Dynamics of trace element concentration during development and excitotoxic cell death in the cerebellum of Lurcher mutant mice.

Elevated concentrations of Zn, Cu and Fe, observed in biopsied or post-mortem tissue from diseased human brains, have often been considered as some of the major factors in the etiology of excitotoxic neuronal death but without any direct evidence for the causal role of metals. Although elevated metal concentrations that precede or coincide with the onset of neurodegeneration may provide such evidence, the dynamics of metal concentrations during excitotoxic cell death has never been established. Hence, we measured time-resolved Zn, Cu and Fe concentrations during the course of excitotoxic cell death in the Lurcher (Lc/+) mutant mouse with neutron activation analysis. In the Lc/+ cerebellum, Fe and Zn but not Cu concentrations were substantially lower than in normal cerebellum before the onset of neurodegeneration; then the concentration of all three metals doubled during excitotoxic Purkinje cell (PC) death, before stabilizing at abnormally high levels at the end of cell death progression. The rise in metal concentrations followed the onset and progression of PC loss after a delay of almost a week. This temporal correlation between neurodegenerative progression and metal concentrations indicates that elevated metal concentrations are the consequence of metabolic overload and glial activation during excitotoxicity rather than the primary cause of PC death.

Radioiodination of mouse anti-III beta-tubulin antibodies and their evaluation with respect to their use as diagnostic agents for peripheral neuropathies.

The monoclonal antibody TU-20 and its scFv fragment were radiolabeled with 125 I in order to develop new imaging agents against the specific neuronal marker III beta-tubulin. The reaction via chloramine-T using thiosulfate as a stopping reductant was determined as the most convenient way for radioiodination. The preserved immunological properties of radioiodinated species were estimated by ELISA, electrophoresis, and immunohistochemistry with autoradiography. Biodistribution studies revealed a different behavior of radioiodinated TU-20 and its scFv.

Translocation of cytochrome c during cerebellar degeneration in Lurcher and weaver mutant mice.

Cytochrome c translocation from the inner mitochondrial membrane into the cytosol is the initial step of the intrinsic apoptotic pathway. As no evidence was ever presented for cytochrome c translocation during cerebellar degeneration in Lurcher (Lc/+) and weaver (wv/wv) mutant mice, we searched for the presence of such a process in cerebellar homogenates of mutant and wild-type mice from postnatal day (P)1 to P56. Here we present the first documented time course of cytochrome c translocation spanning the entire period of neurodegeneration in both mutant types. We identified cytochrome c with Western blotting and monitored cell loss in the cerebellum with Calbindin D-28k immunohistochemistry, Nissl-staining and morphometry. No cytochrome c translocation was ever detected in wild-types at any age investigated. Translocated cytochrome c appeared between P13 and P21 in Lc/+ and between P5 and P6 in wv/wv. These two intervals precisely coincide with the respective periods of maximal neuronal death in the cerebellum. Secondary translocation was also observed at a later stage between P42 and P49 in Lc/+ and from P22 onwards in wv/wv. Since no substantial neuronal loss has ever been observed in Lc/+ and wv/wv mutants at these postnatal ages, the delayed translocation may correspond to cytochrome c of extraneuronal, presumably glial origin. Observations of an increased expression of glial fibrillary acidic protein and sustained remodeling of the astrocytic network in the cerebellum of both mutants, long after the cessation of neuronal death make this assumption rather plausible.

On the variety of cell death pathways in the Lurcher mutant mouse.

Apoptosis as well as autophagy have been implicated in the death of cerebellar Purkinje cells (PCs) in the Lurcher (Lc/+) mutant mouse and at least two different apoptotic pathways participate in the transsynaptic death of granule cells (GC) and inferior olivary (IO) neurones. The relative contribution of these pathways can only be assessed from their momentary involvement at any stage of the complete course of neurodegeneration. Here we used quantitative labelling for activated caspase-3 (Casp-3) and Fluoro-Jade B (FJ-B) to investigate the spatio-temporal pattern of neuronal death from P6 to P67 in Lc/+ mutants. Activated Casp-3 was present only in narrow time intervals (P14 to P22 in PCs; P14 to P28 in GCs) and in small subpopulations of PCs, GCs, and IO neurones. FJ-B positive PCs were detected during a broader period (P14 to P28), and outnumbered Casp-3 labelled PCs by a factor exceeding eight. Nevertheless, FJ-B labelling was restricted to PCs and never found in either GC or IO neurones. In conclusion, we present the first complete time course and extent of Casp-3 activation in Lc/+ mutants and show that the majority of dying neurones in Lc/+ mutants undergo Casp-3 independent cell death. The cellular overload produced by the initial gene defect in Lc/+ mutants apparently activates a variety of apoptotic and non-apoptotic pathways within the same neuronal population. Moreover, we present the first evidence for the ability of FJ-B to selectively label a discrete population of dying PCs, implying a higher selectivity of FJ-B than previously supposed.

TRAIL-related death receptors in normal, Lurcher and weaver mutant mouse brain.

In this study, we searched for murine analogues of the four death-receptor types (TRAIL-R1 to R4), targeted by the tumour necrosis factor related apoptosis inducing ligand (TRAIL), which were recently identified in the human brain. The expression of TRAIL-receptors in the normal murine brain was investigated using antibodies directed against different epitopes of the human TRAIL-receptors. Mouse mutants, in particular weaver and Lurcher with their well defined spatio-temporal patterns of neurodegeneration in the cerebellum, the inferior olive and the substantia nigra, were used as a model for investigating a potential contribution of TRAIL-receptors to the genetically determined cell death observed in these mutants. Although all antibodies used, recognized the respective human antigens, only the murine analogue of the human TRAIL-R2 epitope was also identified in the mouse brain. Antisera against human TRAIL-R1, TRAIL-R3 and TRAIL-R4 failed to reveal any other murine TRAIL-receptor analogue. In normal mice, TRAIL-R2 is not universally expressed throughout the brain but rather restricted to specific neuronal populations predominantly consisting of large neurons. In weaver, the spatial patterns and relative densities of TRAIL-R2 labelling were virtually identical to those seen in wild-types during the period of cell death in the cerebellum and the substantia nigra. In Lurcher, TRAIL-R2 expression in cerebellar granule cells and inferior olivary neurons was identical to that in wildtypes but significantly reduced in Purkinje cells undergoing degeneration. Thus, although TRAIL-R2 is found to be expressed in various cell types of the murine brain, cell death in weaver and Lurcher mutants is apparently not accompanied by an upregulation of TRAIL-receptors.

Management of restless legs syndrome by the partial D2-agonist terguride.

BACKGROUND: Terguride as a partial D2-receptors agonist seems suitable for treatment of restless legs syndrome (RLS). METHODS: Nine RLS patients without previous dopaminergic therapy received a daily dose of terguride (0.25 mg) 29.9+/-16.9 (SD) days. RESULTS: Two patients enrolled in the study failed to turn up for a successive check up. The seven subjects who were re-examined complied with the therapy. Their RLS symptoms improved (as measured on the International RLS intensity scale), decreasing from 24.3+/-5.3 to 14+/-4.7 (p=0.014). However, the terguride treatment did not significantly alter the daytime sleepiness or the subjective duration of nocturnal sleep. The daily dose was doubled in three patients who reported insufficient RLS improvement. One of the three patients later reported augmentation.