倫理創成研究の動向 : シュレーダー＝フレチェットの『リスクと合理性』から

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation

Die Rolle von striatalen Tyrosinhydroxylase-positiven Neuronen bei L-DOPA-induzierten Dyskinesien der Maus

Die Parkinson-Krankheit ist die zweithäufigste neurodegenerative Erkrankung weltweit. Der Goldstandard zur symptomatischen Behandlung ist der Wirkstoff L 3,4 Dihydroxyphenylalanin (L-DOPA), der im Verlauf jedoch bei vielen Patienten ausgeprägte motorische Nebenwirkungen in Form von abnormen unfreiwilligen Bewegungen, sogenannten L-DOPA-induzierten Dyskinesien (LID), hervorruft. Deren genaue Ätiopathogenese liegt bislang noch im Dunkeln. Das Ziel dieser Arbeit war es, die Expression Tyrosinhydroxylase(TH)-positiver und damit L-DOPA-produzierender Zellen im denervierten Corpus striatum (Striatum) als fundamentalen Faktor für die Entstehung und Ausprägung von LID aufzudecken. Hierzu wurden 6-Hydroxydopamin-lädierte Mäuse über 15 Tage mit L-DOPA behandelt, das Auftreten von LID wurde charakterisiert und mit der immunhistochemisch bestimmten Anzahl striataler TH-positiver Zellen korreliert. Bemerkenswerterweise entwickelten 70% der L-DOPA-behandelten Mäuse ausgeprägte LID, die mit einer deutlich gesteigerten Expression TH-positiver Neurone einhergingen und deren Ausprägung eng mit der Zellzahl korrelierte. Die Zunahme TH-positiver Neurone stand im Einklang mit einer gesteigerten Expression von ∆FosB, einem validen molekularen Marker für Dyskinesien. Zudem wiesen dyskinetische Tiere eine erhöhte serotonerge Innervation des Striatums auf – Fasern, die mit ihrer aromatischen Aminosäure-Decarboxylase eine Konversion von L-DOPA in Dopamin vollführen können. Es ist also davon auszugehen, dass die TH-positiven Zellen synergistisch mit serotonergen Terminalen Dopamin synthetisieren und damit erhöhte extrazelluläre Dopaminkonzentrationen begünstigen. Dieser ursprünglich kompensatorische Mechanismus als Antwort auf das striatale dopaminerge Defizit beim Morbus Parkinson scheint im Rahmen einer L-DOPA-Therapie über das Ziel hinaus zu schießen und resultiert in der Entwicklung von LID. Zentraler Entstehungsort ist hierbei das laterale, dem menschlichen Putamen entsprechende Striatum, wohingegen ein prodyskinetischer Effekt der TH-Zellen im Nucleus accumbens und im Cortex ausgeschlossen werden konnte. Die Erkenntnisse dieser Arbeit tragen grundlegend zu einem besseren Verständnis über die Ätiopathogenese von LID und die Funktionalität der weitgehend unerforschten TH positiven Neurone bei. Zukünftige Studien, die sich die TH-positiven Zellen für eine potentielle Parkinsontherapie zunutze machen wollen, sollten deren LID-provozierenden Effekt kritisch berücksichtigen. Auf der anderen Seite könnten diese Neurone langfristig einen hoffnungsvollen Ansatz für neue antidyskinetische Behandlungsstrategien liefern

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[125I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac2[125I]IUdR), 5-[125I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv2[125I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac2[125I]IUdR, Piv2[125I]IUdR and [125I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac2[125I]IUdR and Piv2[125I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [125I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv2[125I]IUdR>Ac2[125I]IUdR>[125I]IUdR. For Ac2[125I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [125I]IUdR, Ac2[125I]IUdR and Piv2[125I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability

Neuregulin-1 receptor tyrosine kinase ErbB4 is upregulated in midbrain dopaminergic neurons in Parkinson disease

Previously we demonstrated that systemically administered neuregulin-1-beta 1, a nerve growth and differentiation factor, passed the blood-brain barrier and accumulated in brain areas with expression of its receptor ErbB4. In substantia nigra (SN), neuregulin-1-beta 1 phosphorylated ErbB4 and protected dopaminergic neurons in a toxin-based mouse model of Parkinson disease (PD). We studied ErbB4 in the context of human midbrain dopaminergic degeneration in vivo and in vitro. Post-mortem ventral midbrain tissue sections of neuropsychiatric healthy individuals and PD patients (matched for age, gender and post-mortem delay) were immunostained for ErbB4. Cultured Lund human mesencephalic (LUHMES) post-mitotic dopaminergic neurons were treated with dopaminergic toxins and analyzed for ErbB4 expression. In control individuals, 85.0 +/- 5.0% of dopaminergic neurons, containing cytoplasmic neuromelanin, expressed ErbB4 in the SN. In PD cases, the percentage of ErbB4-positive nigral dopaminergic neurons was increased to 94.9 +/- 2.5%. The mean ErbB4 immunoreactivity of melanized neurons was higher in PD than controls. LUHMES neurons upregulated ErbB4 when exposed to toxins 1-methyl-4-phenylpyridinium and 6-hydroxydopamine. Increased rate of ErbB4-positive dopaminergic neurons in PD may either reflect a better survival of ErbB4-positive neurons or an increased expression of ErbB4 by remaining neurons to seek trophic support. Enhanced ErbB4 expression in human in vitro toxin-based PD models supports the latter interpretation. Thus, dopaminergic neurons in SN might be susceptible to neuregulin-1 treatment in PD. (C) 2012 Elsevier Ireland Ltd. All rights reserved

