Oral administration of VDAC1-derived small molecule peptides increases circulating testosterone levels in male rats.

Cholesterol is the precursor of all steroid hormones, and the entry of cholesterol into the mitochondria is the rate-limiting step of steroidogenesis. Voltage-dependent anion channel (VDAC1) is an outer mitochondrial protein part of a multiprotein complex that imports cholesterol. We previously reported that intratesticular administration of a 25 amino acid peptide blocking the interaction between 14-3-3ϵ with VDAC1 increased circulating levels of testosterone. This fusion peptide was composed of a HIV-1 transactivator of transcription (TAT) protein transduction domain cell-penetrating peptide, a glycine linker, and amino acids 159-172 of VDAC1 (TV159-172). Here, we describe the development of a family of small molecules that increase circulating testosterone levels after an oral administration. We first characterized an animal model where TV159-172 was delivered subcutaneously. This subcutaneous model allowed us to study the interactions between TV159-172 and the hypothalamus-pituitary-gonadal axis (HPG) and identify the biologically active core of TV159-172. The core consisted of the tetrapeptide RVTQ, which we used as a platform to design synthetic peptide derivatives that can be administered orally. We developed a second animal model to test various derivatives of RVTQ and found 11 active compounds. Dose-response experiments identified 4 synthetic peptides that robustly increased androgen levels in a specific manner. We selected RdVTQ as the leading VDAC1-core derivative and profiled the response across the lifespan of Brown-Norway rats. In summary, we present the development of a new class of therapeutics that act within the HPG axis to increase testosterone levels specifically. This new class of small molecules self-regulates, preventing abuse.

Genome-wide expression analysis of a new class of lncRNAs driven by SINE B2.

Repetitive short interspersed elements B2 (SINE B2) have been shown to possess two promoters: polymerase III promoter for producing short B2-S RNAs and polymerase II promoter for driving the expression of long non-coding RNA (B2-AS lncRNAs). Using a B2-antisense (B2-AS) transcript sequence from the SINE B2 resident in mitochondrial translocator protein gene (Tspo) locus, we constructed a B2-AS specific RNA library and identified 96,862 sequences encoding potential B2-mediated lncRNAs, of which 55,592 lncRNAs with more than 390 nt in length possess a feature of potential genomic locus-specific effect. In addition, small RNA-Northern hybridization showed that the new B2-AS lncRNAs are constantly degraded by the Dicer1 enzyme, a finding further confirmed by in vitro Dicer1 enzyme digestion. B2-AS lncRNAs regulate the expression of target genes in a different fashion than B2-S RNAs. Genome-wide cross-comparison with mRNA mapping showed a total of 904 mRNA loci directly targeted by B2-AS lncRNAs, suggesting a locus-specific effect of the B2-AS lncRNAs and a general effect of B2-S RNAs.

Redox regulation of hormone sensitive lipase: Potential role in the mechanism of MEHP-induced stimulation of basal steroid synthesis in MA-10 Leydig cells.

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a plasticizer with endocrine disruptor activity that has been shown to stimulate basal steroid biosynthesis in Leydig cells. The mechanism by which it does so is unknown. Using MA-10 mouse tumor Leydig cells, we assessed the effects of MEHP on reactive oxygen species (ROS) levels, and on the signal transduction pathways that mobilize cholesterol. Exposure to 0-300 µM MEHP stimulated basal progesterone production in a dose-dependent manner. Progesterone stimulation was correlated with increases in the phosphorylation of hormone-sensitive lipase (HSL; aka cholesteryl ester hydrolase), which is involved in the production of free cholesterol, and of steroidogenic acute regulatory (STAR) protein expression. Co-treating MA-10 cells with MEHP and the ROS scavenger N-acetyl cysteine (NAC) blocked the activation of HSL, blunted MEHP-induced STAR, and reduced basal progesterone formation. These observations suggest that ROS generation by MEHP leads to activation of HSL and increase in STAR which, together, result in increased free-cholesterol bioavailability and progesterone formation.

Fetal Exposure to Low Levels of the Plasticizer DEHP Predisposes the Adult Male Adrenal Gland to Endocrine Disruption.

