Targeting NAAA counters dopamine neuron loss and symptom progression in mouse models of parkinsonism.

The lysosomal cysteine hydrolase N-acylethanolamine acid amidase (NAAA) deactivates palmitoylethanolamide (PEA), a lipid-derived PPAR-α agonist that is critically involved in the control of pain and inflammation. In this study, we asked whether NAAA-regulated PEA signaling might contribute to dopamine neuron degeneration and parkinsonism induced by the mitochondrial neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In vitro experiments showed that 6-OHDA and MPTP enhanced NAAA expression and lowered PEA content in human SH-SY5Y cells. A similar effect was observed in mouse midbrain dopamine neurons following intra-striatal 6-OHDA injection. Importantly, deletion of the Naaa gene or pharmacological inhibition of NAAA activity substantially attenuated both dopamine neuron death and parkinsonian symptoms in mice treated with 6-OHDA or MPTP. Moreover, NAAA expression was elevated in postmortem brain cortex and premortem blood-derived exosomes from persons with Parkinson's disease compared to age-matched controls. The results identify NAAA-regulated PEA signaling as a molecular control point for dopaminergic neuron survival and a potential target for neuroprotective intervention.

N-Acylethanolamine Acid Amidase contributes to disease progression in a mouse model of multiple sclerosis.

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.

Gender specific decrease of a set of circulating N-acylphosphatidyl ethanolamines (NAPEs) in the plasma of Parkinson's disease patients.

INTRODUCTION: Current markers of Parkinson's disease (PD) fail to detect the early progression of disease state. Conversely, current omics techniques allow the investigation of hundreds of molecules potentially altered by disease conditions. Based on evidence previously collected by our group in a mouse model of PD, we speculated that a particular set of circulating lipids might be significantly altered by the pathology. OBJECTIVES: The aim of current study was to evaluate the potential of a particular set of N-acyl-phosphatidylethanolamines (NAPEs) as potential non-invasive plasma markers of ongoing neurodegeneration from Parkinson's disease in human subjects. METHODS: A panel of seven NAPEs were quantified by LC-MS/MS in the plasma of 587 individuals (healthy controls, n = 319; Parkinson's disease, n = 268); Random Forest classification and statistical modeling was applied to compare Parkinson's disease versus controls. All p-values obtained in different tests were corrected for multiplicity by controlling the false discovery rate (FDR). RESULTS: The results indicate that this panel of NAPEs is able to distinguish female PD patients from the corresponding healthy controls. Further to this, the observed downregulation of these NAPEs is in line with the results in plasma of a mouse model of Parkinson's (6-OHDA). CONCLUSIONS: In the current study we have shown the downregulation of NAPEs in plasma of PD patients and we thus speculate that these lipids might serve as candidate biomarkers for PD. We also suggest a molecular mechanism, explaining our findings, which involves gut microbiota.

Endogenous N-acyl taurines regulate skin wound healing.

The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs-N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]-as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy.

Macrophage-derived lipid agonists of PPAR-α as intrinsic controllers of inflammation.

Macrophages are multi-faceted phagocytic effector cells that derive from circulating monocytes and undergo differentiation in target tissues to regulate key aspects of the inflammatory process. Macrophages produce and degrade a variety of lipid mediators that stimulate or suppress pain and inflammation. Among the analgesic and anti-inflammatory lipids released from these cells are the fatty acid ethanolamides (FAEs), which produce their effects by engaging nuclear peroxisome proliferator activated receptor-α (PPAR-α). Two members of this lipid family, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), have recently emerged as important intrinsic regulators of nociception and inflammation. These substances are released from the membrane precursor, N-acylphosphatidylethanolamine (NAPE), by the action of a NAPE-specific phospholipase D (NAPE-PLD), and in macrophage are primarily deactivated by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). NAPE-PLD and NAAA regulate FAE levels, exerting a tight control over the ability of these lipid mediators to recruit PPAR-α and attenuate the inflammatory response. This review summarizes recent findings on the contribution of the FAE-PPAR-α signaling complex in inflammation, and on NAAA inhibition as a novel mechanistic approach to treat chronic inflammatory disorders.

