Merja Lakso

Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human α-synuclein

Overexpression of human α‐synuclein in model systems, including cultured neurons, drosophila and mice, leads to biochemical and pathological changes that mimic synucleopathies including Parkinsons disease. We have overexpressed both wild‐type (WT) and mutant alanine53→threonine (A53T) human α‐synuclein by transgenic injection into Caenorhabditis elegans. Motor deficits were observed when either WT or A53T α‐synuclein was overexpressed with a pan‐neuronal or motor neuron promoter. Neuronal and dendritic loss were accelerated in all three sets of C. elegans dopaminergic neurons when human α‐synuclein was overexpressed under the control of a dopaminergic neuron or pan‐neuronal promoter, but not with a motor neuron promoter. There were no significant differences in neuronal loss between overexpressed WT and A53T forms or between worms of different ages (4 days, 10 days or 2 weeks). These results demonstrate neuronal and behavioral perturbations elicited by human α‐synuclein in C. elegans that are dependent upon expression in specific neuron subtypes. This transgenic model in C. elegans, an invertebrate organism with excellent experimental resources for further genetic manipulation, may help facilitate dissection of pathophysiologic mechanisms of various synucleopathies.

The neuroprotective agent memantine induces brain-derived neurotrophic factor and trkB receptor expression in rat brain.

Memantine is a medium-affinity uncompetitive N-methyl-d-aspartate receptor antagonist and has been clinically used as a neuroprotective agent to treat Alzheimers and Parkinsons diseases. We have examined the effect of memantine (ip 5-50 mg/kg; 4 h) on the expression of brain-derived neurotrophic factor (BDNF) and trkB receptor mRNAs in rat brain by in situ hybridization. Memantine at a clinically relevant dose markedly increased BDNF mRNA levels in the limbic cortex, and this effect was more widespread and pronounced at higher doses. Effects of memantine on BDNF mRNA were also reflected in changes in BDNF protein levels. Moreover, memantine induced isoforms of the BDNF receptor trkB. Taken together, these data suggest that the neuroprotective properties of memantine could be mediated by the increased endogenous production of BDNF in the brain. These findings may open up new possibilities of pharmacologically regulating the expression of neurotrophic factors in the brain.

Antipsychotic drug treatment induces differential gene expression in the rat cortex

Antipsychotic drug treatment is known to modulate gene expression in experimental animals. In this study, candidate target genes for antipsychotic drug action were searched using microarrays after acute clozapine treatment (1, 6 and 24 h) in the rat prefrontal cortex. Microarray data clustering with a self‐organizing map algorithm revealed differential expression of genes involved in presynaptic function following acute clozapine treatment. The differential expression of 35 genes most profoundly regulated in expression arrays was further examined using in situ hybridization following acute clozapine, and chronic clozapine and haloperidol treatments. Acute administration of clozapine regulated the expression of chromogranin A, synaptotagmin V and calcineurin A mRNAs in the cortex. Chronic clozapine treatment induced differential cortical expression of chromogranin A, son of sevenless (SoS) and Sec‐1. Chronic treatment with haloperidol regulated the mRNA expression of inhibitor of DNA‐binding 2 (ID‐2) and Rab‐12. Furthermore, the expression of visinin‐like proteins‐1, ‐2 and ‐3 was regulated by chronic drug treatments in various brain regions. Our data suggest that acute and chronic treatments with haloperidol and clozapine modulate the expression of genes involved in synaptic function and in regulation of intracellular Ca2+ in cortex.

Expression of neurotrophins BDNF and NT-3, and their receptors in rat brain after administration of antipsychotic and psychotrophic agents.

We have investigated the potential role of neurotrophic factors in antipsychotic drug action by examining the effects of antipsychotic and psychotropic treatments on the mRNA expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and their receptors, trkB and trkC, respectively, in rat brain. Neither acute nor chronic clozapine treatment significantly affected the expression of these mRNAs in any brain area investigated, except for a decrease in trkB expression in the granule cells of the olfactory bulb. We then examined the effects of the psychotropic agent MK-801. MK-801 (5 mg/kg; 4h) significantly increased BDNF mRNA in the entorhinal cortex, but did not influence NT-3, trkB, or trkC expression in any brain area except for the olfactory bulb. The induction of BDNF mRNA by MK-801 was attenuated by pre-treatment (1 h prior to MK-801 administration) with the antipsychotics, clozapine (25 mg/kg) and haloperidol (2 mg/kg), but not with the antidepressant desipramine (15 mg/kg). Finally, we confirmed that the effects of MK-801 on BDNF mRNA were reflected in the respective changes in BDNF protein levels: MK-801 significantly increased anti-BDNF reactivity in the entorhinal cortex (126 ± 7% of control) while concomitantly decreasing in the hippocampus (71 ± 2% of control). These data do not support the hypothesis that neurotrophins play an important role in antipsychotic drug action, but rather suggest that induction of BDNF in the entorhinal cortex may play a significant role in the psychotropic action of MK-801.

Global microRNA Expression Profiling of Caenorhabditis elegans Parkinson's Disease Models

MicroRNAs (miRNAs) play an important role in human brain development and maintenance. To search for miRNAs that may be involved in the pathogenesis of Parkinsons disease (PD), we utilized miRNA microarrays to identify potential gene expression changes in 115 annotated miRNAs in PD-associated Caenorhabditis elegans models that either overexpress human A53T α-synuclein or have mutations within the vesicular catecholamine transporter (cat-1) or parkin (pdr-1) ortholog. Here, we show that 12 specific miRNAs are differentially regulated in the animals overexpressing α-synuclein, five in cat-1, and three in the pdr-1 mutants. The family of miR-64 and miR-65 are co-underexpressed in the α-synuclein transgenic and cat-1 strains, and members of let-7 family co-underexpressed in the α-synuclein and pdr-1 strains; mdl-1 and ptc-1 genes are target candidates for miR-64 and miR-65 and are overexpressed in α-synuclein transgenic as well as miR-64/65 (tm3711) knockout animals. These results indicate that miRNAs are differentially expressed in C. elegans PD models and suggest a role for these molecules in disease pathogenesis.

