Intermittent Hypoxic Training as an Effective Tool for Increasing the Adaptive Potential, Endurance and Working Capacity of the Brain.

This review is devoted to the phenomenon of intermittent hypoxic training and is aimed at drawing the attention of researchers to the necessity of studying the mechanisms mediating the positive, particularly neuroprotective, effects of hypoxic training at the molecular level. The review briefly describes the historical aspects of studying the beneficial effects of mild hypoxia, as well as the use of hypoxic training in medicine and sports. The physiological mechanisms of hypoxic adaptation, models of hypoxic training and their effectiveness are summarized, giving examples of their beneficial effects in various organs including the brain. The review emphasizes a high, far from being realized at present, potential of hypoxic training in preventive and clinical medicine especially in the area of neurodegeneration and age-related cognitive decline.

Lactoferrin Induces Erythropoietin Synthesis and Rescues Cognitive Functions in the Offspring of Rats Subjected to Prenatal Hypoxia.

The protective effects of recombinant human lactoferrin rhLF (branded "CAPRABEL™") on the cognitive functions of rat offspring subjected to prenatal hypoxia (7% O2, 3 h, 14th day of gestation) have been analyzed. About 90% of rhLF in CAPRABEL was iron-free (apo-LF). Rat dams received several injections of 10 mg of CAPRABEL during either gestation (before and after the hypoxic attack) or lactation. Western blotting revealed the appearance of erythropoietin (EPO) alongside the hypoxia-inducible factors (HIFs) in organ homogenates of apo-rhLF-treated pregnant females, their embryos (but not placentas), and in suckling pups from the dams treated with apo-rhLF during lactation. Apo-rhLF injected to rat dams either during pregnancy or nurturing the pups was able to rescue cognitive deficits caused by prenatal hypoxia and improve various types of memory both in young and adult offspring when tested in the radial maze and by the Novel Object Recognition (NOR) test. The data obtained suggested that the apo-form of human LF injected to female rats during gestation or lactation protects the cognitive functions of their offspring impaired by prenatal hypoxia.

Caspase Inhibition Restores NEP Expression and Rescues Olfactory Deficit in Rats Caused by Prenatal Hypoxia.

Development of the olfactory system begins early in embryogenesis and is important for the survival of new-borns in postnatal life. Olfactory malfunction in early life disrupts development of behavioural patterns while with ageing manifests development of neurodegenerative disorders. Previously, we have shown that prenatal hypoxia in rats leads to impaired olfaction in the offspring and correlates with reduced expression of a neuropeptidase neprilysin (NEP) in the brain structures involved in processing of the olfactory stimuli. Prenatal hypoxia also resulted in an increased activity of caspases in rat brain and its inhibition restored NEP content in the brain tissue and improved rat memory. In this study, we have analysed effects of intraventricular administration of a caspase inhibitor Ac-DEVD-CHO on NEP mRNA expression, the number of dendritic spines and olfactory function of rats subjected to prenatal hypoxia on E14. The data obtained demonstrated that a single injection of the inhibitor on P20 restored NEP mRNA levels and number of dendritic spines in the entorhinal and parietal cortices, hippocampus and rescued rat olfactory function in food search and odour preference tests. The data obtained suggest that caspase activation caused by prenatal hypoxia contributes to the olfactory dysfunction in developing animals and that caspase inhibition restores the olfactory deficit via upregulating NEP expression and neuronal networking. Because NEP is a major amyloid-degrading enzyme, any decrease in its expression and activity not only impairs brain functions but also predisposes to accumulation of the amyloid-ß peptide and development of neurodegeneration characteristic of Alzheimer's disease.

Angiotensin-converting enzyme 2 (ACE2): Two decades of revelations and re-evaluation.