In utero exposure to endocrine disrupting chemicals (EDCs) may affect adult health. Di-(2-ethylhexyl) phthalate (DEHP) is an EDC widely used in the production of polyvinyl chloride products and an ubiquitous environmental contaminant. We used a rat model system to show that fetal exposure to DEHP decreased levels of major steroid hormones in adulthood and that environmentally-relevant levels of DEHP affected both gene expression and epigenomic loci that were affected by exposure to high levels. In the adrenal gland, we reported that the peroxisome proliferator-activated receptor (PPAR) and cholesterol biosynthesis pathways were sensitive targets of DEHP. We hypothesized that low levels of DEHP exposure insult the endocrine system (a "first hit") and increase its susceptibility to later exposure ("second hit") and subsequent disease. Here, we demonstrate that a second hit in the adult offspring exposed in utero to low levels of DEHP affected serum aldosterone levels. To unveil the first hit influence of early DEHP exposure, we treated in utero DEHP-exposed adult offspring with stressors that targeted the PPAR or cholesterol biosynthesis pathways. Treatment with the PPAR-gamma antagonist T0070907 reduced serum aldosterone compared to animals not exposed to DEHP in utero. Analysis of gene expression in animals that were subjected to both early and late exposure revealed deregulation of genes for the potassium channel Kcnk5 and the retinoid-X receptors (RXR) Rxra and Rxrb, indicating that these entities are linked to endocrine disruption. We propose that early exposure to environmental doses of DEHP predisposes the animal for disease later in life.

Repeated exposures of the male Sprague Dawley rat reproductive tract to environmental toxicants: Do earlier exposures to di-(2-ethylhexyl)phthalate (DEHP) alter the effects of later exposures?

Although exposures to environmental toxicants occur throughout life, little attention has been paid to the possible effects of exposures early in life on later exposure effects. We asked whether DEHP administered in utero (GD14-parturition) affects how male rats respond to later exposures. Neither in utero nor juvenile (PND21-35) exposures to 100mg/kg/day DEHP affected testis weight or histology as assessed on PND35. However, after in utero DEHP, subsequent juvenile exposure resulted in significantly reduced testis weight and altered testicular histology. Both in utero and juvenile exposures resulted in significant reductions in serum testosterone, but there was no effect of earlier on later exposure. Whether or not there had been in utero DEHP exposure, juvenile DEHP exposure had no effect on body, kidney or liver weights. These observations indicate that in utero exposure can, but will not necessarily, alter later exposure effects, with outcomes dependent upon endpoints measured and dose.

De novo steroid biosynthesis in human prostate cell lines and biopsies.

BACKGROUND: Intratumoral androgen formation may be a factor in the development of prostate cancer (PCa), particularly castration-resistant prostate cancer (CRPC). To evaluate the ability of the human prostate to synthesize de novo steroids, we examined the expression of key enzymes and proteins involved in steroid biosynthesis and metabolism. METHODS: Using TissueScan™ Cancer qPCR Arrays and quantitative RT-PCR, we performed comparative gene expression analyses between various prostate cell lines and biopsies, including normal, hyperplastic, cancerous, and androgen-deprived prostate cells lines, as well as normal, benign prostate hyperplasia (BPH), PCa, and CRPC human specimens. These studies were complemented with steroid biosynthesis studies in normal and BPH cells. RESULTS: Normal human prostate WPMY-1 and WPE1-NA22, benign prostate hyperplasia BPH-1, and cancer PC-3, LNCaP, and VCaP cell lines, as well as normal, BPH, PCa, and CRPC specimens, were used. Although all cell lines express mRNA encoding for hydroxymethylglutaryl-CoA reductase (HMGCR), the mitochondrial translocator protein TSPO and cholesterol side chain cleavage enzyme CYP11A1 were only observed in WPMY-1, BPH-1, and LNCaP cells. HSD3B1, HSD3B2, and CYP17A1 are involved in androgen formation and were not found in most cell lines. WPE1-NA22 and BPH-1 cells were unable to synthesize de novo steroids from mevalonate. Moreover, androgen-deprived cells did not have alterations in the expression of enzymes that could lead to de novo steroid formation. All prostate specimens expressed TSPO and CYP11A1. HSD3B1/2, CYP17A1, HSD17B5, and CYP19A1 mRNA expression was distinct to the profile observed in cells lines. The majority of BPH (90.9%) and PCa (83.1%) specimens contained CYP17A1, compared to control (normal) specimens (46.7%). BPH (82%), PCa (59%), normal (40%), and CRPC (34%) specimens expressed the four key enzymes that metabolize cholesterol to androgens. CONCLUSION: These studies question the use of prostate cell lines to study steroid biosynthesis and demonstrate that human prostate samples contain transcripts encoding for key steroidogenic enzymes and proteins indicating that they have the potential to synthesize de novo steroids. We propose CYP17A1 as a candidate enzyme that can be used for patient stratification and treatment in BPH and PCa.

