Pharmacological inhibition of a microRNA family in nonhuman primates by a seed-targeting 8-mer antimiR.

MicroRNAs (miRNAs) regulate many aspects of human biology. They target mRNAs for translational repression or degradation through base pairing with 3' untranslated regions, primarily via seed sequences (nucleotides 2 to 8 in the mature miRNA sequence). A number of individual miRNAs and miRNA families share seed sequences and targets, but differ in the sequences outside of the seed. miRNAs have been implicated in the etiology of a wide variety of human diseases and therefore represent promising therapeutic targets. However, potential redundancy of different miRNAs sharing the same seed sequence and the challenge of simultaneously targeting miRNAs that differ significantly in nonseed sequences complicate therapeutic targeting approaches. We recently demonstrated effective inhibition of entire miRNA families using seed-targeting 8-mer locked nucleic acid (LNA)-modified antimiRs in short-term experiments in mammalian cells and in mice. However, the long-term efficacy and safety of this approach in higher organisms, such as humans and nonhuman primates, have not been determined. We show that pharmacological inhibition of the miR-33 family, key regulators of cholesterol/lipid homeostasis, by a subcutaneously delivered 8-mer LNA-modified antimiR in obese and insulin-resistant nonhuman primates results in derepression of miR-33 targets, such as ABCA1, increases circulating high-density lipoprotein cholesterol, and is well tolerated over 108 days of treatment. These findings demonstrate the efficacy and safety of an 8-mer LNA-antimiR against an miRNA family in a nonhuman primate metabolic disease model, suggesting that this could be a feasible approach for therapeutic targeting of miRNA families sharing the same seed sequence in human diseases.

The use of the African green monkey as a preclinical model for ocular pharmacokinetic studies.

PURPOSE: This investigation evaluated the ocular and systemic pharmacokinetics of besifloxacin in African green monkeys compared with cynomolgus monkeys following topical ocular dosing. METHODS: A suspension formulation containing 0.6% besifloxacin was administered to African green and cynomolgus monkeys. Animals were euthanized at predetermined time intervals, and ocular tissue and systemic blood samples were collected and analyzed by LC/MS/MS. RESULTS: In both African green and cynomolgus monkeys, high concentrations of besifloxacin were detected in anterior segment tissues, while levels in posterior segment tissues and plasma were low. Mean concentration versus time profiles of besifloxacin were generally similar between species, with rapid absorption into ocular tissues after a single dose. In anterior segment tissues, concentrations of besifloxacin were measurable throughout the 24-h sampling period in both species. Quantitatively, concentrations were consistently higher in the conjunctiva of African green monkeys compared with cynomolgus monkeys. Besifloxacin levels were also higher during the first 3 h following dosing in the tear fluid of African green monkeys, but lower in the iris/ciliary body during this timeframe. However after the 3-h time point, concentrations in the tear fluid and iris/ciliary body were similar between species. Exposure in cornea tended to be higher in African green monkeys, but the difference was less pronounced than for conjunctiva. Exposure in aqueous humor was comparable between species. In posterior segment tissues, exposure to besifloxacin tended to be higher in cynomolgus monkeys. Systemic exposure also tended to be higher in cynomolgus monkeys, but measurable levels were present in the plasma of both species throughout the 24-h sampling period. With the exception of iris/ciliary body and vitreous humor, mean ocular tissue weights were generally similar between species although a small, but statistically significant, difference was also observed in the choroid. CONCLUSIONS: African green monkeys may be a suitable model for preclinical ocular pharmacokinetic studies. Additional studies using a variety of compounds would be useful in determining whether the quantitative differences in ocular exposures and ocular tissue weights observed in the present investigation reflect slight variations in the procedures used in these separate experiments, or true physiological and anatomical differences between species.

Optimization of laser-induced choroidal neovascularization in African green monkeys.

