Hematopoietic stem cell gene therapy improves outcomes in a clinically relevant mouse model of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplantation with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Inhibition of insulin-regulated aminopeptidase confers neuroprotection in a conscious model of ischemic stroke.

Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C-C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.

Conversion of Phenolic Acids in Canola Fermentation: Impact on Antimicrobial Activity against Salmonella enterica and Campylobacter jejuni.

Canola meal (CM) is commonly used in poultry feeds. CM has a high protein content but also contains high levels of antimicrobial phenolic acids. Lactic acid bacteria can alter CM phenolic composition during fermentation and influence its antimicrobial activity against pathogens. Fermented CM was analyzed for phenolic composition using tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Sinapic acid and derivatives were the major phenolic acids in CM. Growth of lactobacilli in CM was attenuated when compared to cereal substrates. Glucosides and esters of sinapic acid were extensively hydrolyzed during fermentation with Lactiplantibacillus plantarum and Furfurilactobacillus milii. Lp. plantarum transformed hydroxycinnamic acids to dihydro, 4-vinyl, and 4-ethyl derivatives, Ff. milii reduced hydroxycinnamic acids to dihydroderivatives, but Limosilactobacillus reuteri did not convert hydroxycinnamic acids. The minimum inhibitory concentration of phenolic extracts was assessed with lactobacilli, Salmonella, and Campylobacter jejuni as indicator strains. Fermentation of CM with Lp. plantarum or Ff. milii increased the antimicrobial activity of phenolic extracts against Salmonella enterica and Campylobacter jejuni. Fermentation with Lm. reuteri TMW1.656 but not fermentation with Lm. reuteri TMW1.656ΔrtcN increased the antimicrobial activity of extracts owing to the production of reutericyclin. This study demonstrates that fermentation of CM with lactobacilli converts hydroxycinammic esters and may increase the antimicrobial activity of phenolic compounds in CM against pathogens.

Social behaviour is altered in the insulin-regulated aminopeptidase knockout mouse.

Oxytocin, and the closely related neuropeptide, vasopressin, are both known to modulate social behaviours. The pro-social effects of oxytocin are well-documented and have generated much interest into its suitability as a therapeutic for disorders characterised by social dysfunction. This study investigated the social phenotype of mice with a targeted deletion of the gene for insulin-regulated aminopeptidase, an enzyme involved in the degradation of oxytocin and vasopressin. In the 3-chamber sociability test, a genotype effect was observed and subsequent post hoc analysis revealed that male, but not female, insulin-regulated aminopeptidase knockout mice made significantly more approaches to the enclosure holding a stranger mouse than did wildtype mice (p = 0.0039). Male insulin-regulated aminopeptidase knockout mice also displayed decreased rearing (t = 2.309, df = 24, p = 0.0299) and locomotor activity (t = 2.134, df = 24, p = 0.043) in the open field test, suggestive of a reduced stress response to a novel environment. Our findings provide support for the role of insulin-regulated aminopeptidase in influencing social behaviour, possibly via modulation of oxytocin and vasopressin levels. The increase in social interaction observed in the male, but not female, insulin-regulated aminopeptidase knockout mice is in agreement with reports of sex differences in effects of oxytocin and vasopressin on social behaviours and should be explored further.

Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a.

Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.

Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures.

The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of IRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10 500 drug-like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors. A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.

Cannula implantation into the lateral ventricle does not adversely affect recognition or spatial working memory.

