Activity of Acetylcholinesterase in the Motor-Sensory Cortex in Early Ontogenesis of Rats Exposed to Prenatal Hypoxia

Effects of acute prenatal hypoxia (13–14 days of gestation, 3 h, O₂ = 7%) on acetylcholinesterase (AChE, EC 3.1.1.7) activity in homogenates, synaptosomes, and cytosol of the motor-sensory cortex of Wistar rats were studied on the days 1, 5, 10, 19 and 30 after birth. In homogenates of normally developing cortex, the AChE activity did not significantly change with age. Activity of AChE in synaptosomes increased 4 times throughout the entire period of observation, while in the cytosol, 4.3 times to reach maximum at the 19th day. Maximum rise of the AChE activity in synaptosomes was observed at the period from the 5th to the 10th day. Activity of AChE in homogenate and synaptosomes of rats submitted to prenatal hypoxia decreased during the first five days after birth (p < 0.001) but later, starting from the day 10, it increased in all fractions. A statistically significantly higher activity of AChE than in controls was revealed in homogenate of the motor-sensory cortex on day 19 (p < 0.01), while in synaptosomes, on the days 19 and 26 (p < 0.001 and p < 0.05, respectively), and in cytosol, on the days 10, 26, and 30 (p < 0.05, p < 0.05, and p < 0.001). Maximum change in the ratio of AChE activities in cytosol and synaptosomes was found on the day 19 (p < 0.01). At the same period of development, changes in the ratio of AChE activity in synaptosomes and homogenate of the control and hypoxic animals were also observed. Thus, prenatal hypoxia leads to in changes in the activity both of the cytosol and synaptosomal membrane-bound forms of AChE in the motor-sensory cortex of rats, which agrees with our own and literature data on disorder of neuro- and neuritogenesis in the process of formation of CNS and of behavioral reactions in early postnatal ontogenesis under the effect of pathogenic factors at certain days of prenatal ontogenesis.     

Effect of an inhibitor of alpha-secretase metabolizing amyloid precursor protein on memory in rats

Intracortical administration of 10(-4) M batimastat, a specific inhibitor of one of metalloproteinases metabolizing amyloid precursor protein, namely alpha-secretase, to adult rats resulted in a decrease in the number of correct runs in a one-level 8-arm maze down to 92.78 +/- 1.03% of the control values (p < 0.01) already 60 min after an injection. The effect of a single injection of the inhibitor to adult rats did not have a prolonged character. However, injections of batimastat into the cortex of brain hemispheres of rats during early postnatal ontogenesis (5th and 7th days after birth) resulted in considerable deterioration of 8-arm maze orientation of these animals at adult age (90.92 +/- 2.21% of correct runs, p < 0.001) compared to control animals. The findings suggest an important role of alpha-secretase in memorization. A possible role of alpha-secretase in memory and pathogenesis of Alzheimer's disease is discussed.     

Effect of gangliosides on adenylyl cyclase activity in the cortex and striatum of rats

By studying the effects of gangliosides (G) on learning and memory we have found that i.p. administration of G led to a decrease in AC activity in the cortex (Cx) and striatum (Str) as well as in the threshold of the sensitivity of striatal neurons to the effect of cholinergic agonists. G also modified the sensitivity of AC from the Cx and Str to such agents as Gpp[NH]p and forskolin. The aim of this work was to analyze the correlation between the changes in the activity of AC in the Cx and Str, concentration of G in these brain structures as well as formation of motor reactions of newborn rats during the first month of postnatal ontogenesis. It was found that new born rats develop normal body rotation by sixth day, locomotion by fifteenth day and stabilization of locomotor activity and supporting body balance by 20–22nd day. As shown previously, the concentration of gangliosides in the Cx and Str is gradually increasing during the first month of animal development. The activity of AC (pmol cAMP/min/mg of protein) in the Cx was found to decrease from 34.75 to 4.09 and in the Str from 46.00 to 11.67 during the first week after birth. However in the periods of formation of general behavioural reactions we observed a statistically significant increase in AC activity: in the Str on 10th and 26th days (p < 0.01) and in the Cx from 10th to 19th days (p < 0.05) compared to the AC activity on 5th and 30th days. Thus, formation of locomotor activity and posture‐tonic reactions during development of rats in early postnatal ontogenesis correlates with increasing concentration of G and basal activity of AC. Supported by RFBR (99‐04‐49751) and RAS (99‐06‐287).     

