Gordana Glavan

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.

Synaptotagmins in Neurodegeneration

Synaptotagmins (Syts) are transmembrane proteins involved in the regulation of membrane trafficking. Here, we summarize literature data that provide growing evidence that several Syts are involved in the pathophysiological mechanisms of temporal lobe epilepsy and Parkinsons disease, as well as few reports related to brain ischemia and Alzheimers disease (AD). We also report new data from our laboratories, showing changes of the expression of several Syts in Tg2576 mouse model of AD that may be related to neuroinflammation surrounding the β‐amyloid plaques. Furthermore, we demonstrate N‐methyl‐D‐aspartate receptor‐mediated upregulation of Syt 4 mRNA in a model of excitotoxic striatal lesion induced by unilateral striatal injection of quinolinic acid, associating the upregulation of Syt 4 with mechanisms of excitotoxicity. We propose that phamacological manipulation of Syt expression in animal models of neurodegeneration should be further explored, as it may help to clarify the role of individual Syt isoforms in the regulation of membrane trafficking in neurodegeneration. Anat Rec, 292:1849–1862, 2009.

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.

Neurotoxic potential of ingested ZnO nanomaterials on bees.

The honey bee is among most important pollinators threatened by environmental pollution, pest control and potentially, by products of nanotechnologies. The aim of the current study was an analysis of the neurotoxic potential of ingested zinc oxide nanomaterials (ZnO NMs) or zinc ions (Zn(2+)) on honey bees. We analysed a variety of biomarkers, including metabolic impairment, feeding rate, and survival, as well as the activities of a stress-related enzyme glutathione S-transferase, and the neurotoxicity biomarker acetylcholinesterase. Acetylcholinesterase activity was found to be elevated in bees exposed to either of the tested substances. In addition, we observed increased feeding rate in the group treated with Zn(2+) but not with ZnO NMs or control group. The observed effects we relate primarily to Zn(2+) ions. Here we provide evidence that zinc ions either originating from Zn salt or Zn-based NPs have a neurotoxic potential and thus might contribute to colony survival.

Dopaminergic regulation of synaptotagmin I and IV mRNAs in hemiparkinsonian rats.

Synaptotagmins (Syts) I and IV are synaptic proteins involved in the regulation of neurosecretion. Dopaminergic drugs have been shown to modulate their expression. Here we investigate whether dopaminergic regulation of syt I and syt IV expression could play a role in the hypersensitive striatum of rats with unilateral lesions of dopaminergic nigrostriatal neurons with 6-hydroxydopamine. We show that chronic dopaminergic denervation resulted in a small down-regulation of striatal syt I mRNA, whereas acute treatment with SKF-82958, a dopamine D1 receptor agonist, induced a massive syt IV mRNA up-regulation in the striatum on the lesioned side. We conclude that chronic lack of dopamine and treatment with dopamine D1 receptor agonists alter the synaptic plasticity in dopamine depleted basal ganglia.

Upregulation of synaptotagmin IV protein in kainate-induced seizures

Synaptotagmin IV is a product of immediate early-response gene. It is involved in the regulated neurosecretion in the brain. Its putative role, however, in vesicular transport and localization in secretor y vesicles is still a matter of debate. Here we followed the spatiotemporal pattern of synaptotagmin IV protein upregulation in the hippocampus, caudate putamen, nucleus accumbens, nucleus amygdalae, piriform and entorhinal cortices of rats with kainate-induced seizures. We found that upregulation pattern paralleled the direction of depolarization through the hippocampus and also reflecting seizure activity spreading to other brain regions. We speculate that synaptotagmin IV may have a role in the vesicular transport of the upregulated peptides and proteins involved in the plasticity and/or neurodegeneration provoked by the kainate.

Prochloraz and coumaphos induce different gene expression patterns in three developmental stages of the Carniolan honey bee (Apis mellifera carnica Pollmann)

