SIRT1 is increased in affected brain regions and hypothalamic metabolic pathways are altered in Huntington disease.

AIMS: Metabolic dysfunction is involved in modulating the disease process in Huntington disease (HD) but the underlying mechanisms are not known. The aim of this study was to investigate if the metabolic regulators sirtuins are affected in HD. METHODS: Quantitative real-time polymerase chain reactions were used to assess levels of SIRT1-3 and downstream targets in post mortem brain tissue from HD patients and control cases as well as after selective hypothalamic expression of mutant huntingtin (HTT) using recombinant adeno-associated viral vectors in mice. RESULTS: We show that mRNA levels of the metabolic regulator SIRT1 are increased in the striatum and the cerebral cortex but not in the less affected cerebellum in post mortem HD brains. Levels of SIRT2 are only increased in the striatum and SIRT3 is not affected in HD. Interestingly, mRNA levels of SIRT1 are selectively increased in the lateral hypothalamic area (LHA) and ventromedial hypothalamus (VMH) in HD. Further analyses of the LHA and VMH confirmed pathological changes in these regions including effects on SIRT1 downstream targets and reduced mRNA levels of orexin (hypocretin), prodynorphin and melanin-concentrating hormone (MCH) in the LHA and of brain-derived neurotrophic factor (BDNF) in the VMH. Analyses after selective hypothalamic expression of mutant HTT suggest that effects on BDNF, orexin, dynorphin and MCH are early and direct, whereas changes in SIRT1 require more widespread expression of mutant HTT. CONCLUSIONS: We show that SIRT1 expression is increased in HD-affected brain regions and that metabolic pathways are altered in the HD hypothalamus.

Microglia Involvement into Acute and Chronic Brain Damage in Diabetic Rats: Impact of GLP-1RA and SGLT-2i.

BACKGROUND: Acute and chronic brain damage in type 2 diabetes mellitus (DM) determines the need to investigate the neuroprotective potential of glucose-lowering drugs. The purpose was to directly compare the neuroprotective effects of glucagon-like peptide-1 receptor agonists (GLP-1RAs) with different duration of action and sodium-glucose cotransporter-2 inhibitors (SGLT-2i) in type 2 diabetic rats with and without stroke. METHODS: DM was modelled using high-fat diet and nicotinamide+streptozotocin protocol. The following groups (n = 15 each) were formed: DM without treatment, treatment with liraglutide, dulaglutide, canagliflozin as well as control group without DM and treatment. After 8 weeks, 10 rats from each group underwent middle cerebral artery occlusion. In the reperfusion period neurological deficit, neuroglial damage markers and brain necrosis were evaluated. Brain slices from the remaining 5 animals in each group were histologically examined for microglial activation and neuronal damage. RESULTS: Brain damage was similar in "DM" and "Control" (17.53 [14.23; 26.58] and 15.87 [13.40; 22.68] % of total brain volume, respectively). All study drugs diminished damage volume comparing with "DM" and "Control" whereas the necrosis volume in "DM+Liraglutide" was smaller than in "DM+Canagliflozin" and did not significantly differ from "DM+Dulaglutide" (2.9 [1.83; 4.71], 6.17 [3.88; 8.88] and 4.57 [3.27; 7.90] %). The neurological deficit was more prominent in "DM" than in "Control", while all the drugs demonstrated similar positive effect. Neurofilament light chains (NLC) did not differ between "DM" and "Control". Dulaglutide and canagliflozin caused a marked decrease in NLC. Protein S100BB level was similar in "DM" and "Control". Liraglutide caused the largest S100BB decrease, while canagliflozin did not influence it. In chronic brain ischaemia, all drugs increased the number of normal neurons, but GLP-1RAs had a more pronounced effect. DM was accompanied by increased number of activated microglial cells in Cornu Ammonis (CA)1 hippocampal region. Both GLP-1RAs reduced the number of Iba-1-positive cells, with dulaglutide being more effective than liraglutide, whereas canagliflozin did not affect this parameter. CONCLUSIONS: GLP-1RAs and SGLT-2i have neuroprotective properties against acute and chronic brain damage in diabetic rats, although the infarct-limiting effect of GLP-1RAs may be more pronounced. GLP-1RAs and SGLT-2i exert their protective effects by directly influencing neuronal survival, whereas GLP-1RAs also affect microglia.

Transduced wild-type but not P301S mutated human tau shows hyperphosphorylation in transgenic mice overexpressing A30P mutated human alpha-synuclein.

