Kazutomi Kanemaru

Fetal-Type Phosphorylation of the τ in Paired Helical Filaments

Abstract: To determine the phosphorylation sites of the τ in paired helical filaments (PHF), two types of PHF antisera with different specificities were used: One was a conventional anti‐PHF, and the other was an antiserum to formic acid‐denatured PHF (anti‐HFoPHF). Phosphorylated τ‐specific antibodies, anti‐ptau 1 and anti‐ptau 2, were prepared from anti‐PHF and anti‐HFoPHF, respectively. We found that both anti‐ptau 1 and anti‐ptau 2 labeled fetal or juvenile τ but not adult τ. The anti‐ptau 1‐ and anti‐ptau 2‐recognition sites were immunochemically localized to the fragment Asp313 to Ile328 in the most COOH‐terminal portion of τ. Furthermore, Ser315 was determined as the anti‐ptau 2 recognition site. The sequence surrounding Ser315 was not found in the canonical sequences phosphorylated with known kinases.

Decreased CSF amyloid β42 and normal tau levels in dementia with Lewy bodies

only if present in more than one clone. Results. Notch3 mutational scanning in this family revealed an A-to-G transition in one allele within the 39 acceptor splice of exon 4 (figure). Because 1) no other mutation was detected in the remaining exons, 2) this mutation was not observed in more than 400 control chromosomes, and 3) the mutation segregated with the neuroradiologic phenotype, we concluded that it is the causative mutation in this family. Interestingly, the three family members presenting with migraine and having normal MRI results did not carry the mutation. To investigate whether this 39 acceptor splice site mutation would affect splicing of exon 4, we analyzed reverse-transcribed (RT-PCR) messenger RNA (mRNA) extracted from muscle. Muscle was chosen because the Notch3 transcript is undetectable in peripheral blood leukocytes of both control subjects and patients (A.J., unpublished results). Nucleotide sequence analysis of the cloned RT-PCR products from the patient revealed an in-frame 21-base pair deletion in exon 4, from nucleotides G419 to G439, in approximately 50% of the clones. This deletion most likely results from the use of a cryptic 39 acceptor splice site at position 438-439 within exon 4. This mutation is predicted to lead to an in-frame deletion of seven amino acids (glycine, proline, aspartic acid, cysteine, serine, leucine, proline) in the Notch3 protein, including the sixth cysteine residue of the second EGF domain. Discussion. This is the first report of a splice site mutation within Notch3. This unusual mutation could account for the peculiar phenotypic spectrum observed within this family; namely, the rarity of stroke events, the high prevalence of migraine with aura, and the occurrence in some patients of very unusual confusion or coma episodes, or both. In this family, CADASIL diagnosis was initially suspected chiefly based on neuroimaging data and not on the clinical features; at that time, none of the patients experienced a stroke event. Interestingly, we found that three patients with migraine and normal MRI results did not carry the mutation. This finding, which is likely explained by the high prevalence of migraine within the general population, may be of importance for genetic counseling, given the severity of the prognosis of this disorder. All previously reported Notch3 mutations result in an odd number of cysteine residues within an EGF repeat. Importantly, this small in-frame deletion includes one cysteine residue. Our finding provides additional evidence that the unpaired cysteine residue, which may promote the abnormal oligomerization of the Notch3 receptor, is likely to play a key role in the pathophysiology of CADASIL.

Lewy Body Pathology Involves Cutaneous Nerves

Involvement of the peripheral autonomic nervous system is a core feature of Lewy body (LB) diseases, including Parkinson disease (PD), PD with dementia, and dementia with LBs. To investigate the potential use of skin biopsy for the diagnosis of LB diseases, we assessed anti-phosphorylated &agr;-synuclein immunoreactivity in peripheral nerves in samples of skin from the abdominal wall and flexor surface of the upper arm in 279 prospectively studied consecutively autopsied patients whose data were registered at the Brain Bank for Aging Research between 2002 and 2005. Positive immunoreactivity was demonstrated in the unmyelinated fibers of the dermis in 20 of 85 patients with LB pathology in the CNS and the adrenal glands, the latter representing a substitute for peripheral autonomic nervous system sympathetic ganglia; no reactivity was seen in 194 patients without CNS LB pathology. In 142 retrospectively studied patients autopsied from 1995 onward who had subclinical or clinical LB disease, the sensitivity of the positive skin immunoreactivity was 70% in PD and PD with dementia and 40% in dementia with LBs. Skin immunoreactivity was absent in cases of multiple-system atrophy, progressive nuclear palsy, and corticobasal degeneration. We demonstrate for the first time that the skin is involved and may be a highly specific and useful biopsy site for the pathological diagnosis of LB diseases.

