions are not uniformly spread in the brain. The only way to avoid these two problems and be able to give a precise quantification of inclusions between brains is to do serial EM sections of the entire brain and count the number of non-overlapping inclusions in each brain (Gazi, 2009).
To perform serial EM sections of an entire brain, the following calculations were made. Each EM section is about 100 nm thick and one needs to cut about 1,500 sections to cover the entire brain. The area of the each section is about 150,000 μm2. In order to reliably examine the section for lamellar inclusions, one needs to examine the sections at a magnification of 5,000 times. At this magnification each image will cover 300 μm2 of an area. This means that one needs to take 500 images to cover one entire section at this magnification. Thus, the total number of images one would have to take to examine an entire brain is 1,500 × 500 = 750, 000. This is not practical given that one will have to section a minimum of 3 brains for each sample. Therefore, quantification of inclusions reported in this study should be considered approximations and may or may not be representative of the entire brain. However, it is to be noted that quantification of synaptic connections from EM photomicrographs is somewhat of a different scenario since the synaptic connections are generally spread in a uniform fashion within the neuropile (and the brain overall). Therefore, the error due to imprecision of the location, though still exists, it is at a much lower level. Nevertheless, the quantification of the synaptic connections using EM should be again treated as an approximation. Thus, the number of Syt-positive connections in a discrete structure such as the calyx were examined using the Apotome microscopy between different samples of the brain, a much more accurate analysis (Gazi, 2009).
Because of the technical difficulty with quantifying inclusions between brain samples, this study focused on examining synaptic connections in a discrete structure such as the calyx, where one can quantify the number of Syt-positive terminals using the newly developed method and draw meaningful conclusions. It was found that loss of synaptic terminals is a major contributor to the disease-state. In ptc mutants, there is a significant reduction in the Syt-positive synaptic connections. One can argue that the loss of synaptic connections is due to the degenerative disease. However, it was shown that loss of synaptic connections and the disease-state in ptc mutants can be suppressed by feeding cholesterol; cholesterol appears to prevent the loss of the synaptic connections and also the progression of the disease (Gazi, 2009).
Normally, esterified cholesterol (LDL) is taken up inside the cell via endocytosis by the endosomal/lysosomal (E/L) system and de-esterified in lysosomes; it is then re-esterified in the cytoplasm and then stored or sent to other cellular components. For instance, the most prominent characteristic of the NPC disease at the cellular level is the blockade of intracellular transport of LDL-derived cholesterol between the E/L compartment and the plasma membrane resulting in the accumulation of unesterified cholesterol in the E/L system. This blockade of cholesterol transport appears to be the cause for the formation of inclusion bodies in neurons of NPC human patients or NPC mice. Additionally, the finding that the gene corresponding to NPC2 encodes an ubiquitously-expressed cholesterol binding lysosomal protein, known as HE1, further supports a role for cholesterol in the disease. On the other hand, in human NPC patients, a combination of diet and cholesterol-lowering drugs has been found to have no beneficial effect. Therefore, the nature of cholesterol involvement in the genesis of the disease is not known (Gazi, 2009).
Results from this study suggest that one of the consequences of loss of function for ptc is the accumulation of cholesterol in the cytoplasm leading to the formation of inclusions and membranous material. Furthermore, there is a progressive loss of synaptic connections in the mutant brain. Thus, one possibility is that in ptc mutants, cholesterol becomes limiting, and this leads to a loss of synaptic connections. This is supported by the fact that feeding cholesterol to wild type or mutant flies increases the connections and in mutants, feeding cholesterol suppresses the disease. Results from a few previous studies are also consistent with this possibility. For example, an in vitro study suggests that cholesterol promotes formation of synapses. This was also observed in the adult fly brain. Moreover, apoE has long been suspected to be involved in neurodegenerative loss of synaptic plasticity in the Alzheimer’s disease. Furthermore, the e4 mutant in the gene for ApoE, an important cholesterol transport protein, is associated with an increased risk of late-onset Alzheimer’s disease; this mutant isoform of the protein is less able to promote neurite outgrowth than other apoE isoforms. It is not clear, however, if cholesterol promotes synapse formation by directly influencing the membrane property (i.e., fluidity), as a structural component, via regulating gene activity, or via the synthesis of neurosteroids. A decrease in the available cholesterol may increase the fluidity of the plasma membrane of synaptic terminals and make synaptic connections structurally unstable. Alternatively, limiting amounts of cholesterol may affect gene expression or the synthesis of neurosteroids, contributing to the disease-state. It should also be noted that the suppression of the disease with cholesterol is not complete. Thus, it canbe speculated that loss of function for ptc causes neurodegeneration via two distinct ways: 1) de-repressing genes that interfere with lipid/cholesterol trafficking, and 2) de-repressing genes outside of the lipid trafficking; the functions of both classes of genes ultimately converge on synaptic connections (Gazi, 2009).