In search of the molecular mechanisms mediating the inhibitory effect of the GnRH antagonist degarelix on human prostate cell growth.

Degarelix is a gonadrotropin-releasing hormone (GnRH) receptor (GnRHR) antagonist used in patients with prostate cancer who need androgen deprivation therapy. GnRHRs have been found in extra-pituitary tissues, including prostate, which may be affected by the GnRH and GnRH analogues used in therapy. The direct effect of degarelix on human prostate cell growth was evaluated. Normal prostate myofibroblast WPMY-1 and epithelial WPE1-NA22 cells, benign prostatic hyperplasia (BPH)-1 cells, androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells, as well as VCaP cells derived from a patient with castration-resistant prostate cancer were used. Discriminatory protein and lipid fingerprints of normal, hyperplastic, and cancer cells were generated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The investigated cell lines express GNRHR1 and GNRHR2 and their endogenous ligands. Degarelix treatment reduced cell viability in all prostate cell lines tested, with the exception of the PC-3 cells; this can be attributed to increased apoptosis, as indicated by increased caspase 3/7, 8 and 9 levels. WPE1-NA22, BPH-1, LNCaP, and VCaP cell viability was not affected by treatment with the GnRH agonists leuprolide and goserelin. Using MALDI MS, we detected changes in m/z signals that were robust enough to create a complete discriminatory profile induced by degarelix. Transcriptomic analysis of BPH-1 cells provided a global map of genes affected by degarelix and indicated that the biological processes affected were related to cell growth, G-coupled receptors, the mitogen-activated protein kinase (MAPK) pathway, angiogenesis and cell adhesion. Taken together, these data demonstrate that (i) the GnRH antagonist degarelix exerts a direct effect on prostate cell growth through apoptosis; (ii) MALDI MS analysis provided a basis to fingerprint degarelix-treated prostate cells; and (iii) the clusters of genes affected by degarelix suggest that this compound, in addition to its known use in the treatment of prostate cancer, may be efficacious in BPH.

Mechanisms mediating environmental chemical-induced endocrine disruption in the adrenal gland.

Humans are continuously exposed to hundreds of man-made chemicals that pollute the environment in addition to multiple therapeutic drug treatments administered throughout life. Some of these chemicals, known as endocrine disruptors (EDs), mimic endogenous signals, thereby altering gene expression, influencing development, and promoting disease. Although EDs are eventually removed from the market or replaced with safer alternatives, new evidence suggests that early-life exposure leaves a fingerprint on the epigenome, which may increase the risk of disease later in life. Epigenetic changes occurring in early life in response to environmental toxicants have been shown to affect behavior, increase cancer risk, and modify the physiology of the cardiovascular system. Thus, exposure to an ED or combination of EDs may represent a first hit to the epigenome. Only limited information is available regarding the effect of ED exposure on adrenal function. The adrenal gland controls the stress response, blood pressure, and electrolyte homeostasis. This endocrine organ therefore has an important role in physiology and is a sensitive target of EDs. We review herein the effect of ED exposure on the adrenal gland with particular focus on in utero exposure to the plasticizer di(2-ethylehyl) phthalate. We discuss the challenges associated with identifying the mechanism mediating the epigenetic origins of disease and availability of biomarkers that may identify individual or population risks.

