Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones.

Steroid hormones are critical for organismal development and health. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the cytochrome P450 enzyme CYP11A1 in the inner mitochondrial membrane (IMM). Cholesterol transfer occurs through a complex termed the "transduceosome," in which cytosolic steroidogenic acute regulatory protein interacts with OMM proteins translocator protein and voltage-dependent anion channel (VDAC) to assist with the transfer of cholesterol to OMM. It has been proposed that cholesterol transfer from OMM to IMM occurs at specialized contact sites bridging the two membranes composed of VDAC and IMM adenine nucleotide translocase (ANT). Blue native PAGE of Leydig cell mitochondria identified two protein complexes that were able to bind cholesterol at 66- and 800-kDa. Immunoblot and mass spectrometry analyses revealed that the 800-kDa complex contained the OMM translocator protein (18-kDa) and VDAC along with IMM CYP11A1, ATPase family AAA domain-containing protein 3A (ATAD3A), and optic atrophy type 1 proteins, but not ANT. Knockdown of ATAD3A, but not ANT or optic atrophy type 1, in Leydig cells resulted in a significant decrease in hormone-induced, but not 22R-hydroxycholesterol-supported, steroid production. Using a 22-phenoxazonoxy-5-cholene-3-beta-ol CYP11A1-specific probe, we further demonstrated that the 800-kDa complex offers the microenvironment needed for CYP11A1 activity. Addition of steroidogenic acute regulatory protein to the complex mobilized the cholesterol bound at the 800-kDa complex, leading to increased steroid formation. These results identify a bioactive, multimeric protein complex spanning the OMM and IMM unit that is responsible for the hormone-induced import, segregation, targeting, and metabolism of cholesterol.

Structure-activity relationship (SAR) analysis of a family of steroids acutely controlling steroidogenesis.

Steroids metabolically derive from lipid cholesterol, and vertebrate steroids additionally derive from the steroid pregnenolone. Pregnenolone is derived from cholesterol by hydrolytic cleavage of the aliphatic tail by mitochondrial cytochrome P450 enzyme CYP11A1, located in the inner mitochondrial membrane. Delivery of cholesterol to CYP11A1 comprises the principal control step of steroidogenesis, and requires a series of proteins spanning the mitochondrial double membranes. A critical member of this cholesterol translocation machinery is the integral outer mitochondrial membrane translocator protein (18kDa, TSPO), a high-affinity drug- and cholesterol-binding protein. The cholesterol-binding site of TSPO consists of a phylogenetically conserved cholesterol recognition/interaction amino acid consensus (CRAC). Previous studies from our group identified 5-androsten-3ß,17,19-triol (19-Atriol) as drug ligand for the TSPO CRAC motif inhibiting cholesterol binding to CRAC domain and steroidogenesis. To further understand 19-Atriol's mechanism of action as well as the molecular recognition by the TSPO CRAC motif, we undertook structure-activity relationship (SAR) analysis of the 19-Atriol molecule with a variety of substituted steroids oxygenated at positions around the steroid backbone. We found that in addition to steroids hydroxylated at carbon C19, hydroxylations at C4, C7, and C11 contributed to inhibition of cAMP-mediated steroidogenesis in a minimal steroidogenic cell model. However, only substituted steroids with C19 hydroxylations exhibited specificity to TSPO, its CRAC motif, and mitochondrial cholesterol transport, as the C4, C7, and C11 hydroxylated steroids inhibited the metabolic transformation of cholesterol by CYP11A1. We thus provide new insights into structure-activity relationships of steroids inhibiting mitochondrial cholesterol transport and steroidogenic cholesterol metabolic enzymes.

A lead study on oxidative stress-mediated dehydroepiandrosterone formation in serum: the biochemical basis for a diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, yet irreversible, neurodegenerative disease for which there are limited means for its ante-mortem diagnosis. We previously identified a brain- and cell-specific oxidative stress-mediated mechanism for dehydroepiandrosterone (DHEA) biosynthesis present in rat, bovine, and human brain, independent of the cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) enzyme activity found in the periphery. This alternative pathway is induced by pro-oxidant agents, such as Fe2+ and amyloid-ß peptide. Using brain tissue specimens from control and AD patients we subsequently provided evidence that DHEA is formed in the AD brain by the oxidative stress-mediated metabolism of an unidentified precursor, thus depleting the levels of the precursor present in the blood stream. Here, we tested for the presence of this DHEA precursor in human serum using a simple Fe2+-based reaction and determined the amounts of DHEA formed. A total of 86 subjects were included in this study: 19 male and 20 female AD patients; 18 male and 22 female age-matched controls; and 4 men and 3 women with mild cognitive impairment. Serum oxidation resulted in a dramatic increase of DHEA level in control patients, whereas only a moderate or no increase was observed in the AD patients. The DHEA variation after oxidation correlated with the patients' cognitive and mental status. These results suggest that the comparison of DHEA levels in patient serum before and after oxidation could provide a useful tool to diagnose AD.

Oxidative Stress-Mediated Brain Dehydroepiandrosterone (DHEA) Formation in Alzheimer's Disease Diagnosis.

