Gerda Andringa

Tissue transglutaminase catalyzes the formation of alpha-synuclein crosslinks in Parkinson’s disease

In Parkinsons disease (PD), conformational changes in the α‐synuclein monomer precede the formation of Lewy bodies. We examined postmortem PD and undiseased (control) substantia nigra for evidence of pathological crosslinking of α‐synuclein by tissue transglutaminase (tTG) using immunohistochemistry, immunoprecipitation, and Western blot. Consistent with previous reports, we found that both tTG and its substrate‐characteristic Nε‐(γ‐glutamyl)‐lysine crosslink are increased in PD nigral dopamine neurons. Furthermore, both the tTG protein and its substrate crosslink coprecipitated with α‐synuclein in extracts of PD substantia nigra. Unexpectedly, the isodipeptide crosslink was detected in the α‐synuclein monomer as well as in higher molecular mass oligomers of α‐synuclein. Although the intramolecularly crosslinked α‐synuclein monomer was present in control tissue, it was highly enriched in PD substantia nigra. Conversely, significantly less uncrosslinked α‐synuclein remained in the postimmunoprecipitate lysate of PD tissue than in control. Crosslinked α‐synuclein, formed at the expense of the total α‐synuclein monomer, correlated with disease progression. These results demonstrate that much of the α‐synuclein monomer in PD nigra is crosslinked by tTG and thus may be functionally impaired. This modification appears to be an early step in PD pathogenesis, preceding the aggregation of α‐synuclein in Lewy bodies.

[123I]FP-CIT binds to the dopamine transporter as assessed by biodistribution studies in rats and SPECT studies in MPTP-lesioned monkeys

[123I]FP‐CIT (N‐ω‐fluoropropyl‐2β‐carbomethoxy‐3β‐{4‐iodophenyl}tropane), a radioiodinated cocaine analogue, was evaluated as an agent for the in vivo labeling of dopamine (DA) transporters by biodistribution studies in rats and by single photon emission computed tomography (SPECT) studies in unilateral 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)–lesioned monkeys. In rats, intravenous injection of [123I]FP‐CIT resulted in high accumulation of radioactivity in the striatum. Less pronounced uptake was seen in brain areas with high densities of serotonergic uptake sites. While striatal uptake of radioactivity after injection of [123I]FP‐CIT was displaced significantly by GBR12,909 but not by fluvoxamine, the opposite was observed in brain areas known to be rich of serotonin transporters. Monkeys which were unilaterally treated with neurotoxic doses of MPTP showed severe loss of striatal [123I]FP‐CIT uptake at the side of treatment. The results of this study indicate that [123I]FP‐CIT, although not being a selective radioligand, binds specifically to the striatal DA transporter in vivo and thus suggest that [123I]FP‐CIT promises to be a suitable radioligand for SPECT imaging of DA transporters in humans. Synapse 27:183–190, 1997.

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinsons disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.

The alleged dopamine D1 receptor agonist SKF 83959 is a dopamine D1 receptor antagonist in primate cells and interacts with other receptors

So far, no clear correlation has been found between the effects of dopamine D1 receptor agonists on motor behavior in primate models of Parkinsons disease and their ability to stimulate adenylate cyclase in rats, the benzazepine SKF 83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H- 3-benzazepine) being the most striking example. Since this discrepancy might be attributed to: (A) the different species used to study these effects or (B) the interaction of SKF 83959 with other catecholamine receptors, the aims of this study were: (1) to study the ability of SKF 83959 to stimulate adenylate cyclase in cultured human and monkey glial cells equipped with dopamine D1 receptors and (2) to evaluate the affinity for and the functional interaction of SKF 83959 with other catecholamine receptors. Binding studies revealed that SKF 83959 displayed the highest affinity for the dopamine D1 receptor (pKi=6.72) and the alpha2-adrenoceptor (pKi=6.41) and moderate affinity for the dopamine D2 receptor and the noradrenaline transporter. In monkey and human cells, SKF 83959 did not stimulate cyclic adenosine monophosphate (cAMP) formation to a significant extent, but antagonized very potently the dopamine-induced stimulation of cAMP formation in both cell types. The compound stimulated basal dopamine outflow and inhibited depolarization-induced acetylcholine release only at concentrations > 10 microM. Finally, SKF 83959 concentration dependently increased electrically evoked noradrenaline release, indicating that it had alpha2-adrenoceptor blocking activity and interfered with the noradrenaline transporter. In conclusion, SKF 83959 is a potent dopamine D1 receptor and alpha2-adrenoceptor antagonist. Thus, the anti-parkinsonian effects of SKF 83959 in primates are not mediated by striatal dopamine D1 receptors coupled to adenylate cyclase in a stimulatory way.

Sub-chronic administration of the dopamine D(1) antagonist SKF 83959 in bilaterally MPTP-treated rhesus monkeys: stable therapeutic effects and wearing-off dyskinesia.

