Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson's disease and in MPP+ -treated MN9D cells in vitro.

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson's disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP(+))-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP(+) exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP(+)-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP(+)-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

Dihydropyrimidinase-like 3 regulates the inflammatory response of activated microglia.

Microglia, the resident immune cells of the CNS, are known to respond to injuries, infection and inflammation in the CNS by producing proinflammatory cytokines and phagocytosing cell debris and pathogens. In this study, we investigated the expression pattern and role of dihydropyrimidinase-like 3 (Dpysl3), a member of collapsin response mediator protein family, on the inflammatory reaction of microglia. Microarray analysis comparing the global gene expression profile of ameboid and ramified microglia has shown that Dpysl3 is mainly expressed in ameboid microglia in the 5-day postnatal rat brain. Immunohistochemical analysis revealed that Dpysl3 was intensely expressed in ameboid microglial cells in the rat brain till postnatal 7th day and then gradually diminished in ramified microglia of 2 weeks postnatal rat brain. Further, in vitro analysis confirmed that Dpysl3 expression was induced in activated BV-2 microglia treated with lipopolysaccharide (LPS). It is well documented that microglial activation by LPS increased the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in BV-2 microglia. However, siRNA-mediated knockdown of Dpysl3 prevented the LPS-induced expression of iNOS and cytokines including interleukin-1 beta, and tumor necrosis factor-alpha as well as nuclear translocation of NF-κB in microglia. Remarkably, knockdown of Dpysl3 inhibited the migration of activated microglia coupled with deranged actin filament configuration (as revealed by F-actin cytoskeleton expression) in lamellipodia projecting from the cells. Knockdown of Dpysl3 also inhibited the phagocytic ability of activated microglia. These findings suggest that knockdown of Dpysl3 can inhibit activation, migration and phagocytic capability of microglia and consequently reduce neuroinflammation.

The teaching of anatomy: the first hundred years (1905-2005).

The Straits and Federated Malay States Government Medical School started on 3 July 1905 with the admission of 16 young persons for the full 5-year course. In 1910, 7 successful candidates qualified as medical practitioners and they were no more than 19 years of age. The medical course was based largely on the British system and consisted of 2 years of training in the basic sciences followed by 3 years of clinical clerkships in Medicine, Surgery and Midwifery. Anatomy was taught in the first year and extended into the second year, using cadavers (which were possibly fixed in formalin and glycerin) as study materials. The first Chair of Anatomy was established in 1922 and with the provision of full-time staff, the curriculum was brought in line with those conducted in the British colonies. From the mid-1960s to the mid-1990s, the Anatomy course for medical students spanned 1 1/2 years, with special emphasis on clinical applications, thereby projecting the professional relevance of the course. Big class lectures introduced and previewed important structures that were encountered in dissections and small group tutorials reviewed the tutorial objectives that had been made available earlier. In the late 1990s and early 2000s, the medical curriculum was further revised to meet the challenges of the 21st century. A track system was developed and Human Anatomy came under the "Human Structure and Development Track". The original 1 1/2 -year programme was tailored into a 1-year programme with a drastic reduction in teaching/contact hours, but the big class lectures and small group tutorials plus dissections/prosections were retained. Beginning in the academic year 2003/2004, prosected cadavers (dissected by professional staff) were employed for teaching purposes due to a progressive fall in the availability of cadavers and time constraints imposed by the introduction of several new modules. Teachers demonstrate and students learn on prosected materials and the success of this new mode of teaching-learning can only be seen in the near future.

Differential expression of cytokines in the rat heart in response to sustained volume overload.

OBJECTIVE: The present study aimed to investigate whether sustained volume overload is capable of inducing persistent upregulation of cardiac cytokines including tumor necrosis factor alpha (TNF)-alpha, interleukin (IL)-1beta, interleukin (IL)-6 and transforming growth factor (TGF)-beta(1). METHODS AND RESULTS: Volume overload-induced heart hypertrophy in rats was established by aortacaval fistula, and the cardiac cytokines were measured in the myocardium from 1 to 4 weeks after operation. In the post-fistula rats, cardiac IL-1beta and IL-6 gene and protein levels were upregulated throughout the time of measurement. Immunohistochemistry demonstrated that IL-1beta and IL-6 immunoreactive cells were widely distributed in the myocardium in the earlier time intervals, and mainly localized in the regions close to the endocardium in the later time intervals. The cardiac IL-1beta immunoreactive cells were mainly localized in the blood vessels whereas the IL-6 positive cells were composed of non-myocytes and cardiomyocytes. TGF-beta(1) positive staining was increased in the myocardium up to 3 weeks after aortacaval fistula and then decreased to basal levels thereafter. In contrast to the activation of cardiac IL-1beta and IL-6 in response to volume overload, TNF-alpha expression appeared unaltered in response to sustained volume overload in the transcription and protein levels. CONCLUSION: The results of the present study indicate that sustained volume overload is capable of inducing persistent upregulation of some cardiac cytokines. In addition, the differential expressions of TNF-alpha, IL-1beta and IL-6 suggest that the induction of IL-6 and IL-1beta is independent of TNF-alpha mediated pathways in this animal model.

Induction of cytokine expression in rat post-ischemic sinoatrial node (SAN).

The aim of this study was to determine the spatial and temporal expression of various pro-inflammatory cytokines in the peri-sinoatrial nodal area after atrial infarction. Rats were subjected to permanent atrial infarction, in particular, sinoatrial node (SAN) infarction and sacrificed at various time points up to 7 days. Real-time polymerase chain reaction analysis demonstrated that mRNA levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta, interleukin-6, and transforming growth factor beta 1 (TGF-beta(1)) were upregulated in the peri-sinoatrial nodal area after atrial infarction. Immunostaining for TNF-alpha and TGF-beta(1) proteins revealed that both cytokines were expressed persistently up to 7 days after atrial infarction around the peri-sinoatrial nodal area. Furthermore, the infiltrating inflammatory cells immunoreactive for both cytokines were predominant within the infarct SAN. In situ hybridization analysis showed that TNF-alpha gene expression was enhanced in the inflammatory cells and myocardium within the peri-sinoatrial nodal area in response to the infarction. These results provide evidence for the local expression of cytokines in the post-ischemic peri-sinoatrial nodal area, suggesting that the upregulation of the cytokines might be associated with the atrial arrhythmia observed after acute myocardial infarction.

Retinoic acid influences the expression of the neuronal regulatory genes Mash-1 and c-ret in the developing rat heart.

We analyzed the expression of neuronal regulatory genes Mash-1 and c-ret by immunohistochemistry and reverse transcriptase-polymerase chain reaction in the developing heart of rat embryos following exogenous retinoic acid (RA) treatment of the pregnant dams. On E12, expression of Mash-1 and c-ret was confined to cells migrating via the common cardinal vein. On E16.5, Mash-1 and c-ret expression were restricted to cardiac ganglia around the great vessels and posterior atrial wall. While Mash-1 expression was down-regulated at birth, that of c-Ret was maintained. RA-treated hearts showed a down-regulation of both Mash-1 and c-Ret at the mRNA as well as at the protein level on E16.5. The present results show that differentiation of cardiac ganglionic cells is affected after RA treatment, by the down-regulation of Mash-1 and c-Ret.