Poster Session P3: Related Neurodegenerative Conditions - Vascular Dementia~Multi-infarct Dementia $469

~ 1H-MRS AS A NOVEL TOOL 1N DIFFERENTIAL DIAGNOSIS OF DEMENTIAS. RATIONALE AND FEASIBILITY IN DEMENTIA WITH LEWY BODIES

Iwona Kloszewska*, Arkadiusz Rotkiewicz, Witold Gajewicz, Iwona Karlinska, Bozena Goraj, Radoslaw Magierski. Medical University Of Lodz, Lodz, Poland. Contact e-mail: iklosz@csk.am.lodz.pl

Background: In dementia with Lewy bodies (DLB), clinical studies suggest different pattern of brain metabolites pathology than in Alzheimer's disease (AD) but quantification is largely lacking. Objective(s): Aim of this study was to examine the feasibility and the utility of 1HMRS changes in the differential diagnosis of dementias. Methods: 1H-MRS examination was performed using 1.5 T- scanner in T1 -weigted images in 3 orthogonal planes with the single-voxel (SVS) technique using the STEAM sequence (TR 2000 ms, TE 20 ms) in DLB and AD patients and control subjects. Voxel (8 cm 3) was positioned in the temporal, occipital lobe and centrum semiovale. Results: 20 subjects were examined; MRS scans acquiring was unsuccessful because of movement artifacts in 3 DLB patients. Measurements were successful in all centrum semiovale and occipital regions in AD and control subjects. Acquiring proton spectra from temporal lobes were unsuccessful due to the voxel localization problems connected with brain atrophy in this region. Conclusions: In DLB patients 1HMRS examination is feasible but there are diffÉculties in scanning patients due to tremor and uncooperativeness. In DLB and AD subjects there are difficulties in acquiring proton spectra from temporal lobes in the later stages of the illness due to uncooperativeness and the degree of brain atrophy. 1HMRS is feasible in DLB patients early in the course of dementia.

• EFFECTS OF WILD-TYPE AND MUTANT PARKIN ON STRESS RESPONSES IN SH-SY5Y NEUROBLASTOMA CELLS

Monika Vestling* 1, Linda Sunesson 2, Richard F. Cowburn 2. 1Umed University, Umed, Sweden; 2 Karolinska Institutet, Stockholm, Sweden. Contact e-mail: monika.vestling@molbiol.umu.se

Background: Mutations in the Parkin gene cause early-onset autosomal recessive juvenile Parkinsonism (AR-JP). Parkin has been reported to function as an E3 ubiqnitin ligase. Several studies have indicated that genetic alterations of the protein change its ubiquitin ligase activity, leading to an increased accumulation of abnormal peptides in the cell, which trigger the unfolded protein response (UPR). Objective: We were interested in whether mutations causing AR-JP increase cellular vulnerability to endoplasmic reticulum (ER)-stress. Methods: Human dopaminergic neuroblastoma SH- SY5Y cells were stably transfected with wild-type Parkin and the AR-JP mutants G328E and R42E The cells were treated for 18 h with different stress inducers including tunicamycin (2 Ixg/ml), thapsigargin (500 nM), the calcium ionophore A23187 (1 IxM), hydrogen peroxide (50 IxM) and UV-light (80 kJ/m 2, 30 s) which induce misfolded polypeptides, increase calcium levels or cause other harmful conditions in the cell. The levels of Parkin, Hsp70 and the ER-stress sensitive protein Grp78/BiP were exmnined with immunoblotting. Results & Conclusions: We found that endogenous Parkin is up-regulated following treatment with tunicamycin in SH-SY5Y cells. The up-regulation of Parkin probably protects the cell from unfolded protein stress-induced neurotoxicity, via Parkins involvement in the ubiquitinafion pathway. No differences in the expression of either Hsp70 or Grp78/BiP could be seen between wild-type transfected and mutant parkin transfected cells after any of the treatments. Potential effects of these mutations on cell viability after treatment with the different stress inducers are being further elucidated.

