Kensaku Kasuga

Differential levels of α-synuclein, β-amyloid42 and tau in CSF between patients with dementia with Lewy bodies and Alzheimer's disease

Background The clinical diagnosis of dementia with Lewy bodies (DLB) is made on the basis of consensus criteria; however, the sensitivity of the criteria is relatively low. There are no generally accepted biomarkers to distinguish DLB from other dementias. Here the utility of quantification of α-synuclein, β-amyloid42 (Aβ42) and tau in the CSF of patients with DLB, Alzheimers disease (AD) and other dementias was examined. Methods 86 patients were divided into three age and sex matched groups: DLB (n=34), AD (n=31) and other dementias (n=21). Two patients with α-synuclein gene (SNCA) duplication were also examined. Aβ and tau were quantified using an ELISA kit. A modified sandwich ELISA was developed which enables the sensitive quantification of CSF α-synuclein. Results Total and phosphorylated tau levels as well as Aβ40/42 and tau/Aβ42 ratios were significantly higher in AD patients than in patients with DLB (p<0.01) and other dementias (p<0.01). CSF α-synuclein levels in DLB patients were significantly lower than those in patients with AD (p<0.05) and other dementias (p<0.01). CSF α-synuclein level correlated with the Aβ42 level in DLB patients (p=0.01, r=0.43). Two patients with SNCA duplication exhibited relatively low levels of CSF α-synuclein. Conclusions The study suggests that reduced levels of CSF α-synuclein in DLB may reflect the accumulation of α-synuclein with Lewy pathology in the brain and that quantification of CSF α-synuclein helps in the differentiation of DLB from AD and other dementias in combination with Aβ42 and tau analysis.

Patients Homozygous and Heterozygous for SNCA Duplication in a Family With Parkinsonism and Dementia

BACKGROUND Multiplication of the alpha-synuclein gene (SNCA) (OMIM 163890) has been identified as a causative mutation in hereditary Parkinson disease or dementia with Lewy bodies. OBJECTIVE To determine the genetic, biochemical, and neuropathologic characteristics of patients with autopsy-confirmed autosomal dominant Lewy body disease, with particular reference to the dosage effects of SNCA. DESIGN Four-generation family study. SETTING Academic research. Patients We fractionated samples extracted from frozen brain tissues of 4 patients for biochemical characterization, followed by immunoblot analysis. MAIN OUTCOME MEASURES We determined the dosages of SNCA and its surrounding genes by quantitative polymerase chain reaction analysis. RESULTS Quantitative polymerase chain reaction analysis revealed that 3 patients were heterozygous for SNCA duplication and 1 patient was homozygous for SNCA duplication. The homozygous patient showed earlier age at onset and earlier death, with more severe cognitive impairment than the heterozygous patients. Biochemical analysis revealed that phosphorylated alpha-synuclein accumulated in the sarkosyl-insoluble urea-extracted fraction of the brains of the patients. CONCLUSIONS Pathologically confirmed Lewy body disease clinically characterized by progressive parkinsonism and cognitive dysfunction is caused by SNCA duplication. The homozygous patient demonstrated the most severe phenotype, suggesting that SNCA dosage has a considerable effect on disease phenotype even within a family. SNCA duplication results in the hyperaccumulation of phosphorylated alpha-synuclein in the brains of patients.

Hyperphosphorylation of Tau Induced by Naturally Secreted Amyloid-β at Nanomolar Concentrations Is Modulated by Insulin-dependent Akt-GSK3β Signaling Pathway

Background: Little is known about the underlying mechanisms by which extracellular amyloid-β (Aβ) induces Tau phosphorylation in Alzheimer disease (AD). Results: Naturally secreted Aβ induced hyperphosphorylation of Tau and impaired insulin signal transduction. Conclusion: A disturbed insulin signaling cascade may be implicated in the pathway of Aβ-induced Tau hyperphosphorylation. Significance: These findings may explain the molecular link between Alzheimer disease and insulin signaling. Alzheimer disease (AD) is neuropathologically characterized by the formation of senile plaques from amyloid-β (Aβ) and neurofibrillary tangles composed of phosphorylated Tau. Although there is growing evidence for the pathogenic role of soluble Aβ species in AD, the major question of how Aβ induces hyperphosphorylation of Tau remains unanswered. To address this question, we here developed a novel cell coculture system to assess the effect of extracellular Aβ at physiologically relevant levels naturally secreted from donor cells on the phosphorylation of Tau in recipient cells. Using this assay, we demonstrated that physiologically relevant levels of secreted Aβ are sufficient to cause hyperphosphorylation of Tau in recipient N2a cells expressing human Tau and in primary culture neurons. This hyperphosphorylation of Tau is inhibited by blocking Aβ production in donor cells. The expression of familial AD-linked PSEN1 mutants and APP ΔE693 mutant that induce the production of oligomeric Aβ in donor cells results in a similar hyperphosphorylation of Tau in recipient cells. The mechanism underlying the Aβ-induced Tau hyperphosphorylation is mediated by the impaired insulin signal transduction because we demonstrated that the phosphorylation of Akt and GSK3β upon insulin stimulation is less activated under this condition. Treating cells with the insulin-sensitizing drug rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, attenuates the Aβ-dependent hyperphosphorylation of Tau. These findings suggest that the disturbed insulin signaling cascade may be implicated in the pathways through which soluble Aβ induces Tau phosphorylation and further support the notion that correcting insulin signal dysregulation in AD may offer a potential therapeutic approach.

