Structural properties and role of the endocannabinoid lipases ABHD6 and ABHD12 in lipid signalling and disease.

The endocannabinoid (eCB) system is an important part of both the human central nervous system (CNS) and peripheral tissues. It is involved in the regulation of various physiological and neuronal processes and has been associated with various diseases. The eCB system is a complex network composed of receptor molecules, their cannabinoid ligands, and enzymes regulating the synthesis, release, uptake, and degradation of the signalling molecules. Although the eCB system and the molecular processes of eCB signalling have been studied extensively over the past decades, the involved molecules and underlying signalling mechanisms have not been described in full detail. An example pose the two poorly characterised eCB-degrading enzymes α/ß-hydrolase domain protein six (ABHD6) and ABHD12, which have been shown to hydrolyse 2-arachidonoyl glycerol-the main eCB in the CNS. We review the current knowledge about the eCB system and the role of ABHD6 and ABHD12 within this important signalling system and associated diseases. Homology modelling and multiple sequence alignments highlight the structural features of the studied enzymes and their similarities, as well as the structural basis of disease-related ABHD12 mutations. However, homologies within the ABHD family are very low, and even the closest homologues have widely varying substrate preferences. Detailed experimental analyses at the molecular level will be necessary to understand these important enzymes in full detail.

Biochemical characterization of the PHARC-associated serine hydrolase ABHD12 reveals its preference for very-long-chain lipids.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.

Genetic polymorphisms (Pro197Leu of Gpx1, +35A/C of SOD1, -262C/T of CAT), the level of antioxidant proteins (GPx1, SOD1, CAT) and the risk of distal symmetric polyneuropathy in Polish patients with type 2 diabetes mellitus.

PURPOSE: Oxidative stress and impaired anti-oxidant defense are regarded as contributory factors for distal symmetric polyneuropathy (DSPN). The purpose of the study was to evaluate the plasma level of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) and the association between polymorphic variants in genes encoding for GPx1, SOD, CAT and the risk of DSPN in T2DM patients. MATERIAL/METHODS: We included 401 individuals: 110 T2DM patients with DSPN, 135 T2DM patients without DSPN, and 156 control subjects with normoglycemia, and without DSPN. We employed RFPL-PCR to genotype polymorphic variants Pro197Leu of Gpx1, +35A/C of SOD1, -262C/T of CAT and ELISA tests to measure plasma level of SOD1, GPx1 and CAT. The odds ratios (ORs) and 95% confidence intervals (CIs) for each genotype and allele were calculated. RESULTS: There was a significant decrease in the level of GPx1 (p<0.05), SOD1 (p<0.05) in T2DM patients with DSPN compared to healthy subjects. T2DM patients without DSPN showed a statistically lower serum level of GPX1 (p<0.05) than healthy subjects. SOD 1 and CAT levels were lower in T2DM patients with DSPN compared to T2DM patients without DSPN (p<0.05). The genetic analysis revealed the lack of association between examined polymorphic variants and the risk of DSPN. CONCLUSIONS: The examined polymorphic variants are not associated with DSPN in Polish T2DM patients. The obtained results suggest that disturbances in antioxidant defense system may play significant role in the development and progression of DSPN.

Tumor necrosis factor-α downregulates sodium current in skeletal muscle by protein kinase C activation: involvement in critical illness polyneuromyopathy.

Sepsis is involved in the decrease of membrane excitability of skeletal muscle, leading to polyneuromyopathy. This effect is mediated by alterations of the properties of voltage-gated sodium channels (Na(V)), but the exact mechanism is still unknown. The aim of the present study was to check whether tumor necrosis factor (TNF-α), a cytokine released during sepsis, exerts a rapid effect on Na(V). Sodium current (I(Na)) was recorded by macropatch clamp in skeletal muscle fibers isolated from rat peroneus longus muscle, in control conditions and after TNF-α addition. Analyses of dose-effect and time-effect relationships were carried out. Effect of chelerythrine, a PKC inhibitor, was also studied to determine the way of action of TNF-α. TNF-α induced a reversible dose- and time-dependent inhibition of I(Na). A maximum inhibition of 75% of the control current was observed. A shift toward more negative potentials of activation and inactivation curves of I(Na) was also noticed. These effects were prevented by chelerythrine pretreatment. TNF-α is a cytokine released in the early stages of sepsis. Besides a possible transcriptional role, i.e., modification of the channel type and/or number, we demonstrated the existence of a rapid, posttranscriptional inhibition of Na(V) by TNF-α. The downregulation of the sodium current could be mediated by a PKC-induced phosphorylation of the sodium channel, thus leading to a significant decrease in muscle excitability.