P • - 4 0 • UBIQUITINATION OF ALPHA-SYNUCLEIN

Takashi Nonaka* 1, Takeshi Iwatsubo 2, Masato Hasegawa 1 . 1 Tokyo Institute of Psychiatry, Tokyo, Japan; 2Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, Tokyo, Japan. Contact e-mail: nonakat@prit.go.jp

Background: Filamentous ~-synuclein deposition is the defining hallmarks

of a subset of c~-synucleinopathy brains. We previously reported that c~- synuclein in those brains are extensively phosphorylated at Ser129, and also partially ubiquitinated. Objectlve(s): To study the role of ubiquitination on the formation of filamentous c~-synuclein deposition, we investigate the ubiquitination of c~-synuclein in vitro and in vivo, and analyzed the ubiquitination sites of c~-synucleln, and the effects of familial PD-linked mutations, phosphorylation, and fibril formation of ct-synucleln on the ubiqnitination. Methods: We established the in vitro ubiquitination system using rabbit reticulocyte lyzates and recombinant soluble or filamentous 0t-synuclein as substrate, and performed protein sequence analyses of the ubiquitinated ct-synuclein in vitro for the determination of ubiquitination sites. To study the in vivo ubiquitination of c~-synuclein, we examined the expression system using 293T cells with expression vectors of non-tagged c~-synuclein and hemagglntinin (HA)-tagged ubiqnitin. Results: Protein sequence analysis of the ubiquitinated c~-synuclein revealed that Lys21, Lys23, Lys32 and Lys34 are liable to ubiquitination in vitro. Site-directed mutagensis study confirmed that these are the major ubiquitination sites of c~-synuclein. Pathogenic A53T and A30P mutations had no significant effect on the ubiqnitination of c~-synuclein. Phosphorylation of c~-synuclein at Ser129 did not affect the ubiquitination. We also showed that filamentous ~-synuclein is less ubiqnitinated than soluble form of c~-synucleln, and that the major ubiquitination sites are localized to Lys6, Lysl0 and Lysl2 at the more amino-terminal region. Furthermore, we successfully detected the ubiquitination of non-tagged c~-synuclein in 293T cells by co-transfection with c~-synuclein and HA-ubiquitin. The in vivo ubiquitination sites of c~-synuclein were found to be almost identical to those of filamentous a-synuclein in vitro. Conclusion: Our data showed that even filamentous form of c~-synuclein can be ubiqnitinated, and in vitro nbiquitination sites of filament c~-synuclein are different from those of soluble c~-synuclein. We also found that ubiquitination sites of c~-synuclein in transfected 293T cells are almost identical to those of filamentous c~-synuclein in vitro. These data may have implication for the mechanisms of the formation of a-synuclein deposits in ~-synucleinopathy brains.

Poster Sess ion P3: Related Neurodegenerat ive Condi t ions - Vascular Dement ia /Mul t i - in farc t Dement ia

• VASCULAR DEMENTIA PATIENTS WHO RECEIVE DONEPEZIL TREATMENT FOR 12 TO 18 MONTHS MAINTAIN COGNITIVE BENEFITS

R. Doody* 1, R.D. Pratt 2, H. Posner 2, D. Kumar 3. JDepartment of Neurology, Baylor College of Medicine, Houston, TX, USA," 2 Eisai Medical Research Inc, Teaneck, N J, USA; 3Eisai Inc, Teaneck, N J, USA. Contact e-mail: rsutch@ppsiuk.com

Background: Donepezil treatment provides cognitive and global function benefits to patients with vascular dementia (VaD) for at least 6 months. Objective: To investigate the efficacy and tolerability of donepezil in a group of VaD patients who continued to receive treatment for 12 to 18 months. Methods: A multicenter, open-label (OL) extension study was conducted in patients with VaD (diagnosed according to the NINDS- AJREN criteria). Patients who had completed 1 of 2 identical double-blind, placebo-controlled, 24-week studies with donepezil were enrolled. Patients received donepezil 5 rag/day for the first 6 weeks, increased optionally to 10 mg/day. Results are reported for those patients who were randomized to donepezil treatment (5 or i0 rag/day) in the 24-week double-blind studies, for Week 30 OL and Week 54 OL observed cases. These patients received a total of 12-18 months of continual donepezil therapy. Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) scores are given as mean ± SE change from double-blind baseline scores. Results: 885 patients were enrolled in this open-label extension study; 674 (76%) completed 30 weeks of OL donepezil treatment, while a small group continued for 54 weeks (n = 192, 22%). 584 out of 885 patients were randomized to 24 weeks of active treatment during the double-blind studies and are included in this analysis. Patients who received donepezil for at least 12 months sustained their initial improvement in ADAS-cog scores (Week 30 OL: 5 rag/day, -1 .2 ± 0.4 [n