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The dorsal stream is responsible for visuospatial processing, and visuospatial impairment is common in Alzheimer’s disease (AD). Using magnetoencephalography (MEG) during visuospatial processing, we investigated neural activity in the dorsal stream in patients with clinically diagnosed AD dementia (the dementia group), patients with amnestic mild cognitive impairment (the aMCI group), and cognitively unimpaired subjects (the CU group). We estimated the powers of neural responses induced by optic flow tasks in the five regions that form the dorsal stream, namely medial ventral occipital cortex (MVOcC), lateral occipital cortex, precuneus (Pcun), inferior parietal lobule (IPL), and superior parietal lobule, and compared two-region power ratios in the dementia group (n = 13), aMCI group (n = 11), and CU group (n = 20). Two of the two-region power ratios, namely IPL/Pcun and IPL/MVOcC, were found to be significantly higher in the dementia group than in the CU and aMCI groups, indicating that neural activity during visuospatial processing changes with the progression of AD, and a compensatory mechanism may be present in IPL, Pcun, and MVOcC of patients with AD. Thus, MEG during optic flow tasks may be useful for the investigation of neural activity in the dorsal stream in patients with AD.

軟Ｘ線平面結像型回折格子分光器においては、多層膜をコートすることに より、従来値より小さい入射角で取込み光量と回折効率を同時に増大さ せ、分光分析感度を飛躍的に向上させるさせることができる 。一方、低入射角化は分散の低下を招き検出器のピクセルサイズで制限される波長分解能が低下するが、刻線密度を大きくすることでこの問題を回避できる見込みがある。そこで従来の刻線密度 2400 本/mm と比較して約３割刻線密度が大きい 3200 本/mm のラミナー型回折格子を製作した。回折格子溝を形成する基板に表面粗さの異なる2種類の基板を使用した。AFM 観察による rms 粗さ実測値はそれぞれ 0.3 nm と 1.0 nm であった。各基板面上の一部に Au (30 nm) をコートし、別の場所に 705 eV の Fe L-発光領域で最適設計した C (6.84 nm)/W (4.56 nm) 3 周期多層膜とC 3.00 nm キャップ層を連続してコートした。PFの BL-11D で、入射角 86° および 88° の配置で、それぞれ0.5～1.2 keV および 1.1～1.56 keV の領域で回折効率を測定した。2種類の基板に対していずれも計算値と比較して面粗さ 0.6 nm rms 相当の回折格子実測値が得られた。設計エネルギー 705 eV 付近で 6.8% の回折効率最大値を取り、報告済みの 2400 本/mm W/C 多層膜回折格子での実測値 7.1% と遜色ない結果が得られた。今後、中心刻線密度を 3200 本/mm とする平面結像型不等間隔溝多層膜球面回折格子を製作して回折効率と発光分光器実機での結像特性の評価を行う。, 第35回日本放射光学会年会・放射光科学合同シンポジウム

Parkinson’s disease is one of the most common movement disorders and is characterized by dopaminergic cell loss and the accumulation of pathological α-synuclein, but its precise pathogenetic mechanisms remain elusive. To develop disease-modifying therapies for Parkinson’s disease, an animal model that recapitulates the pathology and symptoms of the disease, especially in the prodromal stage, is indispensable. As subjects with α-synuclein gene (SNCA) multiplication as well as point mutations develop familial Parkinson’s disease and a genome-wide association study in Parkinson’s disease has identified SNCA as a risk gene for Parkinson’s disease, the increased expression of α-synuclein is closely associated with the aetiology of Parkinson’s disease. In this study we generated bacterial artificial chromosome transgenic mice harbouring SNCA and its gene expression regulatory regions in order to maintain the native expression pattern of α-synuclein. Furthermore, to enhance the pathological properties of α-synuclein, we inserted into SNCA an A53T mutation, two single-nucleotide polymorphisms identified in a genome-wide association study in Parkinson’s disease and a Rep1 polymorphism, all of which are causal of familial Parkinson’s disease or increase the risk of sporadic Parkinson’s disease. These A53T SNCA bacterial artificial chromosome transgenic mice showed an expression pattern of human α-synuclein very similar to that of endogenous mouse α-synuclein. They expressed truncated, oligomeric and proteinase K-resistant phosphorylated forms of α-synuclein in the regions that are specifically affected in Parkinson’s disease and/or dementia with Lewy bodies, including the olfactory bulb, cerebral cortex, striatum and substantia nigra. Surprisingly, these mice exhibited rapid eye movement (REM) sleep without atonia, which is a key feature of REM sleep behaviour disorder, at as early as 5 months of age. Consistent with this observation, the REM sleep-regulating neuronal populations in the lower brainstem, including the sublaterodorsal tegmental nucleus, nuclei in the ventromedial medullary reticular formation and the pedunculopontine nuclei, expressed phosphorylated α-synuclein. In addition, they also showed hyposmia at 9 months of age, which is consistent with the significant accumulation of phosphorylated α-synuclein in the olfactory bulb. The dopaminergic neurons in the substantia nigra pars compacta degenerated, and their number was decreased in an age-dependent manner by up to 17.1% at 18 months of age compared to wild-type, although the mice did not show any related locomotor dysfunction. In conclusion, we created a novel mouse model of prodromal Parkinson’s disease that showed RBD-like behaviour and hyposmia without motor symptoms.

