Yu. V. Lyupina

Proteasomes in the brain of β2-microglobulin knockout mice

MHC class I molecules play an important role in synaptic plasticity of the mammalian nervous system. Proteolytic complexes (proteasomes) produce oligopeptides that are presented on cell surfaces in complexes with MHC class I molecules and regulate many cellular processes beside this. The goal of the present work was to study peculiarities in functioning of proteasomes and associated signaling pathways along with evaluation of NeuN and gFAP expression in different sections of the brain in mice with knockout of β2-microglobulin, a constituent of MHC class I molecules. It was found that the frontal cortex and the brainstem, structures with different ratio of NeuN and gFAP expression, are characterized by opposite changes in the proteasome pool under constant total proteasome levels in B2m-knockout mice in comparison with those in control animals. ChTL-activity as well as expression of LMP7 immune subunit and PA28 regulator of proteasomes was elevated in the cortex of B2m-knockout mice, while these indicators were decreased in the brainstem. The concentrations of the signaling molecules nNOS and HSP70 in B2m-knockout mice were increased in the cortex, while being decreased in the brainstem, and this indicates the possibility of control of expression of the LMP7 subunit and the regulator PA28 by these molecules. Changes in the proteasome pool observed in striatum of B2m-knockout mice are similar to those observed in the brainstem. At the same time, the cerebellum is characterized by a specific pattern of proteasome functioning in comparison with that in all other brain structures. In cerebellum the expression of immune subunits LMP7 and LMP2 and the regulator PA28 was increased, while expression of regulator PA700 was decreased. Deficiency of NeuN and gFAP was revealed in most brain compartments of B2m-knockout mice. Thus, increased expression of the above-mentioned immune subunits and the proteasome regulator PA28 in the cortex and cerebellum may compensate disturbances revealed in the brain structures and the absence of MHC class I molecules. Apparently, this promotes production of peptides necessary for cell-to-cell interactions and maintains nervous system plasticity in B2m-knockout mice.

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits during embryonic and early postnatal development of rat spleen and liver was studied in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The distribution of LMP7 and LMP2 immune subunits in spleen and liver cells was also evaluated throughout development. The common tendency of both organs to increase the expression of both LMP7 and LMP2 subunits on the 21st postnatal day (P21) was found. However, the total proteasome level was shown to be constant. At certain developmental stages, the dynamics of immune subunits expression in the spleen and liver was different. While the gradual enhancement of both immune subunits was observed on P1, P18 and P21 in the spleen, the periods of gradual increase observed on E16 (the 16th embryonic day) and E18 gave way to a period of decrease in immune subunits on P5 in the liver. This level did not reliably change until P18 and increased on P21. The revealed changes were accompanied by an increase in chymotrypsin-like activity and a decrease in caspase-like activity in the spleen at P21 compared to the embryonic period. This indicates the increase in proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased compared to the embryonic period by P21. The dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Moreover, it was discovered that the increase in the expression of immune subunits during early spleen development is associated with the process of formation of white pulp by B- and T-lymphocytes enriched with immune subunits. In the liver, the increase in the level of immune subunits by P21 was also accompanied by an increase of their expression in hepatocytes. While the decrease of their level by P5 may be associated with the fact that the liver has lost its function as the primary lymphoid organ in the immune system by this time, as well as with the disappearance of B-lymphocytes enriched with immune proteasomes. In the spleen and the liver, MHC class I molecules were found during the periods of increased levels of proteasome immune subunits. On E21, the liver was enriched with neuronal nitric oxide synthase (nNOS); the level of nNOS decreased after birth and then increased by P18. This fact indicates the possibility of the induction of expression of the LMP7 and LMP2 immune subunits in hepatocytes via a signaling pathway involving nNOS. These results indicate that compared to the rat liver cells, splenic T cell immune response develops in rats starting around P19–P21. First, a T-area of white pulp is formed in the spleen during this period. Second, an increased level of immune proteasomes and MHC class I molecules in hepatocytes can ensure the formation of antigenic epitopes from foreign proteins and their delivery to the cell surface for subsequent presentation to cytotoxic T-lymphocytes.

