Yoshikuni Mizuno

Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism

Parkinsons disease is a common neurodegenerative disease with complex clinical features. Autosomal recessive juvenile parkinsonism (AR-JP), maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253 (ref. 4); the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, we have now isolated a complementary DNA clone of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3–7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and we have therefore named the protein product ‘Parkin’.

Diagnosis and management of dementia with Lewy bodies Third report of the DLB consortium

The dementia with Lewy bodies (DLB) Consortium has revised criteria for the clinical and pathologic diagnosis of DLB incorporating new information about the core clinical features and suggesting improved methods to assess them. REM sleep behavior disorder, severe neuroleptic sensitivity, and reduced striatal dopamine transporter activity on functional neuroimaging are given greater diagnostic weighting as features suggestive of a DLB diagnosis. The 1-year rule distinguishing between DLB and Parkinson disease with dementia may be difficult to apply in clinical settings and in such cases the term most appropriate to each individual patient should be used. Generic terms such as Lewy body (LB) disease are often helpful. The authors propose a new scheme for the pathologic assessment of LBs and Lewy neurites (LN) using alpha-synuclein immunohistochemistry and semiquantitative grading of lesion density, with the pattern of regional involvement being more important than total LB count. The new criteria take into account both Lewy-related and Alzheimer disease (AD)-type pathology to allocate a probability that these are associated with the clinical DLB syndrome. Finally, the authors suggest patient management guidelines including the need for accurate diagnosis, a target symptom approach, and use of appropriate outcome measures. There is limited evidence about specific interventions but available data suggest only a partial response of motor symptoms to levodopa: severe sensitivity to typical and atypical antipsychotics in ∼50%, and improvements in attention, visual hallucinations, and sleep disorders with cholinesterase inhibitors.

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR–JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR–JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR–JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

An Unfolded Putative Transmembrane Polypeptide, which Can Lead to Endoplasmic Reticulum Stress, Is a Substrate of Parkin

A putative G protein-coupled transmembrane polypeptide, named Pael receptor, was identified as an interacting protein with Parkin, a gene product responsible for autosomal recessive juvenile Parkinsonism (AR-JP). When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated in vivo. The insoluble Pael receptor leads to unfolded protein-induced cell death. Parkin specifically ubiquitinates this receptor in the presence of ubiquitin-conjugating enzymes resident in the endoplasmic reticulum and promotes the degradation of insoluble Pael receptor, resulting in suppression of the cell death induced by Pael receptor overexpression. Moreover, the insoluble form of Pael receptor accumulates in the brains of AR-JP patients. Here, we show that the unfolded Pael receptor is a substrate of Parkin, the accumulation of which may cause selective neuronal death in AR-JP.

Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease

Immunoblotting studies on mitochondria prepared from the striata of patients who died of Parkinsons disease were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with Parkinsons disease, the 30-, 25- and 24-kDa subunits of Complex I were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and Parkinsons disease. Deficiencies in Complex I subunits seem to be one of the most important clues to elucidate pathogenesis of Parkinsons disease.

Diagnostic Procedures for Parkinson's Disease Dementia : Recommendations from the Movement Disorder Society Task Force

A preceding article described the clinical features of Parkinsons disease dementia (PD‐D) and proposed clinical diagnostic criteria for “probable” and “possible” PD‐D. The main focus of this article is to operationalize the diagnosis of PD‐D and to propose pratical guidelines based on a two level process depending upon the clinical scenario and the expertise of the evaluator involved in the assessment. Level I is aimed primarily at the clinician with no particular expertise in neuropsychological methods, but who requires a simple, pragmatic set of tests that are not excessively time‐consuming. Level I can be used alone or in concert with Level II, which is more suitable when there is the need to specify the pattern and the severity on the dementia of PD‐D for clinical monitoring, research studies or pharmacological trials. Level II tests can also be proposed when the diagnosis of PD‐D remains uncertain or equivocal at the end of a Level I evaluation. Given the lack of evidence‐based standards for some tests when applied in this clinical context, we have tried to make practical and unambiguous recommendations, based upon the available literature and the collective experience of the Task Force. We accept, however, that further validation of certain tests and modifications in the recommended cut off values will be required through future studies.

Histochemical detection of apoptosis in Parkinson's disease

Apoptosis may be an important mechanism of cell death in some experimental disease models as well as in human neurodegenerative disorders. We report evidence to indicate apoptosis in the substantia nigra of Parkinsons disease patients. We studied the midbrains of 7 patients with late onset PD, 4 patients with young onset PD and 6 control subjects using the nick-end labeling method. Intense nuclear staining indicating apoptotic process was observed in 8 out of 11 parkinsonian patients studied. Apoptosis may be a contributor to the nigral neuronal death in Parkinsons disease (PD).

Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence

A mutant mitochondrial DNA (mtDNA) with a 4,977-bp deletion was detected in the parkinsonian brain by using the polymerase chain reaction. Although the deleted mtDNA was detectable even in the brain of aged controls, the proportion of deleted mtDNA to normal mtDNA in the striatum was higher in the parkinsonian patients than in the controls. In both the parkinsonian patients and the aged controls, the proportion was higher in the striatum than in the cerebral cortex. These results indicate that age-related accumulation of deleted mtDNA is accelerated in the parkinsonian striatum and suggest that the deletion contributes to pathophysiological processes underlying Parkinsons disease.

Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts.

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation ofN α-acetyl-l-arginine with MG at 37 °C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as theN δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-l-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (N δ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine).On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified asN δ-(5-methylimidazolon-2-yl)-l-ornithine (5-methylimidazolone), was found to be identical toN δ-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak atm/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the1H NMR spectrum of this product ind6-Me2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 °C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion.

Parkin Localizes to the Lewy Bodies of Parkinson Disease and Dementia with Lewy Bodies

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkins E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.