軟X線分光分析においては、近年Li-K発光（22.8 nm）近辺でのリチウム電池材料や13.5 nm付近でのEUVリソ材料の状態分析や微量分析などの重要性が益々高まっている。それに伴い、この領域で分光素子として広く使われている軟X線回折格子の分解能や回折効率などの基本性能の向上を図ることが必要となってきている。本研究では、10～30 nm 域で用いる軟X線回折格子において、回折格子溝形状と入射角の最適化とともに、通常の金等の金属表面のラミナー型回折格子に新たな増反射膜を付加し、回折効率と共に分析感度と相関性のある回折格子が取り込む光量と回折効率の積であるSpectral fluxを指標とし、その増大を図ることとした。そのため、鏡面反射での予備検討で22.83 nmにおいて高い反射率を示したB4Cを上記の回折格子面上に積層することとした。回折格子を製作し、実験的に評価するため、現在この領域で実際に分光に供せられているラミナー型回折格子（刻線密度：1200 本/mm，デューティ比：0.3，溝深さ：20 nm，表面物質：Au、推奨入射角：86.0°）を仮定し数値計算で最適化を行った。その結果、回折効率を指標とした場合、入射角：78.2°、B4Cの厚さ：22 nmで、Spectral fluxを指標とした場合、入射角：74.0°、B4Cの厚さ：14 nmでそれぞれの指標が22.83 nmで極大を示し、広い波長範囲でそれぞれ高い数値を示すことが判った。発表では低入射角化で現れる偏光依存性についても触れる。, 第57回X線分析討論会

回折格子分光器を用いた軟Ｘ線発光分光法は高感度微量元素分析への期待が高い。我々は低入射角配置の回折格子で発光収率と回折効率を同時に増大させる分光感度向上を目指している。本講演では183 eV付近のボロンK発光に対する高感度化に成功した例を紹介する。基板溝形状や表面反射材など回折格子に含まれる複数パラメーターの最適化を簡単にするため、反射材の物質選択、入射角と溝深さとデューティー比、反射材の膜厚の順に最適化を行った。高回折効率コート材料の物質を選ぶに際し、解析的な考察が容易なバルク反射率を判断基準とした。その結果に基づき、化学的に安定なLaF3を用いたNi/LaF3膜とLa膜にC保護膜を付加したNi/La/C膜について回折格子パラメーターの最適化設計を行い、回折格子の作製と回折効率の実測を行った。分光器の分光感度と相関がある集光効率と回折効率の積で定義されるspectral fluxの実測値では、入射角87.07°でのNiコート回折格子の値0.138を基準にすると、Ni/LaF3膜とNi/La/C膜を付加した場合、入射角84°で最大値をとり、それぞれ0.548、0.660であった。これはそれぞれ4.0倍、4.8倍のspectral fluxの増大に相当する。, Optics ＆ Photonics Japan 2021

Summary The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects., Graphical abstract, Highlights • Successive light microscopy/electron microscopy in optically cleared tissues • Multi-scale neuronal imaging from the entire brain to individual synapses • Simultaneous interrogation of neural circuit structure and synaptic connectivity • Zooming-in to scarce synaptic contacts with the successive imaging, Imaging anatomy; Optical imaging; Neuroscience; Cell biology

軟Ｘ線平面結像型発光分光器においては、金属膜上に多層膜をコートすることにより、従来値より小さい入射角に於いて、取り込み光量と回折効率が同時に増大し、分光分析感度を飛躍的に向上させることができる。一方、低入射角化は分散の低下を招き検出器のピクセルサイズで制限される波長分解能が低下するが、刻線密度を大きくすることでこの問題を回避できる見込みがある。そこで前回の報告では、従来の刻線密度2400本/mmと比較して約3割刻線密度が大きい 3200 本/mm のラミナー型回折格子基板を製作し、Au をコートして Fe L 発光（705 eV）領域において得られた回折効率を理論値と比較した。その結果、多層膜を付加する回折格子基板としての使用できることが確かめられた。今回は同様の回折格子基板に低入射角においてFe L 発光領域の回折効率の増大を目的に最適設計した C (6.84 nm)/W (4.56 nm) 3 周期多層膜と C 3.00 nm キャップ層を連続してコートすることとした。回折格子基板として用いた2種類の基板の諸元（rms 粗さ: σ、溝深さ: h、デューティ比 : dc）の実測値はそれぞれ（0.3 nm、5.04 nm、0.47）と（ 1.0 nm、5.77 nm、0.53）である。上記のW/C 多層膜の他、比較のためAuを同一基板面上の一部にコートし、 Photon Factory の BL-11D で回折効率を測定した。回折効率は設計エネルギーの705 eV付近で6.8 %の最大値を取り、報告済みの2400 本/mm W/C多層膜回折格子での実測値 7.1% と遜色ない結果が得られた。今後、この中心刻線密度を持つ平面結像型不等間隔溝多層膜球面回折格子を製作し、回折効率、結像特性等の評価を行う予定である。, 2021年第82回応用物理学会秋季講演会