Acid Ceramidase in Melanoma: EXPRESSION, LOCALIZATION, AND EFFECTS OF PHARMACOLOGICAL INHIBITION.

Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 = 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Alleviation of neuropathic pain hypersensitivity by inhibiting neuronal pentraxin 1 in the rostral ventromedial medulla.

Peripheral nerve injury causes spontaneous and long-lasting pain, hyperalgesia, and allodynia. Excitatory amino acid receptor-dependent increases in descending facilitatory drive from the brainstem rostral ventromedial medulla (RVM) contribute to injury-evoked hypersensitivity. Although increased excitability likely reflects changes in synaptic efficacy, the cellular mechanisms underlying injury-induced synaptic plasticity are poorly understood. Neuronal pentraxin 1 (NP1), a protein with exclusive CNS expression, is implicated in synaptogenesis and AMPA receptor recruitment to immature synapses. Its role in the adult brain and in descending pain facilitation is unknown. Here, we use the spared nerve injury (SNI) model in rodents to examine this issue. We show that SNI increases RVM NP1 expression and constitutive deletion or silencing NP1 in the RVM, before or after SNI, attenuates allodynia and hyperalgesia in rats. Selective rescue of RVM NP1 expression restores behavioral hypersensitivity of knock-out mice, demonstrating a key role of RVM NP1 in the pathogenesis of neuropathic pain.

Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF. METHODS: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies. RESULTS: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies. CONCLUSIONS: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

NAPE-specific phospholipase D regulates LRRK2 association with neuronal membranes.

N-acylphosphatidylethanolamines (NAPEs) are glycerophospholipid precursors for bioactive lipid amides and potential regulators of membrane function. They are hydrolyzed by NAPE-specific phospholipase D (NAPE-PLD) and have been implicated in neurodegenerative disorders such as Parkinson's disease. Here, we used siRNA-mediated silencing of NAPE-PLD in human SH-SY5Y cells and NAPE-PLD-/- mice to determine whether NAPEs influence the membrane association of LRRK2, a multifunctional protein kinase that is frequently mutated in persons with sporadic Parkinson's disease. NAPE-PLD deletion caused a significant accumulation of non-metabolized NAPEs, which was accompanied by a shift of LRRK2 from membrane to cytosol and a reduction in total LRRK2 content. Conversely, exposure of intact SH-SY5Y cells to bacterial PLD lowered NAPE levels and enhanced LRRK2 association with membranes. The results suggest that NAPE-PLD activity may contribute to the control of LRRK2 localization by regulating membrane NAPE levels.

A protective role for N-acylphosphatidylethanolamine phospholipase D in 6-OHDA-induced neurodegeneration.

N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) catalyzes the cleavage of membrane NAPEs into bioactive fatty-acid ethanolamides (FAEs). Along with this precursor role, NAPEs might also serve autonomous signaling functions. Here, we report that injections of 6-hydroxydopamine (6-OHDA) into the mouse striatum cause a local increase in NAPE and FAE levels, which precedes neuronal cell death. NAPE, but not FAE, accumulation is enhanced in mice lacking NAPE-PLD, which display a substantial reduction in 6-OHDA-induced neurotoxicity, as shown by increased survival of substantia nigra dopamine neurons, integrity of striatal dopaminergic fibers, and striatal dopamine metabolite content. Reduced damage is accompanied by attenuation of the motor response evoked by apomorphine. Furthermore, NAPE-PLD silencing protects cathecolamine-producing SH-SY5Y cells from 6-OHDA-induced reactive oxygen species formation, caspase-3 activation and death. Mechanistic studies in mice suggest the existence of multiple molecular contributors to the neuroprotective effects of NAPE-PLD deletion, including suppression of Rac1 activity and attenuated transcription of several genes (Cadps, Casp9, Egln1, Kcnj6, Spen, and Uchl1) implicated in dopamine neuron survival and/or Parkinson's disease. The findings point to a previously unrecognized role for NAPE-PLD in the regulation of dopamine neuron function, which may be linked to the control of NAPE homeostasis in membranes.