Organophosphate induced delayed polyneuropathy in man: an overview.

About 80 years have passed since the first cases of organophosphate induced delayed polyneuropathy (OPIDP), as the consequence of human poisoning with certain organophosphorus compounds, were described in the literature. OPIDP is a relatively rare neurodegenerative disorder in humans characterized by loss of function, ataxia and paralysis of distal parts of sensory and motor axons in peripheral nerves and ascending and descending tracts of spinal cord appearing 2-3 weeks after exposure or later. The molecular target for OPIDP is considered to be an enzyme in the nervous system known as neuropathy target esterase (NTE). This review discusses OPIDP in man with emphasis on clinical presentation, pathogenesis, molecular mechanisms, and possibilities for prevention/therapy.

Soluble phenyl valerate esterases of hen sciatic nerve and the potentiation of organophosphate induced delayed polyneuropathy.

Contrary to some organophosphorus esters (OPs), certain esterase inhibitors including sulfonyl halides, carbamates and phosphinates do not cause axonal neuropathy, but they may exacerbate traumatic and some chemical insults to axons. This phenomenon is referred to as the promotion/potentiation of axonopathies. We report here promotion studies of the organophosphate induced delayed polyneuropathy (OPIDP). This neuropathy correlates with inhibition/aging of neuropathy target esterase, but this enzyme is not the target of promotion. Soluble phenyl valerate (PV) esterases in peak I (V(0)) of hen sciatic nerve were analysed. When these activities were inhibited in vitro by a mixture containing mipafox - an OP that causes OPIDP - paraoxon and p-toluene sulfonyl fluoride - two esterase inhibitors that do not cause either neuropathy or promotion-, then the remaining activity was sensitive to classical promoters such as phenylmethane sulfonyl fluoride (PMSF) and phenylmethyl benzyl carbamate. This PV-activity was not inhibited in sciatic nerves of hens treated with di-isopropyl phosphorofluoridate, at a dose that causes OPIDP. When these birds were further dosed with PMSF a dose-response relationship was observed between inhibition of PV-esterases, as above defined, and the severity of clinical responses. These data suggest that the target of promotion is embraced in peak I (V(0)) of soluble proteins of hen sciatic nerve.

Lentivirus infection causes neuroinflammation and neuronal injury in dorsal root ganglia: pathogenic effects of STAT-1 and inducible nitric oxide synthase.

Distal sensory polyneuropathy (DSP) is currently the most common neurological complication of HIV infection in the developed world and is characterized by sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. We previously showed that feline immunodeficiency virus (FIV) infection of cats caused DSP together with immunosuppression in cats, similar to that observed in HIV-infected humans. In this study, we investigated the pathogenic mechanisms underlying the development of FIV-induced DSP using feline dorsal root ganglia (DRG) cultures, consisting of neurons, Schwann cells, and macrophages. FIV-infected cultures exhibited viral Ags (p24 and envelope) in macrophages accompanied by neuronal injury, indicated by neurite retraction, neuronal loss and decreased soma size, compared with mock-infected (control) cultures. FIV infection up-regulated inducible NO synthase (iNOS), STAT-1, and TNF-alpha mRNA levels in DRG cultures. Increased STAT-1 and iNOS mRNA levels were also observed in DRGs from FIV-infected animals relative to mock-infected controls. Similarly, immunolabeling studies of DRGs from FIV-infected animals showed that macrophages were the principal sources of STAT-1 and iNOS protein production. The iNOS inhibitor aminoguanidine reduced nitrotyrosine and protein carbonyl levels, together with preventing neuronal injury in FIV-infected DRG cultures. The present studies indicate that FIV infection of DRGs directly contributes to axonal and neuronal injury through a mechanism involving macrophage immune activation, which is mediated by STAT-1 and iNOS activation.