Angiotensin-converting enzyme-2, or ACE2, is primarily a zinc-dependent peptidase and ectoenzyme expressed in numerous cell types and functioning as a counterbalance to ACE in the renin-angiotensin system. It was discovered 21 years ago more than 40 years after the discovery of ACE itself. Its primary physiological activity is believed to be in the conversion of angiotensin II to the vasodilatory angiotensin-(1-7) acting through the Mas receptor. As such it has been implicated in numerous pathological conditions, largely in a protective mode which has led to the search for ACE2 activatory mechanisms. ACE2 has a diverse substrate specificity allowing its participation in multiple peptide pathways. It also regulates aspects of amino acid transport through its homology with a membrane protein, collectrin. It also serves as a viral receptor for the SARS virus, and subsequently SARS-CoV2, driving the current COVID-19 pandemic. ACE2 therefore provides a therapeutic target for the treatment of COVID and understanding the biological events following viral binding can provide insight into the multiple pathologies caused by the virus, particularly inflammatory and vascular. In part this may relate to the ability of ACE2, like ACE, to be shed from the cell membrane. The shed form of ACE2 (sACE2) may be a factor in determining susceptibility to certain COVID pathologies. Hence, for just over 20 years, ACE2 has provided numerous surprises in the field of vasoactive peptides with, no doubt, more to come but it is its central role in COVID pathology that is producing the current intense interest in its biology.

Effect of Global Brain Ischemia on Amyloid Precursor Protein Metabolism and Expression of Amyloid-Degrading Enzymes in Rat Cortex: Role in Pathogenesis of Alzheimer's Disease.

The incidence of Alzheimer's disease (AD) increases significantly following chronic stress and brain ischemia which, over the years, cause accumulation of toxic amyloid species and brain damage. The effects of global 15-min ischemia and 120-min reperfusion on the levels of expression of the amyloid precursor protein (APP) and its processing were investigated in the brain cortex (Cx) of male Wistar rats. Additionally, the levels of expression of the amyloid-degrading enzymes neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and insulin-degrading enzyme (IDE), as well as of some markers of oxidative damage were assessed. It was shown that the APP mRNA and protein levels in the rat Cx were significantly increased after the ischemic insult. Protein levels of the soluble APP fragments, especially of sAPPß produced by ß-secretase, (BACE-1) and the levels of BACE-1 mRNA and protein expression itself were also increased after ischemia. The protein levels of APP and BACE-1 in the Cx returned to the control values after 120-min reperfusion. The levels of NEP and ECE-1 mRNA also decreased after ischemia, which correlated with the decreased protein levels of these enzymes. However, we have not observed any changes in the protein levels of insulin-degrading enzyme. Contents of the markers of oxidative damage (di-tyrosine and lysine conjugates with lipid peroxidation products) were also increased after ischemia. The obtained data suggest that ischemia shifts APP processing towards the amyloidogenic ß-secretase pathway and accumulation of the neurotoxic Aß peptide as well as triggers oxidative stress in the cells. These results are discussed in the context of the role of stress and ischemia in initiation and progression of AD.

Developmental Profile of Brain Neprilysin Expression Correlates with Olfactory Behaviour of Rats.

A neuropeptidase, neprilysin (NEP), is a major amyloid (Aß)-degrading enzyme involved in the pathogenesis of Alzheimer's disease (AD). The olfactory system is affected early in AD with characteristic Aß accumulation, but data on the dynamics of NEP expression in the olfactory system are absent. Our study demonstrates that NEP mRNA expression in rat olfactory bulbs (OB), entorhinal cortex (ECx), hippocampus (Hip), parietal cortex (PCx) and striatum (Str) increases during the first postnatal month being the highest in the OB and Str. By 3 months, NEP mRNA levels sharply decrease in the ECx, Hip and PCx and by 9 months in the OB, but not in the Str, which correlates with declining olfaction in aged rats tested in the food search paradigm. One-month-old rats subjected to prenatal hypoxia on E14 had lower NEP mRNA levels in the ECx, Hip and PCx (but not in the OB and Str) compared with the control offspring and demonstrated impaired olfaction in the odour preference and food search paradigms. Administration to these rats of a histone deacetylase inhibitor, sodium valproate, restored NEP expression in the ECx, Hip and PCx and improved olfaction. Our data support NEP involvement in olfactory function.

