A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease, Type C1.

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.

Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia.

Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.

Acute Statin Administration Reduces Levels of Steroid Hormone Precursors.

Cholesterol-lowering statin drugs are used by approximately 25% of US adults 45 years of age and older and frequency of use is even higher among the elderly. Cholesterol provides the substrate for steroid hormone synthesis and its intracellular concentrations are tightly regulated. Our aim was to evaluate whether statin use acutely changes the circulating levels of cortisol, other glucocorticoid precursor molecules and their metabolites. Fourteen subjects not taking statins were administered a single oral dose (2 mg) of pitavastatin. Blood samples collected at baseline and 24 h post-treatment were analyzed for plasma cholesterol and steroid hormone profile. A parallel study in mice entailed the administration of atorvastatin (10 mg/kg) via orogastric delivery for three consecutive days. Cholesterol and corticosterone levels were quantified at baseline and at 1-day and 1-week post-treatment. Several precursor molecules in the steroidogenic pathway (corticosterone, cortisone, and 11-deoxycortisol) were significantly decreased 24 h after administration of a single dose of pitavastatin in human study subjects. Their circulating cholesterol concentrations were unchanged. In mice, there were no significant differences in serum cholesterol or corticosterone at 1-day or 1-week post-treatment compared to both pre-treatment baseline levels and control group levels. We conclude that acute dysregulation of the production of certain glucocorticoid precursor molecules was observed after a single treatment with a lipophilic statin drug. This may be of clinical relevance for individuals with underlying or subclinical adrenal insufficiency.

Extracellular vesicles from symbiotic vaginal lactobacilli inhibit HIV-1 infection of human tissues.

The vaginal microbiota, dominated by Lactobacillus spp., plays a key role in preventing HIV-1 transmission. Here, we investigate whether the anti-HIV effect of lactobacilli is mediated by extracellular vesicles (EVs) released by these bacteria. Human cervico-vaginal and tonsillar tissues ex vivo, and cell lines were infected with HIV-1 and treated with EVs released by lactobacilli isolated from vaginas of healthy women. EVs released by L. crispatus BC3 and L. gasseri BC12 protect tissues ex vivo and isolated cells from HIV-1 infection. This protection is associated with a decrease of viral attachment to target cells and viral entry due to diminished exposure of Env that mediates virus-cell interactions. Inhibition of HIV-1 infection is associated with the presence in EVs of several proteins and metabolites. Our findings demonstrate that the protective effect of Lactobacillus against HIV-1 is, in part, mediated by EVs released by these symbiotic bacteria. If confirmed in vivo, this finding may lead to new strategies to prevent male-to-female sexual HIV-1 transmission.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Emissions of DEHP-free PVC flooring.

Degrading 2-ethylhexyl-containing PVC floorings (eg DEHP-PVC floorings) and adhesives emit 2-ethylhexanol (2-EH) in the indoor air. The danger of flooring degradation comes from exposing occupants to harmful phthalates plasticisers (eg DEHP), but not from 2-EH as such. Since the EU banned the use of phthalates in sensitive applications, the market is shifting to use DEHP-free and alternative types of plasticisers in PVC products. However, data on emissions from DEHP-free PVC floorings are scarce. This study aimed at assessing the surface and bulk emissions of two DEHP-free PVC floorings over three years. The floorings were glued on the screed layer of concrete casts at 75%, 85%, and 95% RH. The volatile organic compounds (VOCs) were actively sampled using FLEC (surface emissions) and micro-chamber/thermal extractor (µ-CTE, bulk emissions) onto Tenax TA adsorbents and analyzed with TD-GC-MS. 2-EH, C9-alcohols, and total volatile organic compound (TVOC) emissions are reported. Emissions at 75% and 85% RH were similar. As expected, the highest emissions occurred at 95% RH. 2-EH emissions originated from the adhesive. Because the two DEHP-free floorings tested emitted C9-alcohols at all tested RH, it makes the detection of flooring degradation harder, particularly if the adhesive used does not emit 2-EH.

Cyclodextrin and malto-dextrose collision cross sections determined in a drift tube ion mobility mass spectrometer using nitrogen bath gas.

In this study, we have evaluated a low field limit drift tube ion mobility device for ion mobility-mass spectrometry (IM-MS) measurements that uses nitrogen as a bath gas with electrospray ionization on a modified Q-TOF instrument. We have determined reduced mobility (K0) and collision cross section (CCS) values for a group of analyte ions that have been characterized previously in other drift tube IM-MS instruments. Our determinations of CCS for this set of ions as well as for standards are in agreement with published values. Because of their importance in biophysics and pharmaceuticals, we expanded our analysis to investigate the properties of cyclodextrins in this system. We present CCS data for both positively and negatively charged cyclodextrins and, for purposes of comparison, maltodextrose ions. Our results are the first reports of these materials as negative ions.