400～1000 eV の範囲の 3d 遷移⾦属元素の L 発光は、化合物中のそれらの元素の化学状態だけでなく荷電状態に関する有益な情報を含んでいる。 Lα,β 発光プロファイルとその強度は、それぞれエネルギー分布と 3d 電⼦の量を反映している。また、そのエネルギー位置は、充電状態 (化学シフト) によって影響を受ける。 Ll,ηの放出エネルギーは、3s 準位に空孔がある最終状態の緩和エネルギーを反映している。 さらに、Ll, ηプロファイルは、価数 3d 電⼦の磁気モーメントと、関連する 3d 遷移⾦属元素の 3s との間の相互作⽤を介した磁気モーメントの情報を含んでいる。 しかしながら、従来の電子顕微鏡搭載型の軟X線発光分光器は、Ll,η 線の磁気情報を分析するのに⼗分なエネルギー分解能を持っていなかった。そのため、本研究ではTEM ⽤に設計された⼤きなエネルギー分散を持つ分光器を、EPMA ⽤の分光器に改修を行った。この分光器では試料から回折格子までの距離は従来から市販されている分光器と同じであるが、回折格子から検出器までの距離は約 2 倍であり、このことは、従来比で約 2 倍のエネ ルギー分散が得られることを意味している。検出器は、CMOS カメラ（3.45μm ピッチ） と光学的に結合された MCP（7.5μm ピッチ）で構成されている。この装置を用いてFeのL-発光スペクトルを測定した結果、金属FeとFe2O3のエネルギープロファイルの違いが明確に観測された他、3s3d交換相互作用に起因すると推定されるLl線の内部構造の存在も明らかになった。, Microscopy & Microanalysis 2021

Numerical and experimental studies have been performed to evaluate the enhancement of diffraction efficiency of diffraction gratings around B K -emission by overcoating lanthanum series layers on conventional metal-coated laminar-type gratings. We propose an optical design method based on the concept of spectral flux given by collection efficiency and diffraction efficiency. A diffraction grating with a small angle of incidence provides an advantage to soft x-ray spectrographs because it collects the emission at a larger solid angle compared to that of conventional grazing incidence diffraction gratings. Numerical calculations indicated that La and L a F 3 were promising as overcoating materials on a laminar-type Ni-coated diffraction grating, and we performed an experimental study using L a F 3 and La/C overcoatings, considering their producibility and durability. The diffraction efficiencies were measured using a reflectometer at a synchrotron facility. The diffraction efficiencies observed at 183.4 eV were 29.4% and 34.3% at angles of incidence of 85.1° and 84.9° for N i / L a F 3 and Ni/La/C gratings, respectively.

エネルギー分解が従来より高い電子顕微鏡用の新型軟X線発光分光装置により鉄などの3d遷移金属酸化物のL発光スペクトルを測定した結果とその解析について報告する。従来装置を用いた測定でも内殻準位間の電子遷移（3s→2p）に由来するLl発光スペクトルに構造が存在することが見出されており、これは、終状態における3sホールのスピンと3d電子の磁気モーメントの相互作用に起因すると考えられている。しかし、従来装置ではエネルギー分解が十分でなかったため、これ以上の解析が困難であった。そのため、今回新たに分散の大きな分光光学系に最適化した回折格子を設計製作し、ピクセルサイズが小さく解像度の高い検出器を組み合わせたエネルギー分解が高い新型の分光器を設計製作した。透過型電子顕微鏡にこの分光器を装着し、鉄などの3d遷移金属酸化物のL発光測定を行った結果、以前に比較してLl発光スペクトルの化合物ごとの違いが明瞭にわかる測定ができた。Fe, Fe3O4, Fe2O3のFe-Lℓ,η発光スペクトルの比較ではFeに比べ、Fe3O4, Fe2O3では肩状の構造が見られ、さらに、その位置がFe3O4とFe2O3で異なっているように観察された。発表では、このスペクトルのピーク分離処理し、分離の大きさの定量評価を行った結果についても報告する。, 日本顕微鏡学会第77回学術講演会

Summary Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget’s disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the anti-oxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.

SummaryA series of amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43 and hnRNPA1 has an ability to be liquid-liquid phase separation, and their disease-related mutations cause the transition of their responsible liquid droplets to aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD) and Paget’s disease of bone. SQSTM1/p62 protein forms liquid-droplets through the interaction with ubiquitinated proteins, and the droplet serves as a platform of autophagosome formation and anti-oxidative stress response via the LC3-interacting region (LIR) and Keap1-interacting region (KIR), respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in LIR and KIR form aberrant liquid droplets, cause defective autophagy and stress response or both. To evaluate the effects of ALS/FTD-related p62 mutations in LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway and the autophagic turnover, we developed systems that enable to monitor them with high sensitivity. These systems revealed that some mutants but not all have their less abilities on the Nrf2-activation and show the delayed turnover. By contrast, while the sufficient ability to form liquid droplets, all droplets consisting of p62 mutants showed slower inner fluidity. These results indicate that like other ALS-related mutant proteins, a primary defect in ALS/FTD with p62 missense mutations is a qualitative change of p62-liquid droplets.