Neuropathological and cell culture studies suggest that tau and α-synuclein pathologies may promote each other. To study the relevance and functional implications of these findings in vivo, we transduced hippocampal neurons of wild-type or human A30P α-synuclein transgenic mice with wild-type or P301S mutated human tau using an adeno-associated virus vector. Green fluorescent protein transduction was used as a control. We assessed spontaneous exploratory activity, anxiety and spatial learning and memory 11 weeks after the transduction and perfused the mice for histology. The transduced tau was mainly found in axon terminals and largely restricted within the hippocampi. In addition, neurons around the injection site showed cytoplasmic staining for human tau in both wild-type and A30P mice. Of these tau-positive neurons, 44% in A30P mice but only 3% in wild-type mice receiving human wild-type tau transduction formed paired helical filament-1 (PHF-1)-positive cytoplasmic densities. In contrast, only 1% of tau-positive neurons were also PHF-1 positive after transduction with P301S tau in mice of either genotype. Transduction of P301S tau reduced swimming speed but otherwise tau transduction had no significant behavioral consequences. Cytoplasmic PHF-1 densities were associated with poor spatial memory in wild-type mice but slightly improved memory in A30P mice, indicating that also tau hyperphosphorylation does not necessarily compromise neural functions. These data demonstrate that α-synuclein promotes tau hyperphosphorylation depending on the amino acids on the 301 site.

Generation of the iPSC line FINi002-A from a male Parkinson's disease patient carrying compound heterozygous mutations in the PRKN gene.

The most common cause of autosomal recessive familial Parkinson's disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus. The established line displays typical human primed iPSC morphology and expression of pluripotency-associated markers, normal karyotype without SNP array-detectable copy number variations and can give rise to derivatives of all three embryonic germ layers. We envisage the usefulness of this iPSC line, carrying a common and well-studied missense mutation in the RING1 domain of the PARKIN protein, for the elucidation of PARKIN-dependent mechanisms of PD using in vitro and in vivo models.

Suppression of MAP kinases inhibits microglial activation and attenuates neuronal cell death induced by alpha-synuclein protofibrils.

Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.

Continuous exposure to glial cell line-derived neurotrophic factor to mature dopaminergic transplants impairs the graft's ability to improve spontaneous motor behavior in parkinsonian rats.

Functional recovery following intrastriatal transplantation of fetal dopaminergic neurons in animal models of Parkinson's disease is, at least in part, dependent on the number of surviving dopaminergic neurons and the degree of graft-derived dopaminergic reinnervation of the host striatum. In the present study, we analyzed whether continuous exposure of glial cell line-derived neurotrophic factor (GDNF) to mature dopaminergic grafts could further boost the functional outcome of widespread intrastriatal dopaminergic grafts. Rats with dopamine-denervating lesions received multiple intrastriatal transplants of fetal dopaminergic cells and graft-induced behavioral effects were analyzed in drug-induced and spontaneous motor behaviors. At three months after grafting, animals received intrastriatal injections of recombinant lentiviral vectors encoding for either human GDNF or the green fluorescent protein. Continuous exposure of GDNF to the grafts did not boost the functional recovery beyond what was observed in the control animals. Rather, in some of the spontaneous motor behaviors, animals in the GDNF-group showed deterioration as compared with control animals, and this negative effect of GDNF was associated with a down-regulation of the tyrosine hydroxylase enzyme. Based on these and our earlier results, we propose that intrastriatal administration of GDNF at the time of or shortly after grafting is highly effective in initially promoting the cell survival and fiber outgrowth from the grafts. However, once the grafts are mature, GDNF's ability to boost dopaminergic neurotransmission follows the same dynamics as for the native nigral dopaminergic neurons, which appears to be dependent on the concentration of GDNF. Since rather low doses of glial cell line-derived neurotrophic factor at nanogram levels appear to saturate these effects, it may be critical to adjust GDNF levels using tightly regulated gene expression systems.

Growth and functional efficacy of intrastriatal nigral transplants depend on the extent of nigrostriatal degeneration.

Previous studies have shown that the functional efficacy of intrastriatal transplants of fetal dopamine (DA) neurons in the rat Parkinson model depends on their ability to establish a new functional innervation of the denervated striatum. Here we report that the survival, growth, and function of the grafted DA neurons greatly depend on the severity of the lesion of the host nigrostriatal system. Fiber outgrowth, and to a lesser extent also cell survival, were significantly reduced in animals in which part of the intrinsic DA system was left intact. Moreover, graft-induced functional recovery, as assessed in the stepping, paw-use, and apomorphine rotation tests, was obtained only in severely lesioned animals, i.e., in rats with >70% DA denervation of the host striatum. Functional recovery seen in these animals in which the 6-hydroxydopamine (6-OHDA) lesion was confined to the striatum was more pronounced than that previously obtained in rats with complete lesions of the mesencephalic DA system, indicating that spared portions of the host DA system, particularly those innervating nonstriatal forebrain areas, may be necessary for the grafts to exert their optimal functional effect. These data have implications for the optimal use of fetal nigral transplants in Parkinson patients in different stages of the disease.

