Atso Raasmaja

Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice.

Catechol‐O‐methyltransferase (COMT) catalyses the O‐methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT‐knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase‐dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT‐deficient mice are challenged with l‐dihydroxyphenylalanine, they show an extensive accumulation of 3,4‐dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage‐dependent, brain‐region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinsons disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.

Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) constitute a novel, evolutionarily conserved family of neurotrophic factors (NTF) expressed in vertebrates and invertebrates. The effects of two-week infusions of CDNF, MANF and glial cell line-derived neurotrophic factor (GDNF) were studied in a rat 6-hydroxydopamine (6-OHDA) hemiparkinsonian model. Degeneration of nigrostriatal dopamine nerve tract after toxin injection was assessed by measuring amphetamine-induced rotational behavior, and at the end of the experiment by quantifying tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) and TH-positive fibers in the striatum. The diffusion of the NTFs into the brain tissue following chronic infusion was also studied. Finally, we examined the transportation of intrastriatally injected (125)I-CDNF within the brain. The amphetamine-induced rotational behavior was gradually normalized in rats treated with CDNF for two weeks following the intrastriatal 6-OHDA injection. CDNF was also able to inhibit 6-OHDA-induced loss of TH-immunoreactive cells of the SNpc and TH-positive fibers in the striatum. MANF and GDNF had no statistically significant effect in any of the above measures. The volume of distribution for MANF in the striatum was significantly larger than that of GDNF after 3-day infusions. Both (125)I-CDNF and (125)I-GDNF were retrogradely transported from the striatum to the SN. No behavioral signs of toxicity were observed during treatment with the three NTFs. These results imply that CDNF may have potential as a neuroprotective or even neurorestorative therapy of PD.

Constitutive Ret activity in knock-in multiple endocrine neoplasia type B mice induces profound elevation of brain dopamine concentration via enhanced synthesis and increases the number of TH-positive cells in the substantia nigra.

Ret is the common signaling receptor for glial cell line-derived neurotrophic factor (GDNF) and other ligands of the GDNF family that have potent effects on brain dopaminergic neurons. The Met918Thr mutation leads to constitutive activity of Ret receptor tyrosine kinase, causing the cancer syndrome called multiple endocrine neoplasia type B (MEN2B). We used knock-in MEN2B mice with the Ret-MEN2B mutation to study the effects of constitutive Ret activity on the brain dopaminergic system and found robustly increased concentrations of dopamine (DA) and its metabolites in the striatum, cortex, and hypothalamus. The concentrations of brain serotonin were not affected and those of noradrenaline were slightly increased only in the lower brainstem. Tyrosine hydroxylase (TH) protein levels were increased in the striatum and substantia nigra/ventral tegmental area (SN/VTA), and TH mRNA levels were increased in SN/VTA of MEN2B mice, suggesting that constitutive Ret activity increases DA levels by increasing its synthesis. Also, the striatal DA transporter protein levels in the MEN2B mice were increased, which agrees with increased sensitivity of these mice to the stimulatory effects of cocaine. In the SN pars compacta of homozygous MEN2B mice, we found a 26% increase in the number of TH-positive cells, but no differences were found in the VTA. Thus, we show here that the constitutive Ret activity in mice is sufficient to increase the number of dopaminergic neurons and leads to profound elevation of brain DA concentration. These data clearly suggest that Ret activity per se can have a direct biological function that actively changes and shapes the brain dopaminergic system.

Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease

Cerebral dopamine neurotrophic factor (CDNF) protein has been shown to protect the nigrostriatal dopaminergic pathway when given as intrastriatal infusions in rat and mouse models of Parkinsons disease (PD). In this study, we assessed the neuroprotective effect of CDNF delivered with a recombinant adeno‐associated viral (AAV) serotype 2 vector in a rat 6‐hydroxydopamine (6‐OHDA) model of PD. AAV2 vectors encoding CDNF, glial cell line–derived neurotrophic factor (GDNF), or green fluorescent protein were injected into the rat striatum. Protein expression analysis showed that our AAV2 vector efficiently delivered the neurotrophic factor genes into the brain and gave rise to a long‐lasting expression of the proteins. Two weeks after AAV2 vector injection, 6‐OHDA was injected into the rat striatum, creating a progressive degeneration of the nigrostriatal dopaminergic system. Treatment with AAV2‐CDNF resulted in a marked decrease in amphetamine‐induced ipsilateral rotations while it provided only partial protection of tyrosine hydroxylase (TH)‐immunoreactive cells in the rat substantia nigra pars compacta and TH‐reactive fibers in the striatum. Results from this study provide additional evidence that CDNF can be considered a potential treatment of Parkinsons disease.

DIFFERENT SYNERGISTIC ROLES OF SMALL POLYETHYLENIMINE AND DOSPER IN GENE DELIVERY

Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (<250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.

