| The cerebellum development begins perinatally, continues into the third week of postpartum life and forms three-layered cerebellar cortex: an outer molecular layer (ML), a layer of Purkinje cells (PCL), and an inner layer of granule cells (IGL). Cerebellar cells undergo sequential steps of development in spatially well-defined regions. Proliferating granule cells precursors are prenatally located in the external granular layer (EGL) and followed by inward radial migration to their final destination, the IGL. By the end of the third postnatal week, the EGL has disappeared, then three well-defined neuronal layers form. The radial migration of granule cells toward the IGL is guided by Bergmann glial cells, a specialized form of radial glial cells. There is a mount of evidences shown that mice with Bergmann glial defects during cerebellum development have severe abnormalities including pial rupture, disrupted neuronal migration and layering, and altered connectivity.Bergmann glia are unipolar cerebellar astrocytes in the cerebellar cortex. During postnatal development, Bergmann glia undergo significant morphological changes to accommodate granule neuron differentiation and migration. Bergmann glial fibers serve as scaffolds for granule cells inward migration during cerebellum postnatal development. After the granule neurons migration is completed, most of glial fibers transform into astrocytes. This change highly resembles the cortical radial glia to stellate astrocyte differentiation.The nuclear receptor superfamily consists of a group of transcription factors that are highly homologous in structural components containing six independent functional domains. Many nuclear receptors are involved in the cerebellum development such as the nuclear estrogen receptors (ERs), Rev-erbA?orphan receptor and testicular orphan nuclear receptor 4 (TR4). Liver X receptors (LXRs), LXR?and LXR?, are nuclear receptors belonging to the large family of ligand-activated transcription factors and are both expressed in the mouse fetuses at 11.5 days postcoitum indicating its role in fetal development. It has been demonstrated that both LXR?and LXR?are expressed in the cerebellum, but the function is not clear. Using LXRs knock-out mice, LXRs have been confirmed to regulate the balance between neurons and glia during early midbrain development. Meanwhile, previous study demonstrated that LXR?expression in the mouse cerebral cortex was developmentally regulated, and loss of LXR?in the embryonic brain leaded to abnormal histoarchitecture with obviously layering defects. In LXR?knock-out mice, the radial glial cells appeared to be less organized into radial formations and failed to provide guidance for migrating neurons. These studies indicate that endogenous LXRs affect radial glia development, while the underlying mechanism is unclear.In this study, the role of LXRs agonist T0901317 in granule cells migration was investigated. Early postnatal mice received T0901317 intraperitoneal injection (ip), and the migration of new born granule cells in the EGL was evaluated with 5-bromodeoxyuridine (BrdU) labeling protocol. In addition, whether T0901317 affects Bergmann glial development and the possible underlying mechanism was further explored in this study.The study consisted of three parts:Part 1 The expression of LXRs in the cerebellumAdjacent cerebellum sections were Nissl-stained with cresyl violet for histological analysis.Morphological difference was observed in the cerebellum during all the cerebellar development stage for three postnatal weeks. To study the roles of LXRs in the cerebellum development, we investigated the expression pattern of LXRs.Results:1. Nissl staining showed different morphology in the developing cerebellum for three postnatal weeks. The cerebellum undergoes a period of rapid growth during the first three postnatal weeks. Proliferating granule cells precursors are prenatally located in the external granular layer (EGL) and followed by inward radial migration to their final destination, the IGL. By the end of the third postnatal week, the EGL has disappeared, then three well-defined neuronal layers form.2. The levels of LXRs mRNA were detected using RT-PCR. LXR?and LXR?were expressed in the cerebellum during the first three postnatal weeks. LXR?were expressed in the cerebellum at extremely low levels.However, LXR?were expressed at high levels at P2. Especially, at P5 the expression of LXR?arrived at peak. From P8 to P14, the expression of LXR?was gradually decreased.These results indicate that the expression of LXRs in the mouse cerebellum is developmently regulated, of which LXR?is predominant receptor and could affect cerebellum development.3. The protein levels of LXRs were detected by Immunohistochemistry. LXR?were expressed at extremely low levels. LXR?were expressed