Neurturin enhances the survival of intrastriatal fetal dopaminergic transplants.

We investigated here the effect of the novel glial cell line-derived neurotrophic factor (GDNF)-family member neurturin (NTN) on transplanted fetal dopamine (DA) neurons. Three groups of rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA system received intrastriatal grafts of embryonic ventral mesencephalic tissue. Following transplantation animals received repeated injections of vehicle or NTN (0.3 microg or 3.0 microg) over three weeks posttransplantation. NTN-treated animals had significantly (1.8-fold) more tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Graft volume, TH-IR cell volume and overall dopaminergic host reinnervation remained unchanged. Amphetamine-induced rotation was rapidly compensated in all grafted rats. We conclude that administration of NTN may be a powerful way to increase survival of transplanted fetal DA neurons.

Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model.

During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson's disease.

[The role of the "milk" factor in the transmission of Campylobacter infection]. / Izuchenie roli "molochnogo" faktora peredachi kampilobakterioznoi infektsii.

The epidemiological survey of the foci of campylobacteriosis confirmed the activity of the "milk" factor in the transmission of this enteric infection. The proliferation of Campylobacter in milk corresponded to the general regularities of the growth of bacterial population in the closed system. Most of the Campylobacter strains under study retained their viability in cooled boiled milk for a longer time.

[The cytopathogenic activity of Campylobacter isolated from different sources]. / Tsitopatogennaia aktivnost' kampilobakterii, vydelennykh iz razlichnykh istochnikov.

The data on the cytopathogenic activity of campylobacteria isolated from different sources are presented. 84.7 % of the isolated campylobacteria have been shown to possess cytopathogenicity with respect to Hep-2 cell cultures. The greatest number of highly cytopathogenic strains (52.6 %) has been registered among clinical isolates of campylobacteria. At the same time 44.1 % of highly cytopathogenic strains have been isolated from chickens, which is indicative of the potential danger of poultry as the source of Campylobacter infection. In the course of the epidemiological surveillance of campylobacteriosis the determination of cytopathogenically active Campylobacter strains is necessary.

[The characteristics of the epidemiology of campylobacteriosis under modern conditions]. / Osobennosti épidemiologii kampilobakterioza v sovremennykh usloviiakh.

The results of the clinico-epidemiological study of campylobacteriosis in a concrete area (Kiev and the Kiev region) are presented. The proportion of campylobacteriosis cases was found to be 6.4% among patients hospitalized in connection with acute enteric infections. Hens were most frequently the source of human infection. Thus, at the local poultry farm the proportion of hens contaminated with bacteria of the genus Campylobacter was 44.8%. The possible routes of the spread of Campylobacter infection and the factors of its transmission were established. The most important element of the epidemiological marking of Campylobacter bacteria is the determination of their species and serotype.

[Current parameters of the epidemiological process in campylobacteriosis]. / Nekotorye sovremennye parametry épidemiologicheskogo protsessa kampilobakterioza.

Data on some parameters of the epidemic process of campylobacteriosis in the Ukraine are presented. Campylobacteriosis patients were found to constitute 1.9 +/- 0.095% of all examined patients with acute enteric infections (AEI). No statistically significant difference in the proportion of campylobacteriosis among child and adult AEI patients was established. The proportion of sick persons at the period of the spring-summer rise in morbidity was 71.5%. A definite relationship between the epizootic and epidemic processes in campylobacteriosis was noted. In the Ukraine the predominant infective agent was Campylobacter jejuni belonging mainly to serotype Lio and biotype 1. Quite frequently campylobacteriosis was found to be accompanied by mixed infections.

[Antibiotic resistance of Campylobacter strains and its role in evolutionary processes in bacteria of the genus Campylobacter]. / Antibiotikorezistentnost' kampilobakterii i ee rol' v protsessakh évoliutsii bakterii roda Campylobacter.

The influence of multiple antibiotic resistance of Campylobacter strains determined by R plasmids on their virulence was studied. It was shown that the strains with multiple resistance were mostly isolated from children with campylobacteriosis (26.5 per cent). The number of such strains isolated from the infected adults, hens and environmental objects amounted to 25.8, 23.3 and 21.4 per cent respectively. The difference of the resistance determinants in the tested strains was statistically insignificant. It was suggested that the R plasmids of the human strains could be as well detected in the strains from the infected hens and environmental objects. A chromosome-plasmid pattern of the Campylobacter resistance to kanamycin, tetracycline and erythromycin was determined. The analysis of the cytopathogenic activity of the plasmid-containing strains and their aplasmid clones revealed that this criterion of the virulence statistically significantly increased after the plasmid loss by the strains. It was concluded that the antibiotic resistant strains had a selective superiority while circulating in various ecological niches.

