Hideki Shimura

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.

Immunohistochemical and subcellular localization of parkin protein: Absence of protein in autosomal recessive juvenile parkinsonism patients

Autosomal recessive juvenile parkinsonism (AR‐JP) is a distinct clinical entity characterized by a selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR‐JP has been identified. Now, we report the subcellular localization of Parkin protein in patients with AR‐JP or Parkinsons disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule. Parkin protein was absent in all regions of the brains of patients with AR‐JP. Parkin protein was not decreased in the brains of sporadic PD patients. Immunoreactivity was detected in a few Lewy bodies. Parkin protein was located in both the Golgi complex and cytosol. Ann Neurol 1999;45:668–672

Parkin is associated with cellular vesicles

We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52‐kDa protein with a ubiquitin‐like domain and two RING‐finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans‐Golgi network and the secretory vesicles in U‐373MG or SH‐SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non‐ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein‐tagged deletion mutants of parkin into COS‐1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin‐like domain. The significance of SV localization of parkin is discussed.

Parkin is linked to the ubiquitin pathway

Autosomal recessive juvenile parkinsonism (AR-JP) is one of the most common forms of familial Parkinsons disease. AR-JP is characterized by selective and massive loss of dopaminergic neurons in the substantia nigra of the midbrain and absence of Lewy bodies, the pathological hallmark of idiopathic Parkinsons disease. Parkin, the causative gene of AR-JP, encodes a 52-kDa protein that is a RING-type ubiquitin (Ub) protein ligase (E3) collaborating with a Ub-conjugating enzyme (E2) belonging to a cognate class of UbcH7 or UbcH8. Analysis of parkin mutations in AP-JP patients reveals that the functional loss of parkin as an E3 enzyme is the molecular basis of AR-JP. Thus it is now clear that AR-JP is due to failure of proteolysis mediated by the Ub-proteasome system and accumulation of as yet unidentified protein(s) causes nigral neuronal death without formation of Lewy bodies. These findings should shed new light on the mechanisms underlying neurodegeneration in sporadic Parkinsons disease as well as AR-JP.

Phosphodiesterase III inhibition promotes differentiation and survival of oligodendrocyte progenitors and enhances regeneration of ischemic white matter lesions in the adult mammalian brain

Vascular dementia is caused by blockage of blood supply to the brain, which causes ischemia and subsequent lesions primarily in the white matter, a key characteristic of the disease. In this study, we used a chronic cerebral hypoperfusion rat model to show that the regeneration of white matter damaged by hypoperfusion is enhanced by inhibiting phosphodiesterase III. A rat model of chronic cerebral hypoperfusion was prepared by bilateral common carotid artery ligation. Performance at the Morris water-maze task, immunohistochemistry for bromodeoxyuridine, as well as serial neuronal and glial markers were analyzed until 28 days after hypoperfusion. There was a significant increase in the number of oligodendrocyte progenitor cells in the brains of patients with vascular dementia as well as in rats with cerebral hypoperfusion. The oligodendrocyte progenitor cells subsequently underwent cell death and the number of oligodendrocytes decreased. In the rat model, treatment with a phosphodiesterase III inhibitor prevented cell death, markedly increased the mature oligodendrocytes, and promoted restoration of white matter and recovery of cognitive decline. These effects were cancelled by using protein kinase A/C inhibitor in the phosphodiesterase III inhibitor group. The results of our study indicate that the mammalian brain white matter tissue has the capacity to regenerate after ischemic injury.

Progress in the clinical and molecular genetics of familial parkinsonism.

ABSTRACT¶Parkinsons disease (PD) is a neurodegenerative disease with clinical features resulting from deficiency of dopamine in the nigrostriatal system. Most PD cases are sporadic and the primary cause of the disease is still unknown. Recently, familial PD and parkinsonism have received much attention because these forms of the disease might provide clues to the genetic risk factors involved in the pathogenesis of idiopathic PD. To date, two causative genes, α- synuclein and the parkin gene, have been identified. α- Synuclein is involved in the pathogenesis of an autosomal dominant form of PD and constitutes a major component of the Lewy body, which is a pathological hallmark of idiopathic PD. In addition, mutations in the parkin gene have been identified as the cause of autosomal recessive juvenile parkinsonism (AR-JP). AR-JP manifests itself as a highly selective degeneration of the substantia nigra and the locus coeruleus, but without Lewy body formation. In addition to these two genes, four chromosomal loci have been linked to other forms of familial PD. Furthermore, there are a number of other pedigrees of familial PD in which linkage to known genetic loci has been excluded. Molecular cloning of these disease genes and elucidation of the function of their gene products will greatly contribute to our understanding of the pathogenesis of idiopathic PD.

Impact of diabetes and prediabetes on the short-term prognosis in patients with acute ischemic stroke.

OBJECTIVE We aimed to explore the association between abnormal glucose metabolism such as diabetes, prediabetes, and short-term prognosis in patients with acute ischemic stroke. METHODS Of 242 consecutive acute ischemic stroke patients, a 75-g oral glucose tolerance test was administered to 116 patients without previously diagnosed diabetes. One hundred forty patients were classified into diabetes, 52 patients were prediabetes (impaired glucose tolerance or impaired fasting glucose or both), and 50 patients were normal glucose tolerance (NGT). The association between each glycemic status and early neurological deterioration (END; increase in the NIH Stroke Scale (NIHSS) of ≥2 points during the first 14days after admission) or poor short-term outcome (30-day modified Ranking Scale [mRS] score 2-6) was evaluated. RESULTS In multivariable analysis, the risk of END was significantly higher in the diabetes group than in the NGT group (ORs=11.354; 95% CI, 1.492-86.415; p=0.019), even after adjustment for possible confounding factors (ORs=12.769; 95% CI, 1.361-119.763; p=0.026). Similar but insignificant associations were observed between prediabetes and NGT groups (ORs=6.369; 95% CI, 0.735-55.177; p=0.093). The risk of poor outcome (30-day mRS 2-6) was significantly higher in the diabetes group (ORs=3.667; 95% CI, 1.834-7.334; p<0.001) than in the NGT group, even after adjusting for confounding factors (ORs=3.340; 95% CI, 1.361-8.195; p=0.008). Similar but insignificant associations were observed between prediabetes and NGT groups (ORs=2.058; 95% CI, 0.916-4.623; p=0.08). CONCLUSION In our patient population, both diabetes and prediabetes were associated with a poor early prognosis after acute ischemic stroke.