Identification of independent APP locus duplication in Japanese patients with early-onset Alzheimer disease

Background: The occurrence of duplications of the amyloid precursor protein gene (APP) has been described in European families with early-onset familial Alzheimer disease (EO-FAD) and cerebral amyloid angiopathy. However, the contribution of APP duplication to the development of AD in other ethnic populations remains undetermined. Methods: The occurrence of APP duplication in probands from 25 families with FAD and 11 sporadic EO-AD cases in the Japanese population was examined by quantitative PCR and microarray-based comparative genomic hybridisation analyses. APP expression level was determined by real-time quantitative reverse-transcription (RT) PCR analysis using mRNA extracted from the peripheral blood of the patients. Results: We identified APP locus duplications in two unrelated EO-FAD families. The duplicated genomic regions in two patients of these families differed from each other. No APP duplication was found in the late-onset FAD families or sporadic EO-AD patients. The patients with APP duplication developed insidious memory disturbance in their fifties without intracerebral haemorrhage and epilepsy. Quantitative RT-PCR analysis showed the increased APP mRNA expression levels in these patients compared with those in age- and sex-matched controls. Conclusions: Our results suggest that APP duplication should be considered in patients with EO-FAD in various ethnic groups, and that increased APP mRNA expression level owing to APP duplication contributes to AD development.

The 28‐amino acid form of an APLP1‐derived Aβ‐like peptide is a surrogate marker for Aβ42 production in the central nervous system

Surrogate markers for the Alzheimer disease (AD)‐associated 42‐amino acid form of amyloid‐β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1‐derived Aβ‐like peptides (APL1β) that are generated by β‐ and γ‐cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ‐secretase modulators (GSMs) and familial AD‐associated presenilin1 mutants that up‐regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non‐AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non‐AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

Mutational analysis in early-onset familial dementia in the Japanese population. The role of PSEN1 and MAPT R406W mutations.

Background: Three major causative genes have been implicated as the cause of early-onset familial Alzheimer’s disease (AD): the amyloid precursor protein gene (APP), presenilin-1 (PSEN1) and PSEN2. Although rare, a tau-related dementia with mutations in the microtubule-associated protein tau gene (MAPT) has been identified in patients showing clinical presentations similar to those of AD. Methods: We performed mutational analysis of APP, PSEN1, PSEN2, and MAPT in 10 Japanese families with early-onset dementia clinically diagnosed as probable Alzheimer’s disease. Results: In 4 index patients, we identified 4 missense PSEN1 mutations, namely, L286V, G378E, L381V, and L392V. The mean age at onset in the patients with PSEN1 mutations was 39 years. In 2 families, we found the R406W mutation in MAPT. The mean age at onset of the patients carrying the R406W mutation was 52 years, and they presented with the peculiar AD-like phenotype without apparent behavioral or language problems. Conclusion: These observations suggest that although PSEN1 mutations are the most frequent cause, the MAPT R406W mutation is an important cause of early-onset familial dementia clinically diagnosed as AD. Differentiation of patients with the MAPT mutation from AD patients by genetic testing would be meaningful, considering that a different therapeutic approach should be applied.

Increased Levels of Soluble LR11 in Cerebrospinal Fluid of Patients with Alzheimer Disease

Background: Recent genetic and pathological studies have suggested that a lipoprotein receptor, LR11, is intricately implicated in the pathogenesis of Alzheimer disease (AD). We have recently established a novel sandwich ELISA, which enabled the sensitive quantification of a soluble LR11 (sLR11). By this ELISA, we attempted to determine the difference in the levels of CSF sLR11 in AD patients. Methods: We examined CSF from 29 AD patients, 20 frontotemporal lobar degeneration patients and 27 age-matched control subjects. The CSF sLR11 level as well as the levels of tau and β-amyloid42 (Aβ42) were determined by sandwich ELISA. Results: The CSF tau level and tau/Aβ42 ratio were significantly increased (p < 0.01) in the AD patients. The CSF sLR11 level in the AD patients was significantly higher (p < 0.01) than that of the frontotemporal lobar degeneration patients and the controls. The APOE-Ε4-positive AD patients have higher sLR11 levels than the APOE-Ε4-negative patients (p < 0.01). Conclusions: These results suggest that the quantification of CSF sLR11 may serve as a biomarker of AD, although the diagnostic value for individual patients is limited. An elevated CSF sLR11 level in AD patients may be relevant to AD pathogenesis.