Aim Previous reports showed associations between oxytocin induced labor and mental disorders in offspring. However, those reports are restricted in epidemiological analyses and its mechanism remains unclear. In this study, we hypothesized that induced labor directly causes brain damage in newborns and results in the development of mental disorders. Therefore we aimed to investigate this hypothesis with animal model. Methods The animal model of induced labor was established by subcutaneous oxytocin administration to term-pregnant C57BL/6J mice. We investigated the neonatal brain damage with evaluating immediate early gene expression (c-Fos, c-Jun and JunB) by quantitative polymerase reaction and TdT-mediated dUTP nick end labeling staining. To investigate the injured brain cell types, we performed double-immunostaining with TdT-mediated dUTP nick end labeling staining and each brain component specific protein, such as Oligo2, NeuN, GFAP and Iba1. Results Brain damage during induced labor led to cell death in specific brain regions, which are implicated in mental disorders, in only male offspring at P0. Furthermore, oligodendrocyte precursors were selectively vulnerable compared to the other cell types. This oligodendrocyte-specific impairment during the perinatal period led to an increased numbers of Olig2-positive cells at P5. Expression levels of oxytocin and Oxtr in the fetal brain were not affected by the oxytocin administered to mothers during induced labor. Conclusion Oligodendrocyte cell death in specific brain regions, which was unrelated to the oxytocin itself, was caused by induced labor in only male offspring. This may be an underlying mechanism explaining the human epidemiological data suggesting an association between induced labor and mental disorders.

Molecular isomerism has been discussed from the viewpoint of the tiniest switch and memory elements in electronics. Here, we report an overcrowded ethylene-based molecular conductance switch, which fulfills all the essential requirements for implementation into electronic devices, namely, electric-field-controllable reversible conductance change with a molecular-level spatial resolution, robust conformational bistability under ambient conditions, and ordered monolayer formation on electrode surfaces. The conformational state of this overcrowded ethylene, represented by a folded or twisted conformer, is susceptible to external environments. Nanoscopic measurements using scanning tunneling microscopy techniques, together with theoretical simulations, revealed the electronic properties of each conformer adsorbed on Au(111). While the twisted conformer prevails in the molecularly dispersed state, upon self-assembly into a monolayer, a two-dimensional network structure of the folded conformer is preferentially formed due to particular intermolecular interaction. In the monolayer state, folded-to-twisted and its reverse isomerization can be controlled by the modulation of electric fields.

Summary A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner., Graphical Abstract, Highlights • CFLARs Tg mice develop severe ileitis in utero • Intestinal epithelial cells die by apoptosis and necroptosis in CFLARs Tg mice • Blockade of necroptosis rescues lethality of CFLARs Tg mice • Necroptosis activates type 3 innate lymphoid cells, resulting in severe ileitis, Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

Parkinson's disease (PD) is characterized by dopaminergic (DA) cell loss and the accumulation of pathological alpha synuclein (asyn), but its precise pathomechanism remains unclear, and no appropriate animal model has yet been established. Recent studies have shown that a heterozygous mutation of glucocerebrosidase (gba) is one of the most important genetic risk factors in PD. To create mouse model for PD, we crossed asyn Bacterial Artificial Chromosome transgenic mice with gba heterozygous knockout mice. These double-mutant (dm) mice express human asyn in a physiological manner through its native promoter and showed an increase in phosphorylated asyn in the regions vulnerable to PD, such as the olfactory bulb and dorsal motor nucleus of the vagus nerve. Only dm mice showed a significant reduction in DA cells in the substantia nigra pars compacta, suggesting these animals were suitable for a prodromal model of PD. Next, we investigated the in vivo mechanism by which GBA insufficiency accelerates PD pathology, focusing on lipid metabolism. Dm mice showed an increased level of glucosylsphingosine without any noticeable accumulation of glucosylceramide, a direct substrate of GBA. In addition, the overexpression of asyn resulted in decreased GBA activity in mice, while dm mice tended to show an even further decreased level of GBA activity. In conclusion, we created a novel prodromal mouse model to study the disease pathogenesis and develop novel therapeutics for PD and also revealed the mechanism by which heterozygous gba deficiency contributes to PD through abnormal lipid metabolism under conditions of an altered asyn expression in vivo.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscope/electron microscope (LM/EM) imaging with an ultrastructurally-preserved tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates 1) brain-wide macroscopic observation, 2) mesoscopic circuit mapping, 3) microscopic subcellular imaging, and 4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied the technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in the simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