Indwelling cannulas are often used to deliver pharmacological agents into the lateral ventricles of the brain to study their effects on memory and learning, yet little is known about the possible adverse effects of the cannulation itself. In this study, the effect of implanting an indwelling cannula into the right lateral ventricle was examined with respect to cognitive function and tissue damage in rats. Specifically, the cannula passed through sections of the primary motor (M1) and somatosensory hind limb (S1HL) cortices. One week following implantation, rats were impaired on the rotarod task, implying a deficit in fine motor control, likely caused by the passage of the cannula through the aforementioned cortical regions. Importantly, neither spatial working nor recognition memory was adversely affected. Histological examination showed immune cell activation only in the area immediately surrounding the cannulation site and not spreading to other brain regions. Both GFAP and CD-11b mRNA expression was elevated in the area immediately surrounding the cannulation site, but not in the contralateral hemisphere or the hippocampus. Neither of the inflammatory cytokines, TNF-α or IL-6, were upregulated in any region. These results show that cannulation into the lateral ventricle does not impair cognition and indicates that nootropic agents delivered via this method are enhancing normal memory rather than rescuing deficits caused by the surgery procedure.

Binding to and Inhibition of Insulin-Regulated Aminopeptidase by Macrocyclic Disulfides Enhances Spine Density.

Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09, and of Ang IV in either the extended or γ-turn conformation at the C terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

Insulin-regulated aminopeptidase deficiency provides protection against ischemic stroke in mice.

Recent studies have demonstrated that angiotensin IV (Ang IV) provides protection against brain injury caused by cerebral ischemia. Ang IV is a potent inhibitor of insulin-regulated aminopeptidase (IRAP). Therefore, we examined the effect of IRAP gene inactivation on neuroprotection following transient middle cerebral artery occlusion (MCAo) in mice. IRAP knockout mice and wild-type controls were subjected to 2 h of transient MCAo using the intraluminal filament technique. Twenty-four hours after reperfusion, neurological deficits of the stroke-induced mice were assessed and infarct volumes were measured by TTC staining. The cerebral infarct volume was significantly reduced in the IRAP knockout mice compared to wild-type littermates with corresponding improvement in neurological performance at 24 h post-ischemia. An increase in compensatory cerebral blood flow during MCAo was observed in the IRAP knockout animals with no differences in cerebral vascular anatomy detected. The current study demonstrates that deletion of the IRAP gene protects the brain from ischemic damage analogous to the effect of the IRAP inhibitor, Ang IV. This study indicates that IRAP is potentially a new therapeutic target for the development of treatment for ischemic stroke.

Identification and development of specific inhibitors for insulin-regulated aminopeptidase as a new class of cognitive enhancers.

Two structurally distinct peptides, angiotensin IV and LVV-haemorphin 7, both competitive high-affinity inhibitors of insulin-regulated aminopeptidase (IRAP), were found to enhance aversion-associated and spatial memory in normal rats and to improve performance in a number of memory tasks in rat deficits models. These findings provide compelling support for the development of specific, high-affinity inhibitors of the enzyme as new cognitive enhancing agents. Different classes of IRAP inhibitors have been developed including peptidomimetics and small molecular weight compounds identified through in silico screening with a homology model of the catalytic domain of IRAP. The proof of principal that inhibition of IRAP activity results in facilitation of memory has been obtained by the demonstration that the small-molecule IRAP inhibitors also exhibit memory-enhancing properties.

Distinct distribution of GLUT4 and insulin regulated aminopeptidase in the mouse kidney.

The physiological importance of the insulin responsive glucose transporter GLUT4 in adipocytes and muscle in maintaining glucose homeostasis is well established. A key protein associated with this process is the aminopeptidase IRAP which co-localizes with GLUT4 in specialized vesicles, where it plays a tethering role. In this study, we investigated the distribution of both GLUT4 and IRAP in the kidney to gain insights into the potential roles of these proteins in this organ. Both IRAP and GLUT4 immunostaining was observed in the epithelial cells of the proximal and distal tubules and thick ascending limbs in the cortex, but very little overlap between GLUT4 and IRAP immunoreactivity was observed. GLUT4 staining was consistent with a vesicular localization, whereas IRAP staining was predominantly on the luminal surface. In the principal cells of the inner medulla collecting duct (IMCD), IRAP immunoreactivity was detected throughout the cell, with limited overlap with the vasopressin responsive water channel aquaporin-2 (AQP-2). AQP-2 levels were observed to be two-fold higher in IRAP knockout mice. Based on our results, we propose that GLUT4 plays a role in shunting glucose across epithelial cells. In the kidney cortex, IRAP, in concert with other peptidases, may be important in the generation of free amino acids for uptake, whereas in the principal cells of the inner medulla IRAP may play a localized role in the regulation of vasopressin bioactivity.

