The first author of the article has duely admitted that he is mainly responsible for the misconduct.

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative. METHODS: CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats. RESULTS: Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1alpha (IL-1alpha), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals. CONCLUSIONS: From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1alpha, CCL5, and TIMP-1 levels may play important roles in this regard.
Animals ; Apoptosis ; Culture Media ; Culture Media, Conditioned ; Deoxyuridine ; Fetal Blood* ; Gene Expression ; Humans ; Hypertension* ; In Situ Nick-End Labeling ; Interleukin-1alpha ; Lung ; Mesenchymal Stromal Cells ; Models, Animal ; Monocrotaline ; Pulmonary Artery ; Rats* ; Rats, Sprague-Dawley ; Survival Rate ; Tissue Inhibitor of Metalloproteinase-1 ; Umbilical Cord* ; Ventricular Pressure

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.
Amyotrophic Lateral Sclerosis ; Animals ; Autophagy ; Brain ; Central Nervous System* ; Cerebellum ; Cerebral Cortex ; Hippocampus ; Homeostasis ; Mice ; Mice, Transgenic* ; Spinal Cord ; Superoxide Dismutase ; Thalamus ; Tissue Distribution

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.
Amyotrophic Lateral Sclerosis ; Animals ; Autophagy ; Brain ; Central Nervous System* ; Cerebellum ; Cerebral Cortex ; Hippocampus ; Homeostasis ; Mice ; Mice, Transgenic* ; Spinal Cord ; Superoxide Dismutase ; Thalamus ; Tissue Distribution

Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca2+ levels. Ca2+ binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS-/- mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca2+ levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca2+ homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca2+ homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.
Animals ; Calcium ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Carrier Proteins ; Cerebral Cortex ; Homeostasis ; Immunohistochemistry ; Mice ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase ; Synaptic Transmission

Nitric Oxide (NO) actively participates in the regulation of neuronal intracellular Ca2+ levels by modulating the activity of various channels and receptors. To test the possibility that modulation of Ca2+ buffer protein expression level by NO participates in this regulatory effect, we examined expression of calbindin-D28k, calretinin, and parvalbumin in the cerebellum of neuronal NO synthase knock-out (nNOS(-/-)) mice using immunohistochemistry. We observed that in the cerebellar cortex of the nNOS(-/-) mice, expression of calbindin-D28k and parvalbumin were significantly increased while expression of calretinin was significantly decreased. These results suggest another mechanism by which NO can participate in the regulation of Ca2+ homeostasis.
Animals ; Calcium ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Cerebellar Cortex ; Cerebellum ; Homeostasis ; Immunohistochemistry ; Mice ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase

In the present study, we investigated influences of glycogen synthase kinase (GSK) 3beta on the development and/or progression of amyotrophic lateral sclerosis (ALS). We used transgenic mice expressing a human Cu/Zn superoxide dismutase mutant (SOD1G93A) as an in vivo model of ALS and examined expressional changes of GSK3beta immunohistochemically in the spinal cord, brain stem and cerebellum. With these experiments we demonstrate that the neurons in these regions of symptomatic SOD1G93A transgenic mice showed increased GSK3beta immunoreactivities compared with wild-type SOD1 transgenic mice. In contrast to symptomatic SOD1G93A transgenic mice, few GSK3beta immunoreactivity changes were detected in 8w- and 13w-old presymptomatic SOD1G93A transgenic mice. These data suggest the possibility that GSK3 functions as a modulating factor of apoptosis-related alterations in ALS and that GSK3beta exert differential functions in the development and/or progression of ALS. But the exact functional significances of these changes require further elucidation.
Amyotrophic Lateral Sclerosis ; Animals ; Brain Stem ; Central Nervous System* ; Cerebellum ; Glycogen Synthase Kinases* ; Glycogen Synthase* ; Glycogen* ; Humans ; Mice ; Mice, Transgenic* ; Neurons ; Spinal Cord ; Superoxide Dismutase