at extremely low levels in the EGL, but localized mainly in the PCL and IGL. Especially, consistent with mRNA level detection in the cerebellum, the peak level of LXR?expression was observed at P5, the strongest signal was detected in the IGL.Part 2 The effects of LXRs agonist on the migration of granule neurons during cerebellar developmentLXRs are ligand-activated nuclear receptors. The natural ligands for LXRs are oxysterols, the oxygenated derivatives of cholesterol such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid. In addition, two nonsteroidal synthetic compounds, GW3965 and T0901317, have been identified as potent and orally active agonists for both LXRs, and widely been used to study the function of endogenous LXRs.In this study, Early postnatal mice received T0901317 intraperitoneal injection (ip), and the migration of new born granule cells in the EGL was evaluated with 5-bromodeoxyuridine (BrdU) labeling protocol. In addition, whether T0901317 affects Bergmann glial development and the possible underlying mechanism was further explored in this study. It has been reported that TGF-?1/Smad signaling might be an important factor involved in radial glia-astrocyte transformation in vitro. This provides direct proof that TGF-?1 might drive radial glia towards an astrocyte phenotype. To examine this hypothesis, the levels of TGF-?1 and Smad4 mRNA in vivo were detected in both vehicle and T0901317 groups using RT-PCR.Results:1. Nissl staining showed that T0901317 treatment did not affect cerebellar global morphology and the thickness of EGL. Purkinje cells were identified by immunostaining with the antibody against calbindin, a marker for Purkinje cells. T0901317 treatment is more effective in dendrites growth of Purkinje cells than the change of Purkinje cells number.2. T0901317 treatment promoted the migration of granule neurons migration transiently by using BrdU labeling protocol.The number of PCNA labeled cells in the EGL did not change significantly after administration of T0901317.3. T0901317 treatment resulted in delaying premature differentiation of Bergmann glia and leaded to promoting granule neurons migration. Then, we demonstrated that LXRs agonist T0901317 downregulated the level of TGF-?1 and Smad4 mRNA expression in the cerebellum.Part 3 LXRs agonist protect against the neurotoxicity of cisplatin in the developing cerebellumMany adverse neurological effects often accompanied with treatment of both adult and childhood cancers range from abnormalities detected by central nervous system images to clinical symptoms including seizures, cerebral infarctions and cognitive impairments. Cisplatin is a therapeutically effective anti-cancer agent; despite its adverse side-effect of neurotoxicity, the survival rate of children with cancer has increased drastically over the past few decades through the use of this agent. However, the effective prevention approach of cisplatin-induced neurotoxicity has not been discovered. Cisplatin induced cell death of cerebellar granule neurons in vitro and interfered with morphological and molecular events in postnatal rat cerebellum development. In addition, early after the administration of cisplatin changed in the migration of the external granular layer and glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet.Results:1. The granule neurons and Purkinje cells presented apoptotic features in cisplatin-treatment group with Nissl staining.But pretreatment with T0901317, the cell apoptosis decreased obviously.In cisplatin-treatment group, the dendrite length of Purkinje cells was shorter than that in vehicle treated mice.In contrast, pretreatment with T0901317, the dendrite length of Purkinje cells was longer than that in cisplatin-treatment group.2. In cisplatin- treated mice, glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet present. But pretreatment with T0901317 the morphology of glial fibers recovered partially.3. Rotating rod test indicated that cisplatin- treated mice stayed on the rod with shorter time than vehicle- treated mice. Howerver, mice pretreatment with T0901317 stayed on the rod with longer time than cisplatin- treated mice. Taken together, these findings suggested that the distribution pattern of LXRs in cerebellar cortex was developmentally regulated. T0901317, a potent LXRs receptor agonist, increased dendritic growth of Purkinje cell and promoted the migration of granule neurons from the external granular layer to the internal granular layer during cerebellum development. T0901317 treatment also inhibited premature differentiation of Bergmann glia during cerebellum development, which is related to the decreased levels of TGF-?1 and Smad4 in the cerebellum. LXRs agonist could protect against the neurotoxicity of cisplatin in the developing cerebellum... |
PDF Full Text Request