Effect of long-term treatment with pramipexole or levodopa on presynaptic markers assessed by longitudinal [123I]FP-CIT SPECT and histochemistry

A previous clinical trial studied the effect of long-term treatment with levodopa (LD) or the dopamine agonist pramipexole (PPX) on disease progression in Parkinson disease using SPECT with the dopamine transporter (DAT)-radioligand [(123)I]β-CIT as surrogate marker. [(123)I]β-CIT binding declined to significantly lower levels in patients receiving LD compared to PPX. However, the interpretation of this difference as LD-induced neurotoxicity, PPX-induced neuroprotection/-regeneration, or only drug-induced regulatory changes of DAT-availability remained controversial. To address this question experimentally, we induced a subtotal lesion of the substantia nigra in mice by bilateral injection of the neurotoxin 6-hydroxydopamine. After 4 weeks, mice were treated for 20 weeks orally with LD (100mg/kg/day) or PPX (3mg/kg/day), or water (vehicle) only. The integrity of nigrostriatal projections was assessed by repeated [(123)I]FP-CIT SPECT in vivo and by immunostaining for DAT and the dopamine-synthesizing enzyme tyrosine hydroxylase (TH) after sacrifice. In sham-lesioned mice, we found that both LD and PPX treatment significantly decreased the striatal FP-CIT binding (LD: -21%; PPX: -14%) and TH-immunoreactivity (LD: -42%; PPX: -45%), but increased DAT-immunoreactivity (LD: +42%; PPX: +33%) compared to controls without dopaminergic treatment. In 6-hydroxydopamine-lesioned mice, however, neither LD nor PPX significantly influenced the stably reduced FP-CIT SPECT signal (LD: -66%; PPX: -66%; controls -66%), TH-immunoreactivity (LD: -70%; PPX: -72%; controls: -77%) and DAT-immunoreactivity (LD: -70%; PPX: -75%; controls: -75%) in the striatum or the number of TH-positive cells in the substantia nigra (LD: -88%; PPX: -88%; controls: -86%), compared to lesioned mice without dopaminergic treatment. In conclusion, chronic dopaminergic stimulation with LD or PPX induced similar adaptive presynaptic changes in healthy mice, but no discernible changes in severely lesioned mice. These findings allow to more reliably interpret the results from clinical trials using neuroimaging of DAT as surrogate parameter. Copyright © 2013 Elsevier Inc. All rights reserved

Additional file 1: of Loss of cerebellar neurons in the progression of lentiviral disease: effects of CNS-permeant antiretroviral therapy

Figure S1. Colocalization of IBA-1/SIV gp41. Figure S2. Breakdown of BBB. Table S1. Neuroinflammatory activity score. PDF 526 kb

Toll like receptor 4 mediates cell death in a mouse MPTP model of Parkinson disease

International audienceIn mammalians, toll-like receptors (TLR) signal-transduction pathways induce the expression of a variety of immune-response genes, including inflammatory cytokines. It is therefore plausible to assume that TLRs are mediators in glial cells triggering the release of cytokines that ultimately kill DA neurons in the substantia nigra in Parkinson disease (PD). Accordingly, recent data indicate that TLR4 is up-regulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in a mouse model of PD. Here, we wished to evaluate the role of TLR4 in the acute mouse MPTP model of PD: TLR4-deficient mice and wild-type littermates control mice were used for the acute administration way of MPTP or a corresponding volume of saline. We demonstrate that TLR4-deficient mice are less vulnerable to MPTP intoxication than wild-type mice and display a decreased number of Iba1+ and MHC II+ activated microglial cells after MPTP application, suggesting that the TLR4 pathway is involved in experimental PD

Susceptibility to N-glycosidic bond cleavage by thymidine phosphorylase after 0.5 and 6 h (n = 4) of incubation.

<p>values are given as percentage related to the amount of incubated iodonucleoside (mean ± SEM).</p><p>*experimentally determined detection minimum of the HPLC system.</p