We developed and validated a new nonhuman primate model of laser-induced choroidal neovascularization (CNV) that addresses study design limitations prevalent in laser-induced CNV-based efficacy studies. Laser-induced Bruch's membrane disruption triggers CNV and has been widely utilized in animals to model neovascular ("wet") age-related macular degeneration (AMD). Despite widespread use of the approach, detailed assessment of experimental parameters and their influence on pathophysiological endpoints critical for disease modeling has been extremely limited and largely based on anecdotal observations. We evaluated laser power parameters and endpoint measures to optimize methods for CNV formation and quantification to facilitate drug efficacy screening in African green monkeys. Six laser spots of 350, 550, 750, 950 or 1500 mW laser power were positioned bilaterally 1.5 disc diameters from the fovea, within the macula. Fluorescein angiograms were collected 3-5 weeks later and scored by trained masked investigators using graded (I-IV) and densitometric methods. Histopathology assessments were also performed, including determination of CNV area. Test system sensitivity to angiogenesis inhibition was subsequently assessed by evaluating the effect of intravitreal bevacizumab (Avastin) pretreatment (one day prior to laser photocoagulation) on incidence of CNV. Grade III and grade IV lesions were considered clinically relevant, demonstrating early hyperfluorescence and late leakage within or beyond the lesion borders. By 4 weeks post-laser all treatment groups demonstrated evidence of grade III lesions with greatest incidence observed in lesions induced by 750 and 950 mW laser power (72.9% and 69.4% respectively). Grade IV lesions were confined to eyes receiving 550 mW laser power or higher, with highest incidence of grade IV lesions observed in eyes receiving 950 (19.4%) and 1500 mW (31%) laser spots, incidence peaking 4 weeks post-laser photocoagulation. Densitometric analyses of angiograms corroborated visual scoring. Bevacizumab completely abolished grade IV lesion development and significantly lowered lesion fluorescein signal intensity (P < 0.0001) and CNV area (P = 0.038) compared to vehicle-treated controls. Our studies demonstrate that laser power of 950-1500 mW and angiography analysis 4 weeks post-laser are optimal parameters to evaluate treatment effects on CNV induction following laser photocoagulation. Bevacizumab significantly attenuated CNV development, as determined by fluorescein angiography and histopathology assessments in this model, supporting the application of African green monkeys in preclinical modeling of CNV. Laser parameters and time points for therapeutic dosing and angiography endpoints are critical factors to the laser-induced CNV model and must be validated for robust assessment of efficacy. The newly optimized nonhuman primate model described will facilitate preclinical efficacy assessments of novel therapeutics for CNV.

Clinical chemistry and hematology values in a Caribbean population of African green monkeys.

BACKGROUND: Hematology and clinical chemistry (HCC) reference values are critical in veterinary practice and in vivo pre-clinical research, enabling detection of health abnormalities, response to therapeutic intervention or adverse toxicological effects, as well as monitoring of clinical management. METHODS: In this report, reference ranges for 46 HCC parameters were characterized in 331 wild-caught and colony-bred African green monkeys. Effects of sex, weight and duration of captivity were determined by one-way analysis of variance. RESULTS: Significant sex differences were observed for several HCC parameters. Significant differences were also observed for select HCC variables between newly caught animals and those held in captivity for 1-12 months or longer. CONCLUSIONS: Comparison of this data with other non-human primate species and humans highlights similarities and disparities between species. Potential causes of interpopulation variability and relevance to the use of the African green monkey as a non-human primate model are discussed.

Experimental reovirus-induced acute flaccid paralysis and spinal motor neuron cell death.

Acute flaccid paralysis (AFP) describes the loss of motor function in 1 or more limbs commonly associated with viral infection and destruction of motor neurons in the anterior horns of the spinal cord. Therapy is limited, and the development of effective treatments is hampered by a lack of experimental models. Reovirus infection of neonatal mice provides a model for the study of CNS viral infection pathogenesis. Injection of the Reovirus serot Type 3 strains Abney (T3A) or Dearing (T3D) into the hindlimb of 1-day-old mice resulted in the development of AFP in more than 90% of infected mice. Acute flaccid paralysis began in the ipsilateral hindlimb at 8 to 10 days postinfection and progressed to paraplegia 24 hours later. Paralysis correlated with injury, neuron loss, and spread of viral antigen first to the ipsilateral and then to the contralateral anterior horns. As demonstrated by the activation of caspase 3 and its colocalization with viral antigen in the anterior horn and concomitant cleavage of poly-(adenosine diphosphate-ribose) polymerase, AFP was associated with apoptosis. Calpain activity and inducible nitric oxide synthase expression were both elevated in the spinal cords of paralyzed animals. This study represents the first detailed characterization of a novel and highly efficient experimental model of virus-induced AFP that will facilitate evaluation of therapeutic strategies targeting virus-induced paralysis.

JAK-STAT signaling pathways are activated in the brain following reovirus infection.