Enhanced Accumulation of Phosphorylated α-Synuclein and Elevated β-Amyloid 42/40 Ratio Caused by Expression of the Presenilin-1 ΔT440 Mutant Associated with Familial Lewy Body Disease and Variant Alzheimer's Disease

Mutations in the PSEN1 gene encoding presenilin 1 (PS1) are linked to a vast majority of pedigrees with early-onset, autosomal dominant forms of familial Alzheimers disease (FAD). Lewy body (LB) pathology is frequently found in the brains of FAD patients harboring PSEN1 mutations. We recently reported on a novel PS1 mutation with the deletion of threonine at codon 440 (ΔT440) in a familial case diagnosed as having the neocortical type of dementia with LBs (DLB) and variant AD. In this report, we investigated the possible involvement of PS1 ΔT440 mutation in aberrant α-synuclein accumulation. We established cell lines that stably express either wild-type (WT) PS1 or the FAD-linked PS1 H163R, E280A, ΔE9, and PS1 ΔT440 mutants and now demonstrate that the expression of the PS1 ΔT440 mutant led to a marked elevation in the ratio of β-amyloid (Aβ) 42/40 peptides in a conditioned medium. More importantly, we report here that the levels of phosphorylated α-synuclein increase in neuronal and non-neuronal cells expressing the PS1 ΔT440 mutant compared with cells that express WT PS1 or the PS1 H163R and E280A variants that are not associated with LB pathology. This finding is consistent with our demonstration of elevated levels of phosphorylated α-synuclein in the detergent-resistant fraction prepared from a patients brain with PS1 ΔT440 mutation. These observations raise the intriguing suggestion that the mechanism(s) by which the PS1 ΔT440 mutant causes DLB and variant AD are by enhancing the phosphorylation of α-synuclein and the ratio of Aβ42/40 peptides, respectively, in the brain.

α-Synuclein as CSF and Blood Biomarker of Dementia with Lewy Bodies

Dementia with Lewy bodies (DLB) is a common subtype of dementia in the elderly. DLB is neuropathologically characterized by the presence of Lewy bodies and Lewy neurites, both of which are composed of α-synuclein. Although α-synuclein was initially considered to be an exclusively intracellular protein, it has been found to be secreted into biological fluids. α-Synuclein in biological fluids such as cerebrospinal fluid (CSF) and blood has been discussed as a potential biomarker of DLB and α-synuclein-related disorders, because α-synuclein is characteristically accumulated in the brain of patients with these disorders. The α-synuclein level in CSF has been examined by several investigators, and the majority of studies have shown a reduction in CSF α-synuclein level in DLB and α-synuclein-related disorders. Discrepant findings of studies of plasma α-synuclein level in patients with DLB have been reported. Because the level of α-synuclein stored in red blood cells is considerably high, blood contamination and haemolysis during sample collection and processing should be considered as a confounding factor for quantification of α-synuclein. Here, the recent progress in the studies of α-synuclein as a biomarker of DLB and their potential clinical applications are reviewed.

A Japanese family with idiopathic basal ganglia calcification with novel SLC20A2 mutation presenting with late-onset hallucination and delusion

OBJECTIVE: To describe a Japanese family with idiopathic basal ganglia calcification (IBGC) with novel SLC20A2 mutation presenting with late-onset hallucination and delusion. BACKGROUND: IBGC, also known as Fahr disease, is a neuropsychiatric disorder characterized by symmetric calcification in the basal ganglia and other regions in central nervous system without apparent metabolic disorders. Clinical features include a variable combination of movement disorders and neuropsychiatric symptoms. Recently, SLC20A2 mutations have been identified in several IBGC families with autosomal dominant inheritance. DESIGN/METHODS: Case: A 73-year-old Japanese woman developed an auditory hallucination and delusion. Neuropsychological tests revealed that she had short-term memory loss, working memory deficit, and impaired cognitive flexibility. Her brain CT images revealed symmetric calcification in the basal ganglia, thalamus, subcortical white matter, and cerebellar dentate nuclei. Her elder sister aged 80 years and her brother aged 78 were also examined. Although both of them were asymptomatic, brain CT images revealed that her brother had calcification in similar regions with higher severity than the patient. RESULTS: We found the novel SLC20A2 missense mutation of p.L108P both in the patient and her asymptomatic brother with brain calcification. The missense mutation of p.L108P was predicted as probably damaging by PolyPhen-2 and deleterious by SIFT. Leucine at position 108 is well conserved among species. The missense mutation causes an amino acid substitution located in ProDom domain of SLC20A2, which is critical for Pi transport function. This mutation was absent in her asymptomatic sister without brain calcification and 196 unrelated Japanese controls. 99 mTc-ECD single-photon emission CT revealed hypoperfusion at the ventrolateral prefrontal cortex of the proband. CONCLUSIONS: We identified the first case of genetically proven IBGC in a Japanese family with a SLC20A2 mutation. Our case may emphasize the importance of evaluating calcification by brain CT in elderly patients with hallucination and delusion. Disclosure: Dr. Kasuga has nothing to disclose. Dr. Konno has nothing to disclose. Dr. Nishizawa has nothing to disclose. Dr. Ikeuchi has nothing to disclose.