Changes in the proteasome function after induction of donor-specific tolerance in rats with ovarian allograft

Induction of donor-specific tolerance in a recipient is one of the methods for enhancing acceptance of the grafts of endocrine glands in the absence of immunodepressants, which interfere with hormone production. This paper describes changes in the proteasome pool in the rat liver, spleen, and graft during the development of donor-specific tolerance after intraportally infusing the recipient with donor splenocytes with subsequent allografting of ovarian tissue into the renal capsule. It has been demonstrated that the shift in the balance in the liver and graft proteasome pools towards the variants with the LMP2 subunit determines the development of immunological tolerance and graft retention. On the contrary, an increase in the forms with the LMP7 subunit induces the immune response and graft rejection.

Proteasomes on thyroid tissue allotransplantation under induction of donor-specific tolerance in rats

Proteasomes in the liver of August rats (RT1c) were investigated 30 days after allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donor-specific tolerance by donor splenocyte intraportal administration. The levels of total proteasome pool, immune proteasomes containing subunits LMP2 and/or LMP7, and proteasome regulators 19S and 11S were defined. Intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups, which indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of nontolerant rats compared to tolerant animals. In the liver of tolerant rats with accepted grafts, the number of mononuclear cells expressing the immune subunit LMP2 greatly increased in comparison with control and nontolerant animals. Study of accepted grafts showed an increase in the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them, compared to the tissue replacing the rejected grafts. Immune proteasomes were almost completely absent from the control intact thyroid tissue, while 19S/11S ratio was maximal in it. Thus, the development of the immune reaction or its suppression are accompanied by a change in the balance between different proteasome forms. Immune subunit LMP7 and 11S regulator are associated with the response against donor tissue. On the contrary, immune subunit LMP2 and 19S regulator are likely to be important for the development of immune tolerance and surviving tissue functioning. Immunofluorescence assay revealed a low content of the immune proteasomes in the follicle cells. Probably, formation of antigens for the major histocompatibility complex class I molecules was impaired by the low content of immune proteasomes, which led to immunological tolerance of hormone-producing follicle cells.

Effects of Acute and Chronic Caffeine Intake on Intravenous Self-Administration of Morphine in Two Rat Strains

Effects of acute and chronic caffeine intake on the level and pattern of morphine self-administration behavior in WAG/G and Fisher-344 rats were studied. Both acute and chronic caffeine intake decreased morphine self-administration only in WAG/G rats, which attested to increased sensitivity of these rats to reinforcing effects of morphine. Possible relationship between the observed changes and increased anxiety in rats receiving caffeine is discussed.

Native structure of rat liver immune proteasomes

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of “rotated” ubiquitinated proteins.

Distinctive features of immune proteasome expression during the development of the central nervous system in rats

Formation of the central nervous system in ontogeny and its functioning in adult mammals are controlled by the universal ubiquitin-proteasome proteolytic system. The aim of the present work was to study the dynamics of immune proteasome expression as compared with the dynamics of chymotrypsin-like and caspase-like activities of the proteasome (ChLA and CLA) and expression of the transcription factor Zif268 in selected brain structures (cortex, hippocampus, and brainstem) in embryonic (days E19 and E21 of embryonic development) and early postnatal (postnatal days P1, P3, P4, P5, P7, and P15) development in rats. ChLA and CLA were determined in clarified homogenates of rat brain structures using commercial fluorogenic oligopeptide substrates Suc-LLVY-AMC and Z-LLG-AMC, respectively. Levels of constitutive (β1 and β5) and immune (LMP7 and LMP2) proteasome subunits, as well as those of proteasome activators PA700 and PA28 and the transcription factor Zif268, were evaluated by Western blotting. Increased expression of the immune subunit LMP7 in the cerebral cortex and hippocampus was observed during the period of active formation of the biochemical mediator structure of neurons (P7-P15). Increased ChLA and CLA were registered in the cortex during this period. The content of the immune subunit LMP2 was significantly elevated in all brain structures between P7 and P15. The content of the constitutive proteolytic subunit β1 in all structures was lower on P4 than on P1 and increased to the same level as on P1 by P15. However, the expression level of the constitutive proteolytic β5 subunit of the proteasome in the cortex, hippocampus, and brainstem increased starting from E21 and reached maximal values at P3, P5 and P1, respectively, with a sharp decline in all structures by P7. Expression of the immune subunit LMP2 and the constitutive subunit β1 in all structures increased sharply around P15 concomitantly to the increase in the content of the immune subunit LMP7. Moreover, a positive correlation of increased expression of the activator PA28 and the constitutive β5 subunit in the hippocampus during P3-P5 and in the brain stem during P1-P5 has been demonstrated. Elevation of proteasome CLA and expression levels of immune proteasome subunits LMP2 and LMP7 during days P7-P15 of postnatal development in the brain regions investigated were related to the expression of the transcription factor Zif268. Immune proteasomes probably play an important role in the regulation of key biochemical processes during early ontogenesis of the central nervous system and are necessary for the emergence and maintenance of synaptic plasticity in rat brain structures analyzed.

