Phenotypic Correction of Murine Mucopolysaccharidosis Type II by Engraftment of Ex Vivo Lentiviral Vector-Transduced Hematopoietic Stem and Progenitor Cells.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS-deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon-optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV-transduced cells showed supraphysiological levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells, and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS-engrafted animals were restored to 10-20% than that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of ex vivo MNDU3-IDS LVV-transduced HSPCs for treatment of MPS II.

Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain.

Hematopoietic stem cell transplantation (HSCT) is a therapy used for multiple malignant and nonmalignant diseases, with chemotherapy used for pretransplantation myeloablation. The post-HSCT brain contains peripheral engrafted parenchymal macrophages, despite their absence in the normal brain, with the engraftment mechanism still undefined. Here we show that HSCT chemotherapy broadly disrupts mouse brain regenerative populations, including a permanent loss of adult neurogenesis. Microglial density was halved, causing microglial process expansion, coinciding with indicators of broad senescence. Although microglia expressed cell proliferation markers, they underwent cell cycle arrest in S phase with a majority expressing the senescence and antiapoptotic marker p21. In vivo single-cell tracking of microglia after recovery from chemical depletion showed loss of their regenerative capacity, subsequently replaced with donor macrophages. We propose that HSCT chemotherapy causes microglial senescence with a gradual decrease to a critical microglial density, providing a permissive niche for peripheral macrophage engraftment of the brain.

A New Chapter in Genetic Medicine: RNA Editing and its Role in Disease Pathogenesis.

The transfer of genomic information from DNA to mRNA to protein usually occurs with high fidelity, but can also be subverted by a programmed RNA sequence alteration termed 'RNA editing', involving deamination of adenosine to inosine (decoded as guanosine), or of cytosine to uracil. These sequence changes can lead to cellular heterogeneity by generating variable sets of transcripts within otherwise identical cells. Recent studies have demonstrated that editing is most prevalent in cells and tissues with high propensity for plasticity. Within those, RNA editing reproducibly targets transcripts of related function, altering the outcomes of entire pathways at once. In ongoing work, changes in patterns of editing have been correlated with neuronal disease pathogenesis, suggesting that RNA editing harbors diagnostic potential.

Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function.

Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells.

Loss of APOBEC1 RNA-editing function in microglia exacerbates age-related CNS pathophysiology.

Microglia (MG), a heterogeneous population of phagocytic cells, play important roles in central nervous system (CNS) homeostasis and neural plasticity. Under steady-state conditions, MG maintain homeostasis by producing antiinflammatory cytokines and neurotrophic factors, support myelin production, and remove synapses and cellular debris, as well as participating in "cross-correction," a process that supplies neurons with key factors for executing autophagy-lysosomal function. As sentinels for the immune system, MG also detect "danger" signals (pathogenic or traumatic insult), become activated, produce proinflammatory cytokines, and recruit monocytes and dendritic cells to the site of damage through a breached blood-brain barrier or via brain lymphatics. Failure to effectively resolve MG activation can be problematic and can lead to chronic inflammation, a condition proposed to underlie CNS pathophysiology in heritable brain disorders and age-related neurodegenerative and cognitive decline. Here, we show that APOBEC1-mediated RNA editing occurs within MG and is key to maintaining their resting status. Like bone marrow-derived macrophages, RNA editing in MG leads to overall changes in the abundance of edited proteins that coordinate the function of multiple cellular pathways. Conversely, mice lacking the APOBEC1 editing function in MG display evidence of dysregulation, with progressive age-related signs of neurodegeneration, characterized by clustering of activated MG, aberrant myelination, increased inflammation, and lysosomal anomalies that culminate in behavioral and motor deficiencies. Collectively, our study identifies posttranscriptional modification by RNA editing as a critical regulatory mechanism of vital cellular functions that maintain overall brain health.

Estrogens, androgens and generalized behavioral arousal in gonadectomized female and male C57BL/6 mice.

General arousal has been operationally defined as an enhanced motor activity and enhanced intensity of response to sensory stimuli. Even though the effects of gonadal hormones on mating behavior have been much studied, their potential effect on generalized arousal, as defined above, has never been evaluated. In the present study we employed a thoroughly validated assay of general arousal to determine the effects of estradiol (E) and testosterone (T) in gonadectomized female and male mice, respectively. The steroids were administered in three different ways: A fast-acting, water soluble preparation given intraperitoneally, an oil solution given subcutaneously, and an oil solution in a subcutaneous Silastic capsule. Motor activity and responses to sensory stimuli were recorded for 24h, 91h, and seven days following hormone administration, respectively. All measures of arousal varied according to the day/night cycle. The water soluble steroid preparation had no reliable effect. When the same doses of estradiol and testosterone were administered subcutaneously in an oil vehicle no effect of either treatment on arousal was observed. The subcutaneously implanted capsule containing estradiol or testosterone had a delayed effect on motor activity in females (four to seven days) but no effect in males. The long time required by the gonadal hormones for affecting arousal would be consistent with, but does not prove, a genomic action. The limited effects of E and T in our arousal assay suggest to us that the strongest actions of these hormones on arousal occur in the context of sequences of responses to sexually relevant stimuli.

Impact of Thyroid Hormones on Estrogen Receptor α-Dependent Transcriptional Mechanisms in Ventromedial Hypothalamus and Preoptic Area.

