Atg9a deficiency causes axon-specific lesions including neuronal circuit dysgenesis

Conditional knockout mice for Atg9a, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in Atg9a-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed Atg9a-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from atg9a-KO mouse brains, but not in those from atg7-KO and atg16l1-KO brains. Moreover, this tendency was also confirmed in Atg9a-knockdown neurons under an atg7-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that Atg9a deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of Atg9a impaired nerve fiber tract formation

Purified Mesenchymal Stem Cells Are an Efficient Source for iPS Cell Induction

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the forced expression of defined transcription factors. Although most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear.Here, we generated iPS cells from PDGFRα+ Sca-1+ (PαS) adult mouse mesenchymal stem cells (MSCs) and PDGFRα⁻ Sca-1⁻ osteo-progenitors (OP cells), and compared the induction efficiency and quality of individual iPS clones. MSCs had a higher reprogramming efficiency compared with OP cells and Tail Tip Fibroblasts (TTFs). The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency.Our findings suggest that a purified source of undifferentiated cells from adult tissue can produce high-quality iPS cells. In this context, prospectively enriched MSCs are a promising candidate for the efficient generation of high-quality iPS cells

Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFRα+Sca-1+CD45−TER119−) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities

Lack of Cathepsin D in the Renal Proximal Tubular Cells Resulted in Increased Sensitivity against Renal Ischemia/Reperfusion Injury

Cathepsin D is one of the major lysosomal aspartic proteases that is essential for the normal functioning of the autophagy-lysosomal system. In the kidney, cathepsin D is enriched in renal proximal tubular epithelial cells, and its levels increase during acute kidney injury. To investigate how cathepsin D-deficiency impacts renal proximal tubular cells, we employed a conditional knockout CtsDflox/−; Spink3Cre mouse. Immunohistochemical analyses using anti-cathepsin D antibody revealed that cathepsin D was significantly decreased in tubular epithelial cells of the cortico-medullary region, mainly in renal proximal tubular cells of this mouse. Cathepsin D-deficient renal proximal tubular cells showed an increase of microtubule-associated protein light chain 3 (LC3; a marker for autophagosome/autolysosome)-signals and an accumulation of abnormal autophagic structures. Renal ischemia/reperfusion injury resulted in an increase of early kidney injury marker, Kidney injury molecule 1 (Kim-1), in the cathepsin D-deficient renal tubular epithelial cells of the CtsDflox/−; Spink3Cre mouse. Inflammation marker was also increased in the cortico-medullary region of the CtsDflox/−; Spink3Cre mouse. Our results indicated that lack of cathepsin D in the renal tubular epithelial cells led to an increase of sensitivity against ischemia/reperfusion injury

AAV-based gene therapy ameliorated CNS-specific GPI defect in mouse models

Thirty genes are involved in the biosynthesis and modification of glycosylphosphatidylinositol (GPI)-anchored proteins, and defects in these genes cause inherited GPI deficiency (IGD). PIGA is X-linked and involved in the first step of GPI biosynthesis, and only males are affected by variations in this gene. The main symptoms of IGD are neurological abnormalities, such as developmental delay and seizures. There is no effective treatment at present. We crossed Nestin-Cre mice with Piga-floxed mice to generate CNS-specific Piga knockout (KO) mice. Hemizygous KO male mice died by P10 with severely defective growth. Heterozygous Piga KO female mice are mosaic for Piga expression and showed severe defects in growth and myelination and died by P25. Using these mouse models, we evaluated the effect of gene replacement therapy with adeno-associated virus (AAV). It expressed efficacy within 6 days, and the survival of male mice was extended to up to 3 weeks, whereas 40% of female mice survived for approximately 1 year and the growth defect was improved. However, liver cancer developed in all three treated female mice at 1 year of age, which was probably caused by the AAV vector bearing a strong CAG promoter

GFP transgenic mice reveal active canonical Wnt signal in neonatal brain and in adult liver and spleen

In the past decades, the function of the Wnt canonical pathway during embryogenesis has been intensively investigated; however, little survey of neonatal and adult tissues has been made, and the role of this pathway remains largely unknown. To investigate its role in mature tissues, we generated two new reporter transgenic mouse lines, ins-TOPEGFP and ins-TOPGAL, that drive EGFP and β-galactosidase expression under TCF/β-catenin, respectively. To obtain the accurate expression pattern, we flanked these transgenes with the HS4 insulator to reduce chromosomal positional effects. Analysis of embryos showed that the reporter genes were activated in regions where canonical Wnt activity has been implicated. Furthermore, their expression patterns were consistent in both lines, indicating the accuracy of the reporter signal. In the neonatal brain, the reporter signal was detected in the mesencephalon and hippocampus. In the adult mice, the reporter signal was found in the mature pericenteral hepatocytes in the normal liver. Furthermore, during inflammation the number of T cells expressing the reporter gene increased in the adult spleen. Thus, in this research, we identified two organs, i.e., the liver and spleen, as novel organs in which the Wnt canonical signal is in motion in the adult. These transgenic lines will provide us broader opportunities to investigate the function of the Wnt canonical pathway in vivo

<i>Atg9a</i> deficiency causes axon-specific lesions including neuronal circuit dysgenesis

<p>Conditional knockout mice for <i>Atg9a</i>, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in <i>Atg9a</i>-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed <i>Atg9a</i>-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from <i>atg9a</i>-KO mouse brains, but not in those from <i>atg7</i>-KO and <i>atg16l1</i>-KO brains. Moreover, this tendency was also confirmed in <i>Atg9a</i>-knockdown neurons under an <i>atg7</i>-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that <i>Atg9a</i> deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of <i>Atg9a</i> impaired nerve fiber tract formation.</p