Yu. F. Pastukhov

Exogenous protein HSP70 blocks neurodegeneration in the rat model of the clinical stage of Parkinson’s disease

According to modern views, impairment of protein folding is a serious danger for cell survival and a molecular basis for neurodegeneration (1, 2). A decrease in the activity of the ubiquitin proteasome system, which is responsible for disposal of misfolded proteins, may cause accumulation of toxic oligomers of α synuclein and degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars com� pacta (SNpc) and their axons in the striatum. This mechanism plays the key role in the pathogenesis of Parkinsons disease (PD) (3-6). This disease still has no cure due to commonly late diagnosis and symp� tomatic treatment, which cannot stop neuron death (7, 8). In recent years, the use of chaperones, particu� larly heat shock proteins of the 70�kDa family (HSP70), which are able to recover misfolded pro� teins, is considered as a promising therapeutic strategy, including treatment of PD (9-13). Neurons of SNpc during a moderate decrease in proteasome function survive mainly when they have increased content of inducible chaperone Hsp70i (14). The protective effects of exogenous Hsp70i in PD have not been stud� ied. The aim of this study was to investigate if intrana� sal administration of recombinant Hsp70i could atten� uate neurodegeneration and reduce motor impair� ment in the model of the clinical stage of PD. The rat model of a prolonged clinical stage of PD (up to 21 days) was created using the modern views on the molecular mechanisms of neurodegeneration by diminishing the proteasome activity in the nigrostri� atal system. The protective effects of exogenous Hsp70 were evaluated in this model. We discovered for the first time that intranasal administration of recombi� nant Hsp70 in the dynamics of clinical stage reduced the loss of DAergic neurons in SNpc by a factor of two and recovered motor functions. Our results can be used for further clinical study of chaperones drugs and their inductors elevating the Hsp70 level in the brain to develop methods of preventive treatment and to search new ways of blocking neurodegeneration at the clinical stage of PD. These results can be considered to be a priority. The study was carried out on male Wistar rats

Lipopolysaccharide-Free 70-kDa Heat Shock Protein Has Hypotherimic and Somnogenic Effects

The intracellular expression of 70-kDa heat-shockprotein (HSP70) is one of the most important molecularmechanisms that protect cells and various organisms(from bacteria to humans) from heat and many otherstresses (see [1, 2] for review). After the appearance ofexogenous HSP70 preparations (both natural andrecombinant), the development of new HSP70-baseddrugs is attracting much attention. However, the thera-peutic potential of HSP70 has not been evaluated thusfar, because some problems have not been solved. First,

Chaperone Hsp70 Content in Dopaminergic Neurons of the Substantia Nigra Increases in Proteasome Dysfunction

Decreases in the activity of the ubiquitin-proteasome system (UPS), which uses up to 90% of cell protein, are regarded as a key mechanism in the development of age-related conformational diseases (Parkinson’s disease (PD), Alzheimer’s disease, and others). Studies in a model of the preclinical stage of PD in Wistar rats showed that the specific UPS inhibitor lactacystin induced degeneration of 24% of dopaminergic neurons in the compact zone of the substantia nigra (czSN), which was almost the same as the proportion of neurons (23%) which in control conditions did not contain the chaperone 70-kDal Heat Shock Protein (Hsp70), which has neuroprotective properties. Of the 77% of neurons which contained Hsp70 in control conditions, 15% lost it in response to lactacystin (which may reduce their resistance to neurotoxin). However, 62% of surviving dopaminergic neurons in the czSN showed a 47% increase in Hsp70 content (which may protect them from degeneration). The increase in Hsp70 content (in czSN tissue) was verified by immunoblotting. Confocal microscopy studies identified partial colocalization of Hsp70 with the key dopamine (DA) synthesis enzyme tyrosine hydroxylase. Thus, moderate weakening of UPS function in czSN, typical of this model of the preclinical stage of PD, was characterized by an increase in the activity of the chaperone system. It is suggested that this process is directed to restoring UPS activity and preserving the dopaminergic neuron population.