Incidence and Extent of Lewy Body-Related α-Synucleinopathy in Aging Human Olfactory Bulb

We investigated the incidence and extent of Lewy body (LB)-related &agr;-synucleinopathy (LBAS) in the olfactory bulb (OB) in 320 consecutive autopsy patients from a general geriatric hospital (mean age, 81.5 ± 8.5 years). Paraffin sections were immunostained with anti-phosphorylated &agr;-synuclein, tyrosine hydroxylase, phosphorylated tau, and amyloid &bgr; antibodies. LBAS was found in 102 patients (31.9%) in the central nervous system, including the spinal cord; the OB was involved in 85 (26.6%). Among these 85 patients, 2 had LBAS only in the anterior olfactory nucleus, 14 in the peripheral OB only, and 69 in both areas. In 5 patients, Lewy bodies were found only in the OB by hematoxylin and eosin stain; 3 of these patients had Alzheimer disease, and all had LBAS. Very few tyrosine hydroxylase-immunoreactive periglomerular cells exhibited LBAS. All 35 LBAS patients with pigmentation loss in the substantia nigra had LBAS in the OB. LBAS in the amygdala was more strongly correlated with LBAS in the anterior olfactory nucleus than with that in the OB periphery. LBAS did not correlate with systemic tauopathy or amyloid &bgr; amyloidosis. These results indicate a high incidence of LBAS in the aging human OB; they also suggest that LBAS extends from the periphery to the anterior olfactory nucleus and results in clinical manifestations of LB disease.

Analysis of the adrenal gland is useful for evaluating pathology of the peripheral autonomic nervous system in lewy body disease.

Abstract Lewy body disease is defined as Lewy body-related neuronal degeneration involving the nigrostriatal system, limbic-neocortical system, and peripheral autonomic nervous system (PANS). We investigated whether the adrenal gland, which is evolutionarily related to sympathetic ganglia and is routinely examined in general autopsy, could be used to assess pathology of the PANS in Lewy body disease. Brains, spinal cords, and adrenal glands from 783 consecutive autopsy cases from a general geriatric hospital were examined immunohistochemically with antiphosphorylated &agr;-synuclein antibodies and routine staining. Parkinson disease (PD) with dementia and dementia with Lewy bodies (DLB) were defined using 1996 Consensus Guidelines for DLB and the secondary Lewy body-related &agr;-synucleinopathy or amygdala variants using previously established criteria. Lewy body-related &agr;-synucleinopathy was found in 207 (26.4%) of 783 cases, with 1 case solely in the adrenal gland. In all 18 PD cases with or without dementia and in 33 of 38 DLB cases, the adrenal gland was involved, but it was spared in all cases of amygdala variants. Our results indicate that the adrenal gland can provide useful information for evaluation of the PANS in Lewy body disease.

Validation of cardiac 123I-MIBG scintigraphy in patients with Parkinson’s disease who were diagnosed with dopamine PET

PurposeThe aim of this study was to evaluate the diagnostic potential of cardiac 123I-labelled metaiodobenzylguanidine (123I-MIBG) scintigraphy in idiopathic Parkinson’s disease (PD). The diagnosis was confirmed by positron emission tomography (PET) imaging with 11C-labelled 2β-carbomethoxy-3β-(4-fluorophenyl)-tropane (11C-CFT) and 11C-raclopride (together designated as dopamine PET).MethodsCardiac 123I-MIBG scintigraphy and dopamine PET were performed for 39 parkinsonian patients. To estimate the cardiac 123I-MIBG uptake, heart to mediastinum (H/M) ratios in early and delayed images were calculated. On the basis of established clinical criteria and our dopamine PET findings, 24 patients were classified into the PD group and 15 into the non-PD (NPD) group.ResultsBoth early and delayed images showed that the H/M ratios were significantly lower in the PD group than in the NPD group. When the optimal cut-off levels of the H/M ratio were set at 1.95 and 1.60 in the early and delayed images, respectively, by receiver-operating characteristic analysis, the sensitivity of cardiac 123I-MIBG scintigraphy for the diagnosis of PD was 79.2 and 70.8% and the specificity was 93.3 and 93.3% in the early and delayed images, respectively. In the Hoehn and Yahr 1 and 2 PD patients, the sensitivity decreased by 69.2 and 53.8% in the early and delayed images, respectively.ConclusionIn early PD cases, cardiac 123I-MIBG scintigraphy is of limited value in the diagnosis, because of its relatively lower sensitivity. However, because of its high specificity for the overall cases, cardiac 123I-MIBG scintigraphy may assist in the diagnosis of PD in a complementary role with the dopaminergic neuroimaging.