Changes in the Expression of Immune Proteasomes in the Liver after the Induction of Portal Tolerance Depending on Donor—Recipient Differences in Rats

Induction of donor-specific tolerance in outbred Wistar rats (RT1u) and inbred August rats (RT1c) increased the expression of immune proteasome subunits in liver with a peak on day 7 after beginning of the induction. The increase in the level of immune subunits LMP2 and LMP7 was more pronounced in the liver of August rats in comparison with Wistar rats (by 2 and 6 times, respectively), which was associated with higher concentrations of monoamines in the CNS of August rats. After induction of donor-specific tolerance in August and Wistar rats, the immune subunits were in cells of sinusoidal lining and in cells located in sinusoid lumens. It can be suggested that immune proteasomes in these cells producing antigenic peptides for presentation to immunocompetent participate in the suppression of their activity and form the molecular basis for the development of donor-specific tolerance at very early stages of this process.

Proteasomes and transplantology: Current state of the problem and the search for promising trends

Basic research of the structure and functions of multiple forms of proteasomes is overviewed. The proteasomes of liver, acting as a primary lymphoid organ in embryogenesis, displaying nonspecific local immunity, and providing development of tolerance to food and other foreign antigens during the lifespan, are described. The role of individual forms of liver proteasomes in transplantology is considered as well as their utility as markers for endocrine transplant survival.

Synthesis of dopamine in non-dopaminergic neurons of the mediobasal hypothalamus of adult rats

286 It has been considered for many years that dopamm ine (DA), one of the most important and widely diss tributed neurotransmitters, is synthesized in DAAergic bienzymatic neurons only. These neurons contain two enzymes: tyrosine hydroxylase (TH) converting LL tyrosine to LLdioxyphenylalanine (LLDOPA) and aroo matic LLamino acid decarboxylase (AADC) cataa lyzing DA formation from LLDOPA [1–3]. However, in the middle of the 1980s, the method of double immunocytochemical labeling [3] was developed that allowed to detect, in addition to bienzymatic neurons, monoenzymatic neurons, which expressed only one of the enzymes of DA synthesis, TH or AADC, in various brain regions of many mammalian species [4–8]. The number and distribution of monoenzymatic neurons within the brain is higher than those of bienzymatic neurons, which indirectly indicates their considerable involvement in brain functioning. Mostly neurons studied are located in the mediobasal hypothalamus, including the arcuate nucleus containing the bodies of DAAproducing neuu rons and the median eminence, which contains axonal projections of these cells. In the mediobasal hypothalamus of adult animals, monoenzymatic neurons account for about 50% of total number of neurons expressing the enzymes of DA synthesis [8]. At the end of prenatal development, the arcuate nucleus of the rat fetus contains more than 99% of monoenzymatic neurons and less than 1% of DAergic neurons [8, 10]. However, it has been shown in ex vivo experiments performed in dissociated cell culture that neurons of the arcuate nucleus of rat fetuses synthesize DA and release it in amounts suffii cient for inhibitory control of prolactin secretion in the pituitary gland [11, 12]. The above data allowed us to suggest involvement of monoenzymatic neurons containing the complii mentary enzymes of DA synthesis in cooperative synn thesis of this neurotransmitter [13]. During testing of this hypothesis, we directly demonstrated cooperative DA synthesis in neurons of the mediobasal hypothalaa mus; however, this process was observed in rat fetuses, in which monoenzymatic neurons predominated [14]. The question on hypothetical DA synthesis in monoenzymatic neurons of adult animals remained open. In the present study, we tested our hypothesis on DA synthesis in monoenzymatic nonnDAAergic neuu rons not only during early stages of ontogeny, but also in adult animals. The experiments were performed on 30 adult male Wistar rats weighing 200–300 g. The animals were anesthetized with 40 mg/kg of pentobarbital and decapitated; the brains were removed. Vibratome slices of the brain (3000µm) were prepared at 4°C …