Age-Dependent Electrocorticogram Dynamics and Epileptogenic Responsiveness in Rats Subjected to Prenatal Hypoxia.

Using electrocorticogram (ECoG) analysis, we compared age-related dynamics of general neuronal activity and convulsive epileptiform responsiveness induced by intracortical microinjections of 4-aminopyridine (4-AP) in control Wistar rats and those subjected to prenatal hypoxia (Hx; E14; 7% O2, 3 h). The studies were carried out in three age periods roughly corresponding to childhood (P20-27), adolescence (P30-45), and adulthood (P90-120). It was found that in the process of postnatal development of the control rats, the peak of the ECoG power spectrum density (PSD) of the theta rhythm during wakefulness shifted from the low to the higher frequency, while in the Hx rats this shift had the opposite direction. Moreover, the Hx rats had different frequency characteristics of the ECoG PSD and longer episodes of spike-and-wave discharges caused by 4-AP injections compared to the controls. The total ECoG PSD of slow-wave sleep (1-5 Hz) was also dramatically decreased in the process of development of the Hx rats. Such alterations in PSD could be explained by the changes in balance of the excitation and inhibition processes in the cortical networks. Analyzing protein levels of neurotransmitter transporters in the brain structures of the Hx rats, we found that the content of the glutamate transporter EAAT1 was higher in the parietal cortex in all age groups of Hx rats while in the hippocampus it decreased during postnatal development compared to controls. Furthermore, the content of the vesicular acetylcholine transporter in the parietal cortex, and of the inhibitory GABA transporter 1 in the hippocampus, was also affected by prenatal Hx. These data suggest that prenatal Hx results in a shift in the excitatory and inhibitory balance in the rat cortex towards excitation, making the rat's brain more vulnerable to the effects of proconvulsant drugs and predisposing animals to epileptogenesis during postnatal life.

Targeting amyloid clearance in Alzheimer's disease as a therapeutic strategy.

Targeting the amyloid-ß (Aß) peptide cascade has been at the heart of therapeutic developments in Alzheimer's disease (AD) research for more than 25 years, yet no successful drugs have reached the marketplace based on this hypothesis. Nevertheless, the genetic and other evidence remains strong, if not overwhelming, that Aß is central to the disease process. Most attention has focused on the biosynthesis of Aß from its precursor protein through the successive actions of the ß- and γ-secretases leading to the development of inhibitors of these membrane proteases. However, the levels of Aß are maintained through a balance of its biosynthesis and clearance, which occurs both through further proteolysis by a family of amyloid-degrading enzymes (ADEs) and by a variety of transport processes. The development of late-onset AD appears to arise from a failure of these clearance mechanisms rather than by overproduction of the peptide. This review focuses on the nature of these clearance mechanisms, particularly the various proteases known to be involved, and their regulation and potential as therapeutic targets in AD drug development. The majority of the ADEs are zinc metalloproteases [e.g., the neprilysin (NEP) family, insulin-degrading enzyme, and angiotensin converting enzymes (ACE)]. Strategies for up-regulating the expression and activity of these enzymes, such as genetic, epigenetic, stem cell technology, and other pharmacological approaches, will be highlighted. Modifiable physiological mechanisms affecting the efficiency of Aß clearance, including brain perfusion, obesity, diabetes, and sleep, will also be outlined. These new insights provide optimism for future therapeutic developments in AD research. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

Role of Prenatal Hypoxia in Brain Development, Cognitive Functions, and Neurodegeneration.

