Marko Živin

Nephrotoxic effects of chronic administration of microcystins -LR and -YR

Acute intoxication with MC-LR induces cytoskeletal alterations, apoptosis and necrosis of hepatocytes resulting in intrahepatic hemorrhage. Preliminary results have shown that chronic treatment of rats with intraperitoneal injections of sublethal doses of microcystins MC-LR and MC-YR could induce not only liver, but also kidney injuries. We aimed to investigate whether the induction of the cytoskeletal changes, apoptosis and necrosis could be the mechanisms involved in the injury of kidney cells in the chronic model of microcystin intoxication. Experimental rats were receiving intraperitoneal injections of MC-LR (10 microg/kg) or MC-YR (10 microg/kg) every second day for 8 months, while control rats were receiving only the vehicle. The histopathological investigation revealed collapsed glomeruli with thickened basement membranes and dilated tubuli filled with eosinophilic casts. Rhodamine-phalloidin labeling showed cytoplasmic aggregation and accumulation of fibrilar actin filaments within the epithelial tubular cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed increased number of TUNEL-positive cells in the kidney cortex and medulla. The pathological changes induced by MC-LR appeared more severe than those induced by MC-YR. The results support the view that at the cellular level, the mechanisms that underly the chronic nephrotoxicity are similar to the mechanisms of the acute hepatotoxicity of microcystins.

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.

Exploiting microRNAs for cell engineering and therapy

MicroRNAs (miRNAs) form a large class of non-coding RNAs that function in repression of gene expression in eukaryotes. By recognizing short stretches of nucleotides within the untranslated regions of mRNAs, miRNAs recruit partner proteins to individual transcripts, leading to mRNA cleavage or hindering of translation. Bioinformatic predictions and a wealth of data from wet laboratory studies indicate that miRNAs control expression of a large proportion of protein-coding genes, implying involvement of miRNAs in regulation of most biologic processes. In this review we discuss the biology of miRNAs and present examples of how manipulation of miRNA expression or activity can be exploited to attain the desired phenotypic traits in cell engineering as well as achieve therapeutic outcomes in treatment of a diverse set of diseases.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.

The dopamine D1 receptor agonist and D2 receptor antagonist LEK-8829 attenuates reinstatement of cocaine-seeking in rats

Various dopaminergic drugs have been studied for their efficacy in the treatment of cocaine addiction. Pretreatment with either selective dopamine D1 receptor agonists or selective dopamine D2 receptor antagonists prevents reinstatement of cocaine-seeking in animal models of drug craving and relapse. We tested a novel ergoline derivative with combined D1 agonistic and D2 antagonistic effects, 9,10-didehydro-N-methyl-N-(2-propynyl)-6-methyl-8β-aminomethylergoline bimaleate (LEK-8829), for its effects on cocaine-seeking in the intravenous cocaine self-administration model in rats. Pretreatment with systemic injections of LEK-8829 attenuated reinstatement of cocaine-seeking induced by cocaine priming injections and diminished cocaine intake in cocaine self-administration sessions. LEK-8829 itself did not induce reinstatement of cocaine-seeking and did not maintain intravenous self-administration. The results of our study indicate that LEK-8829 is a candidate medication for the treatment of cocaine craving in cocaine addiction.

Antiparkinsonian potential of interaction of LEK-8829 with bromocriptine

The ergoline derivative, LEK-8829 (9,10-didehydro-N-methyl-(2-propynyl)-6-methyl-8-aminomethylerg oline), has been proposed as a potential atypical antipsychotic drug with antagonistic actions at dopamine D2 and serotonin 5-HT2 and 5-HT1A receptors (Krisch et al., 1994, 1996). LEK-8829 also induces contralateral turning in rats with 6-hydroxydopamine-induced unilateral lesion of dopamine nigrostriatal neurons. Turning is blocked by SCH-23390 (R(+)-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaze pine), a dopamine D1 receptor antagonist. It has been suggested that LEK-8829 could have beneficial effects in parkinsonian patients suffering from psychotic episodes induced as a side-effect of antiparkinsonian treatment with dopamine D2 receptor agonists. Therefore, we now investigated the interaction of LEK-8829 with the dopamine D2 receptor agonist bromocriptine (2-bromo-alpha-ergokryptine) in 6-hydroxydopamine-lesioned rats. Treatment with either LEK-8829 (3 mg kg(-1)) or bromocriptine (3 mg kg(-1)) induced a vigorous contralateral turning response. The cumulated number of turns induced by the treatment with both drugs combined was not significantly different from the cumulated number of turns induced by single-drug treatment. The pretreatment with SCH-23390 (1 mg kg(-1)) did not have a significant effect on the bromocriptine-induced turning but significantly decreased the turning observed after the combined LEK-8829/bromocriptine treatment. We conclude that in the 6-hydroxydopamine model, the turning behaviour mediated by the LEK-8829/bromocriptine combination may be the result of opposing activity of both drugs at dopamine D2 receptors with concomitant stimulation of dopamine D1 receptors by LEK-8829. Therefore, LEK-8829 may have a potential for the therapy of parkinsonism complicated by dopamine D2 receptor agonist drug-induced psychosis.