Correction: Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1.

[This corrects the article DOI: 10.1371/journal.pone.0175478.].

Daily Rhythm in Plasma N-acetyltryptamine.

Normal physiology undergoes 24-h changes in function that include daily rhythms in circulating hormones, most notably melatonin and cortical steroids. This study focused on N-acetyltryptamine, a little-studied melatonin receptor mixed agonist-antagonist and the likely evolutionary precursor of melatonin. The central issue addressed was whether N-acetyltryptamine is physiologically present in the circulation. N-acetyltryptamine was detected by LC-MS/MS in daytime plasma of 3 different mammals in subnanomolar levels (mean ± SEM: rat, 0.29 ± 0.05 nM, n = 5; rhesus macaque, 0.54 ± 0.24 nM, n = 4; human, 0.03 ± 0.01 nM, n = 32). Analysis of 24-h blood collections from rhesus macaques revealed a nocturnal increase in plasma N-acetyltryptamine (p < 0.001), which varied from 2- to 15-fold over daytime levels among the 4 animals studied. Related RNA sequencing studies indicated that the transcript encoding the tryptamine acetylating enzyme arylalkylamine N-acetyltransferase (AANAT) is expressed at similar levels in the rhesus pineal gland and retina, thereby indicating that either tissue could contribute to circulating N-acetyltryptamine. The evidence that N-acetyltryptamine is a physiological component of mammalian blood and exhibits a daily rhythm, together with known effects as a melatonin receptor mixed agonist-antagonist, shifts the status of N-acetyltryptamine from pharmacological tool to candidate for a physiological role. This provides a new opportunity to extend our understanding of 24-h biology.

Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1.

2-Hydroxypropyl-beta-cyclodextrin (HPßCD) has gained recent attention as a potential therapeutic intervention in the treatment of the rare autosomal-recessive, neurodegenerative lysosomal storage disorder Niemann-Pick Disease Type C1 (NPC1). Notably, HPßCD formulations are not comprised of a single molecular species, but instead are complex mixtures of species with differing degrees of hydroxypropylation of the cyclodextrin ring. The degree of substitution is a critical aspect of the complex mixture as it influences binding to other molecules and thus could potentially modulate biological effects. VTS-270 (Kleptose HPB) and Trappsol® Cyclo™ are HPßCD products under investigation as novel treatments for NPC1. The purpose of the present work is to compare these two different products; analyses were based on ion distribution and abundance profiles using mass spectrometry methodology as a means for assessing key molecular distinctions between products. The method incorporated electrospray ionization and analysis with a linear low-field ion mobility quadrupole time-of-flight instrument. We observed that the number of hydroxypropyl groups (the degrees of substitution) are substantially different between the two products and greater in Trappsol Cyclo than in VTS-270. The principal ions of both samples are ammonium adducts. Isotope clusters for each of the major ions show doubly charged homodimers of the ammonium adducts. In addition, both products show doubly charged homodimers from adduction of both a proton and ammonium. Doubly charged heterodimers are also present, but are more intense in Trappsol Cyclo than in VTS-270. Based on the analytical differences observed between VTS-270 and Trappsol Cyclo with respect to the degree of substitution, the composition and fingerprint of the complex mixture, and the impurity profiles, these products cannot be considered to be the same; the potential biological and clinical implications of these differences are not presently known.

ProteinProcessor: A probabilistic analysis using mass accuracy and the MS spectrum.

Current approaches to protein identification rely heavily on database matching of fragmentation spectra or precursor peptide ions. We have developed a method for MALDI TOF-TOF instrumentation that uses peptide masses and their measurement errors to confirm protein identifications from a first pass MS/MS database search. The method uses MS1-level spectral data that have heretofore been ignored by most search engines. This approach uses the distribution of mass errors of peptide matches in the MS1 spectrum to develop a probability model that is independent of the MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional corroboration enables us to confirm protein identifications to MS/MS-based scores that are otherwise considered to be only of moderate quality. Straightforward and easily applicable to current proteomic analyses, this tool termed "ProteinProcessor" provides a robust and invaluable addition to current protein identification tools.

Hormonal circadian rhythms in patients with congenital adrenal hyperplasia: identifying optimal monitoring times and novel disease biomarkers.

