A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely—not only in the nucleus, but also in the cytoplasm—and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well

末梢神経慢性伸張に伴う神経活動停滞化の病態について

取得学位：博士(医学), 学位授与番号：医博乙第1522号, 学位授与年月日：平成12年9月6日, 学位授与年：200

Desulfation of Heparan Sulfate by Sulf1 and Sulf2 Is Required for Corticospinal Tract Formation

Heparan sulfate (HS) has been implicated in a wide range of cell signaling. Here we report a novel mechanism in which extracellular removal of 6-O-sulfate groups from HS by the endosulfatases, Sulf1 and Sulf2, is essential for axon guidance during development. In Sulf1/2 double knockout (DKO) mice, the corticospinal tract (CST) was dorsally displaced on the midbrain surface. In utero electroporation of Sulf1/2 into radial glial cells along the third ventricle, where Sulf1/2 mRNAs are normally expressed, rescued the CST defects in the DKO mice. Proteomic analysis and functional testing identified Slit2 as the key molecule associated with the DKO phenotype. In the DKO brain, 6-O-sulfated HS was increased, leading to abnormal accumulation of Slit2 protein on the pial surface of the cerebral peduncle and hypothalamus, which caused dorsal repulsion of CST axons. Our findings indicate that postbiosynthetic desulfation of HS by Sulfs controls CST axon guidance through fine-tuning of Slit2 presentation

Differentiation of banding patterns between Streptococcus mutans and Streptococcus sobrinus isolates in rep-PCR using ERIC primer

Streptococcus mutans and Streptococcus sobrinus are considered to be important bacterial species in the initiation of human dental caries. Therefore, the establishment of a reliable genotyping method to distinguish S. mutans from S. sobrinus is of central importance.We assessed the usefulness of repetitive extragenic palindromic polymerase chain reaction (rep-PCR) using ERIC primer banding patterns in differentiating S. mutans and S. sobrinus.Five S. mutans and two S. sobrinus prototype strains and 50 clinical isolates (38 S. mutans serotype c, 4 S. sobrinus serotype d, and 8 S. sobrinus serotype g) were examined. The banding patterns of amplicons generated were compared among the prototype strains and clinical isolates, to find common bands that distinguish S. mutans and S. sobrinus.Multiple banding patterns were seen with all strains tested. The representative strains of S. mutans tested revealed six unique, strong bands at 2,000 bp, 1,700 bp, 1,400 bp, 1,100 bp, 850 bp, and 250 bp, whereas S. sobrinus had seven strong bands at 2,000 bp, 1,800 bp, 1,100 bp, 900 bp, 800 bp, 600 bp, and 550 bp. The band at 1,100 bp was the only band that was observed in both S. mutans and S. sobrinus. Furthermore, most clinical S. mutans isolates revealed identical banding patterns. All S. mutans had amplicons at 1,700 bp, 850 bp, and 250 bp, whereas those of S. sobrinus were at 1,100 bp, 900 bp, and 800 bp.These results indicate that using rep-PCR with the ERIC primers can distinguish between S. mutans and S. sobrinus

Remarkable complexity and variability of corticospinal tract defects in adult Semaphorin 6A knockout mice

The corticospinal tract (CST) has a complex and long trajectory that originates in the cerebral cortex and ends in the spinal cord. Semaphorin 6A (Sema6A), a member of the semaphorin family, is an important regulator of CST axon guidance. Previous studies have shown that postnatal Sema6A mutant mice have CST defects at the midbrain–hindbrain boundary and medulla. However, the routes the aberrant fibers take throughout the Sema6A mutant brain remain unknown. In this study, we performed 3D reconstruction of immunostained CST fibers to reevaluate the details of the abnormal CST trajectories in the brains of adult Sema6A mutant mice. Our results showed that the axon guidance defects reported in early postnatal mutants were consistently observed in adulthood. Those abnormal trajectories revealed by 3D analysis of brain sections were, however, more complex and variable than previously thought. In addition, 3D analysis allowed us to identify a few new patterns of aberrant projections. First, a subset of fibers that separated from and descended in parallel to the main bundle projected laterally at the caudal pons, subsequently changed direction by turning caudally, and extended to the medulla. Second, some abnormal fibers returned to the correct trajectory after deviating substantially from the original tract. Third, some fibers reached the pyramidal decussation normally but did not enter the dorsal funiculus. Section immunostaining combined with 3D reconstruction is a powerful method to track long projection fibers and to examine the entire nerve tracts of both normal and abnormal animals

Abnormal Pyramidal Decussation and Bilateral Projection of the Corticospinal Tract Axons in Mice Lacking the Heparan Sulfate Endosulfatases, Sulf1 and Sulf2

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CONTROLS FOR MONITORING THE DETERIORATION OF STORED BLOOD SAMPLES IN THE JAPAN MULTI-INSTITUTIONAL COLLABORATIVE COHORT STUDY (J-MICC STUDY)

2008-08Cohort studies commonly store blood samples to measure the associations of biomarkers with disease risks for a long time after the study subjects are enrolled. To obtain valid measurements of the stored samples, monitoring their degree of deterioration is essential. The first stage of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study launched in 2005 included a project to validate the quality of stored blood samples. This project will compare the measurements of representative molecules over different storage periods (1, 4, and 8 years after sampling, and when a nested case-control study is conducted), different storage temperatures (–80 and –150°C), and different separation conditions (temperature and time) before storage. For these purposes, 28 ml of peripheral blood from 10 people was sampled four times annually, using two tubes for serum and two EDTA -Na tubes for plasma. These samples were treated using the process adopted for the J-MIC study protocol, and stored in tubes containing 300 μl of serum or plasma labeled with two-dimensional bar codes. The sampling was started in 2006, and some of the specimens will be stored until the end of the J-MICC Study in 2035. The resulting findings will produce valuable information on the stability of the molecules, not only for the J-MICC Study, but also for other cohort studies.departmental bulletin pape