In utero exposure to the endocrine disruptor di(2-ethylhexyl) phthalate targets ovarian theca cells and steroidogenesis in the adult female rat.

Di-2-ethylhexyl phthalate (DEHP) is an endocrine disruptor used in industry as an additive to polyvinyl chloride-based products. Pregnant dams were gavaged with oil, 1, 20, 50, or 300mg of DEHP/kg/day from gestational day 14 until birth in order to characterize the effects of DEHP in the adult female offspring. In utero exposure to DEHP resulted in reduced estrogen levels at proestrus. Theca cell layer thickness was decreased starting at 50mg DEHP/kg/day dose. Follicle-stimulating hormone levels were significantly increased at proestrus and estrus. F1 reproduction using a known breeder was not affected. F3 generation showed a decreased pregnancy rate and weight, and increased litter size in the animals exposed to 20mg DEHP/kg/day. The data presented herein suggest that in utero exposure to DEHP targets the theca cell layer and decreases the estrus cycle steroid surge, but despite these effects, does not cause infertility.

Steroidogenesis in MA-10 mouse Leydig cells is altered via fatty acid import into the mitochondria.

Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation.

In silico analysis identifies novel restriction enzyme combinations that expand reduced representation bisulfite sequencing CpG coverage.

BACKGROUND: Epigenetics is the study of gene expression changes that are not caused by changes in the deoxyribonucleic acid (DNA) sequence. DNA methylation is an epigenetic mark occurring in C-phosphate-G sites (CpGs) that leads to local or regional gene expression changes. Reduced-representation bisulfite sequencing (RRBS) is a technique that is used to ascertain the DNA methylation of millions of CpGs at single-nucleotide resolution. The genomic coverage of RRBS is given by the restriction enzyme combination used during the library preparation and the throughput capacity of the next-generation sequencer, which is used to read the generated libraries. The four-nucleotide cutters, MspI and TaqαI, are restriction enzymes commonly used in RRBS that, when combined, achieve ~12% genomic coverage. The increase in throughput of next-generation sequencers allows for novel combinations of restriction enzymes that provide higher CpG coverage. RESULTS: We performed a near-neighbor analysis of the four nucleotide sequences most frequently found within 50 nt of all genomic CpGs. This resulted in the identification of seven methylation-insensitive restriction enzymes (AluI, BfaI, HaeIII, HpyCH4V, MluCI, MseI, and MspI) that shared similar restriction conditions suitable for RRBS library preparation. We report that the use of two or three enzyme combinations increases the theoretical epigenome coverage to almost half of the human genome. CONCLUSIONS: We provide the enzyme combinations that are more likely to increase the CpG coverage in human, rat, and mouse genomes.

Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

Functional characterization of the human translocator protein (18kDa) gene promoter in human breast cancer cell lines.

The translocator protein (18kDa; TSPO) is a mitochondrial drug- and cholesterol-binding protein that has been implicated in several processes, including steroidogenesis, cell proliferation, and apoptosis. Expression of the human TSPO gene is elevated in several cancers. To understand the molecular mechanisms that regulate TSPO expression in human breast cancer cells, the TSPO promoter was identified, cloned, and functionally characterized in poor-in-TSPO hormone-dependent, non-aggressive MCF-7 cells and rich-in-TSPO hormone-independent, aggressive, and metastatic MDA-MB-231 breast cancer cells. RNA ligase-mediated 5'-rapid amplification of cDNA ends analysis indicated transcription initiated at multiple sites downstream of a GC-rich promoter that lacks functional TATA and CCAAT boxes. Deletion analysis indicated that the region from -121 to +66, which contains five putative regulatory sites known as GC boxes, was sufficient to induce reporter activity up to 24-fold in MCF-7 and nearly 120-fold in MDA-MB-231 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1, Sp3 and Sp4 bind to these GC boxes in vitro and to the endogenous TSPO promoter. Silencing of Sp1, Sp3 and Sp4 gene expression reduced TSPO levels. In addition, TSPO expression was epigenetically regulated at one or more of the identified GC boxes. Disruption of the sequence downstream of the main start site of TSPO differentially regulated TSPO promoter activity in MCF-7 and MDA-MB-231 cells, indicating that essential elements contribute to its differential expression in these cells. Taken together, these experiments constitute the first in-depth functional analysis of the human TSPO gene promoter and its transcriptional regulation.