Magnetic Resonance Imaging for Rapid Screening for the Nephrotoxic and Hepatotoxic Effects of Microcystins

In vivo visualization of kidney and liver damage by Magnetic Resonance Imaging (MRI) may offer an advantage when there is a need for a simple, non-invasive and rapid method for screening of the effects of potential nephrotoxic and hepatotoxic substances in chronic experiments. Here, we used MRI for monitoring chronic intoxication with microcystins (MCs) in rat. Male adult Wistar rats were treated every other day for eight months, either with MC-LR (10 μg/kg i.p.) or MC-YR (10 μg/kg i.p.). Control groups were treated with vehicle solutions. T1-weighted MR-images were acquired before and at the end of the eight months experimental period. Kidney injury induced by the MCs presented with the increased intensity of T1-weighted MR-signal of the kidneys and liver as compared to these organs from the control animals treated for eight months, either with the vehicle solution or with saline. The intensification of the T1-weighted MR-signal correlated with the increased volume density of heavily injured tubuli (R2 = 0.77), with heavily damaged glomeruli (R2 = 0.84) and with volume density of connective tissue (R2 = 0.72). The changes in the MR signal intensity probably reflect the presence of an abundant proteinaceous material within the dilated nephrons and proliferation of the connective tissue. T1-weighted MRI-is a valuable method for the in vivo screening of kidney and liver damage in rat models of intoxication with hepatotoxic and nephrotoxic agents, such as microcystins.

Application of the nonradioactive in situ hybridization for the localization of acetylcholinesterase mRNA in the central nervous system of the rat; comparison to the radioactive technique.

In this preliminary report nonradioactive digoxigenine — based and radioactivein situ hybridization procedures for the localization of acetylcholinesterase mRNA were tested and compared in rat brain. General patterns ofAche mRNA localization observed by both techniques did not differ significantly and were practically the same as reported in previousin situ studies on the mammalian brain. Shorter procedure time and avoidance of precautions necessary at work with radioactive materials are major advantages of nonradioactive technique. Under-and over- staining can be prevented by direct examination of coloring reaction. Faint staining in the control experiment with heterologous DNA suggests that proper stringency is essential for the specificity of staining.

Modelling FUS Mislocalisation in an In Vitro Model of Innervated Human Muscle

Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS. This model was first characterised for endogenous expression and distribution of ALS-related proteins TDP-43 and FUS. Then, wild-type FUS and its mutants were introduced into these co-cultures to determine how FUS defects in nuclear transport modulate the pathological conditions. FUS-bearing plasmids were introduced by classical transfection and electroporation, as novel approaches to deliver plasmids into explants, and their cellular distributions were characterised. Endogenous nuclear expression of TDP-43 and FUS was observed in explants and myoblasts/myotubes. After transfection, wild-type FUS was expressed in nuclei of myoblasts, myotubes and explants, although with low transfection rates. Following successful electrotransfection into explants, the localisation of wild-type FUS was nuclear, and it was detected in neurons, astrocytes, Schwann cells and oligodendrocyte precursors, whereas the FUS∆Y, FUSY526A and FUSY526E mutants were cytoplasmic, and the FUSY526F mutant was nuclear and cytoplasmic. This co-culture model is applicable to the study of neuronal and non-neuronal cell contributions to ALS and other neurodegenerative diseases, and it can be used to investigate drug targets amenable to intervention.

Upregulation and axonal transport of synaptotagmin-IV in the direct-pathway medium spiny neurons in hemi-parkinsonian rats induced by dopamine D1 receptor stimulation

Synaptotagmin‐IV (Syt‐IV) may function as a regulator of Ca2+‐dependent synaptic transmission. In the hemi‐parkinsonian rats with unilateral lesions of dopaminergic nigrostriatal neurons Syt‐IV and substance‐P (SP) mRNAs could be upregulated within the dopaminergically hypersensitive striatum of the lesioned brain hemisphere via the stimulation of striatal dopamine D1 (D1‐R), but not D2 receptors. The hypersensitive D1‐R‐mediated transmission may be the culprit for the undesired expression of levodopa‐induced dyskinesia, implying the involvement of Syt‐IV and SP in the process. First, striatal cellular phenotypes expressing Syt‐IV were determined. It was found to be expressed in all striatal neurons and a small population of astrocytes. Then it was examined, if the D1‐R‐mediated upregulation of Syt‐IV mRNA may result in the upregulation of the translated protein. It was found that, after acute stimulation with a selective D1 agonist, (±)‐6‐chloro‐7,8‐dihydroxy‐3‐allyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine hydrobromide (SKF‐82958), Syt‐IV was elevated within the SP‐expressing striatal neurons of the lesioned side. This was followed by the upregulation of Syt‐IV, but not of its mRNA, within the ipsilateral target nuclei of the direct‐pathway medium spiny neurons, indicating axonal transport of de novo synthesized protein to their SP‐positive synaptic terminals. However, despite the striatal upregulation of SP and Syt‐IV following a similar time‐course, their subcellular co‐localization within the axonal terminals was not found. It was therefore suggested that Syt‐IV may regulate the hypersensitive striatal synaptic transmission, although via a SP‐independent mechanism.