This review focuses on the role of prenatal hypoxia in the development of brain functions in the postnatal period and subsequent increased risk of neurodegenerative disorders in later life. Accumulating evidence suggests that prenatal hypoxia in critical periods of brain formation results in significant changes in development of cognitive functions at various stages of postnatal life which correlate with morphological changes in brain structures involved in learning and memory. Prenatal hypoxia also leads to a decrease in brain adaptive potential and plasticity due to the disturbance in the process of formation of new contacts between cells and propagation of neuronal stimuli, especially in the cortex and hippocampus. On the other hand, prenatal hypoxia has a significant impact on expression and processing of a variety of genes involved in normal brain function and their epigenetic regulation. This results in changes in the patterns of mRNA and protein expression and their post-translational modifications, including protein misfolding and clearance. Among proteins affected by prenatal hypoxia are a key enzyme of the cholinergic system-acetylcholinesterase, and the amyloid precursor protein (APP), both of which have important roles in brain function. Disruption of their expression and metabolism caused by prenatal hypoxia can also result, apart from early cognitive dysfunctions, in development of neurodegeneration in later life. Another group of enzymes affected by prenatal hypoxia are peptidases involved in catabolism of neuropeptides, including amyloid-ß peptide (Aß). The decrease in the activity of neprilysin and other amyloid-degrading enzymes observed after prenatal hypoxia could result over the years in an Aß clearance deficit and accumulation of its toxic species which cause neuronal cell death and development of neurodegeneration. Applying various approaches to restore expression of neuronal genes disrupted by prenatal hypoxia during postnatal development opens an avenue for therapeutic compensation of cognitive dysfunctions and prevention of Aß accumulation in the aging brain and the model of prenatal hypoxia in rodents can be used as a reliable tool for assessment of their efficacy.

Role of Ageing and Oxidative Stress in Regulation of Amyloid-Degrading Enzymes and Development of Neurodegeneration.

The accumulation of cerebral amyloid ßpeptide (Aß) is a key precipitating factor for neuronal cell death in Alzheimer's Disease (AD). However, brain Aß levels are modifiable since there is a balance between its formation from the Amyloid Precursor Protein (APP) and its removal by clearance mechanisms, which can be either through proteolysis or by protein binding and subsequent transport). Among the major enzymes degrading brain Aß are several zinc-proteases: neprilysin (NEP), its homologues NEP2 and the Endothelin Converting Enzymes (ECE-1 and -2) and also the Insulin-Degrading Enzyme (IDE). During the ageing process, and under certain pathological conditions (e.g. ischemia and stroke), the expression and activity of these enzymes decline, which leads to a deficit of Aß clearance and its accumulation in the brain. Some of these changes in the enzyme properties are due to their reduced expression and/or structural modification by reactive oxygen species. In this review paper we shall discuss some mechanisms of regulation of Amyloid-Degrading Enzymes (ADEs) and possible therapeutic approaches which might prevent their decline with age and after pathology.

Reflections on 60 years of publication of the Journal of Neurochemistry.

This review reflects on the origins, development, publishing trends, and scientific directions of the Journal of Neurochemistry over its 60 year lifespan as seen by key contributors to the Journal's production. The Journal first appeared in May 1956 with just two issues published in that inaugural year. By 1963, it appeared monthly and, by 2002, 24 hard copy issues were published yearly. In 2014, the Journal became online only. For much of its time, the Journal was managed through two separate editorial offices each with their respective Chief Editor (the 'Western' and 'Eastern' hemispheres). The Journal was restructured to operate through a single editorial office and Editor-in-Chief from 2013. Scientifically, the Journal progressed through distinct scientific eras with the first two decades generally centered around developments in methodology followed by a period when publications delved deeper into underlying mechanisms. By the late 1980s, the Journal had entered the age of genetics and beyond, with an increasing focus on neurodegenerative diseases. Reviews have played a regular part in the success of J Neurochem with focused special and virtual issues being a highlight of recent years. Today, 60 years and onwards, J Neurochem continues to be a leading source of top-quality, original and review articles in neuroscience. We look forward to its continued success at the forefront of neurochemistry in the decades to come. This article celebrates 60 years of publication of Journal of Neurochemistry including personal reminiscences from some of the Chief Editors, past and present, as well as input from some of the key contributors to the Journal over this period. We highlight the scientific, technological, and publishing developments along the way, with reference to key papers published in the Journal. The support of the Journal toward the aims and objectives of the International Society for Neurochemistry (ISN) is also emphasized. This article is part of the 60th Anniversary special issue.