Neurosteroids are steroids made by brain cells independently of peripheral steroidogenic sources. The biosynthesis of most neurosteroids is mediated by proteins and enzymes similar to those identified in the steroidogenic pathway of adrenal and gonadal cells. Dehydroepiandrosterone (DHEA) is a major neurosteroid identified in the brain. Over the years we have reported that, unlike other neurosteroids, DHEA biosynthesis in rat, bovine, and human brain is mediated by an oxidative stress-mediated mechanism, independent of the cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) enzyme activity found in the periphery. This alternative pathway is induced by pro-oxidant agents, such as Fe(2+) and ß-amyloid peptide. Neurosteroids are involved in many aspects of brain function, and as such, are involved in various neuropathologies, including Alzheimer's disease (AD). AD is a progressive, yet irreversible neurodegenerative disease for which there are limited means for ante-mortem diagnosis. Using brain tissue specimens from control and AD patients, we provided evidence that DHEA is formed in the AD brain by the oxidative stress-mediated metabolism of an unidentified precursor, thus depleting levels of the precursor in the blood stream. We tested for the presence of this DHEA precursor in human serum using a Fe(2+)-based reaction and determined the amounts of DHEA formed. Fe(2+) treatment of the serum resulted in a dramatic increase in DHEA levels in control patients, whereas only a moderate or no increase was observed in AD patients. The DHEA variation after oxidation correlated with the patients' cognitive and mental status. In this review, we present the cumulative evidence for oxidative stress as a natural regulator of DHEA formation and the use of this concept to develop a blood-based diagnostic tool for neurodegenerative diseases linked to oxidative stress, such as AD.

Dimethyl-carbamic acid 2,3-Bis-Dimethylcarbamoyloxy-6-(4-Ethyl-Piperazine-1-Carbonyl)-phenyl ester: a novel multi-target therapeutic approach to neuroprotection.

We report herein the synthesis and biological evaluation of dimethyl-carbamic acid 2,3-bis-dimethylcarbamoyloxy-6-(4-ethyl-piperazine-1-carbonyl)-phenyl ester (SP-04), a new drug candidate that is designed to offer a multi-target therapeutic neuroprotective approach as a treatment for Alzheimer's disease (AD). SP-04 inhibits acetylcholinesterase (AchE) activity both in vitro and in vivo, and induces a dose-dependent increase in Ach levels. SP-04 releases the metabolite 4-(4-ethyl-piperazin-1-yl)-1-(2,3,4-trihydroxy-phenyl)-butan-1-one (SP-04m). Both SP-04 and SP-04m are s1-receptor antagonists supporting their interest in relieving symptoms related to psychosis, a non-cognitive condition often associated with AD. SP-04m displays important antioxidant properties and both SP-04 and SP-04m offers neuroprotection against Ab42 toxicity in various neuronal cell lines. In addition, both SP-04 and SP-04m protect neuronal cells and rat brain mitochondria exposed to various mitochondrial respiratory chain complex toxins. Taken together these data suggest that the SP-04 multi-targeting approach might offer a novel therapeutic strategy for the treatment of AD.

Caprospinol: moving from a neuroactive steroid to a neurotropic drug.

In search of new drugs for Alzheimer's disease, we departed from the classic concepts and investigated the ability of normal and Alzheimer's disease brain to convert cholesterol to steroids, otherwise known as neurosteroids. We identified 22R-hydroxycholesterol to be present in much lower levels in the hippocampus and frontal cortex of Alzheimer's disease than in tissue from age-matched controls. 22R-hydroxycholesterol was shown to protect against beta-amyloid (A beta(42))-induced neurotoxicity and block the formation of A beta oligomers. In search of a 22R-hydroxycholesterol stable analog, we identified the naturally occurring heterospirostenol, (22R,25R)-20 alpha-spirost-5-en-3beta-yl hexanoate (caprospinol). The mechanism of action underlying the neuroprotective properties of caprospinol involves, first, the ability of the compound to bind A beta(42) and, second, its interaction with components of the mitochondria respiratory chain. Samaritan Pharmaceuticals is developing caprospinol as a disease-modifying drug for the treatment of Alzheimer's disease. Samaritan Pharmaceuticals filed for an Investigational New Drug application with the FDA in 2006. The pharmacokinetic and pharmacodynamic parts of the application were found satisfactory, and the FDA has requested that additional information is submitted in support of caprospinol's safety prior to initiating the Phase I clinical study.

The use of naphthalene-2,3-dicarboxaldehyde for the analysis of primary amines using high-performance liquid chromatography and capillary electrophoresis.

This paper reviews analytical methods, instrumental developments and applications for derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde using fluorescence and chemiluminescence detection with capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). The use of lasers as well as lamps as the excitation source for fluorescence detection is discussed. The detection limit observed with naphthalene-2,3-dicarboxaldehyde derivatization is often lower and better than those obtained with other analytical separations and other fluorescent dyes. In addition, this paper describes the crucial points that influence the stability of NDA primary amine derivatives, and summarize the separation, derivatization and migration conditions of the different techniques, with their advantages and drawbacks.

Analysis of sphingosine 1-phosphate by capillary electrophoresis coupled to laser-induced fluorescence detection: use of a transparent fused-silica capillary.

C18-sphingosine 1-phosphate (C18-SPP) is a sphingolipid with important functional and structural roles in cells. In this paper we report a new capillary electrophoresis technique that is coupled to a laser-induced fluorescence detection (CE-LIF) method to quantify the level of C18-SPP in biological samples. The method utilizes a commercial standard C17-sphingosine 1-phosphate (C17-SPP) derivatized with naphthalene-2,3-dicarboxaldehyde (NDA), a fluorogenic dye used to label primary amines. The detection limit for the C17-SPP NDA was 0.54 fmol. We quantified the C18-SPP in leukemic human cells before and after irradiation by gamma rays. We demonstrated that the amounts of this sphingolipid decreased after the irradiation. In a second part of this work, we used the technique to evaluate the ability of a novel transparent fused-silica capillary, which allows the use of fused-silica capillaries without burning a window. Solarization, homogeneity, and sensitivity were studied using this capillary. The results demonstrate that this new durable capillary can provide a sensitive and reproducible quantitation procedure for CE-LIF studies.