Acute perinatal asphyxia impairs non-spatial memory and alters motor coordination in adult male rats.

A large body of clinical evidence suggests a possible association between perinatal asphyxia and the onset of early, as well as long-term, neurological and psychiatric disorders including cognitive deficits. The present study investigated cognitive and motor function modifications in a well characterized and clinically relevant experimental rat model of human perinatal asphyxia. The results reported here show that adult rats exposed to a single (20 min) asphyctic episode at delivery displayed: (a) a deficit in non-spatial memory, assessed in a novel object recognition task; (b) an impaired motor coordination, measured by the rotarod test. On the other hand, gross motor activity and spatial memory, evaluated in both the Y maze and the Barnes maze, were not affected by perinatal asphyxia. The results of this study provide further insights into the long-term effects of perinatal asphyxia on neurobehavioural functions.

Direct and indirect striatal efferent pathways are differentially influenced by low and high dyskinetic drugs: behavioural and biochemical evidence.

Clinical evidence suggests that stimulation of the D(1) rather than D(2) dopamine receptor is related to the development of dyskinesias in Parkinson's disease (PD). We evaluated, in the 6-hydroxydopamine rat model of PD, sensitization of contralateral turning (SCT) behaviour and abnormal involuntary movements (AIMs) as behavioural parameters of dyskinetic response, and changes in zif-268 mRNA expression in striatonigral and striatopallidal neurons on subchronic administration of the D(2)/D(3) agonist ropinirole, defined as a mild dyskinetic drug in the clinic. Results were compared with previous findings on repeated L-dopa treatment. Ropinirole displayed a mild dyskinetic response characterized by SCT only, which contrasted with the presence of SCT in association with AIMs elicited by repeated L-dopa. Zif-268 mRNA levels were decreased in both striatonigral and striatopallidal neurons by ropinirole, in contrast to hyper-expression of zif-268 mRNA selectively induced by L-dopa in striatonigral neurons. Unbalanced responsiveness of striatal efferent neurons might represent a molecular correlate of high dyskinetic potential and AIMs in rats; in contrast, a balanced striatal output might underlie the low dyskinetic potential displayed by ropinirole.

The N-Acylethanolamine Acid Amidase Inhibitor ARN077 Suppresses Inflammation and Pruritus in a Mouse Model of Allergic Dermatitis.

N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase highly expressed in macrophages and B lymphocytes, catalyzes the degradation of palmitoylethanolamide. Palmitoylethanolamide is an agonist of PPAR-α and an important regulator of pain and innate immunity. In this study, we investigated the properties of the NAAA inhibitor, ARN077, in a mouse model of allergic contact dermatitis. Acute topical applications of ARN077 attenuated key signs of DNFB-induced dermatitis in a dose-dependent manner. Moreover, ARN077 increased tissue palmitoylethanolamide content and normalized circulating levels of cytokines and immunoglobulin E. No such effect was seen in PPAR-α-deficient mice. Moreover, mice lacking NAAA failed to develop edema or scratching behavior after challenge with DNFB, confirming that this enzyme plays an important role in dermatitis. Consistent with this conclusion, subchronic applications of ARN077 suppressed DNFB-induced inflammation when administered either before or after the DNFB challenge. The effects of subchronic ARN077 were dose dependent and comparable in size to those produced by the steroids clobetasol and dexamethasone. Unlike the latter, however, ARN077 did not cause skin atrophy. The results identify NAAA as a promising target for the development of effective and safe treatments for atopic dermatitis and other inflammatory disorders of the skin.