Identification of gene expression changes in transgenic C. elegans overexpressing human α-synuclein

Alpha-synuclein containing cellular inclusions are a hallmark of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. A genome wide expression screen was performed in C. elegans overexpressing both wild-type and A53T human alpha-synuclein. 433 genes were up- and 67 genes down-regulated by statistical and fold change (> or <2) criteria. Gene ontology (GO) categories within the regulated gene lists indicated over-representation of development and reproduction, mitochondria, catalytic activity, and histone groups. Seven genes (pdr-1, ubc-7, pas-5, pas-7, pbs-4, RPT2, PSMD9) with function in the ubiquitin-proteasome system and 35 mitochondrial function genes were up-regulated. Nine genes that form histones H1, H2B, and H4 were down-regulated. These results demonstrate the effects of alpha-synuclein on proteasome and mitochondrial complex gene expression and provide further support for the role of these complexes in mediating neurotoxicity. The results also indicate an effect on nuclear protein genes that suggests a potential new avenue for investigation.

Selective sensitivity of Caenorhabditis elegans neurons to RNA interference.

RNA interference is a new approach to knockdown gene expression, but effectiveness varies depending on the organism, cell type or target sequence. Studies with Caenorhabditis elegans have shown that subsets of cells including neurons are often resistant to RNA interference. We measured RNA interference using green fluorescent protein reporter strains and feeding, soaking and injection delivery methods in a number of Caenorhabditis elegans neuron subtypes (dopaminergic, GABAergic, cholinergic, glutamatergic, touch). The sensitivity to RNA interference varied: GABAergic and dopaminergic neurons showed greater resistance while cholinergic, glutamatergic and touch neurons were more sensitive. Dysfunctional RRF-3, a putative RNA-directed RNA polymerase, had a significant effect on increasing neuron sensitivity in most subtypes. These results demonstrate that Caenorhabditis elegans neurons vary in their sensitivity to RNA interference.

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry

A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.

Chronic Antipsychotic Drug Treatment Induces Long-lasting Expression of fos and jun Family Genes and Activator Protein 1 Complex in the Rat Prefrontal Cortex

We have characterized the effects of chronic clozapine and haloperidol treatments on the expression of fos (c-fos, fosB, fra-2) and jun (c-jun, junB, junD) family genes in the rat forebrain. The effects of chronic (17d) clozapine and haloperidol on mRNA expression were determined two hours, 24 hours, and six days after the last drug injection, and the DNA-binding activity of the activator protein-1 (AP-1) complex was studied after washout periods of 24 hours and six days. Chronic clozapine treatment with a 6 d washout period induced the expression of several fos and jun family genes in cortical regions, including the prefrontal cortex (PFC), and in the caudate putamen and nucleus accumbens. Moreover, the DNA-binding activity of the AP-1 complex was greatly increased in the anterior cingulate cortex-PFC in mobility shift assays already after 24 h, and remained increased after a 6d washout period. Chronic administration of haloperidol upregulated fos and jun family mRNA expression that was detectable 24 h and 6 d after cessation of the treatment mainly in the cortex. However, the DNA-binding activity of the AP-1 complex was not altered in the anterior cingulate cortex-PFC by chronic haloperidol administration at any of the time points studied. Thus, chronic treatments with clozapine and haloperidol induce a long-lasting enhancement of fos and jun family transcription factors that continues for several days after the cessation of the treatments in the cortex. These lasting effects might represent events that are potentially involved in the mechanisms of antipsychotic drug action.

Identification of Genes Regulated by Memantine and MK-801 in Adult Rat Brain by cDNA Microarray Analysis

In this study, we monitored gene expression profiles using cDNA microarrays after an acute systemic administration of the high affinity N-methyl-D-aspartate (NMDA) uncompetitive antagonist MK-801 (1 mg/kg; 4 h), and the clinically used moderate affinity antagonist memantine (25 mg/kg; 4 h) in adult rat brains. From a microarray containing 1090 known genes, 13 genes were regulated by both treatments of which 12 were upregulated and one was downregulated. In addition, 28 and 34 genes were regulated (⩾1.5- or ⩽0.67-fold change) by either memantine or MK-801, respectively. Genes commonly regulated by both treatments and not previously reported were confirmed by in situ hybridization (ISH) and include regenerating liver inhibitory factor-1 (RL/IF-1), GDP-dissociation inhibitor 1 (GDI-1), neural visinin Ca2+-binding protein 2 (NVP-2), neuromedin B receptor, and Na+/K+ transporting ATPase 2β. ISH with memantine (5–50 mg/kg) revealed regulation of these genes in other cortical and hippocampal regions. RL/IF-1 induction occurred at 1 h and returned to basal levels by 8 h, consistent with the profile of an immediate early gene. Western blot analysis showed increases (∼30–65%) in GDI-1 protein present in both cytosolic and membrane fractions that were significant in the 84-kDa Rab bound form, suggesting that memantine influences Ras-like GTPase function. Genes regulated by a 5 mg/kg dose of memantine might be important in its therapeutic effects. These findings increase the number of known, differentially altered genes after treatment of uncompetitive NMDA receptor antagonists and suggest broader actions of these agents than previously realized.