Effects of Quercetin on the Severity of Chemically Induced Convulsions and 70-kDal Heat Shock Protein Content in Brain Structures in Rats

Behavioral methods were used to study the effects of the 70-kDal heat shock protein (Hsp70) expression inhibitor quercetin on convulsions and motor disorders evoked by N-methyl-D-aspartic acid (N-methyl- D-aspartate (NMDA)) or corazol in adult Wistar rats. These experiments showed that intraperitoneal administration of quercetin 4 h before microinjection of NMDA into the third ventricle of the brain increased the duration of the toxic component of convulsive seizures and the severity of convulsions and ataxic symptoms. The same dose and route of quercetin increased the duration of clonic and tonic convulsions but did not alter the severity of convulsive seizures or ataxia symptoms induced by intraperitoneal injection of corazol. Immunoblotting studies showed that administration of quercetin decreased the content of the inducible form of Hsp70 in the hippocampus, thalamus, and corpus callosum. These data provide evidence that the Hsp70 expression inhibitor quercetin has anticonvulsant properties. It is suggested that Hsp70 has a role in the central mechanisms regulating behavioral convulsions and motor disorders induced by NMDA and corazol in rats.

Microinjection of 70-kDal heat shock protein into the oral reticular nucleus of the pons suppresses rapid eye movement sleep in pigeons

Previous studies have demonstrated that increases in the duration of slow-wave sleep and decreases in somatovisceral measures in response to microinjections of 70-kDal heat shock protein (Hsp70) into the third ventricle in pigeons may be due to activation of GABAA receptors in the preoptic area of the hypothalamus. With the aim of identifying the transmitter mechanisms whose activation is temporally (2–3 h) linked with suppression of rapid eye movement sleep, the present studies were based on injection of Hsp70 into the oral reticular pontine nucleus (nucleus reticularis pontis oralis, NRPO), whose cholinergic neurons are critical for generating rapid eye movement sleep. Hsp70 was found to induce earlier (within the first 2 h) decreases in the number of episodes and the total duration of rapid eye movement sleep, with decreases in electroencephalogram (EEG) spectral power in the range 9–14 Hz, the level of muscle contractile activity, and brain temperature. It is hypothesized that the effects of Hsp70 are mediated by activation of GABAA receptors in the NRPO, evoking suppression of the cholinergic mechanisms initiating rapid eye movement sleep. The increase in the total duration of slow-wave sleep occurring with a long latent period (8–12 h after injection of Hsp70 into the NRPO) may be due to the influence of Hsp70 on the population of neurons responsible for maintaining slow-wave sleep outside the NRPO.

Participation of GABAergic Mechanisms of Hypothalamus Ventrolateral Preoptic Area in Regulation of Sleep and Wakefulness and Temperature Homeostasis in the Pigeon Columba livia

A comparative analysis of awakening, somnogenic, and thermoregulating effects of agonists and antagonists of GABAA,B-receptors after microinjections to neuronal populations of ventrolateral preoptic area (VLPA) of hypothalamus was carried out for the first time in representatives of the avian class (pigeon). It has bee established that: (1) VLPA of hypothalamus contains populations of neurons differing by their function and representation of receptor types and participating in control of wakefulness, sleep, and thermoregulation; (2) executive GABAA-ergic mechanisms of maintenance of the slow-wave sleep (SWS) are located predominantly in the caudal part of the hypothalamic VLPA; (3) GABAA,B-ergic mechanisms of control of thermal homeostasis are located predominantly in the caudal part of VLPA. It is suggested that the maintenance of SWS depends on an increase of activity of inhibitory GABAergic VLPA mechanisms leading to inactivation of neuronal networks of wakefulness outside VLPA and on a reduction of activity of the stimulatory aminergic systems present in the preoptic area and outside it.

Impairment of non-associative learning in a rat experimental model of preclinical stage of Parkinson’s disease

An experimental model of the preclinical stage of Parkinson’s disease was induced by double intranasal administration of the proteasome inhibitor lactacystin. The results demonstrated signs of cognitive impairments expressed as impaired non-associative learning. This was related to degeneration of one-third of dopaminergic neurons in the ventral tegmental area of the midbrain and their axons in the dorsolateral prefrontal cortex. Impairment of non-associative learning may be an early non-motor marker of Parkinson’s disease indicating the start of neurodegenerative processes in the dopaminergic mesocortical system of the brain.