Phillips, S.E., Woodruff, E.A. 3rd, Liang, P., Patten, M. and Broadie, K. (2008). Neuronal loss of Drosophila NPC1a causes cholesterol aggregation and age-progressive neurodegeneration. J Neurosci 28: 6569-6582. PubMed ID: 18579730
95% of NPC disease cases are caused by mutation of NPC1, a 13-pass transmembrane protein that resides in a unique class of endosomal organelles, binds cholesterol and has the hallmarks of a transporter involved in sphingolipid/cholesterol trafficking. Loss of the Drosophila ortholog, dNPC1a, has been previously reported to result in early lethality owing to the loss of cholesterol-dependent ecdysone production, a steroid hormone required for molting. This insect-specific requirement has been a distraction, and has prevented detailed characterization of the Drosophila model. Fortunately, this early block in development is easily bypassed through a diet of excess ecdysone precursors (cholesterol or 7-dehydrocholesterol) or by targeted expression of dNPC1a in the ring gland, the endocrine organ which produces ecdysone. Null dnpc1a mutants rescued to adulthood show progressive locomotor defects, greatly reduced life-span, intracellular accumulation of cholesterol aggregates and age-progressive neurodegeneration; a constellation of phenotypes the closely mimic the human NPC disease condition. Importantly, all of these phenotypes are rescued by targeted neuronal expression of dNPC1a in the null mutant, demonstrating a neural requirement (Philips, 2008).
Previous reports conclude that loss of dNPC1a does not cause neurodegeneration. In contrast, this study shows that the lack of dNPC1a within the brain causes the age-progressive accumulation of massive intracellular membranous structures, which are never observed in wildtype neurons, and brain degeneration starting as small vacuoles within the retina and progressing to massive tissue loss within both the retina and central brain. Similar neurodegeneration occurs in dnpc1a null mutants fed high levels of cholesterol during larval growth or expressing wildtype dnpc1a in the ring gland. Thus, neither cholesterol feeding nor ecdysone function contributes to the dnpc1a neurodegeneration phenotypes. In Drosophila, dNPC1a-dependent steroid hormone expression clearly explains the requirement for dNPC1a during development. Similarly in mammals, loss of NPC1 is associated with lower neurosteroid levels and administration of the neurosteroid allopregnanolone reportedly delays onset of neurological symptoms in NPC1-/- mice. These data clearly argue for NPC1-dependent generation of cholesterol-derived signaling steroids. However, characterized steroid hormones in Drosophila derive from the ring gland, and this study shows that dNPC1a function in the ring gland provides no protection against neurodegeneration (Philips, 2008).
Targeted neuronal expression of wildtype NPC1 in both the murine and Drosophila models prevents neurodegeneration associated with NPC1 dysfunction. The elav-GAL4 driven expression of wildtype dNPC1a does not lead to glial cell transgene expression. The neurodegenerative process in NPC cases has been linked to glial cell and astrocyte cellular dysfunction because of NPC1 localization to these cell types. However, in the neuronal dNPC1a rescue animals, glial cells lacking dNPC1a adjacent to neurons expressing wildtype dNPC1a still accumulate massive MLBs and filipin positive puncta. It is presently not known whether these glial cells die. Nevertheless, targeted glial expression of dNPC1a clearly provides significant rescue for the early onset lethality of dnpc1a mutants, demonstrating that dNPC1a function in glia plays a substantial role in this disease model. Thus, in Drosophila dNPC1a function in both glia and neurons is important for prolonged survival during aging (Philips, 2008).