電子顕微鏡搭載用小型軟Ｘ線発光分光計の波長分解能向上、および軟Ｘ線回折格子分光器の高エネルギー化のために 3000 本/mm 以上の高刻線密度軟Ｘ線回折格子の開発が必要とされている。しかしながら、一般的には刻線密度が高くなるほど格子溝形状の製作精度が回折効率に与える影響が大きくなることから、現状では刻線密度が 2400 本/mm までの回折格子が多く利用される。我々は今回刻線密度 3200 本/mm のラミナー型回折格子を試作して性能評価を行った。基板には 1 nm rms 以下の面粗さを伴う通常研磨の BK7 平面基板を用いた。製作した回折格子の AFM 観察により得られた溝深さ、デューティー比はそれぞれ 5.77nm、0.53 であった。Au を 30 nm の厚さでコートして Photon Factory の BL-11D で回折効率を測定した。入射角 86° および 87.95° の配置で、それぞれ 0.5–1.2 keV および 1.1–1.56 keV の領域で測定した。ビームライン分光器出力に含まれる EUV 成分の影響で 、0.6 keV 以下では下方に、1.1 keV 以上では上方にそれぞれ測定誤差が発生している。入射角 87.95° の測定ではアルミ箔フィルターで EUV 光を除去した。数値計算で測定結果を再現するには面粗さを 0.6 nm rms と仮定すればよく、刻線プロセスに問題がないことがわかった。今後、基板面粗さの低減、多層膜コート、異なるエネルギー領域での溝形状最適化等により性能向上と仕様拡張を図る, 2021年第68回応用物理学会春季講演会

Summary We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020)., Graphical Abstract, Highlights • Exclusive labeling of two groups of neurons with different fluorescent proteins • Detailed protocols from stereotaxic virus injection to immunostaining methods • A signal enhancement method via peroxidase activity, BT-GO reaction • Significant improvement in a signal-to-noise ratio by the cost-effective reaction, We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization.

Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson’s disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

Cytotoxic T lymphocyte antigen-2 (CTLA-2) alpha has been reported to suppress the activities of cathepsin L (Cath L), which is deeply involved in angiogenesis. Therefore, we assessed whether CTLA-2 alpha plays a role in angiogenesis in ocular tissue. To establish models of corneal inflammation and experimental choroidal neovascularization (CNV), male C57BL/6J mice (n = 5) underwent corneal suture placement or laser-induced CNV, respectively. Mice were then injected with recombinant CTLA-2 alpha (1 µg) into the peritoneal cavity at day 0 and every 2 days after operation. In vitro experiments were performed to assess the inflammatory response by measuring TNF-alpha secretion in peritoneal cavity exudate cells (PECs) or the proliferation of mouse vascular endothelial cells (mVECs). CTLA-2 alpha treatment dramatically suppressed corneal angiogenesis, as well as laser-induced CNV. Moreover, CTLA-2 alpha inhibited the proliferation of mVECs in vitro, while CTLA-2 alpha abolishment was able to rescue proliferation. However, CTLA-2 alpha could not suppress cytokine secretion from inflammatory cells such as PECs. In summary, CTLA-2 alpha was able to suppress angiogenesis by suppressing endothelial cell proliferation. Further studies are needed to investigate its usefulness as a new antiangiogenic treatment for a variety of conditions, including age-related macular degeneration.

Dorsal stream, which has a neuronal connection with dorsolateral prefrontal cortex (DLPFC), is known to be responsible for detection of motion including optic flow perception. Using magnetoencephalography (MEG), this study aimed to examine neural responses to optic flow stimuli with looming motion in the DLPFC in patients with mild cognitive impairment due to Alzheimer’s disease (AD-MCI) compared with cognitively unimpaired participants (CU). We analyzed the neural responses by evaluating maximum source-localized power for the AD-MCI group (n = 11) and CU (n = 20), focusing on six regions of interest (ROIs) that form the DLPFC: right and left dorsal Brodmann area 9/46 (A9/46d), Brodmann area 46 (A46) and ventral Brodmann area 9/46 (A9/46v). We found significant differences in the maximum power between the groups in the left A46 and A9/46v. Moreover, in the left A9/46v, the maximum power significantly correlated with the Wechsler Memory Scale-Revised general memory score and delayed recall score. The maximum power in the left A9/46v also revealed high performance in AD-MCI versus CU classification with the area under the ROC curve of 0.90. This study demonstrated that MEG during the optic flow task can be useful in discriminating AD-MCI from CU.

Based on the assumption that some progenitor cells in an organ might reside in neighboring adipose tissue, we investigated whether melanocyte progenitor cells reside in human subcutaneous adipose tissue. First, we examined the expression of human melanoma black 45 (HMB45) and microphthalmia-associated transcription factor (MITF) in undifferentiated adipose-derived stem cells (ADSCs) by immunostaining, RT-PCR, and western blotting. These two markers were detected in undifferentiated ADSCs, and their expression levels were increased in differentiated ADSCs in melanocyte-specific culture medium. Other melanocytic markers (Melan A, MATP, Mel2, Mel EM, tyrosinase, KIT, and PAX3) were also detected at variable levels in undifferentiated ADSCs, and the expression of some markers was increased during differentiation into the melanocyte lineage. We further showed that ADSCs differentiated in melanocyte-specific culture medium localized in the basal layer and expressed tyrosinase and HMB45 in a 3D epidermal culture system. Melanin deposits were also induced by ultraviolet-light-B (UVB) irradiation. These results demonstrate that melanocyte progenitor cells reside in human subcutaneous adipose tissue and that these cells might have the potential to differentiate into mature melanocytes. Melanocyte and keratinocyte progenitors residing in human subcutaneous tissue can be used for the treatment of skin diseases and skin rejuvenation in the future.