In utero exposure to the antiandrogen di-(2-ethylhexyl) phthalate decreases adrenal aldosterone production in the adult rat.

We previously reported that in utero exposure of the male fetus to the plasticizer di-(2-ethylhexyl) phthalate (DEHP) resulted in decreased circulating levels of testosterone in the adult without affecting Leydig cell numbers, luteinizing hormone levels, or steroidogenic enzyme expression. Fetal exposure to DEHP resulted in reduced mineralocorticoid receptor (MR; NR3C2) expression in adult Leydig cells. In the present studies, treatment of pregnant Sprague-Dawley dams from Gestational Day 14 until birth with 20, 50, 100, 300, or 750 mg kg(-1) day(-1) of DEHP resulted in significant sex-specific decreases in serum aldosterone but not corticosterone levels at Postnatal Day 60 (PND60) but not at PND21. There was no effect on circulating levels of potassium, angiotensin II or adrenocorticotropin hormone (ACTH). However, there was reduced expression of AT receptor Agtr1a, Agtr1b, and Agtr2 mRNAs. The mRNA levels of proteins and enzymes implicated in aldosterone biosynthesis were not affected by in utero DEHP treatment except for Cyp11b2, which was decreased at high (≥ 500 mg kg(-1) day(-1)) doses. The data presented herein, together with our previous observation that aldosterone stimulates testosterone production via an MR-mediated mechanism, suggest that in utero exposure to DEHP causes reduction in both adrenal aldosterone synthesis and MR expression in Leydig cells, leading to reduced testosterone production in the adult. Moreover, these results suggest the existence of a DEHP-sensitive adrenal-testis axis regulating androgen formation.

Epigenetic regulation of the expression of genes involved in steroid hormone biosynthesis and action.

Steroid hormones participate in organ development, reproduction, body homeostasis, and stress responses. The steroid machinery is expressed in a development- and tissue-specific manner, with the expression of these factors being tightly regulated by an array of transcription factors (TFs). Epigenetics provides an additional layer of gene regulation through DNA methylation and histone tail modifications. Evidence of epigenetic regulation of key steroidogenic enzymes is increasing, though this does not seem to be a predominant regulatory pathway. Steroid hormones exert their action in target tissues through steroid nuclear receptors belonging to the NR3A and NR3C families. Nuclear receptor expression levels and post-translational modifications regulate their function and dictate their sensitivity to steroid ligands. Nuclear receptors and TFs are more likely to be epigenetically regulated than proteins involved in steroidogenesis and have secondary impact on the expression of these steroidogenic enzymes. Here we review evidence for epigenetic regulation of enzymes, transcription factors, and nuclear receptors related to steroid biogenesis and action.

In utero exposure to di-(2-ethylhexyl) phthalate exerts both short-term and long-lasting suppressive effects on testosterone production in the rat.