Reovirus infection provides a classic experimental model system for studying the pathogenesis of viral infections of the central nervous system (CNS), with apoptosis acting as the major mechanism of cell death. The authors have examined the role of signal transducer and activator of transcription (STAT)1, a component of Janus-activated kinase (JAK)-STAT signaling, a pathway implicated in antiviral responses and pathways regulating apoptosis, following reovirus infection. Infection of primary cortical neuron cultures with reovirus serotype 3 strain Abney (T3A) resulted in phosphorylation of STAT1 at sites critical for transcriptional activity. Activated STAT1 was also detected in the brain of neonatal mice following T3A infection, with a nuclear pattern of expression in areas of virus-induced injury. Activation of STAT proteins is typically mediated by JAKs. The authors observed JAK2 phosphorylation (Tyr 1007/1008) in brain lysates from T3A-infected mice. Inhibition of JAK activity with the inhibitor AG-490 blocked reovirus-induced STAT1 activation in neuronal cultures, indicating reovirus-induced STAT activation is JAK dependent. Pretreatment of neuronal cultures with antibody raised against interferon (IFN)-alpha/betaR2 inhibited T3A-induced STAT1 phosphorylation, whereas neither IFN-gamma or IFN-gammaR2 antibody pretreatment had any effect on T3A-induced STAT1 phosphorylation. Mice lacking the STAT1 gene demonstrated increased susceptibility to reovirus infection, with increased mortality and higher viral titers in the brain compared to wild-type animals. The results demonstrate activation of a type I IFN-mediated, JAK-dependent STAT signaling pathway following reovirus infection and suggest that STAT1 is a key component of host defense mechanisms against reovirus infection in the brain.

Novel strategy for treatment of viral central nervous system infection by using a cell-permeating inhibitor of c-Jun N-terminal kinase.

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.

Reovirus infection of the CNS enhances iNOS expression in areas of virus-induced injury.

Nitric oxide (NO) has been implicated as a contributor to the host's innate defense against viral infections including those affecting the CNS. Reovirus infection of the CNS is a classic experimental system for understanding the pathogenesis of neurotropic viral infection. Infection with serotype 3 strains is associated with perturbations in various cellular signaling pathways including NF-kappaB and NO plays a regulatory role in many of these same pathways. We therefore examined whether NO production is dysregulated following reovirus serotype 3 strain Abney (T3A) infection of the mouse CNS. Nitric oxide synthase (NOS) activity was significantly higher in brain homogenates from T3A-infected animals compared to mock infected. Increased NOS activity correlated with inducible NOS (iNOS) expression in brain homogenates of T3A-infected animals. Expression of iNOS was confined to areas of viral infection and injury. T3A infection of primary neuronal and glial cultures was also associated with enhanced expression of iNOS. Immunocytochemical studies of primary glial cultures demonstrated that, in addition to its known neuronotropism, T3A was also capable of infecting immature microglial cells. T3A infection did not alter expression of either neuronal or endothelial NOS isoforms in neuronal or glial cultures or in mouse brain. The NO donor S-Nitroso-N-acetyl penicillamine (SNAP) significantly inhibited T3A growth in neuronal cultures, conversely the NOS inhibitor N-omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) augmented viral growth. Our findings provide the first evidence of reovirus-induced iNOS expression and the first demonstration that NO inhibits mammalian reovirus replication, suggesting that NO may play an antiviral role during reovirus infection.

Nonstructural protein sigma1s is a determinant of reovirus virulence and influences the kinetics and severity of apoptosis induction in the heart and central nervous system.

The mechanisms by which viruses kill susceptible cells in target organs and ultimately produce disease in the infected host remain poorly understood. Dependent upon the site of inoculation and strain of virus, experimental infection of neonatal mice with reoviruses can induce fatal encephalitis or myocarditis. Reovirus-induced apoptosis is a major mechanism of tissue injury, leading to disease development in both the brain and heart. In cultured cells, differences in the capacity of reovirus strains to induce apoptosis are determined by the S1 gene segment, which also plays a major role as a determinant of viral pathogenesis in both the heart and the central nervous system (CNS) in vivo. The S1 gene is bicistronic, encoding both the viral attachment protein sigma-1 and the nonstructural protein sigma-1-small (sigma1s). Although sigma1s is dispensable for viral replication in vitro, we wished to investigate the expression of sigma1s in the infected heart and brain and its potential role in reovirus pathogenesis in vivo. Two-day-old mice were inoculated intramuscularly or intracerebrally with either sigma1s(-) or sigma1s(+) reovirus strains. While viral replication in target organs did not differ between sigma1s(-) and sigma1s(+) viral strains, virus-induced caspase-3 activation and resultant histological tissue injury in both the heart and brain were significantly reduced in sigma1s(-) reovirus-infected animals. These results demonstrate that sigma1s is a determinant of the magnitude and extent of reovirus-induced apoptosis in both the heart and CNS and thereby contributes to reovirus pathogenesis and virulence.