Induction and augmentation of labor is one of the most common obstetrical interventions. However, this intervention is not free of risks and could cause adverse events, such as hyperactive uterine contraction, uterine rupture, and amniotic-fluid embolism. Our previous study using a new animal model showed that labor induced with high-dose oxytocin (OXT) in pregnant mice resulted in massive cell death in selective brain regions, specifically in male offspring. The affected brain regions included the prefrontal cortex (PFC), but a detailed study in the PFC subregions has not been performed. In this study, we induced labor in mice using high-dose OXT and investigated neonatal brain damage in detail in the PFC using light and electron microscopy. We found that TUNEL-positive or pyknotic nuclei and Iba-1-positive microglial cells were detected more abundantly in infralimbic (IL) and prelimbic (PL) cortex of the ventromedial PFC (vmPFC) in male pups delivered by OXT-induced labor than in the control male pups. These Iba-1-positive microglial cells were engulfing dying cells. Additionally, we also noticed that in the forceps minor (FMI) of the corpus callosum (CC), the number of TUNEL-positive or pyknotic nuclei and Iba-1-positive microglial cells were largely increased and Iba-1-positive microglial cells phagocytosed massive dying cells in male pups delivered by high-dose OXT-induced labor. In conclusion, IL and PL of the vmPFC and FMI of the CC, were susceptible to brain damage in male neonates after high-dose OXT-induced labor.

400～1000 eV の範囲の 3d 遷移⾦属元素の L 発光は、化合物中のそれらの元素の化学状態だけでなく荷電状態に関する有益な情報を含んでいる。 Lα,β 発光プロファイルとその強度は、それぞれエネルギー分布と 3d 電⼦の量を反映している。また、そのエネルギー位置は、充電状態 (化学シフト) によって影響を受ける。 Ll,ηの放出エネルギーは、3s 準位に空孔がある最終状態の緩和エネルギーを反映している。 さらに、Ll, ηプロファイルは、価数 3d 電⼦の磁気モーメントと、関連する 3d 遷移⾦属元素の 3s との間の相互作⽤を介した磁気モーメントの情報を含んでいる。 しかしながら、従来の電子顕微鏡搭載型の軟X線発光分光器は、Ll,η 線の磁気情報を分析するのに⼗分なエネルギー分解能を持っていなかった。そのため、本研究ではTEM ⽤に設計された⼤きなエネルギー分散を持つ分光器を、EPMA ⽤の分光器に改修を行った。この分光器では試料から回折格子までの距離は従来から市販されている分光器と同じであるが、回折格子から検出器までの距離は約 2 倍であり、このことは、従来比で約 2 倍のエネ ルギー分散が得られることを意味している。検出器は、CMOS カメラ（3.45μm ピッチ） と光学的に結合された MCP（7.5μm ピッチ）で構成されている。この装置を用いてFeのL-発光スペクトルを測定した結果、金属FeとFe2O3のエネルギープロファイルの違いが明確に観測された他、3s3d交換相互作用に起因すると推定されるLl線の内部構造の存在も明らかになった。

The aging process is accompanied by various neurophysiological changes, and the severity of neurodegenerative disorders such as Parkinson’s disease increases with aging. However, the precise neuroanatomical changes that accompany the aging process in both normal and pathological conditions remain unknown. This is in part because there is a lack of high-resolution imaging tool that has the capacity to image a desired volume of neurons in a high-throughput and automated manner. In the present study, focused ion beam/scanning electron microscopy (FIB/SEM) was used to image striatal neuropil in both wild-type (WT) mice and an A53T bacterial artificial chromosome human α-synuclein (A53T-BAC-SNCA) transgenic mouse model of Parkinson’s disease, at 1 month, 3 months, 6 months, and 22 months of age. We demonstrated that spine density gradually decreases, and average spine head volume gradually increases with age in WT mice; suggesting a homeostatic balance between spine head volume and spine density. However, this inverse relationship between spine head volume and spine density was not observed in A53T-BAC-SNCA transgenic mice. Taken together, our data suggest that Parkinson’s disease is accompanied by an abnormality in the mechanisms that control synapse growth and maturity. Significance statement Currently, the clinical diagnosis of Parkinson’s disease is based on the presence of motor symptoms. However, these symptoms only manifest after a significant proportion of dopaminergic cells have degenerated. The latent, prodromal phase is therefore of particular interest for the development of disease-modifying therapies to slow down or reverse the course of neurodegeneration. Although clinical markers to diagnose patients in the prodromal phase are gradually emerging, a structural marker that defines this phase has not yet been identified. Structural changes likely occur in the synapse before the onset of neurodegeneration. Thus, this study performed an ultrastructural analysis of synapses in a mouse model of prodromal Parkinson’s disease and revealed distinct, age-dependent structural changes.