Neuropathologic analysis of Lewy‐related α‐synucleinopathy in olfactory mucosa

We analyzed the incidence and extent of Lewy‐related α‐synucleinopathy (LBAS) in the olfactory mucosa, as well as the central and peripheral nervous systems of consecutive autopsy cases from a general geriatric hospital. The brain and olfactory mucosa were immunohistochemically examined using antibodies raised against phosphorylated α‐synuclein. Thirty‐nine out of 105 patients (37.1%) showed LBAS in the central or peripheral nervous systems. Seven patients presented LBAS (Lewy neurites) in the olfactory lamina propria mucosa. One out of the seven cases also showed a Lewy neurite in a bundle of axons in the cribriform plate, but α‐synuclein deposits were not detected in the olfactory receptor neurons. In particular, high incidence of α‐synuclein immunopositive LBAS in the olfactory mucosa was present in the individuals with clinically as well as neuropathologically confirmed Parkinsons disease and dementia with Lewy bodies (6/8 cases, 75%). However, this pathologic alteration was rare in the cases with incidental or subclinical Lewy body diseases (LBD) (one out of 31 cases, 3.2%). In the olfactory bulb, the LBAS was usually present in the glomeruli and granular cells of most symptomatic and asymptomatic cases with LBD. Our studies further confirmed importance of the olfactory entry zone in propagation of LBAS in the human aging nervous system.

Comparable amyloid β-protein (Aβ) 42(43) and Aβ40 deposition in the aged monkey brain

Two distinct species of amyloid beta-protein (A beta), A beta 42(43) and A beta 40, are deposited in the brains of patients with Alzheimers disease and normal aged individuals. A beta 42(43), the long tailed A beta, is the initially and predominantly deposited species in senile plaques. Deposition of A beta is also observed in the aged monkey brains. We investigated the A beta species in the aged monkey brains immunocytochemically using monoclonal antibodies that discriminate between the C-termini of A beta 42(43) and A beta 40. We report here that A beta 40 as well as A beta 42(43) deposit in various types of senile plaques, including diffuse plaques of the aged monkey brain and that the ratio of A beta 40 to A beta 42(43) is higher compared with that in human brain.

Neuropathological Asymmetry in Argyrophilic Grain Disease

The presence of argyrophilic grains in the neuropil is associated with a form of dementia. We investigated morphological asymmetry in 653 consecutive autopsy patients from a general geriatric hospital (age [mean ± SD] = 81.1 ± 8.9 years), focusing on those from patients with advanced argyrophilic grain disease. Paraffin sections of the bilateral posterior hippocampi were immunostained with anti-phosphorylated tau and anti-4-repeat tau antibodies and by the Gallyas-Braak method. In a side-to-side comparison, asymmetry was defined when either the extent or the density of argyrophilic grains was different. Of the 653 subjects, 65 (10%) had Stage 3 argyrophilic grain disease, and 59 (90.8%) showed histopathological asymmetry. Antemortem computed tomographic images (n = 24), magnetic resonance imaging scans (n = 8), and combined computed tomographic and magnetic resonance images (n = 15) were available; images from 20 of the 47 subjects showed asymmetry that correlated with the histopathological asymmetry. Cerebral cortical asymmetry consistent with the histopathology was also visible in N-isopropyl-123I-p-iodoamphetamine single photon emission computed tomographic images from 6 patients and 18F-labeled fluorodeoxyglucose positron emission tomographic images from 2 patients. Thus, asymmetric involvement of the medial temporal lobe in patients with advanced argyrophilic grain disease may represent a diagnostic feature and contribute to distinguishing dementia with grains from Alzheimer disease.

Association of Estrogen Receptor α Gene Polymorphisms with Neurofibrillary Tangles

Estrogen receptor α (ERα) may be implicated in the pathogenesis of Alzheimer’s disease (AD). The aim of this study was to clarify the association between ERα gene polymorphisms and AD-related pathologic changes. The staging of neurofibrillary tangles (NFT) and senile plaques (SP) was performed according to the method by Braak and Braak and two polymorphisms, PvuII (P or p) and XbaI (X or x), of the ERα gene were typed in 551 Japanese cadavers (294 men and 257 women; mean age, 80.8 years). Distributions of the NFT and SP stages significantly correlated with age (NFT: r = 0.306, p < 0.0001; SP: r = 0.237, p < 0.0001) and were significantly higher in patients with the apolipoprotein E Ε4 allele (p < 0.0001). Possession of the P allele showed a trend to be associated with a more serious NFT stage, but had no relationship with the SP stage. In men, a significant association between PvuII polymorphism and the NFT stage (p = 0.002) was found, revealing a gene- dose effect of the P allele. Similar results were obtained in the men without the Ε4 allele (p = 0.011). Multiple regression analyses demonstrated that age was the strongest determinant of the NFT stage, possession of the Ε4 allele was the next strongest, and PvuII polymorphism was the third strongest (p < 0.0001, R2 = 0.144). The XbaI polymorphism did affect neither the NFT stage nor the SP stage. In conclusion, the PvuII polymorphism of the ERα gene is associated with Braak NFT stages and possession of the P allele may act as a risk factor for AD in Japanese men, especially in those without the Ε4 allele.