OBJECTIVES: The treatment goal in congenital adrenal hyperplasia (CAH) is to replace glucocorticoids while avoiding androgen excess and iatrogenic Cushing's syndrome. However, there is no consensus on how to monitor disease control. Our main objectives were to evaluate hormonal circadian rhythms and use these profiles to identify optimal monitoring times and novel disease biomarkers in CAH adults on intermediate- and long-acting glucocorticoids. DESIGN: This was an observational, cross-sectional study at the National Institutes of Health Clinical Center in 16 patients with classic CAH. METHODS: Twenty-four-hour serum sampling for ACTH, 17-hydroxyprogesterone (17OHP), androstenedione (A4), androsterone, DHEA, testosterone, progesterone and 24-h urinary pdiol and 5ß-pdiol was carried out. Bayesian spectral analysis and cosinor analysis were performed to detect circadian rhythmicity. The number of hours to minimal (TminAC) and maximal (TmaxAC) adrenocortical hormone levels after dose administration was calculated. RESULTS: A significant rhythm was confirmed for ACTH (r(2), 0.95; P<0.001), 17OHP (r(2), 0.70; P=0.003), androstenedione (r(2), 0.47; P=0.043), androsterone (r(2), 0.80; P<0.001), testosterone (r(2), 0.47; P=0.042) and progesterone (r(2), 0.64; P=0.006). The mean (s.d.) TminAC and TmaxAC for 17OHP and A4 were: morning prednisone (4.3 (2.3) and 9.7 (3.5) h), evening prednisone (4.5 (2.0) and 10.3 (2.4) h), and daily dexamethasone (9.2 (3.5) and 16.4 (7.2) h). AUC0-24 h progesterone, androsterone and 24-h urine pdiol were significantly related to 17OHP. CONCLUSION: In CAH patients, adrenal androgens exhibit circadian rhythms influenced by glucocorticoid replacement. Measurement of adrenocortical hormones and interpretation of results should take into account the type of glucocorticoid and time of dose administration. Progesterone and backdoor metabolites may provide alternative disease biomarkers.

An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags.

Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.

BORC, a multisubunit complex that regulates lysosome positioning.

The positioning of lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multisubunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the kinesin-dependent movement of lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of lysosomes.

Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

BACKGROUND: Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. METHODOLOGY/PRINCIPAL FINDINGS: The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. CONCLUSIONS/SIGNIFICANCE: The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight.

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Warburg effect's manifestation in aggressive pheochromocytomas and paragangliomas: insights from a mouse cell model applied to human tumor tissue.

A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.

De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila.

The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.

Quantitative proteomics analysis of inborn errors of cholesterol synthesis: identification of altered metabolic pathways in DHCR7 and SC5D deficiency.

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.

Activation of Rho GTPases in Smith-Lemli-Opitz syndrome: pathophysiological and clinical implications.

Smith-Lemli-Opitz syndrome (SLOS) is a malformation syndrome with neurocognitive deficits due to mutations of DHCR7 that impair the reduction of 7-dehydrocholesterol to cholesterol. To investigate the pathological processes underlying the neurocognitive deficits, we compared protein expression in Dhcr7(+/+) and Dhcr7(Delta3-5/Delta3-5) brain tissue. One of the proteins identified was cofilin-1, an actin depolymerizing factor which regulates neuronal dendrite and axon formation. Differential expression of cofilin-1 was due to increased phosphorylation. Phosphorylation of cofilin-1 is regulated by Rho GTPases through Rho-Rock-Limk-Cofilin-1 and Rac/Cdc42-Pak-Limk-Cofilin-1 pathways. Pull-down assays were used to demonstrate increased activation of RhoA, Rac1 and Cdc42 in Dhcr7(Delta3-5/Delta3-5) brains. Consistent with increased activation of these Rho GTPases, we observed increased phosphorylation of both Limk and Pak in mutant brain tissue. Altered Rho/Rac signaling impairs normal dendritic and axonal formation, and mutations in genes encoding regulators and effectors of the Rho GTPases underlie other human mental retardation syndromes. Thus, we hypothesized that aberrant activation of Rho/Rac could have functional consequences for dendrite and axonal growth. In vitro analysis of Dhcr7(Delta3-5/Delta3-5) hippocampal neurons demonstrated both axonal and dendritic abnormalities. Developmental abnormalities of neuronal process formation may contribute to the neurocognitive deficits found in SLOS and may represent a potential target for therapeutic intervention.