Neurons in the central nervous system display a great diversity of synaptic architecture. While much of our knowledge on the excitatory synapse morphology derives from the prototypical asymmetric synapses, little has been studied about the atypical crest-type synapse that exists in the restricted brain regions. Here, we used focused ion beam scanning electron microscopy (FIB/SEM) to image a neuropil volume of interpeduncular nucleus (IPN) and manually reconstructed several dendrites to obtain an insight about the topography and quantitative features of crest synapses. Three-dimensional reconstruction showed numerous U-shaped structures protruding from the IPN dendrites. On either faces of the U-shaped structure, a pair of crest synapses are aligned in parallel such that there exists a positive correlation between the postsynaptic density (PSD) area of synapses that participate in pair formation. Interestingly, mitochondria are excluded from the site of crest synapses. Several presynaptic axons run through the hollow, cylindrical space of the U-shape grooves such that the plasma membrane of the axon and the dendrite are organized in a tight opposition without any intervening glial membrane. Unlike the peculiar dendritic morphology, IPN neurons possess typical somatic morphology with an oval, centrally located nucleus. In conclusion, our data reveals a hitherto unknown unique topographical feature of crest synapses in the IPN.

Cells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. After osmotic stress-enforced cell swelling or shrinkage, the cells regulate intracellular osmolality to recover their volume. However, the mechanisms recognizing osmotic stress remain obscured. We previously clarified that apoptosis signal-regulating kinase 3 (ASK3) bidirectionally responds to osmotic stress and regulates cell volume recovery. Here, we show that macromolecular crowding induces liquid-demixing condensates of ASK3 under hyperosmotic stress, which transduce osmosensing signal into ASK3 inactivation. A genome-wide small interfering RNA (siRNA) screen identifies an ASK3 inactivation regulator, nicotinamide phosphoribosyltransferase (NAMPT), related to poly(ADP-ribose) signaling. Furthermore, we clarify that poly(ADP-ribose) keeps ASK3 condensates in the liquid phase and enables ASK3 to become inactivated under hyperosmotic stress. Our findings demonstrate that cells rationally incorporate physicochemical phase separation into their osmosensing systems., Cells experience various osmotic perturbation, but cellular osmosensing mechanisms remain obscure. Here, the authors report that cells recognize osmotic stress from the inside through macromolecular crowding-driven and poly(ADP-ribose)-conditioned liquid–liquid phase separation.

p62/SQSTM1 is a multivalent protein that has the ability to cause liquid–liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non‐canonical activation of the Keap1‐Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62‐liquid droplet formation and Keap1‐Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid‐like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62‐mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1‐liquid droplets, but also of p62‐dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1‐liquid droplet formation to activate the Keap1‐Nrf2 pathway.

Significance LRRK2, a protein kinase related to Parkinson’s disease, is implicated in the maintenance of lysosomes, and a subset of Rab GTPases has been identified as bona fide substrates of LRRK2. Here, we reveal a key stress-responsive pathway composed of Rab7L1, LRRK2, and phosphorylated Rab8/10 involved in lysosomal homeostasis. Lysosomal overload stress induces translocation of Rab7L1 and LRRK2 to lysosomes, where LRRK2 is activated, and stabilizes Rab8 and Rab10 through phosphorylation. The activation of this machinery protects against lysosomal enlargement and upregulates lysosomal secretion through Rab effectors, EHBP1 and EHBP1L1. These findings elucidate a novel regulatory mechanism of Rab GTPases by phosphorylation by LRRK2 in stressed lysosomes, which may also be involved in the pathomechanism of LRRK2-related disorders., Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson’s disease and Crohn’s disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

Autophagy regulates fatty acid and triglyceride synthesis at the transcriptional level by fine-tuning the levels of NCoR1, a negative regulator of nuclear receptors. Defective autophagy impairs physiological steatosis both under fasting conditions and after hepatectomy., Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, such as after a meal. LDs serve as an energy reservoir during fasting and have a buffering capacity that prevents lipotoxicity. Autophagy and the autophagic machinery have been proposed to play a role in LD biogenesis, but the underlying molecular mechanism remains unclear. Here, we show that when nuclear receptor co-repressor 1 (NCoR1), which inhibits the transactivation of nuclear receptors, accumulates because of autophagy suppression, LDs decrease in size and number. Ablation of ATG7, a gene essential for autophagy, suppressed the expression of gene targets of liver X receptor α, a nuclear receptor responsible for fatty acid and triglyceride synthesis in an NCoR1-dependent manner. LD accumulation in response to fasting and after hepatectomy was hampered by the suppression of autophagy. These results suggest that autophagy controls physiological hepatosteatosis by fine-tuning NCoR1 protein levels.

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.

Apples are well known to have various benefits for the human body. Procyanidins are a class of polyphenols found in apples that have demonstrated effects on the circulatory system and skeletal organs. Osteoarthritis (OA) is a locomotive syndrome that is histologically characterized by cartilage degeneration associated with the impairment of proteoglycan homeostasis in chondrocytes. However, no useful therapy for cartilage degeneration has been developed to date. In the present study, we detected beneficial effects of apple polyphenols or their procyanidins on cartilage homeostasis. An in vitro assay revealed that apple polyphenols increased the activities of mitochondrial dehydrogenases associated with an increased copy number of mitochondrial DNA as well as the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), suggesting the promotion of PGC-1α-mediated mitochondrial biogenesis. Apple procyanidins also enhanced proteoglycan biosynthesis with aggrecan upregulation in primary chondrocytes. Of note, oral treatment with apple procyanidins prevented articular cartilage degradation in OA model mice induced by mitochondrial dysfunction in chondrocytes. Our findings suggest that apple procyanidins are promising food components that inhibit OA progression by promoting mitochondrial biogenesis and proteoglycan homeostasis in chondrocytes.