The role of neutral sphingomyelinase in the interleukin-1beta signal transduction in cells of the mouse cerebral cortex

Involvement of the sphingomyelin cascade in Interleukin 1 beta (IL-1) signal transduction pathway in membrane fraction P2 of the murine brain cortex, was found. A key role of the membrane enzyme neutral sphingomyelinase (nSMase) in triggering the sphingomyelin pathway for IL-1 beta, was confirmed. The IL-1 beta was shown to activate in a dose-dependent manner nSMase in the P2 fraction of the brain cortex. Employment of both brain cortex membranes from the mice deficient in the type I IL-1 receptor and of IL-1 receptor antagonist made it possible to obtain evidence on the necessity of the IL-1 beta binding to the type I IL-1 receptor for the nSMase activation. It appears that the IL-1 beta effects on the CNS are realized via IL-1 receptor type I and activation of the nSMase as an initiating enzyme of the sphingomyelin cascade.     

Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression. Special issue dedicated to Dr. Moussa Youdim.     

Regulation of endothelin-converting enzyme-1 expression in human neuroblastoma cells

In recent years endothelin-converting enzyme (ECE-1) has been suggested to play an important role in amyloid-beta peptide metabolism as one of the amyloid-degrading enzymes. In this connection, the analysis of the levels of expression and distribution of ECE-1 in the brain under normal and pathologic conditions could be important in neurodegeneration and pathogenesis of Alzheimer disease. In our previous studies, we have demonstrated that expression of ECE-1 was significantly reduced in the cortex of adult rats after 15 mins of global ischemia. It was also significantly reduced in the striatum of rats subjected to prenatal hypoxia. In the present study, we analyzed effects of hypoxia and oxidative stress on ECE-1 in human neuroblastoma NB7 cells and effects of the cholinergic agonist carbachol and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). We have found that chronic (24 hrs) hypoxia and oxidative stress resulted in 30% and 20% decrease in expression of ECE-1 at the protein level, respectively, although at the level of ECE-1 mRNA there were no statistically significant changes. Serum withdrawal from the incubation medium as well as addition of carbachol or PMA for 24 hrs also led to a significant reduction of the levels of ECE-1 protein in NB7 cells. Further study of the downstream signaling cascades involved in downregulation of ECE expression in NB7 cells and primary neuronal cells might provide us with new insights into possible therapeutic strategies for prevention or treatment of Alzheimer disease in elderly patients and those who suffer from stroke or cerebrovascular disorders.     

The content and composition of gangliosides in brain subcellular fractions of the frog Rana temporaria

Ganglioside distribution in various frog brain subcellular fractions (myelin, microsomes, mitochondria, synaptosomes, plasma membranes of nerve endings and synaptic vesicles) was investigated. The synaptosomes and plasma membranes of nerve endings were found to be the main places of ganglioside localization, ganglioside concentration being 2.42 and 1.79 times higher than that in homogenates. Gangliosides were shown to be present in synaptic vesicles. The characteristic features of gangliosides from frog brain and its subcellular fractions are the predominance of polysialogangliosides with 3-5 sialic acid residues (up to 57.4%), low content of monosialogangliosides (not more than 7%) and the presence of disialogangliosides with short carbohydrate chain. The increase of ganglioside content per one nerve cell during phylogenetic development of vertebrates is discussed.     

New Insights into Epigenetic and Pharmacological Regulation of Amyloid-Degrading Enzymes

Currently, deficit of amyloid β-peptide (Aβ) clearance from the brain is considered as one of the possible causes of amyloid accumulation and neuronal death in the sporadic form of Alzheimer’s disease (AD). Aβ clearance can involve either specific proteases present in the brain or Aβ-binding/transport proteins. Among amyloid-degrading enzymes the most intensively studied are neprilysin (NEP) and insulin-degrading enzyme (IDE). Since ageing and development of brain pathologies is often accompanied by a deficit in the levels of expression and activity of these enzymes in the brain, there is an urgent need to understand the mechanisms involved in their regulation. We have recently reported that NEP and also an Aβ-transport protein, transthyretin are epigenetically co-regulated by the APP intracellular domain (AICD) and this regulation depends on the cell type and APP₆₉₅ isoform expression in a process that can be regulated by the tyrosine kinase inhibitor, Gleevec. We have now extended our work and shown that, unlike NEP, another amyloid-degrading enzyme, IDE, is not related to over-expression of APP₆₉₅ in neuroblastoma SH-SY5Y cells but is up-regulated by APP₇₅₁ and APP₇₇₀ isoforms independently of AICD but correlating with reduced HDAC1 binding to its promoter. Studying the effect of the nuclear retinoid X receptor agonist, bexarotene, on NEP and IDE expression, we have found that both enzymes can be up-regulated by this compound but this mechanism is not APP-isoform specific and does not involve AICD but, on the contrary, affects HDAC1 occupancy on the NEP gene promoter. These new insights into the mechanisms of NEP and IDE regulation suggest possible pharmacological targets in developing AD therapies.