Hiroyuki Hatsuta

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.

SOD1 (Copper/Zinc Superoxide Dismutase) Deficiency Drives Amyloid β Protein Oligomerization and Memory Loss in Mouse Model of Alzheimer Disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282–11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of Nϵ-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.

Spliceosome integrity is defective in the motor neuron diseases ALS and SMA

Two motor neuron diseases, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are caused by distinct genes involved in RNA metabolism, TDP‐43 and FUS/TLS, and SMN, respectively. However, whether there is a shared defective mechanism in RNA metabolism common to these two diseases remains unclear. Here, we show that TDP‐43 and FUS/TLS localize in nuclear Gems through an association with SMN, and that all three proteins function in spliceosome maintenance. We also show that in ALS, Gems are lost, U snRNA levels are up‐regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP‐43 pathology. This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome. These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell‐type specific vulnerability of motor neurons.

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.

An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

The β‐site amyloid precursor protein cleaving enzyme‐1 (BACE1), an essential protease for the generation of amyloid‐β (Aβ) peptide, is a major drug target for Alzheimers disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N‐acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT‐III (Mgat3), revealed that cleavage of Aβ‐precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin‐2, are normally cleaved in GnT‐III‐deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT‐III‐deficient mice remain healthy, GnT‐III may be a novel and promising drug target for AD therapeutics.

Evaluation of SLC20A2 mutations that cause idiopathic basal ganglia calcification in Japan

Objective: To investigate the clinical, genetic, and neuroradiologic presentations of idiopathic basal ganglia calcification (IBGC) in a nationwide study in Japan. Methods: We documented clinical and neuroimaging data of a total of 69 subjects including 23 subjects from 10 families and 46 subjects in sporadic cases of IBGC in Japan. Mutational analysis of SLC20A2 was performed. Results: Six new mutations in SLC20A2 were found in patients with IBGC: 4 missense mutations, 1 nonsense mutation, and 1 frameshift mutation. Four of them were familial cases and 2 were sporadic cases in our survey. The frequency of families with mutations in SLC20A2 in Japan was 50%, which was as high as in a previous report on other regions. The clinical features varied widely among the patients with SLC20A2 mutations. However, 2 distinct families have the same mutation of S637R in SLC20A2 and they have similar characteristics in the clinical course, symptoms, neurologic findings, and neuroimaging. In our study, all the patients with SLC20A2 mutations showed calcification. In familial cases, there were symptomatic and asymptomatic patients in the same family. Conclusion: SLC20A2 mutations are a major cause of familial IBGC in Japan. The members in the families with the same mutation had similar patterns of calcification in the brain and the affected members showed similar clinical manifestations.

Molecular analysis and biochemical classification of TDP-43 proteinopathy

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kDa pathology are progressive neurodegenerative diseases that are characterized by intracytoplasmic aggregates of hyperphosphorylated TAR DNA-binding protein of 43 kDa. These TAR DNA-binding protein 43 proteinopathies can be classified into subtypes, which are closely correlated with clinicopathological phenotypes, although the differences in the molecular species of TAR DNA-binding protein 43 in these diseases and the biological significance thereof, remain to be clarified. Here, we have shown that although the banding patterns of abnormally phosphorylated C-terminal fragments of TAR DNA-binding protein 43 differ between the neuropathological subtypes, these are indistinguishable between multiple brain regions and spinal cord in individual patients. Immunoblot analysis of protease-resistant TAR DNA-binding protein 43 demonstrated that the fragment patterns represent different conformations of TAR DNA-binding protein 43 molecular species in the diseases. These results suggest a new clinicopathological classification of TAR DNA-binding protein 43 proteinopathies based on their molecular properties.

[Monoclonal antibody against the turn of the 42-residue amyloid β-protein at positions 22 and 23].

Aggregation of the 42-mer amyloid β-protein (Aβ42) plays a critical role in the pathogenesis of Alzheimers disease (AD). We have proposed a toxic conformer with a turn at positions 22 and 23, as well as a nontoxic conformer with a turn at positions 25 and 26, in Aβ42 aggregates from systematic proline scanning and solid-state NMR studies. Although recent clinical trials of immunization targeting Aβ42 aggregates have proved useful, some adverse effects were reported. One of the reasons was hypothesized to be excessive immunoreactions derived from the unintended removal of nontoxic Aβ42, which plays an important role in the physiological function. To develop a monoclonal antibody for toxic Aβ42, E22P-Aβ10-35, a minimum moiety for neurotoxicity containing the turn at positions 22 and 23, was used for the generation of antibodies, following the selection of clones using Aβ42 mutants of E22P (turn-inducing) and E22V (turn-preventing). The obtained clone (11A1) showed a high binding affinity (K(D) = 10.3 nM) for Aβ42 using surface plasmon resonance. 11A1 also inhibited the neurotoxicity of Aβ42 in PC12 cells. Immunohistochemical studies showed that not only extracellular but intracellular amyloid was stained in human AD brains. In Western blotting analyses using human brains, low-molecular weight-oligomers rather than the monomer of Aβ were readily recognized by 11A1. These results imply that 11A1 could detect toxic Aβ42 oligomers with the turn at positions 22 and 23 and that 11A1 could be applicable for the therapeutic targeting of toxic Aβ42 in AD.

Lack of genetic association between TREM2 and late-onset Alzheimer's disease in a Japanese population.

Rare non-synonymous variants of TREM2 have recently been shown to be associated with Alzheimers disease (AD) in Caucasians. We here conducted a replication study using a well-characterized Japanese sample set, comprising 2,190 late-onset AD (LOAD) cases and 2,498 controls. We genotyped 10 non-synonymous variants (Q33X, Y38C, R47H, T66M, N68K, D87N, T96K, R98W, H157Y, and L211P) of TREM2 reported by Guerreiro et al. (2013) by means of the TaqMan and dideoxy sequencing methods. Only three variants, R47H, H157Y, and L211P, were polymorphic (range of minor allele frequency [MAF], 0.0002-0.0059); however, no significant association with LOAD was observed in these variants. Considering low MAF of variants examined and our study sample size, further genetic analysis with a larger sample set is needed to firmly evaluate whether or not TREM2 is associated with LOAD in Japanese.

Altered γ‐secretase activity in mild cognitive impairment and Alzheimer's disease

We investigated why the cerebrospinal fluid (CSF) concentrations of Aβ42 are lower in mild cognitive impairment (MCI) and Alzheimers disease (AD) patients. Because Aβ38/42 and Aβ40/43 are distinct product/precursor pairs, these four species in the CSF together should faithfully reflect the status of brain γ‐secretase activity, and were quantified by specific enzyme‐linked immunosorbent assays in the CSF from controls and MCI/AD patients. Decreases in the levels of the precursors, Aβ42 and 43, in MCI/AD CSF tended to accompany increases in the levels of the products, Aβ38 and 40, respectively. The ratios Aβ40/43 versus Aβ38/42 in CSF (each representing cleavage efficiency of Aβ43 or Aβ42) were largely proportional to each other but generally higher in MCI/AD patients compared to control subjects. These data suggest that γ‐secretase activity in MCI/AD patients is enhanced at the conversion of Aβ43 and 42 to Aβ40 and 38, respectively. Consequently, we measured the in vitro activity of raft‐associated γ‐secretase isolated from control as well as MCI/AD brains and found the same, significant alterations in the γ‐secretase activity in MCI/AD brains.