Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.     

Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity

Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process.     

Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP)

Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N-terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D-NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.     

The role of astroglia on the survival of dopamine neurons

Glial cells play a key role in the function of dopamine (DA) neurons and regulate their differentiation, morphology, physiological and pharmacological properties, survival, and resistance to different models of DA lesion. Several studies suggest that glial cells may be important in the pathogenesis of Parkinson’s disease (PD), a common neurodegenerative disorder characterized by degeneration of the nigrostriatal DA system. In this disease the role of glia could be due to the excessive production of toxic products such as nitric oxide (NO) or cytokines characteristic of inflammatory process, or related to a defective release of neuroprotective agents, such as small antioxidants with free radical scavenging properties or peptidic neurotrophic factors.     

Taurine-induced attenuation of MPP+ neurotoxicity in vitro: a possible role for the GABA(A) subclass of GABA receptors

Taurine is a sulphur-containing beta-amino acid found in high (millimolar) concentrations in excitable tissues such as brain and heart. Its suggested roles include osmoregulator, thermoregulator, neuromodulator, and potential neurotransmitter. This amino acid has also been shown to be released in large concentrations during ischaemia and excitotoxin-induced neuronal damage. Here we report a protective effect of taurine against MPP(+)-induced neurotoxicity in coronal slices from rat brain. Significant protective effects were observed at taurine concentrations of 20 and 1 mM, suggesting a potential role for taurine in cases of neuronal insult. Studies with the synthetic taurine analogues taurine phosphonate, guanidinoethane sulphonate, and trimethyltaurine suggested the observed effect to be mediated via an extracellular mechanism. The use of GABA receptor ligands muscimol and bicuculline indicated the effect to be mediated through activation of GABA(A) receptors.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

Distinct effects of atypical 1,4-dihydropyridines on 1-methyl-4-phenylpyridinium-induced toxicity

Our previous data obtained from in vivo experiments demonstrated high neuroprotective effects of three novel atypical neuronal non-calcium antagonistic 1,4-dihydropyridine (DHP) derivatives cerebrocrast, glutapyrone and tauropyrone. The present studies were carried out in vitro to clarify, at least in part, their mechanism of action in primary culture of cerebellar granule cells by use of 1-methyl-4-phenylpyridinium (MPP+) as a neurotoxic agent which causes dramatic oxidative stress. Cerebrocrast (highly lipophilic, with a classical two-ring structure) dose-dependently (0.01-10.0 microM, EC50 = 13 nM) reduced MPP+-induced cell death. At the same time, the calcium antagonist nimodipine (reference drug) protected cell death at much higher concentrations (EC50 = 12.4 microM). Cerebrocrast decreased also the generation of reactive oxygen species and loss of mitochondrial membrane potential. In contrast, low lipophilic amino acid-containing DHPs glutapyrone and tauropyrone (glutamate- and taurine-containing, correspondingly) were without significant effects indicating their distinct mode of action in comparison to cerebrocrast. We have demonstrated for the first time an ability of atypical non-calcium antagonistic DHP cerebrocrast (which has classical DHP structure elements and high lipophilicity) to protect MPP+-induced deterioration of mitochondrial bioenergetics. One may suggest mitochondria as an essential intracellular target for the neuroprotective action of cerebrocrast and indicate its usefulness in the treatment of Parkinson's disease.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.     

Efficiently Tracking Selection in a Multiparental Population: The Case of Earliness in Wheat

Multiparental populations are innovative tools for fine mapping large numbers of loci. Here we explored the application of a wheat Multiparent Advanced Generation Inter-Cross (MAGIC) population for QTL mapping. This population was created by 12 generations of free recombination among 60 founder lines, following modification of the mating system from strict selfing to strict outcrossing using the ms1b nuclear male sterility gene. Available parents and a subset of 380 SSD lines of the resulting MAGIC population were phenotyped for earliness and genotyped with the 9K i-Select SNP array and additional markers in candidate genes controlling heading date. We demonstrated that 12 generations of strict outcrossing rapidly and drastically reduced linkage disequilibrium to very low levels even at short map distances and also greatly reduced the population structure exhibited among the parents. We developed a Bayesian method, based on allelic frequency, to estimate the contribution of each parent in the evolved population. To detect loci under selection and estimate selective pressure, we also developed a new method comparing shifts in allelic frequency between the initial and the evolved populations due to both selection and genetic drift with expectations under drift only. This evolutionary approach allowed us to identify 26 genomic areas under selection. Using association tests between flowering time and polymorphisms, 6 of these genomic areas appeared to carry flowering time QTL, 1 of which corresponds to Ppd-D1, a major gene involved in the photoperiod sensitivity. Frequency shifts at 4 of 6 areas were consistent with earlier flowering of the evolved population relative to the initial population. The use of this new outcrossing wheat population, mixing numerous initial parental lines through multiple generations of panmixia, is discussed in terms of power to detect genes under selection and association mapping. Furthermore we provide new statistical methods for use in future analyses of multiparental populations.