The Carniolan honey bee, Apis mellifera carnica, is a Slovenian autochthonous subspecies of honey bee. In recent years, the country has recorded an annual loss of bee colonies through mortality of up to 35%. One possible reason for such high mortality could be the exposure of honey bees to xenobiotic residues that have been found in honey bee and beehive products. Acaricides are applied by beekeepers to control varroosis, while the most abundant common agricultural chemicals found in honey bee and beehive products are fungicides, which may enter the system when applied to nearby flowering crops and fruit plants. Acaricides and fungicides are not intrinsically highly toxic to bees but their action in combination might lead to higher honey bee sensitivity or mortality. In the present study we investigated the molecular immune response of honey bee workers at different developmental stages (prepupa, white-eyed pupa, adult) exposed to the acaricide coumaphos and the fungicide prochloraz individually and in combination. Expression of 17 immune-related genes was examined by quantitative RT-PCR. In treated prepupae downregulation of most immune-related genes was observed in all treatments, while in adults upregulation of most of the genes was recorded. Our study shows for the first time that negative impacts of prochloraz and a combination of coumaphos and prochloraz differ among the different developmental stages of honey bees. The main effect of the xenobiotic combination was found to be upregulation of the antimicrobial peptide genes abaecin and defensin-1 in adult honey bees. Changes in immune-related gene expression could result in depressed immunity of honey bees and their increased susceptibility to various pathogens.

Intermittent l-DOPA treatment differentially alters synaptotagmin 4 and 7 gene expression in the striatum of hemiparkinsonian rats

Long-term use of L-DOPA in Parkinsons disease (PD) is frequently associated with side effects that are reflected in changed neurotransmitter/neuropeptide secretion in basal ganglia. These side effects could be connected with synaptotagmins (syts) because syts are involved in regulation of membrane trafficking. We have previously reported that acute L-DOPA treatment upregulated the expression of Syt 4 and Syt 7 mRNAs in hypersensitive striatum of 6-OHDA rat model for PD. Here we investigate whether intermittent L-DOPA treatment that produces behavior sensitization affects the Syt 1, Syt 2, Syt 4, Syt 7 and Syt 10 mRNAs in striatum of 6-OHDA rats killed 4 and 12 h after the last L-DOPA injection. We verified behavioral sensitization by increased intensity of contralateral turning. 6-OHDA lesion caused Syt 2 mRNA downregulation and Syt 10 mRNA upregulation in striatum, but failed to alter Syt 4, Syt 7 and Syt 1 mRNAs. Acute l-DOPA induced an increase of Syt 4 and Syt 7 mRNAs in the denervated striatum leaving the levels of Syt 1, Syt 2 and Syt 10 mRNAs unaffected. Intermittent L-DOPA treatment did not alter Syt 1, Syt 2 and Syt 10 mRNA striatal levels, suggesting that 6-OHDA-induced Syt 2 and Syt 10 mRNA changes reflect a persistent striatal abnormality caused by dopamine depletion. On contrary, intermittent L-DOPA treatment downregulated Syt 4 mRNA and prolonged the elevation of Syt 7 mRNA in the denervated striatum. We conclude that Syt 4 and Syt 7 might be specifically involved in striatal plasticity caused by repeated L-DOPA administration that accompanies sensitization.

Effect of apomorphine on striatal synaptotagmin 7 mRNA levels in reserpinized rats.

Synaptotagmin 7 (Syt 7) is a Ca2+ sensor implicated in the regulation of membrane fusion in vesicular transport, but its precise role in neurons is still a matter of controversy. Dopaminergic drugs have been shown to modulate its expression in the striatum. Here we investigate whether dopamine receptor agonist-up-regulation of Syt 7 mRNA is specifically involved in the pathophysiological adaptations of hypersensitive striatum by analyzing other dopaminergic neurons containing brain regions. We treated rats with systemic reserpine injections that rapidly depletes dopamine throughout the brain, but leaves dopaminergic neurons spared from destruction. We analyzed the effects of apomorphine, a D1 and D2 receptor agonist on Syt 7 mRNA expression in caudate putamen, nucleus accumbens, cingulate cortex, substantia nigra compacta, ventral tegmental area and hippocampus. The treatment with reserpine resulted in akinesia, catalepsy and rigidity and up-regulation of proenkephalin and down-regulation of preprotachykinin mRNA in caudate putamen, indicating a severe depletion. By acute treatment with apomorphine proenkephalin mRNA was down-regulated and preprotachykinin mRNA up-regulated in the caudate putamen of reserpinized rats. Apomorphine increased Syt 7 mRNA levels only in striatum (caudate putamen and nucleus accumbens) of reserpinized rats, while in other brain regions it did not have such effect. The reserpinization and/or apomorphine treatment had no effect on Syt 1 mRNA expression in caudate putamen. It may be concluded, that in the striatum depleted of biogene amines, such as occurs after reserpine treatment, the up-regulation of Syt 7 could play a specific role as part of hypersensitive response to dopaminergic agonists.

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.