Matrix metalloproteinases-9 and -2 in secondary vasculitic neuropathies.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases that play an important role in inflammation and tissue degradation. MMP-9 and MMP-2 are gelatinases that have been implicated in the degradation of the blood-brain or blood-nerve barrier. We present an immunohistochemical study on 11 nerve biopsy samples of inflammatory and non-inflammatory polyneuropathies. Perineurium and endothelium were positive for MMP-2 in all tissue sections. In addition, there was a specific up-regulation of MMP-2 in stromal cells of chronic inflammatory demyelinating polyneuropathy (CIDP) and even more in vasculitic neuropathies. MMP-9-positive cells were detected in vessel walls, infiltrates, epineurium and endoneurium of vasculitic neuropathies. In CIDP, MMP-9-positive cells were prominent in vessel walls. Only a few MMP-9-positive cells were detected in noninflammatory controls in blood vessels and adhering to vessel walls. Double staining indicated that the infiltrating cells were T cells and macrophages. Our findings suggest that MMP-9 plays an important role in inflammatory peripheral neuropathy probably as means for inflammatory cell invasion.

The relationship between isofenphos cholinergic toxicity and the development of polyneuropathy in hens and humans.

Species differences have been observed between hen and human clinical manifestations of isofenphos toxicities. Hens treated with the insecticide isofenphos (90 mg/kg p.o.) developed severe cholinergic toxicity followed by mild organophosphate-induced delayed polyneuropathy (OPIDP). However, a patient developed severe OPIDP, which was preceded by very mild cholinergic signs, after an attempted suicide with a commercial formulation containing isofenphos and phoxim, an insecticide not causing OPIDP (estimated doses were 500 and 125 mg/kg, respectively). To explain this difference the following hypotheses were tested: (1) phoxim is a promoter of isofenphos-induced OPIDP; (2) whereas neuropathy target esterase (NTE) is thought to be the target of OPIDP, activation of isofenphos by liver microsomes causes the formation of more potent NTE inhibitor(s) in humans than in hens; (3) in contrast to hen NTE, the sensitivity of the human enzyme to such inhibitor(s) is higher than that of acetylcholinesterase (AChE), the target of cholinergic toxicity. Results showed that phoxim (22.5 mg/kg p.o.) was not a promoter of OPIDP in hens and that the ratio AChE inhibition:NTE inhibition by microsome-activated isofenphos was similar for both hen and human enzymes. The schedule of antidotal treatment in hens is the likely explanation for the observed difference from the patient. Peak AChE inhibition was maintained in hen brain up to 6 days after a single dose of isofenphos, suggesting prolonged pharmacokinetics. However, the AChE reactivator pyridine-2-aldoxime (2-PAM) was given to hens before isofenphos and then every 8 h, whereas continuous 2-PAM infusion was provided to the patient. When 2-PAM was given to hens every hour after isofenphos (90 mg/kg p.o.), the birds remained asymptomatic. Since other organophosphates may have a prolonged pharmacokinetics, testing procedures for the potential of these insecticides to cause OPIDP may underestimate the risk for humans.

Long-term follow-up of children with chronic relapsing polyneuropathy.

Long-term follow-up of three children with early-onset chronic inflammatory demyelinating polyneuropathy is presented. A 3-year-old male (Patient 1) manifested initially progressive muscle weakness during 6 months with spontaneous regression, followed by two severe relapses at 5 and 6 years of age. Decreased arylsulfatase A activity was present in Patient 1 (17.6) and his family members (24.1-40 nmol/mg/hour). Arterial hypertension up to 20/12 kPa was present in two patients in the initial phase associated with muscle stiffness, occasional meningism, and left ventricular hypertrophy in one of them (Patient 3). Subsequently, they both developed two mild relapses at 3.5 and 6 years of age. Clinical outcome was excellent in all three cases, although clinical course, therapy response, and electrophysiologic outcome was quite different in the only patient with low arylsulfatase A activity. The significance of this difference is discussed.

Organophosphate induced delayed polyneuropathy.