Abstract  Rationale: SKF 83959 acts as a D1 antagonist in vitro but has been claimed to induce anti-parkinsonian effects after acute administration in MPTP-treated marmosets. Objective: The aim of the present study was to evaluate the therapeutic and undesired effects of sub-chronic administration of SKF 83959 in bilaterally MPTP-treated rhesus monkeys and to compare these effects with the effects of l-dopa and the dopamine agonist SKF 82958. Methods: MPTP was given in the left carotid artery (2.5 mg) and 6 weeks later, the right carotid artery (1.25 mg). The monkeys (n=4) had previously been treated chronically with l-dopa (22 days, 10 mg/kg) and SKF 82958 (22 days, 1 mg/kg). Three months after the last administration of SKF 82958, SKF 83959 was given in a dose of 0.5 mg/kg from day 1 to day 15 and in a dose of 1.0 mg/kg from day 16 to day 18. Results:SKF 83959 increased goal-directed limb movements in all animals, including those unresponsive to l-dopa. This therapeutic effect did not diminish during treatment. With respect to body displacement and undesired effects, a large variation in the response to SKF 83959 was found: a large increase in body displacement co-occurred with oro-facial dyskinesia (n=2), whereas a small increase in body displacement co-occurred with dystonia (n=2). In contrast to the undesired effects of l-dopa, the dyskinetic effects of SKF 83959 were primarily limited to the first treatment day. Unlike l-dopa and SKF 82958, SKF 83959 did not induce epileptoid behaviour. Conclusion: Sub-chronic administration of SKF 83959 induced both clear-cut therapeutic effects that remained stable in time, and a limited number of dyskinetic effects that wore off during the treatment. The dopamine D1 antagonist SKF 83959 may be considered as an alternative treatment in Parkinson’s disease, especially in those patients who do not respond to l-dopa.

Pinhole SPECT imaging of dopamine transporters correlates with dopamine transporter immunohistochemical analysis in the MPTP mouse model of Parkinson's disease.

The in vivo analysis of dopaminergic degeneration in animal models of Parkinsons disease (PD), using pinhole single photon emission computed tomography (SPECT), ideally should afford a serial study design, enabling the analysis of the degenerative process as well as the potential neuroprotective and/or restorative properties of drugs over time in living animals. Previously, we demonstrated that striatal dopamine transporter (DAT) levels in rats could be analyzed reproducibly, using pinhole SPECT with the DAT probe [(123)I]N-omega-fluoropropyl-2beta-carbomethoxy-3beta-{4-iodophenyl}nortropane (FP-CIT). However, the capacity of this approach to accurately detect a range of striatal DAT levels in the most widely used animal model of PD, i.e., the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse, remains to be determined. For this purpose, various levels of DAT were induced by treating c57BL/6J mice for 1, 3, or 5 days with MPTP (25 mg/kg ip), respectively. [(123)I]FP-CIT SPECT scans were performed 5 days after the last MPTP injection. Mice were perfused 6 days after the last MPTP injection, and the SPECT data were compared to ex vivo striatal and nigral DAT levels as measured by immunohistochemistry within the same animals. The analysis of striatal DAT levels using SPECT and DAT immunohistochemistry yielded highly comparable results on the percentage of DAT reduction in each MPTP group. The in vivo data showed a decrease of specific striatal to non-specific binding ratios by 59%, 82%, and 76% in mice treated for 1, 3, and 5 days, respectively. Moreover, a strong, positive correlation was observed between the in vivo and ex vivo parameters. The present study provides the first evidence that [(123)I]FP-CIT pinhole SPECT allows the accurate detection of a range of striatal DAT (i.e., losses of approximately 60-80%) levels in mice. Since such large dopaminergic lesions could be detected, this SPECT method may at least be useful for analyzing neuroprotective treatment with a clear-cut positive (i.e., complete protection) or negative (i.e., not any protection) effect. Whether this method is also useful for analyzing more subtle effects of neuroprotective treatment (partial protection) remains to be established, by studying mice with small dopaminergic lesions.

TCH346 prevents motor symptoms and loss of striatal FDOPA uptake in bilaterally MPTP-treated primates

The neuroprotective efficacy of the propargylamine TCH346 was studied in the primate model of Parkinsons disease, the bilaterally MPTP-treated monkey. Male rhesus monkeys received 2.5 mg MPTP into the left carotid artery and, 8 weeks later, 1.25 mg MPTP into the right carotid artery. Starting 2 h after the second MPTP infusion, either 0.014 mg/kg TCH346 or its solvent was subcutaneously injected twice per day for 14 days. The first MPTP treatment induced mild Parkinson symptoms, reduced right limb movements, and reduced FDOPA uptake in the left striatum. The second MPTP treatment made Parkinson symptoms worse, reduced left limb movements, and reduced FDOPA uptake in the right striatum of solvent-treated monkeys. In contrast, the second MPTP treatment did not further worsen motor symptoms and did not decrease FDOPA uptake in the right striatum of TCH346-treated monkeys. Although the effects of the second MPTP treatment were largely prevented, the effects of the first MPTP treatment were not reversed by TCH346. Immunohistochemical examination confirmed the dramatic loss of dopamine cells in vehicle-treated monkeys and the preservation of these neurons in the right brain side of the TCH346-treated animals. In conclusion, systemic administration of TCH346 prevented motor symptoms and nigrostriatal degeneration induced by MPTP in primates.

