アルツハイマー病における神経細胞特異的DNAメチル化解析による新規病態基盤の探索

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 尾藤 晴彦, 東京大学准教授 垣内 千尋, 東京大学講師 武笠 晃丈, 東京大学教授 油谷 浩幸, 東京大学教授 秋下 雅弘University of Tokyo(東京大学

Control of Cortical Axon Elongation by a GABA-Driven Ca<sup style="margin: 0px; padding: 0px; border: 0px; outline-style: none; font-weight: inherit; font-style: inherit; font-size: 0.85em; font-family: inherit; line-height: 0; text-align: inherit; vertical-align: super;">2+/Calmodulin-Dependent Protein Kinase Cascade</sup>

Ca(2+) signaling plays important roles during both axonal and dendritic growth. Yet, whether and how Ca(2+) rises may trigger and contribute to the development of long range cortical connections remains largely unknown. Here we demonstrate that two separate limbs of CaMK kinase (CaMKK) - CaMKI cascades, CaMKK-CaMKIα and CaMKK-CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABA(A) receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK-CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension, and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK-CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process which may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the central nervous system

Autocorrelation-based method to identify disordered rhythm in Parkinson's disease tasks: A novel approach applicable to multimodal devices.

ObjectiveWe aim to propose a novel method of evaluating the degree of rhythmic irregularity during repetitive tasks in Parkinson's disease (PD) by using autocorrelation to extract serial perturbation in the periodicity of body part movements as recorded by objective devices.MethodsWe used publicly distributed sequential joint movement data recorded during a leg agility task or pronation-supination task. The sequences of body part trajectory were processed to extract their short-time autocorrelation (STACF) matrices; the sequences of single task conducted by participants were then divided into two clusters according to their similarity in terms of their STACF representation. The Unified Parkinson's Disease Rating Scale sub-score rated for each task was compared with cluster membership to obtain the area under the curve (AUC) to evaluate the discrimination performance of the clustering. We compared the AUC with those obtained from the clustering of the raw sequence or short-time Fourier transform (STFT).ResultsIn classifying the pose estimator-based trajectory data of the knee during the leg agility task, the AUC was the highest when the STACF sequence was used for clustering instead of other types of sequences with up to 0.815, being comparable to the results reported in the original analysis of the data using an approach different from ours. In addition, in classifying another dataset of accelerometer-based trajectory data of the wrist during a pronation-supination task, the AUC was again highest up to 0.785 when clustering was performed using the STACF rather than other types of sequence.ConclusionOur autocorrelation-based method achieved a fair performance in detecting sequences with irregular rhythm, suggesting that it might be used as another evaluation strategy that is potentially widely applicable to qualify the disordered rhythm of PD regardless of the kinds of task or the modality of devices, although further refinement is needed

Quantifying normal and parkinsonian gait features from home movies: Practical application of a deep learning-based 2D pose estimator.

OBJECTIVE:Gait movies recorded in daily clinical practice are usually not filmed with specific devices, which prevents neurologists benefitting from leveraging gait analysis technologies. Here we propose a novel unsupervised approach to quantifying gait features and to extract cadence from normal and parkinsonian gait movies recorded with a home video camera by applying OpenPose, a deep learning-based 2D-pose estimator that can obtain joint coordinates from pictures or videos recorded with a monocular camera. METHODS:Our proposed method consisted of two distinct phases: obtaining sequential gait features from movies by extracting body joint coordinates with OpenPose; and estimating cadence of periodic gait steps from the sequential gait features using the short-time pitch detection approach. RESULTS:The cadence estimation of gait in its coronal plane (frontally viewed gait) as is frequently filmed in the daily clinical setting was successfully conducted in normal gait movies using the short-time autocorrelation function (ST-ACF). In cases of parkinsonian gait with prominent freezing of gait and involuntary oscillations, using ACF-based statistical distance metrics, we quantified the periodicity of each gait sequence; this metric clearly corresponded with the subjects' baseline disease statuses. CONCLUSION:The proposed method allows us to analyze gait movies that have been underutilized to date in a completely data-driven manner, and might broaden the range of movies for which gait analyses can be conducted

Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

<div><p>Histone deacetylases (HDACs) are potential therapeutic targets of polyglutamine (pQ) diseases including Huntington’s disease (HD) that may function to correct aberrant transcriptional deactivation caused by mutant pQ proteins. HDAC3 is a unique class 1 HDAC found in both the cytoplasm and in the nucleus. However, the precise functions of HDAC3 in the two cellular compartments are only vaguely known. HDAC3 directly binds to huntingtin (Htt) with short pQ and this interaction is important for suppressing neurotoxicity induced by HDAC3. With long pQ Htt, the interaction with HDAC3 is inhibited, and this supposedly promotes neuronal death, indicating that HDAC3 would be a good therapeutic target for HD. However, the knockout of one HDAC3 allele did not show any efficacy in reducing neurodegenerative symptoms in a mouse model of HD. Therefore, the role of HDAC3 in the pathogenesis of HD has yet to be fully elucidated. We attempted to resolve this issue by focusing on the different roles of HDAC3 on cytoplasmic and nuclear Htt aggregates. In addition to supporting the previous findings, we found that HDAC3 preferentially binds to nuclear Htt over cytoplasmic ones. Specific HDAC3 inhibitors increased the total amount of Htt aggregates by increasing the amount of nuclear aggregates. Both cytoplasmic and nuclear Htt aggregates were able to suppress endogenous HDAC3 activity, which led to decreased nuclear proteasome activity. Therefore, we concluded that Htt aggregates impair nuclear proteasome activity through the inhibition of HDAC3. Our findings provide new insights regarding cross-compartment proteasome regulation.</p></div

Effect of HDAC3 on cytoplasmic and nuclear Htt aggregates.

<p><b>A:</b> Aspartate at the 166^th and 168^th amino acid of HDAC3 is crucial for its activity. An empty plasmid (–), FLAG tagged wild-type (wt), or D166A + D168A mutant (DA) of HDAC3 were overexpressed in 293T cells. After immunoprecipitation using anti-FLAG antibodies, pan-histone deacetylase activity was measured by fluorometric analysis. *P≤0.05 vs. empty plasmid by ANOVA and multiple comparisons. N = 3. Anti-FLAG and anti-actin western blots from cell lysates are shown below. <b>B–C:</b> HDAC3 overexpression reduces nuclear Htt-ex1 aggregates. Empty vector (–), FLAG-tagged wild-type or DA mutant HDAC3 were transfected to E3 and N3 cells. Amount of aggregate measured by filter trap assay are shown in B and C. *significant against – and DA by ANOVA and multiple comparisons. N = 3. <b>D–G</b>: Empty vector (–), FLAG-tagged wild-type or DA mutant HDAC3 were transfected to E3 and N3 cells. Cells harboring inclusion bodies are counted and their fraction in total cells was plotted in 2D and E. Representative GFP images of low powered magnification fields are shown in 2F and 2G. *significant against – and DA by ANOVA and multiple comparisons. <b>H:</b> HDAC3 shRNA reduces HDAC3 amount by 70%. Molecular weight markers are shown at the left side. <b>I–J:</b> HDAC3 knockdown increases nuclear aggregates. HDAC3 shRNA was transfected into E3 or N3 cells and the 1% TritonX-100 insoluble fraction was subjected to filter trap assay. *P = 0.0003 by <i>t</i>-test. N = 3.</p