This review discusses the current understanding of organophosphate induced delayed polyneuropathy (OPIDP) with emphasis on molecular mechanisms, pathogenesis and possibilities for prevention/therapy. OPIDP is a rare toxicity caused by certain organophosphorus compounds (OP) characterized by degeneration of some long axons in the central and peripheral nervous system that appear about 2-3 weeks after exposure. The molecular target for OPIDP is considered to be an enzyme in the nervous system known as neuropathy target esterase (NTE). NTE can be inhibited by two types of inhibitors: a) phosphates, phosphonates, and phosphoramidates, which cause OPIDP when >70% of the enzyme is inhibited, and b) phosphinates, carbamates, and sulfonyl halides which inhibit NTE and cause either protection from, or promotion, of OPIDP when given before or after a neuropathic OP, respectively. The ability of a NTE inhibitor to cause OPIDP, besides its affinity for the enzyme, is related to its chemical structure and the residue left attached to the NTE. If such residues undergo the aging reaction i.e. the loss of an alkyl group bound to the enzyme, those OPs usually have a high likelihood of causing OPIDP. Protection from neuropathic doses of OP inhibitors is obtained when NTE is inhibited with nonageable inhibitors. Promotion of OPIDP involves another site besides NTE because it can occur when all NTE is affected. It is now known that this other site is similar to NTE in that it is also sensitive to mipafox but at much higher concentrations. Promotion affects either the progression or expression of OPIDP after the initial biochemical effect on NTE. Some recent observations suggest that development of OPIDP in hens can be influenced by atropine, oximes and methylprednisolone when they are given before or soon after neuropathic OPs.

Testing for organophosphate-induced delayed polyneuropathy.

Organophosphorous compounds may cause two distinct types of toxicity: acute cholinergic toxicity and organophosphate-induced delayed polyneuropathy (OPIDP). The ability of a compound to cause OPIDP is assessed as described by administering the compound to hens and screening the brain, spinal cord, and peripheral nerves for neuropathy target esterase activity to detect OPIDP and acetylcholinesterase activity to rule out the acute toxicity. This assay can also be used as part of a screen for protective agents.

[The ultrastructural localization of NO-synthase NADPH diaphorase in a peripheral nerve and its change in diphtheritic polyneuropathy]. / Ul'trastrukturnaia lokalizatsiia NO-sintaznoi NADPH-diaforazy v perifericheskom nerve i ee izmenenie pri difteriinoi polineiropatii.

Light and electron microscopy was used to study the distribution and changes of NADPH-diaphorase in the cutaneous nerve biopsy specimens in different periods of diphtheritic polyneuropathy (DP). there was a reduction in the reaction rate of the enzyme in Schwann's cells of the destructively changed nerve fibers and an increase in the remyelinated nerve fibers. The enzyme is located on the nuclear and endoplasmic reticulum membranes and ribosomes. It is suggested that there is an association of the synthesis of nitric oxide with the myelin-producing function of Schwann's cells.

Estudios enzimáticos en pacientes con Neuropatía Epidémica en su forma clínica de Neuritis Optica

A 28 enfermos con neuropatía epidémica en su forma clínica de neuritis óptica y 15 individuos aparentemente sanos (familiares o convivientes de los pacientes), se les determinó la actividad de glucosa 6 fosfato deshidrogenasa (G6PD), 6 fosfoglucónico deshidrogenasa (G6PD), glutatión reductasa (GR), deshidrogenasa láctica (LDH) y muramidasa. Los resultados se compararon con los valores normales establecidos con anterioridad a la aparición de la enfermeda. No se observaron diferencias significativas en 6 PGD, LDH y muramidasa entre los e grupos ni con los controles. La G6PD y la GR mostraron diferencias significativas entre pacientes y controles (p < 0,01 y p < 0,001) y entre convivientes y controles (p <0,001 y p <0,05). El 54,2 de los pacientes y el 38,5 de los convivientes mostraron niveles de G6PD disminuidos y el 53,6 de los pacientes y el 46,7 de los convivientes presentaron actividad de GR por debajo del límite inferior normal.

Giant axonal degeneration: determination of essential glycolytic enzymes.

Aggregation and accumulation of intermediate filaments (IF) in different cell types is an ultrastructural hallmark of autosomal recessively inherited giant axonal degeneration (GAD) as well as of experimentally induced or occupationally acquired toxic polyneuropathies. Insufficient glycolysis caused by glycolytic enzyme deficiencies has been proposed as a likely cause of the IF accumulation. In this report data are presented on essential glycolytic enzymes in erythrocytes of a GAD patient and family. No significant changes were noted.

Neurological disorders and phytanic acid metabolism.

Fifty patients with neurological symptoms and signs resembling those of Refsum's disease were analyzed for phytanic acid in serum. In addition, the phytanic acid oxidase capacity in skin fibroblasts was determined. The patients suffered from retinitis pigmentosa, cerebellar ataxia and/or chronic polyneuropathy of unknown cause. The serum level of phytanic acid was not increased, and the alpha-oxidation of (1-14C) phytanic acid was found to be normal in all patients. The present investigation lends no support to the theory that so-called atypical or mild cases of Refsum's disease exist. This disorder appears to be a disease entity with a specific genetic phenotype.