Lack of robust protective effect of quercetin in two types of 6-hydroxydopamine-induced parkinsonian models in rats and dopaminergic cell cultures

In the present study, we examined the ability of a flavonoid quercetin to prevent 6-hydroxydopamine (6-OHDA)-induced oxygen radical formation and cytotoxicity in vitro and neurotoxicity in vivo. Quercetin (10-100 microM) had an acute significant antioxidant effect against the 6-OHDA-induced (30 microM) oxygen radical formation in catecholaminergic SH-SY5Y neuroblastoma cells. Moreover, in these cells, quercetin at 10-50 microM had a significant protective effect against 6-OHDA though at 100 microM it was itself harmful to the cells. The possible effect of quercetin in preventing neurotoxicity in unilateral medial forebrain bundle (full nigral lesion) or striatal (partial lesion) 6-OHDA rat lesion models of Parkinsons disease was studied in three treatment schedules: a 7-day pre- or post-treatment or their combination. Rotational responses to apomorphine (0.1 mg/kg, subcutaneously) and d-amphetamine (2.5 mg/kg, intraperitoneally) were assessed at weeks 1 and 2 post-lesion. Quercetin had no consistent neuroprotective effect in either model at 50-200 mg/kg once a day or 100 mg/kg twice a day. Furthermore, no protection was observed in tyrosine hydroxylase positive nigral cell numbers, striatal fiber density or in striatal levels of dopamine. These in vitro and in vivo results cast doubt on the theory that quercetin exerts reliable neuroprotective effects against 6-OHDA-induced toxicity. In vitro, quercetin seems to be protective at low doses but damaging at high doses.

The use of low-molecular-weight PEIs as gene carriers in the monkey fibroblastoma and rabbit smooth muscle cell cultures

Polyethylenimines (PEIs) and cationic polymers have been used successfully in gene delivery. In earlier reports, only large PEIs (MW>10 000) have shown significant transfection efficiency. In the present study, the roles of small PEIs (MW 700 and 2000) were studied as additional compounds to see if they can improve gene delivery with cationic liposomes.

Mechanisms of polyethylenimine-mediated DNA delivery: free carrier helps to overcome the barrier of cell-surface glycosaminoglycans

Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/− charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell‐surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear.

Anti-obesity effect of MPV-1743 A III, a novel imidazoline derivative, in genetic obesity

MPV-1743 A III ((+/-)-4-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a novel imidazoline derivative. In this study, it was shown to bind with high affinity to alpha2-adrenoceptor subtypes alpha2A (IC50) = 0.66 +/- 0.06 nM), alpha2B (IC50) = 3.8 +/- 0.53 nM), alpha2C (IC50) = 3.1 +/- 0.61 nM) in the recombinant S115 cells and to alpha2D (IC50 = 0.94 +/- 0.10 nM) in the rat submandibular gland. MPV-1743 A III also showed remarkably high affinity to alpha1-adrenoceptors (IC50 = 150 +/- 12 nM) in the rat cerebral cortex and to imidazoline I2b-binding sites (IC50) = 150 +/- 5.0 nM) in the rat liver. The functional alpha2-adrenoceptor antagonistic effect of MPV-1743 A III was demonstrated by studying the ability of orally administered MPV-1743 A III to reverse and prevent the alpha2-adrenoceptor agonist detomidine-induced mydriasis in rat. The anti-obesity effect of MPV-1743 A III was investigated in genetically obese (fa/fa) Zucker rats in two different phases of obesity. Chronic treatment with MPV-1743 A III (0.3 3 mg/kg per day p.o. for 3 weeks) dose dependently decreased weight gain in early-phase obesity. In fully established obesity, GDP binding to mitochondria and expression of uncoupling protein mRNA were increased in brown adipose tissue by MPV-1743 A III indicating an activation of non-shivering thermogenesis. The present study shows that MPV- 1743 A III has a modest anti-obesity effect in the genetic rodent model of obesity. The relative importance of alpha2- and alpha1-adrenoceptors and imidazoline I2b-binding sites in mediating the effects of MPV-1743 A III needs further evaluation.

The role of PEI structure and size in the PEI/liposome-mediated synergism of gene transfection.

Polyethylenimines (PEIs) and cationic liposomes are widely used for nonviral gene delivery. When PEIs have been used alone, the transfection efficiency has been higher for larger or linear than smaller or branched PEIs. We have reported previously that a combination of small PEIs and liposomes results in a potentiation of transfection efficiency in vitro. Here, the role of PEI size and structure in this synergism has been clarified further. Therefore, two structurally different high MW PEIs, i.e. the linear PEI22K and branched PEI25K, were studied in the SMC cells. We found that both linear PEI22K and branched PEI25K resulted in a similar synergism and comparable transfection efficiencies. However, the potentiation for larger PEIs found in the present study was weaker than that for smaller PEIs obtained in our previous studies. In conclusion, our present and previous results demonstrate that the increment of PEI/liposome-mediated gene transfection by different types of PEIs in vitro is a common attribute that is rather associated with their size than the structure. Interestingly, the effect of PEI size seems to be opposite when combined with liposome or given alone, i.e. the small PEIs are more effective when combined and less effective when alone than the larger ones.