[SLOW-WAVE SLEEP AND MOLECULAR CHAPERONES].

Since long ago, one of the most vital issues mankind is concerned about is why spending almost one-third of human lives for sleep. This review addresses the major function of slow-wave sleep (SWS) and molecular mechanisms of its regulation. The main conclusions are presented below as the following generalizations and hypotheses. 1. SWS performs an energy-conserving function which developed parallel to the evolution of tachimetabolism and endothermy/homoiothermy. 2. Most significant reduction in the brain energy demands during deep SWS, characterized by increased EEG delta power, creates optimal conditions for the enhancement of anabolic processes and actualization of the major biological function of sleep—accelerating protein synthesis in the brain. 3. Conditions of paradoxical sleep (PS) as an “archeowakefulness”, containing the elements of endogenous stress, seem acceptable for chaperone expression required to fix misfolded proteins synthesized de novo during deep SWS. 4. Close integration of the HSP70 and HSP40 molecular systems, contained in the sleep center of the preoptic area of the hypothalamus, and their compensatory interrelationship contribute significantly to the maintenance of sleep homeostasis and implementation of its functions under non-stress conditions and during a long-term chaperone deficiency intrinsic to ageing and varied neuropathologies. 5. Cyclic changes in the protein synthesis rate (during deep SWS) and HSP70 chaperone expression (during wakefulness and, probably, PS), which occur on a daily basis throughout the entire lifetime, are critical for all vital functions of homeothermic organisms, including recovery of the nervous system structure and functions.

Changes in sleep characteristics of rat preclinical model of Parkinson’s disease based on attenuation of the ubiquitin—proteasome system activity in the brain

Numerous experimental and epidemiological data indicate a high significance of environmental neurotoxins, specifically, inhibitors of the ubiquitin–proteasome system, in pathogenesis of Parkinson’s disease (PD). To develop a preclinical model of PD in rats we used a technique of intranasal administration of lactacystin, a natural proteasome inhibitor, into the brain. It was found that three weeks after the first lactacystin administration it induced a little degeneration of dopaminergic neurons in the olfactory bulb and substantia nigra pars compacta without any olfactory dysfunction and motor behavior disorders. Besides, its effect led to the appearance of some signs of sleep disorders: increased somnolence (especially in the dark, active daily phase), fragmentation of slow-wave sleep, decreased EEG delta rhythm during slow-wave sleep. These signs share some similarity with PD and could be useful in clinical studies for the quick search for polysomnographic markers of the early PD stage.

The role of adenosine A 2A receptors of the preoptic area in somnogenic activity of 70 kDa protein in pigeons

The effects of adenosine A2A receptor antagonist microinjections into the hypothalamic ventrolateral preoptic area (VLPA) on the natural sleep-wake cycle and somnogenic activity of a 70 kDa heat shock protein (Hsp70) are analyzed electrophysiologically in birds, specifically, pigeons (Columba livia) for the first time. We report that: (1) at the beginning of the rest period, microinjections of 8-(3-Chlorostyryl)caffeine (ChC), an adenosine A2A receptor antagonist, into VLPA increase wake duration and inhibited sleep in a dose-dependent manner; (2) Hsp70 injections into VLPA evoke the somnogenic effect manifested as an increase in total slow-wave sleep (SWS) duration and enhancement of the mechanisms responsible for SWS triggering and maintenance; (3) ChC blockade of adenosine A2A receptors inhibits Hsp70-induced SWS. These data suggest that adenosine A2A receptors located in VLPA are involved in the modulation of the natural sleep-wake cycle and promotion of the Hsp70 somnogenic effect in pigeons. It is hypothesized that the somnogenic effect of Hsp70 is mediated by the modulating influence of this chaperone on the functioning of adenosine receptor proteins.