Elevated levels of thyroid hormones (TH) reduce estradiol (E2)-dependent female sexual behavior. E2 stimulates progesterone receptor (Pgr) and oxytocin receptor (Oxtr) within the ventromedial hypothalamus and preoptic area, critical hypothalamic nuclei for sexual and maternal behavior, respectively. Here, we investigated the impact of TH on E2-dependent transcriptional mechanisms in female mice. First, we observed that triiodothyronine (T3) inhibited the E2 induction of Pgr and Oxtr. We hypothesized that differences in histone modifications and receptor recruitment could explain the influence of TH on E2-responsive Pgr and Oxtr expression. We observed that histone H3 acetylation (H3Ac) and methylation (H3K4me3) was gene and brain-region specific. We then analyzed the recruitment of estrogen receptor α (ERα) and TH receptor α (TRα) on the putative regulatory sequences of Pgr and Oxtr. Interestingly, T3 inhibited E2-induced ERα binding to a specific Pgr enhancer site, whereas TRα binding was not affected, corroborating our theory that the competitive binding of TRα to an ERα binding site can inhibit ERα transactivation and the subsequent E2-responsive gene expression. On the Oxtr promoter, E2 and T3 worked together to modulate ERα and TRα binding. Finally, the E2-dependent induction of cofactors was reduced by hypothyroidism and T3. Thus, we determined that the Pgr and Oxtr promoter regions are responsive to E2 and that T3 interferes with the E2 regulation of Pgr and Oxtr expression by altering the recruitment of receptors to DNA and changing the availability of cofactors. Collectively, our findings provide insights into molecular mechanisms of response to E2 and TH interactions controlling sex behavior in the hypothalamus.

GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding.

Multiple peptide systems, including neuropeptide Y, leptin, ghrelin, and others, are involved with the control of food intake and body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body weight. In the present study, we used a combination of ligand-binding and receptor-activity assays to characterize a Gαi/o protein-coupled receptor activated by BigLEN in the mouse hypothalamus and Neuro2A cells. We then selected orphan G protein-coupled receptors expressed in the hypothalamus and Neuro2A cells and tested each for activation by BigLEN. G protein-coupled receptor 171 (GPR171) is activated by BigLEN, but not by the C terminally truncated peptide LittleLEN. The four C-terminal amino acids of BigLEN are sufficient to bind and activate GPR171. Overexpression of GPR171 leads to an increase, and knockdown leads to a decrease, in binding and signaling by BigLEN and the C-terminal peptide. In the hypothalamus GPR171 expression complements the expression of BigLEN, and its level and activity are elevated in mice lacking BigLEN. In mice, shRNA-mediated knockdown of hypothalamic GPR171 leads to a decrease in BigLEN signaling and results in changes in food intake and metabolism. The combination of GPR171 shRNA together with neutralization of BigLEN peptide by antibody absorption nearly eliminates acute feeding in food-deprived mice. Taken together, these results demonstrate that GPR171 is the BigLEN receptor and that the BigLEN-GPR171 system plays an important role in regulating responses associated with feeding and metabolism in mice.

AT1R-CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II.

The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB(1)R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB(1)R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB(1)R is upregulated. We found a significant upregulation of AT1R-CB(1)R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB(1)R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.

Gene expression in neuroendocrine cells during the critical period for sexual differentiation of the brain.

Following transcription of the SRY gene on the Y chromosome of genetic males, a cascade of genomic and biochemical events causes the developing brain to be influenced by two testosterone metabolites, the potent androgen dihydrotestosterone and the aromatization product estradiol (E2). These steroid hormones binding to their cognate nuclear receptors produce differential gene expression profiles between male and female brains, and as a result, male-typical sex behaviors appear in adulthood and female-typical sex behaviors are suppressed. Although anatomical and cellular substrates underlying sexually dimorphic brain and behavior have been identified, still very little information is available about the molecular mechanisms involved. Microarray technology is a powerful technique that can be a used to assess the changes in thousands of gene transcripts simultaneously. Thus such high-throughput screening may be a useful initial step in the identification of estrogen-responsive genes involved in the sexual differentiation of brain.

Identification of an endothelin-converting enzyme-2-specific fluorigenic substrate and development of an in vitro and ex vivo enzymatic assay.

Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.

Sex on the brain.

Release of testosterone during early development is necessary for masculinization of brain structures in rodents. Wu et al. (2009) now elucidate the neuronal changes caused by aromatase-mediated conversion of testosterone into estrogen in perinatal mice and the ensuing effects on adult behavior.

Homology modeling and site-directed mutagenesis to identify selective inhibitors of endothelin-converting enzyme-2.

Endothelin-converting enzyme-2 (ECE-2), a member of M13 family of zinc metallopeptidases, has previously been shown to process a number of neuropeptides including those derived from prodynorphin, proenkephalin, proSAAS, and amyloid precursor protein. ECE-2, unlike ECE-1, exhibits restricted neuroendocrine distribution and acidic pH optimum; it is consistent with a role in the regulation of neuropeptide levels in vivo. Here, we report the generation of a three-dimensional (3D) molecular model of ECE-2 using the crystal structure of neprilysin (EC 3.4.24.11) as a template. On the basis of the predictions made from the molecular model, we mutated and tested two residues, Trp 148 and Tyr 563, in the catalytic site. The mutation of Tyr 563 was found to significantly affect the catalytic activity and inhibitor binding. The molecular model was used to virtually screen a small molecule library of 13 000 compounds. Among the top-scoring compounds three were found to inhibit ECE-2 with high affinity and exhibited specificity for ECE-2 compared to neprilysin. Thus, the model provides a new useful tool to probe the active site of ECE-2 and design additional selective inhibitors of this enzyme.