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. N- terminal fragments of huntingtin with polyglutamine produce aggregates in the endosome-lysosomal system, where proteolytic fragments of huntingtin is generated. Heat shock protein 70 (HSP70) prevents the formation of protein aggregates, but the effect of HSP70 on the huntingtin in the endosome-lysosomal system is unknown. This study was to determine whether HSP70 alters the distribution of huntingtin in endosome-lysosomal system. HSP70 expressing stable cells (NIH/3T3 or cerebral hybrid cell line A1) were generated, and mutant [(CAG)100] huntingtin was transiently overexpressed. Analysis of subcellular distribution by immnuocytochemistry or proteolysis cleavage by Western blotting was performed. 18 CAG repeat wild type [WT; (CAG)18] huntingtin was used as a control. Cells with huntingtin showed patterns of endosome- lysosomal accumulation, from a 'dispersed vacuole (DV)' type into a coalescent 'perinuclear vacuole (PV)' type over time. In WT huntingtin, HSP70 increased the cells with the PV types that enhanced the proteolytic cleavage of huntingtin. However, HSP70 reduced cells of the DV and PV types expressing mutant huntingtin, that result in less proteolysis than that of control. In addition, intranuclear inclusions were formed only in mutant cells, which was not affected by HSP70. These results suggest that HSP70 alters the distribution of huntingtin in the endosome-lysosomal system, and that this contributes to huntingtin proteolysis.
Peptide Hydrolases/metabolism ; Nuclear Proteins/genetics/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; NIH 3T3 Cells ; Mice ; Lysosomes/*metabolism ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Endosomes/*metabolism ; Cytoplasm/metabolism ; Cell Survival ; Animals

Corticotropin releasing factor (CRF) is known to be involved in the stress response and in some degenerative brain disorders. In addition, CRF has a role as a neuromodulator in adult cerebellar circuits. Data from developmental studies suggest a putative role for CRF as a trophic factor during cerebellar development. In this study, we investigated the trophic role for CRF family of peptides by culturing cerebellar neurons in the presence of CRF, urocortin or urocortin II. Primary cell cultures of cerebella from embryonic day 18 mice were established, and cells were treated for either 1, 5 or 9 days with Basal Medium Eagles complete medium alone or complete medium with 1 micrometer CRF, urocortin, or urocortin II. The number of GABA-positive neurons in each treatment condition was counted at each culture age for monitoring the changes in neuronal survival. Treatment with 1 micrometer CRF or 1 micrometer urocortin increased the survival of GABAergic neurons at 6 days in vitro and 10 days in vitro, and this survival promoting effect was abolished by treatment with astressin in the presence of those peptides. Based on these data, we suggest that CRF or urocortin has a trophic role promoting the survival of cerebellar GABAergic neurons in cultures.
gamma-Aminobutyric Acid/*metabolism ; Time Factors ; Receptors, Corticotropin-Releasing Hormone/*metabolism ; Peptides/chemistry ; Neurons/*metabolism ; Mice, Inbred C57BL ; Mice ; Immunohistochemistry ; Image Processing, Computer-Assisted ; Corticotropin-Releasing Hormone/biosynthesis/*physiology ; Cerebellum/*embryology/*metabolism ; Cells, Cultured ; Cell Survival ; Animals

Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, bAPP secretion was up-regulated by caveolin-1 over-expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by beta-secretase via PKC downregulation.
Up-Regulation ; Receptors, Cell Surface/*metabolism ; Rats ; Protein Kinase C/metabolism ; Middle Aged ; Microscopy, Electron ; Humans ; Caveolin 1/*metabolism/physiology ; Caveolae/*metabolism/ultrastructure ; Brain/metabolism/pathology/ultrastructure ; Animals ; Amyloid beta-Protein Precursor/*metabolism ; Amyloid beta-Protein/*metabolism ; Alzheimer Disease/*metabolism ; Aging/metabolism ; Aged, 80 and over ; Aged