[Biochemical studies on muscles in neurogenic atrophies and central paralysis. Studies of the trophic functions of neurons]. / Biochemische Untersuchungen in der Muskulatur bei neurogenen Atrophien und zentralen Paresen. Untersuchungen zur trophischen Funktion des Neurons.

Enzyme activities of the energy supplying metabolism were investigated in muscle specimens of brachial biceps, deltoid or anterior tibial muscles of patients with traumatic nerve lesions, polyneuropathies, Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, spinal muscular atrophy and hemiparesis. The key enzymes of glycogenolysis (glycogen phosphorylase), glycolysis (triosephosphate dehydrogenase, lactate dehydrogenase), alpha-glycerophosphate cycle (alpha-glycerophosphate dehydrogenase), beta-oxidation of fatty acids (beta-hydroxy-acyl-CoA-dehydrogenase), citrate acid cycle (citrate synthase, malate dehydrogenase), hexokinase reaction (hexokinase) and pentosephosphate shunt (6-phosphogluconate dehydrogenase) were measured. The present study shows that in case of disorders of the lower motor neuron--especially those with impaired axoplasmic transport--changes in the enzyme patterns of muscles occur at an early stage. The glycolytic enzyme activities are of particular significance because they are the most sensitive indicators of the onset, extent and course of neurogenic atrophy. There is a good correlation between severity of the lesion, functional state of the muscles and reduction of these enzyme activities. In case of traumatic nerve lesions re-innervation can prevent a permanent reduction of glycolytic enzymes only if it occurs during the first months after denervation. In all cases in which operative revision is considered, it is therefore not advisible to wait since the regenerative capacity of the motor neuron is not the only limiting factor but also the biochemical and morphological changes in the muscle fibre. These are permanent after long lasting denervation without re-innervation within the first months. Primary neuroaxonal degeneration of the nerve fibre which was found in the majority of our alcoholic patients obviously impairs the metabolism of the muscle to a greater extent than primary demyelination most frequently observed in diabetics with polyneuropathy. Corresponding to the chronic course of the illness over years and to the severity of the pareses, drastic reduction in the activities of glycolytic enzymes was found in patients with Charcot-Marie-Tooth disease. Simultaneously the activity of 6-phosphogluconate dehydrogenase was significantly increased as a result of the chronic neurogenic lesion of the muscle fibres. Follow-up during the treatment of diseases of the lower motor neuron can be performed because the enzyme activities can be measured even in small muscle specimens. In patients with hemiparesis slight but not significant reduction in the glycolytic enzyme activities was found by comparison with a normal control group. We assume that this reduction is due to general inactivity which is caused by the movement disorder rather than to the particular influence of the upper motor neuron.

Histochemical and electron microscopic observations on the myoneural junctions of rats with carbon disulfide induced polyneuropathy.

Myoneural junctions in the tibialis anterior muscle of rats with clinical signs of polyneuropathy induced with carbon disulfide were studied by light and electron microscopy. Histochemically demonstrable acetylcholinesterase (AChE; E.C. 3.1.1.7) activity was distributed similarly in the myoneural junctions of both the exposed and the control rats. In both groups intense enzyme activity was localized at the level of the post-synaptic membrane of the myoneural junction. The postsynaptic infoldings of the myoneural junctions of the exposed rats appeared normal. No enzyme activity was seen outside the zone of the myoneural junctions. The ultrastructure of the sub-sarcolemmal space, as well as the postsynaptic membranes of the myoneural junctions of the exposed animals, was normal. In the terminal axons signs of various degrees of degeneration were present, e.g., disappearance of the preterminal axoplasmic neurotubules, partial disappearance of synaptic vesicles, appearance of dense bodies, and even total disappearance or destruction of the terminal axons. Synaptic clefts were often widened with Schwann cell interposition. It thus seems that systemic carbon disulfide poisoning primarily alters the presynaptic structures of the myoneural junctions, while the postsynaptic side remains relatively intact, especially since the histochemical distribution of AChE in myoneural junctions was normal.

Cerebrospinal fluid fructose-biphosphate aldolase (aldolase) activity in infectious diseases of the central nervous system.

Aldolase levels were estimated in the cerebrospinal fluid of patients with infectious diseases of the central nervous system. A significant rise was found in bacterial and cryptococcal meningitis but the investigation failed to elucidate the source and clinical significance of the elevated activity.