Successful treatment with Yokukansan for behavioral and psychological symptoms of Parkinsonian dementia.

OBJECTIVE To evaluate the efficacy and safety of Yokukansan, a traditional Chinese herbal medicine, for treating behavioral and psychological symptoms of dementia (BPSD) in patients with Parkinson disease (PD; n=7) and those with PD with dementia (PDD; n=7). BACKGROUND BPSD are often seen in patients with senile dementia and have serious deleterious effects on the lives of patients and caregivers. Recent studies indicate that the traditional Chinese herbal medicine Yokukansan may be safe and beneficial for the treatment of BPSD patients. METHODS We treated 7 PD and 7 PDD patients for 4 weeks with Yokukansan and observed them without Yokukansan for 4 weeks. Changes in behavioral and psychological symptoms were evaluated every 4 weeks according to the Neuropsychiatric Inventory (NPI) scale. RESULTS Significant improvements in behavioral and psychological symptoms, particularly in the incidence and duration of hallucinations, were observed in most PD and PDD patients after 4 weeks of Yokukansan treatment. No significant changes were observed in the laboratory tests, cognitive function, activities of daily living, or parkinsonism. CONCLUSION Our results suggest that Yokukansan improves BPSD in both PD and PDD patients without worsening their cognitive function, ability to perform activities of daily living, or parkinsonism.

Autosomal recessive juvenile parkinsonism: a key to understanding nigral degeneration in sporadic Parkinson's disease.

The contribution of genetic factors to the pathogenesis of Parkinsons disease (PD) is supported by the demonstration of the high concordance in twins studies using positron emission tomography (PET), the increased risk among relatives of PD patients in case–control and family studies, and the existence of familial PD and parkinsonism by single gene defect. Recently several genes have been mapped and/or identified. α‐Synuclein is involved in a rare dominant form of familial PD with dopa‐responsive parkinsonism features and Lewy body‐positive pathology. In contrast, parkin is responsible for the autosomal recessive form (AR‐JP) of early onset PD with Lewy body‐negative pathology. The clinical features of this form include early onset (in the 20s), levodopa‐responsive parkinsonism, diurnal fluctuation, and slow progression of the disease. Parkin consists of 12 exons and the estimated size is over 1.5 Mb. To date, variable mutations such as deletions or point mutations resulting in missense and nonsense changes have been reported in AR‐JP patients. In addition, the localization of parkin indicates that parkin may be involved in the axonal transport system. More recently we have found that parkin interacts with the ubiquitin‐conjugating enzyme E2 and is functionally linked to the Ub‐proteasome pathway as a ubiquitin ligase, E3. These findings fit the characteristics of a lack of Lewy bodies (these are cytoplasmic inclusions that are considered to be a pathological hallmark). Our findings should enhance the exploration of the mechanisms of neuronal death in PD as well as other neurodegenerative disorders of which variable inclusion bodies are observed.

Crucial role for Ser133-phosphorylated form of cyclic AMP-responsive element binding protein signaling in the differentiation and survival of neural progenitors under chronic cerebral hypoperfusion.

Various stimuli, such as ischemia/hypoxia enhance newborn cell survival in the subventricular zone and their migration tangentially in chains toward the olfactory bulb. The present study assessed the fate of newborn neurons from subventricular zone to olfactory bulb under conditions of chronic cerebral hypoperfusion, and examined the role of cAMP-responsive element binding protein signaling on the survival of these neurons by using cilostazol, a potent inhibitor of type III phosphodiesterase. Rats underwent bilateral common carotid artery ligation. They were divided into sham-operated (n=70), vehicle- (n=70), and type III phosphodiesterase inhibitor-treated (n=70) groups. Immunohistochemically-stained section for 5-bromodeoxyuridine and a series of neuronal and glial markers were analyzed at days 7, 14, 21 and 28 after hypoperfusion. The reduction of olfactory bulb size gradually progressed in the vehicle group (P<0.05), but not in the sham-operated and type III phosphodiesterase inhibitor-treated group. The subventricular zone of the vehicle-treated rats contained significantly larger numbers of newborn neuroblasts after hypoperfusion, compared with sham-operated rats (P<0.05), but significantly lower numbers in the rostral migratory stream and olfactory bulb (P<0.05). Treatment of rats with type III phosphodiesterase inhibitor increased the number of neuroblasts and enhanced the survival and differentiation of cells (P<0.05). Phosphorylated cAMP-responsive element binding protein within neuroblasts was markedly decreased in the subventricular zone, rostral migratory stream, and olfactory bulb of vehicle-treated rats (P<0.05), but treatment with type III phosphodiesterase inhibitor resulted in recovery of this expression throughout hypoperfusion, leading to enhanced neurogenesis (P<0.05). These effects were abrogated by protein kinase A and C inhibitor. Our results indicated that cAMP-responsive element binding protein signaling is a key mediator of neurogenesis after prolonged hypoperfusion and provide the basis for new regenerative therapies for ischemic brain injury.