Presence of tissue transglutaminase in granular endoplasmic reticulum is characteristic of melanized neurons in Parkinson's disease brain.

Parkinsons disease (PD) is characterized by the accumulation of α‐synuclein aggregates and degeneration of melanized neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross‐linking. In PD brain, tTG‐induced cross‐links have been identified in α‐synuclein monomers, oligomers and α‐synuclein aggregates. However, whether tTG and α‐synuclein occur together in PD affected neurons remains to be established. Interestingly, using immunohistochemistry, we observed a granular distribution pattern of tTG, characteristic of melanized neurons in PD brain. Apart from tTG, these granules were also positive for typical endoplasmic reticulum (ER)‐resident chaperones, that is, protein disulphide isomerase, ERp57 and calreticulin, suggesting a direct link to the ER. Additionally, we observed the presence of phosphorylated pancreatic ER kinase (pPERK), a classical ER stress marker, in tTG granule positive neurons in PD brain, although no subcellular colocalization of tTG and pPERK was found. Our data therefore suggest that tTG localization to granular ER compartments is specific for stressed melanized neurons in PD brain. Moreover, as also α‐synuclein aggregates were observed in tTG granule positive neurons, these results provide a clue to the cellular site of interaction between α‐synuclein and tTG.

Changed distribution pattern of the constitutive rather than the inducible HSP70 chaperone in neuromelanin‐containing neurones of the Parkinsonian midbrain

Aberrant protein aggregation has been recognized as an important factor in the degeneration of melanized dopaminergic neurones in Parkinsons disease (PD). The constitutive (HSP73) and (heat)‐inducible (HSP72) proteins of the heat shock 70 family form a major defence system against pathological protein aggregation. However, the distribution patterns of these chaperones in nigral neuromelanin‐laden neurones are largely unknown. The present study determined the distribution of HSP72 and HSP73 in control and Parkinsonian substantia nigra, using immunohistochemistry. In the neuromelanin‐laden neurones of controls, HSP72 was nondetectable, whereas HSP73 was weakly expressed in both the cytosol and the nucleus. Surprisingly, in PD subjects, marked nuclear HSP73, but not HSP72 immunoreactivity was observed, while cytosolic immunoreactivity of the two chaperones resembled the labelling pattern observed in controls. Furthermore, HSP73 immunoreactivity was observed in a subset of the Lewy bodies (LBs) detected in the substantia nigra of PD subjects, whereas only few of these LBs were labelled with HSP72. Interestingly, HSP72 and to a lesser extent HSP73 immunoreactivity was much stronger in nonmelanized neurones as compared with melanized neurones in this area. Thus, we conclude that the distribution pattern of HSP73 rather than HSP72 is changed in the nigral neuromelanin‐laden neurones of PD subjects as compared with control subjects. The impaired ability of aged, dopaminergic neurones to express high levels of chaperones, may contribute to the preferential vulnerability of the latter cells in PD.

The thiol antioxidant 1,2-dithiole-3-thione stimulates the expression of heat shock protein 70 in dopaminergic PC12 cells

In Parkinsons disease (PD), the pathogenic factors oxidative stress and protein aggregation interact and culminate in the apoptotic death of (mainly catecholaminergic) neurons. The dithiolethiones comprise thiol antioxidants that are well known for their activation of the expression of a wide collection of cytoprotective genes, including genes coding for antioxidant enzymes. Given the observation that heat shock proteins (HSPs), in particular the heat shock protein 72 (HSP72), protects against cellular degeneration in various models of PD, the ability of the unsubstituted dithiolethione 1,2-dithiole-3-thione (D3T) to stimulate heat shock protein gene and protein expression was studied using the dopaminergic PC12 cell line. As anticipated, D3T stimulated the expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). Quantitative PCR analysis revealed that D3T stimulates the expression of the inducible, cytoplasmic HSP72. Moreover, D3T strongly potentiated HSP72 gene and protein expression in heat-stressed cells. Taken together, our data show that, in addition to antioxidant enzymes, D3T stimulates the expression of HSP72, a chaperone shown to be neuroprotective in various models of PD, in particular under conditions of cellular stress. Thus, the broad range manipulation of endogenous cellular defense mechanisms, through D3T, may represent an innovative approach to therapeutic intervention in PD.