Chimeric mice with functional dNPC1 expressed in a few cells still manifest death of nearby npc1 mutant neurons, at least suggesting a cell-autonomous role for NPC1 in neuronal cell survival. However, in Drosophila MARCM clonal results showed that MB neurons apparently do not require dNPC1a for long-term survival. MARCM analyses of aged day 50 dnpc1a null neurons show intact neuronal perikarya, elaborate dendrites and structurally mature axons. These data, along with the neurodegenerative delay associated with neurosteroid application to npc1-/- mutants early in murine development, argue for a non-cell autonomous role for NPC1 function. In contrast, MARCM analysis of randomly-induced dnpc1a null clones throughout the brain clearly demonstrates a cell-autonomous accumulation of cholesterol aggregates, as revealed by filipin staining, and the formation of extensive MLBs in neuronal soma, characteristic of ailing cells. It remains formally possible that steroids produced by neurons depend on dNPC1a function, and that such neurosteroids maintain neuronal viability and so guard against complete neurodegeneration. Furthermore, while almost all neurons in the NPC-/- mice are filipin positive, not all neurons are equally vulnerable, as there are low levels of neuron loss in the thalamous and prefrontal cortex in the face of nearly complete PC loss. Similarly, in the Drosophila brain it appears that some neuron populations are also more sensitive to the loss of dNPC1a, at least at the level of full cellular death (Philips, 2008).
Loss of dNPC1a function in Drosophila neurons strongly impacts cholesterol trafficking. Null dnpc1a brains accumulate highly elevated levels of cholesterol and cholesterol aggregates at exceedingly high levels within aberrant neuronal organelles. These multilamellar structures are often composed of hundreds of layers of wrapped membrane, and accumulate progressively in both size and abundance in all neurons in the absence of dNPC1a. While the existence and formation of MLBs is not always a pathological symptom, the massive accumulation of MLBs seen in a variety of disease conditions is clearly an indication of severe cellular dysfunction. It has been proposed that sphingolipid storage disease cells upregulate a Beclin-1 regulated autophagic process (self-digestion) to promote cell survival. The formation of cholesterol-rich MLBs seen in NPC1-deficient cells is consistent with such autophagic cell death (Philips, 2008).
The cytoplasmic accumulation of MLBs in neurons is observed in a variety of other Drosophila mutants, also associated with adult lethality, neurodegeneration and synaptic dysfunction. The formation of MLBs in the retina and neuromuscular synaptic junction are neuronal phenotypes in the benchwarmer (bnch) mutant, caused by loss of a predicted lysosomal sugar carrier. The eggroll mutant exhibits similar MLB structures in neurons and glial cells, with associated brain vacuolization and early onset lethality. A Drosophila disease model for infantile neuronal ceroid lipofuscinoses (Ppt1 mutants) displays the same accumulation of MLBs throughout the brain, with associated early lethality but without reported signs of brain vacuolization or neuronal tissue loss. The genetic lesion causing eggroll has yet to be determined, but dnpc1a, bnch, and Ppt1 are all lysosomal/endosomal proteins, clearly linking neuronal MLB formation with endosomal trafficking defects (Philips, 2008).
Sterols (ergosterol and cholesterol) together with sphingolipids are the major components of lipid rafts. Drosophila lipid rafts are important for the light-induced recruitment of INAD-signaling phototransduction complexes in photoreceptors and also act as positive regulators of the Drosophila metabotropic glutamate receptor (DmGluRA) signaling between neurons. Drosophila lipid rafts also regulate voltage-gated ion channel signaling and the synaptic vesicle cycling underlying neurotransmission. Altering cholesterol levels and trafficking within neurons presumably has a great impact on membrane lipid rafts and their dependent mechanisms. Loss of NPC1 is linked to the depletion of lipid rafts in endocytic membranes. Loss of dNPC1a causes profound changes in cholesterol distribution and thus likely also disrupts lipid rafts. This study shows that loss of dNPC1a disrupts relevant neuronal processes, including phototransduction in retinal photoreceptors and synaptic transmission to the optic lamina. The Drosophila NPC1 model is a good system to investigate whether NPC1 regulates formation or maintenance of lipid rafts in neurons, which may in turn be required for neuronal function and viability. Genetic and proteomic screens can tease out other players in the lipid trafficking pathway, and thus provide insight into the dysfunction driving intracellular cholesterol/sphingolipid accumulation and identify potential drug targets for alleviating the catastrophic consequences of the NPC disease (Philips, 2008).
Huang, X., Warren, J.T., Buchanan, J., Gilbert, L.I. and Scott, M.P. (2007). Drosophila Niemann-Pick type C-2 genes control sterol homeostasis and steroid biosynthesis: a model of human neurodegenerative disease. Development 134: 3733-3742. PubMed ID: 17804599
Niwa, R. and Niwa, Y.S. (2011). The fruit fly Drosophila melanogaster as a model system to study cholesterol metabolism and homeostasis. Cholesterol 2011: 176802. PubMed ID: 21512589
Role of dispatched in Niemann-Pick diseaseGo to top
Date revised: 18 August 2015
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