Phenylalanine-544 plays a key role in substrate and inhibitor binding by providing a hydrophobic packing point at the active site of insulin-regulated aminopeptidase.

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn(2+) ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn(2+) ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the K(i) in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

Gene knockout of insulin-regulated aminopeptidase: loss of the specific binding site for angiotensin IV and age-related deficit in spatial memory.

The AT(4) ligands, angiotensin IV and LVV-hemorphin 7, elicit robust effects on facilitating memory by binding to a specific site in the brain historically termed the angiotensin AT(4) receptor. The identification of the AT(4) receptor as insulin-regulated aminopeptidase (IRAP) is controversial, with other proteins speculated to be the target(s) of these peptides. In this study we have utilized IRAP knockout mice to investigate IRAP in the brain. We demonstrate that the high-affinity binding site for angiotensin IV is absent in IRAP knockout mice brain sections in parallel with the loss of IRAP immunostaining, providing irrefutable proof that IRAP is the specific high-affinity binding site for AT(4) ligands. However, our characterization of the behavioural phenotype of the IRAP knockout mice revealed a totally unexpected finding. In contrast to the acute effects of IRAP inhibitors in enhancing memory, deletion of the IRAP gene resulted in mice with an accelerated, age-related decline in spatial memory that was only detected in the Y maze paradigm. Moreover, no alterations in behaviour of the IRAP knockout mice were observed that could assist in elucidating the endogenous substrate(s). Our results highlight the importance of analysing the behavioural phenotype of knockout mice across different ages and in distinct memory paradigms.

Reproduction and maternal behavior in insulin-regulated aminopeptidase (IRAP) knockout mice.

During human pregnancy, a circulating form of insulin-regulated aminopeptidase (IRAP EC 3.4.11.3), often termed oxytocinase or placental leucine aminopeptidase (PLAP), is present in plasma. It is proposed that circulating IRAP plays an important role in regulating the circulating levels of oxytocin and/or vasopressin during pregnancy. We assessed the reproductive and maternal profile of global IRAP knock out mice. No differences in the reproductive profile were observed, with normal gestational period, litter size and parturition recorded. However, western blot analysis of pregnant mouse serum, failed to detect IRAP, a result which was confirmed by fluorimetric IRAP enzyme assay. A review of the literature revealed that the presence of IRAP in the maternal circulation during pregnancy has been only reported in humans. Moreover, the sequence, Phe154 Ala155, identified as the cleavage site for the release of soluble IRAP, is restricted to members of the homindae family. Therefore the absence of IRAP from the circulation in mice, and other species during pregnancy, is due to the inability of a secretase to cleave placental IRAP to produce a soluble form of the enzyme. Given the expression of IRAP in areas of the brain associated with oxytocin modulated maternal behavior, we also investigated whether the IRAP global knockout mice had improved maternal responses. Using standard tests to assess maternal behavior, including pup retrieval, feeding and nurturing, no differences between knock out and wild type dams were observed. In conclusion, the physiological significance of circulating IRAP during human pregnancy cannot be addressed by investigations on mice.

Identification and characterization of a new cognitive enhancer based on inhibition of insulin-regulated aminopeptidase.

Approximately one-quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effective cognitive-enhancing agents. Insulin-regulated aminopeptidase (IRAP) is potentially an innovative target for the development of cognitive enhancers, as its peptide inhibitors exhibit memory-enhancing effects in both normal and memory-impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small-molecule inhibitors of IRAP. Structure-based computational development of an initial "hit" resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities (K(i) 20-700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 microl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibitors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cognitive enhancers. Notably, this study also provides unequivocal proof of principal that inhibition of IRAP results in memory enhancement.

Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase.

Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.

Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor.

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.