Novel activity-based probes for N-acylethanolamine acid amidase.

The cysteine hydrolase, N-acylethanolamine acid amidase (NAAA) is a promising target for analgesic and anti-inflammatory drugs. Here, we describe the development of two unprecedented NAAA-reactive activity-based probes as research tools for application in the discovery of new inhibitors and for the in-depth characterization of NAAA in its cellular environment.

Expression of dyskinetic movements and turning behaviour in subchronic L-DOPA 6-hydroxydopamine-treated rats is influenced by the testing environment.

Sensitisation in contralateral turning behaviour and induction of abnormal involuntary movements (AIMs) after subchronic intermittent L-DOPA were compared for their predictive validity as model of parkinsonian dyskinetic movements. L-DOPA treatment produced sensitisation in turning behaviour in 6-hydroxydopamine-lesioned rats, when animals were evaluated in hemispherical bowls but not in cages. In contrast, sensitisation in AIMs was obtained both in hemispherical bowls and cages. Results provide evidence that the choice of the environment used in evaluation of AIMs and turning behaviour is of crucial importance.

Ion mobility mass spectrometry enhances low-abundance species detection in untargeted lipidomics.

We describe a simple method for the detection of low intensity lipid signals in complex tissue samples, based on a combination of liquid chromatography/mass spectrometry and ion mobility mass spectrometry. The method relies on visual and software-assisted analysis of overlapped mobilograms (diagrams of mass-to-charge ratio, m/z, vs drift time, DT) and was successfully applied in untargeted lipidomics analyses of mouse brain tissue to detect relatively small variations in a scarce class of phospholipids (N-acyl phosphatidylethanolamines) generated during neural tissue damage, against a background of hundreds of lipid species. Standard analytical tools, including Principal Component Analysis, failed to detect such changes.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme's active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

A Potent Systemically Active N-Acylethanolamine Acid Amidase Inhibitor that Suppresses Inflammation and Human Macrophage Activation.

Fatty acid ethanolamides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are lipid-derived mediators that potently inhibit pain and inflammation by ligating type-α peroxisome proliferator-activated receptors (PPAR-α). These bioactive substances are preferentially degraded by the cysteine hydrolase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Here, we describe a new class of ß-lactam derivatives that are potent, selective, and systemically active inhibitors of intracellular NAAA activity. The prototype of this class deactivates NAAA by covalently binding the enzyme's catalytic cysteine and exerts profound anti-inflammatory effects in both mouse models and human macrophages. This agent may be used to probe the functions of NAAA in health and disease and as a starting point to discover better anti-inflammatory drugs.

Behavioral, neurochemical, and electrophysiological changes in an early spontaneous mouse model of nigrostriatal degeneration.

In idiopathic Parkinson's disease, clinical symptoms do not emerge until consistent neurodegeneration has occurred. The late appearance of symptoms implies the existence of a relatively long preclinical period during which several disease-induced neurochemical changes take place to mask the existence of the disease and delay its clinical manifestations. The aim of this study was to examine the neurochemical, neurophysiological, and behavioral changes induced by the loss of nigrostriatal innervation in the En1+/-;En2-/- mouse, in the 10 months following degeneration, compared to En2 null mutant mice. Behavioral analysis (Pole-test, Beam-walking test, and Inverted grid test) and field potential recordings in the striatum indicated that loss of ~70% of nigrostriatal neurons produced no significant functional effects until 8 months of age, when En1+/-;En2-/- animals started to show frank motor deficits and electrophysiological alterations in corticostriatal plasticity. Similarly, alterations in dopamine homeostasis, dopamine turnover, and dopamine innervation were observed in aged animals compared to young En1+/-;En2-/- mice. These data suggests that in En1+/-;En2-/- mice nigrostriatal degeneration in the substantia nigra is functionally compensated.