[Current concepts of the biology of bacteria in genus Campylobacter and their role in human pathology]. / Sovremennye predstavleniia o biologii bakterii roda Campylobacter i ikh roli v patologii cheloveka.

An analytical review of literature on the problem of biological properties of campylobacteria is presented. Taxonomic characteristics of campylobacteriosis agents are given. Morphological and cultural peculiarities of campylobacteria are considered, their ability to form coccal forms is emphasized. Peculiar attention is paid to serotype and biotype characteristics of bacteria of the Campylobacter genus. It is shown expedient to develop the home schemes of sero- and biotypization. Data on biological properties of "new" agents of human campylobacteriosis (Campylobacter cinaedi, C. hypointestinalis, C. upsaliensis) are generalized for the first time in home literature. A conclusion is made that the study of biological properties of campylobacteria strains circulating in the Ukrainian territory as well as the development of efficient prophylactic and antiepidemic measures on this basis are urgent now.

[The epidemiological characteristics of the immunological response in campylobacteriosis]. / Epidemiologicheskie osobennosti immunologicheskogo otveta pri kampilobakterioze.

An analytical survey of literature on the problem of immunological aspects of campylobacteriosis is presented. Antigenic nature of various components of a bacterial cell of these agents is described. Schemes of serotyping of campylobacteria on the basis of thermolabile and thermostable antigens are analyzed. It is shown expedient to use serotyping for determination of an infection source in the epidemiological practice. Proceeding from the published data presented, a conclusion is made that it is necessary to develop the home schemes of serotyping. At the same time a problem on development of new proximate methods promoting immunological indication of campylobacteriosis antigens in various ecological niches is not less urgent and is to be taken into account when planning antiepidemiological measures against the campylobacteriosis infection.

[The antibiotic resistance of strains of Campylobacter of different origins]. / Izuchenie antibiotikorezistentnosti shtammov kampilobakterii razlichnogo proiskhozhdeniia.

Antibioticograms of different campylobacteria strains have been analyzed. It is shown possible to develop a system of epidemiological marking on this basis. With this purpose sensitivity of campylobacteria to gentamycin, canamycin, carbenicyllin, tetracylin and erythromycin has been studied. No statistical difference in the average markers of resistance in the studied groups of strains was observed. This permitted supposing that R-plasmids in "human" strains may be isolated not only from the human intestine microflora, but also from other sources (animals, birds, environmental objects) as well. There are found common R-spectra in different groups of strains (Gm Kb Tc Er; Kb and Kb Tc), which confirms the same infection source. The study of antibioticograms of campylobacteria which circulate among people, animals, birds and environmental objects permits revealing regularities of epidemic process in case of campylobacteriosis.

[The epidemiological and clinical characteristics of campylobacteriosis in Ukraine]. / Epidemiologicheskie i klinicheskie osobennosti kampilobakteroza v Ukraine.

Clinical course and epidemiological features of campylobacteriosis in Ukraine are described. The disease accounts for 11.2% of the total number of acute enteric infectious cases. Ratio of bacterial contamination of hens and their role in spread of campylobacteriosis have been established. Possible ways of transfer of the infection are discussed.

[The fatty acid composition of the total lipids in bacteria of the genus Campylobacter]. / Zhirnokislotnyi sostav summarnykh lipidov bakterii roda Campylobacter.

Composition of fatty acids of total lipids in home strains of campylobacteria has been studied. Lipids of all the strains of C. jejuni and C. coli mainly consist of saturated fatty acids (from 75.7 to 78.7%) with predominance of tetradecanoic and hexadecanoic fatty acids. The level of unsaturated fatty acids is considerably lower (from 21.0 to 22.5%); These acids are mainly presented by hexadecene acid. Qualitative composition of fatty acids of total lipids in C. jejuni and C. coli does not permit using it for differentiation within these species.

Long-term polarization of microglia upon α-synuclein overexpression in nonhuman primates.

We have previously shown that persistent α-synuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) α-synuclein. However, A53T α-synuclein overexpression induced neurodegeneration that resulted in nigral dopaminergic cell death. Here, we analyze the microglia population in the midbrain of these animals by stereological quantification of Iba1+ cells. Our data here show that monkeys overexpressing A53T α-synuclein showed a long-term increase in microglia presenting macrophagic morphology. However, wt α-synuclein overexpression, despite the absence of dopaminergic cell death, resulted in a permanent robust increase of the microglia population characterized by a range of distinct morphological types that persisted after 1 year. These results confirm that the microglial response differs depending on the type of α-synuclein (wt/A53T) and/or whether α-synuclein expression results in cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, the microglial response is modulated by events related to α-synuclein expression in substantia nigra and persists in the long term. The data presented here is in agreement with that previously observed in a recombinant adeno-associated virus (rAAV) α-synuclein rat model, thereby validating both the findings and the model, and highlighting the translational potential of the rodent model to higher species closer to humans.