AChE and the amyloid precursor protein (APP) - Cross-talk in Alzheimer's disease.

The amyloid precursor protein (APP) and acetylcholinesterase (AChE) are multi-faceted proteins with a wide range of vital functions, both crucially linked with the pathogenesis of Alzheimer's disease (AD). APP is the precursor of the Aß peptide, the pathological agent in AD, while AChE is linked to its pathogenesis either by increasing cholinergic deficit or exacerbating Aß fibril formation and toxicity. As such, both proteins are the main targets in AD therapeutics with AChE inhibitors being currently the only clinically available AD drugs. In our studies we have demonstrated an important inter-relation in functioning of these proteins. Both can be released from the cell membrane and we have shown that AChE shedding involves a metalloproteinase-mediated mechanism which, like the α-secretase dependent cleavage of APP, is stimulated by cholinergic agonists. Overexpression of the neuronal specific isoform APP695 in neuronal cells substantially decreased levels of the AChE mRNA, protein and catalytic activity accompanied by a similar decrease in mRNA levels of the AChE membrane anchor, PRiMA (proline rich membrane anchor). We further established that this regulation does not involve APP processing and its intracellular domain (AICD) but requires the E1 region of APP, specifically its copper-binding domain. On the contrary, siRNA knock-down of APP in cholinergic SN56 cells resulted in a significant upregulation of AChE mRNA levels. Hence APP may influence AChE physiology while released AChE may regulate amyloidogenesis through multiple mechanisms suggesting novel therapeutic targets.

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

Currently, deficit of amyloid ß-peptide (Aß) 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). Aß clearance can involve either specific proteases present in the brain or Aß-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 Aß-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.

Hypoxia Affects Neprilysin Expression Through Caspase Activation and an APP Intracellular Domain-dependent Mechanism.

While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid ß-peptide (Aß) in the brain causing neurodegeneration and familial Alzheimer's disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aß in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aß production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aß-degrading enzyme neprilysin (NEP) which might deregulate Aß clearance. Aß itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment (CTF) termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8, and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischaemia leading to a deficit of Aß clearance and increasing the risk of development of AD.

Effects of ageing and experimental diabetes on insulin-degrading enzyme expression in male rat tissues.

Due to an increasing life expectancy in developing countries, cases of type 2 diabetes and Alzheimer's disease (AD) in the elderly are growing exponentially. Despite a causative link between diabetes and AD, general molecular mechanisms underlying pathogenesis of these disorders are still far from being understood. One of the factors leading to cell death and cognitive impairment characteristic of AD is accumulation in the brain of toxic aggregates of amyloid-ß peptide (Aß). In the normally functioning brain Aß catabolism is regulated by a cohort of proteolytic enzymes including insulin-degrading enzyme (IDE) and their deficit with ageing can result in Aß accumulation and increased risk of AD. The aim of this study was a comparative analysis of IDE expression in the brain structures involved in AD, as well as in peripheral organs (the liver and kidney) of rats, during natural ageing and after experimentally-induced diabetes. It was found that ageing is accompanied by a significant decrease of IDE mRNA and protein content in the liver (by 32 and 81%) and brain structures (in the cortex by 58 and 47% and in the striatum by 53 and 68%, respectively). In diabetic animals, IDE protein level was increased in the liver (by 36%) and in the striatum (by 42%) while in the brain cortex and hippocampus it was 20-30% lower than in control animals. No significant IDE protein changes were observed in the kidney of diabetic rats. These data testify that ageing and diabetes are accompanied by a deficit of IDE in the brain structures where accumulation of Aß was reported in AD patients, which might be one of the factors predisposing to development of the sporadic form of AD in the elderly, and especially in diabetics.