We examined the effects of fetal exposure to a wide range of di-(2-ethylhexyl) phthalate (DEHP) doses on fetal, neonatal, and adult testosterone production. Pregnant rats were administered DEHP from Gestational Day (GD) 14 to the day of parturition (Postnatal Day 0). Exposure to between 234 and 1250 mg/kg/day of DEHP resulted in increases in the absolute volumes of Leydig cells per adult testis. Despite this, adult serum testosterone levels were reduced significantly compared to those of controls at all DEHP doses. Organ cultures of testes from GD20 rats exposed in utero to DEHP showed dose-dependent reductions in basal testosterone production. Surprisingly, however, no significant effect of DEHP was found on hCG-induced testosterone production by GD20 testes, suggesting that the inhibition of basal steroidogenesis resulted from the alteration of molecular events upstream of the steroidogenic enzymes. Reduced fetal and adult testosterone production in response to in utero DEHP exposure appeared to be unrelated to changes in testosterone metabolism. In view of the DEHP-induced reductions in adult testosterone levels, a decrease in the expression of steroidogenesis-related genes was anticipated. Surprisingly, however, significant increases were seen in the expression of Cyp11a1, Cy17a1, Star, and Tspo transcripts, suggesting that decreased testosterone production after birth could not be explained by decreases in steroidogenic enzymes as seen at GD20. These changes may reflect an increased number of Leydig cells in adult testes exposed in utero to DEHP rather than increased gene expression in individual Leydig cells, but this remains uncertain. Taken together, these results demonstrate that in utero DEHP exposure exerts both short-term and long-lasting effects on testicular steroidogenesis that might involve distinct molecular targets in fetal and adult Leydig cells.

Neurotensin polyplex as an efficient carrier for delivering the human GDNF gene into nigral dopamine neurons of hemiparkinsonian rats.

Recently we showed that the neurotensin polyplex is a nanoparticle carrier system that targets reporter genes in nigral dopamine neurons in vivo. Herein, we report its first practical application in experimental parkinsonism, which consisted of transfecting dopamine neurons with the gene coding for human glial cell line-derived neurotrophic factor (hGDNF). Hemiparkinsonism was induced in rats by a single dose of 6-hydroxydopamine (30 microg) into the ventrolateral part of the striatum. We showed that transfection of the hGDNF gene into the substantia nigra of rats 1 week after the neurotoxin injection produced biochemical, anatomical, and functional recovery from hemiparkinsonism. RT-PCR analysis showed mRNA expression of exogenous hGDNF in the transfected substantia nigra. Western blot analysis verified transgene expression by recognizing the flag epitope added at the C-terminus of the hGDNF polypeptide, which was found mainly in dopamine neurons by double immunofluorescence techniques. These data indicate that the neurotensin polyplex holds great promise for the neuroprotective therapy of Parkinson disease.

Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection.

Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.

Improved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal.

Recently we reported that neurotensin-SPDP-poly-L-lysine (NT-vector) is able to bind plasmid DNA (NT-polyplex) and polyfect cells expressing the high-affinity neurotensin receptor (NTRH) although with low transfecting efficiency: in vitro, 6.5+/-1.5%, and in vivo, 5+/-4%. In this work, we attempted to increase the transfecting efficiency by integrating the hemagglutinin HA2 fusogenic peptide and the Vp1 nuclear localization signal of SV40 to the NT-polyplex (fusogenic-karyophilic-NT-polyplex). Confocal microscopy and flow cytometry analysis showed that the fusogenic-karyophilic-NT-polyplex produced mostly nuclear localization of the plasmid DNA in NTRH-bearing N1E-115 cells. About 50% of N1E-115 cells internalized and expressed the reporter gene when the plasmid DNA was transferred by the fusogenic-karyophilic-NT-polyplex. Although to a less extent, the addition of each viral peptide separately to NT-polyplex (fusogenic-NT-polyplex or karyophilic-NT-polyplex) improved polyfection. Fusogenic-NT-polyplex produced 22.41+/-5.96% of internalization and 20.35+/-0.82% of expression in N1E-115 cells, whereas karyophilic-NT-polyplex yielded 13.75+/-3.88% and 10.94+/-2.04%, respectively. Basal internalization and expression were detected in N1E-115 cells in the presence of 100 nM SR-48692 and in NTRH-lacking cells. The fusogenic-karyophilic-NT-polyplex was microinjected into the substantia nigra to test its ability for gene transfer in vivo. Fusogenic-karyophilic-NT-polyplex internalization was observed within dopamine neurons only. Reporter gene expression was observed in a high proportion of dopamine neurons up to 60 days after NT-polyfection. Both internalization and expression were prevented by SR-48692. Our results show that the fusogenic-karyophilic-NT-polyplex is a highly efficient and specific gene vector and encourage its use to transfer gene of physiological interest to NTRH-bearing neurons.