Apoptotic death of striatal neurons induced by human immunodeficiency virus-1 Tat and gp120: Differential involvement of caspase-3 and endonuclease G.

Human immunodeficiency virus-1 (HIV-1) infection affects the striatum, resulting in gliosis and neuronal losses. To determine whether HIV-1 proteins induce striatal neurotoxicity through an apoptotic mechanism, mouse striatal neurons isolated on embryonic day 15 and the effects of HIV-1 Tat(1-72) and gp120 on survival were assessed in vitro. Mitochondrial release of cytochrome c, caspase-3 activation, and neuron survival, as well as an alternative apoptotic pathway involving endonuclease G (endo G), were assessed at 4 h, 24 h, 48 h, and/or 72 h using enzyme assays and immunoblotting. Both HIV-1 Tat and gp120 significantly increased caspase-3 activation in a concentration-dependent manner in striatal neurons at 4 h following continuous exposure in vitro. Tat(1-72) and gp120 caused significant neuronal losses at 48 h and/or 72 h. Tat(1-72) increased cytochrome c release, and caspase-3 and endo G activation at 4 h, 24 h, and/or 72 h. By contrast, gp120 increased caspase-3 activation, but failed to increase cytochrome c or endo G levels in the cytoplasm at 4 h, 24 h, and/or 72 h. The cell permeant caspase inhibitor Z-DEVD-FMK significantly attenuated gp120-induced, but not Tat(1-72)-induced, neuronal death, suggesting that gp120 acts in large part through the activation of caspase(s), whereas Tat(1-72)-induced neurotoxicity was accompanied by activating an alternative pathway involving endo G. Thus, although Tat(1-72) and gp120 induced significant neurotoxicity, the nature of the apoptotic events preceding death differed. Collectively, our findings suggest that HIV-1 proteins are intrinsically toxic to striatal neurons and the pathogenesis is mediated through separate actions involving both caspase-3 and endo G.

Selective vulnerability of cerebellar granule neuroblasts and their progeny to drugs with abuse liability.

Cerebellar development is shaped by the interplay of genetic and numerous environmental factors. Recent evidence suggests that cerebellar maturation is acutely sensitive to substances with abuse liability including alcohol, opioids, and nicotine. Assuming substance abuse disrupts cerebellar maturation, a central question is: what are the basic mechanisms underlying potential drug-induced developmental defects? Evidence reviewed herein suggests that the maturation of granule neurons and their progeny are intrinsically affected by several classes of substances with abuse liability. Although drug abuse is also likely to target directly other cerebellar neuron and glial types, such as Purkinje cells and Bergmann glia, findings in isolated granule neurons suggest that they are often the principle target for drug actions. Developmental events that are selectively disrupted by drug abuse in granule neurons and/or their neuroblast precursors include proliferation, migration, differentiation (including neurite elaboration and synapse formation), and programmed cell death. Moreover, different classes of drugs act through distinct molecular mechanisms thereby disrupting unique aspects of development. For example, drug-induced perturbations in: (i) neurotransmitter biogenesis; (ii) ligand and ion-gated receptor function and their coupling to intracellular effectors; (iii) neurotrophic factor biogenesis and signaling; and (iv) intercellular adhesion are all likely to have significant effects in shaping developmental outcome. In addition to identifying therapeutic strategies for drug abuse intervention, understanding the mechanisms by which drugs affect cellular maturation is likely to provide a better understanding of the neurochemical events that normally shape central nervous system development.

Quantitative autoradiographic mapping of opioid receptors in the brain of delta-opioid receptor gene knockout mice.

Using quantitative receptor autoradiography we have determined if deletion of the delta-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse delta-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. Delta-opioid receptors were labeled with [(3)H]deltorphin I (7 nM), mu- with [(3)H]DAMGO (4 nM), and kappa- with [(3)H]CI-977 (2.5 nM) or [(3)H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [(3)H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [(3)H]deltorphin I binding correlated significantly with those demonstrating high levels of the mu-receptor and were abolished in the presence of the mu-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [(3)H]DAMGO and [(3)H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [(3)H]bremazocine binding were higher in brains from -/- than +/+ mice. Our findings suggest that residual [(3)H]deltorphin I binding in the brain of delta-receptor gene knockout mice is the result of cross-reactivity with mu-sites and that there are no delta-receptor subtypes derived from a different gene. Changes in mu- and kappa-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the delta-receptor. The differences in [(3)H]CI-977 and [(3)H]bremazocine binding indicate these ligands show differential recognition of the kappa-receptor.