TDP-43 is an RNA-binding protein important for many aspects of RNA metabolism. Abnormal accumulation of TDP-43 in the cytoplasm of affected neurons is a pathological hallmark of the neurodegenerative diseases frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Several transgenic mouse models have been generated that recapitulate defects in TDP-43 accumulation, thus causing neurodegeneration and behavioural impairments. While aging is the key risk factor for neurodegenerative diseases, the specific effect of aging on phenotypes in TDP-43 transgenic mice has not been investigated. Here, we analyse age-dependent changes in TDP-43 transgenic mice that displayed impaired memory. We found the accumulation of abundant poly-ubiquitinated protein aggregates in the hippocampus of aged TDP-43 transgenic mice. Intriguingly, the aggregates contained some interneuron-specific proteins such as parvalbumin and calretinin, suggesting that GABAergic interneurons were degenerated in these mice. The abundance of aggregates significantly increased with age and with the overexpression of TDP-43. Gene array analyses in the hippocampus and other brain areas revealed dysregulation in genes linked to oxidative stress and neuronal function in TDP-43 transgenic mice. Our results indicate that the interneuron degeneration occurs upon aging, and TDP-43 accelerates age-dependent neuronal degeneration, which may be related to the impaired memory of TDP-43 transgenic mice.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce inflammation by suppressing cyclooxygenases (COXs). NSAID eye drops are frequently prescribed after ocular surgery to reduce inflammation and pain, but this treatment has clinically significant side effects, including corneal ulcer and perforation. The molecular mechanisms underlying these side effects remain unknown. Recently, the COX product 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) was identified as an endogenous ligand for leukotriene B4 receptor 2 (BLT2), which is important in maintenance of epithelial homeostasis. We hypothesized that NSAID-dependent corneal damage is caused by reduced production of 12-HHT. Diclofenac eye drops decreased the abundance of downstream products of COX and delayed corneal wound healing in BALB/c mice. Expression of BLT2 was observed in murine ocular tissues including cornea, and in human corneal epithelial cell line and human primary corneal epithelial cells. In BLT2-knockout mice, corneal wound healing was delayed, but the diclofenac-dependent delay in corneal wound healing disappeared. 12-HHT accelerated wound closure both in BLT2-transfected corneal cell line and human primary corneal epithelial cells. Thus, our results reveal that NSAIDs delay corneal wound healing by inhibiting 12-HHT production, and suggest that stimulation of the 12-HHT/BLT2 axis represents a novel therapeutic approach to corneal wound healing.

Neurologic phenotypes of cathepsin D (CTSD)-deficient mice, a murine model of neuronal ceroid lipofuscinoses, indicate the importance of CTSD for the maintenance of metabolism in central nervous system neurons. To further understand the role of CTSD in central nervous system neurons, we generated mice with a CTSD deficiency specifically in the Purkinje cells (PCs) (CTSDFlox/Flox;GRID2-Cre) and compared their phenotypes with those of PC-selective Atg7-deficient (Atg7Flox/Flox;GRID2-Cre) mice. In both strains of mice, PCs underwent degeneration, but the CTSD-deficient PCs disappeared more rapidly than their Atg7-deficient counterparts. When CTSD-deficient PCs died, the neuronal cell bodies became shrunken, filled with autophagosomes and autolysosomes, and had nuclei with dispersed small chromatin fragments. The dying Atg7-deficient PCs also showed similar ultrastructures, indicating that the neuronal cell death of CTSD- and Atg7-deficient PCs was distinct from apoptosis. Immunohistochemical observations showed the formation of calbindin-positive axonal spheroids and the swelling of vesicular GABA transporter-positive presynaptic terminals that were more pronounced in Atg7-deficient PCs than in CTSD-deficient PCs. An accumulation of tubular vesicles may have derived from the smooth endoplasmic reticulum; nascent autophagosome-like structures with double membranes was a common feature in the swollen axons of these PCs. These results suggested that PCs were more vulnerable to CTSD deficiency in lysosomes than to autophagy impairment, and this vulnerability does not depend on the severity of axonal swelling.

Mutations of the retromer component Vps35 and endosomal kinase LRRK2 are linked to autosomal dominant forms of familial Parkinson's disease (PD). However, the physiological and pathological roles of Vps35 and LRRK2 in neuronal functions are poorly understood. Here, we demonstrated that the loss of Drosophila Vps35 (dVps35) affects synaptic vesicle recycling, dopaminergic synaptic release and sleep behavior associated with dopaminergic activity, which is rescued by the expression of wild-type dVps35 but not the PD-associated mutant dVps35 D647N. Drosophila LRRK2 dLRRK together with Rab5 and Rab11 is also implicated in synaptic vesicle recycling, and the manipulation of these activities improves the Vps35 synaptic phenotypes. These findings indicate that defects of synaptic vesicle recycling in which two late-onset PD genes, Vps35 and LRRK2, are involved could be key aspects of PD etiology.