In the developing spinal cord, early progenitor cells of the oligodendrocyte lineage are induced in the motor neuron progenitor (pMN) domain of the ventral neuroepithelium by the ventral midline signal Sonic hedgehog (Shh). The ventral generation of oligodendrocytes requires Nkx6-regulated expression of the bHLH gene Olig2 in this domain. In the absence of Nkx6 genes or Shh signaling, the initial expression of Olig2 in the pMN domain is completely abolished. In this study, we provide the in vivo evidence for a late phase of Olig gene expression independent of Nkx6 and Shh gene activities and reveal a brief second wave of oligodendrogenesis in the dorsal spinal cord. In addition, we provide genetic evidence that oligodendrogenesis can occur in the absence of hedgehog receptor Smoothened, which is essential for all hedgehog signaling.

Pigment epithelium-derived factor (PEDF) has been identified as one of the most potent of endogenous negative regulators of blood vessel growth in the body. Here we report that PEDF is able to inhibit growth factor-induced angiogenesis in microvascular endothelial cells through a novel pathway requiring cleavage and intracellular translocation of the transmembrane domain of the VEGFR-1. Analysis of the subcellular distribution of VEGFR-1 revealed the appearance of an 80-kDa C-terminal domain in the cytosol of cells treated with VEGF and PEDF that correlated with a decrease of the full-length receptor in the nuclear and cytoskeletal fractions. This regulated intramembrane proteolysis is dependent on ␥-secretase because inhibition of ␥-secretase abolished the inhibitory effect of PEDF on VEGF-induced angiogenesis as well as VEGFR-1 cleavage. The addition of PEDF to microvascular endothelial cells significantly increases ␥-secretase activity even in the absence of VEGF, showing that VEGF binding to VEGF-R1 is essential for substrate availability. This increase in activity was associated with translocation of presenilin 1 from the perinuclear region to the cell membrane. PEDF was also able to inhibit VEGF-induced phosphorylation of VEGFR-1. Taken together we have identified two novel pathways by which PEDF inhibits VEGF-induced angiogenesis: regulated intramembrane proteolysis and inhibition of phosphorylation. This confirms the importance of PEDF and VEGFR-1 in the negative regulation of angiogenesis. Pigment epithelium-derived factor (PEDF) 2 was identified in the late 1980s by Tombran-Tink and Johnson (1) as a novel neurotrophic activity in conditioned medium from fetal pigmented epithelial cells. PEDF, a 50-kDa noninhibitory member of the serine protease inhibitor (Serpin) gene family (2, 3) has now been identified in a wide variety of tissues throughout the body. The finding that PEDF is essential for maintaining avascularity (4), coupled with recent observations that PEDF plays a critical role in preventing aberrant neovascularization (5), has suggested that PEDF is a potent regulator of angiogenesis. PEDF has been shown to act by inhibiting the angiogenic response to factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (4). This inhibitory effect was more profound than any of the other anti-angiogenic factors tested (i.e. angiostatin, thrombospondin-1, and endostatin). PEDF selectively inhibits the formation of new vessels from endothelial cells but does not appear to harm the existing vascular structure (6). The inhibitory effect of PEDF on vessel formation appears to be reversible when a transient and regulated angiogenesis occurs in situations including tissue repair after injury (7). A recent study by Ogata et al. (8) has provided clinical evidence to show that expression of VEGF and PEDF are inversely correlated during the development of retinal neovascularization, further supporting an anti-angiogenic role for PEDF in vivo. Although numerous studies have shown that VEGF ...

Hyperglycaemia appears to be a critical factor in the aetiology of diabetic retinopathy and initiates downstream events including: basement membrane thickening, pericyte drop out and retinal capillary non-perfusion. More recently, focus has been directed to the molecular basis of the disease process in diabetic retinopathy. Of particular importance in the development and progression of diabetic retinopathy is the role of growth factors (eg vascular endothelial growth factor, placenta growth factor and pigment epithelium-derived factor) together with specific receptors and obligate components of the signal transduction pathway needed to support them. Despite these advances there are still a number of important questions that remain to be answered before we can confidently target pathological signals. How does hyperglycaemia regulate retinal vessels? Which growth factors are most important and at what stage of retinopathy do they operate? What is the preferred point in the growth factor signalling cascade for therapeutic intervention? Answers to these questions will provide the basis for new therapeutic interventions in a debilitating ocular condition.

Although many agents have therapeutic potentials for central nervous system (CNS) diseases, few of these agents have been clinically used because of the brain barriers. As the protective barrier of the CNS, the blood–brain barrier and the blood–cerebrospinal fluid barrier maintain the brain microenvironment, neuronal activity, and proper functioning of the CNS. Different strategies for efficient CNS delivery have been studied. This article reviews the current approaches to open or facilitate penetration across these barriers for enhanced drug delivery to the CNS. These approaches are summarized into three broad categories: noninvasive, invasive, and miscellaneous techniques. The progresses made using these approaches are reviewed, and the associated mechanisms and problems are discussed.

Recent studies have suggested that oligodendrocyte development is likely to be under the control of a hierarchy of lineage-specific transcription factors. In the developing mouse spinal cord, expression of Olig2, Sox10 and Nkx2.2 is sequentially up-regulated in cells of oligodendrocyte lineage. These transcription factors play essential roles in oligodendrocyte specification and differentiation. However, the regulatory relationship and functional interactions among these transcription factors have not been determined. In this study, we systematically investigated the function and hierarchical relationship of Olig2, Sox10 and Nkx2.2 transcription factors in the control of oligodendrocyte differentiation. It was found that over-expression of Olig2 is sufficient to induce Sox10, Nkx2.2 and precocious oligodendrocyte differentiation in embryonic chicken spinal cord. Sox10 expression alone is also sufficient to stimulate ectopic oligodendrocyte differentiation and weakly induce Nkx2.2 expression. Although genetic evidence indicated that Sox10 functions downstream of Olig2, Sox10 activity can modulate Olig2 expression. In addition, we presented evidence that the control of oligodendrocyte differentiation by Olig2, Sox10 and Nkx2.2 is a dosage-dependent developmental process and can be affected by both haploinsufficiency and over-dosage.

Forward genetic screens in genetically accessible invertebrate organisms such as

β-Secretase (BACE1) is a major drug target for combating Alzheimer's disease (AD). Here we show that BACE1−/− mice develop significant retinal pathology including retinal thinning, apoptosis, reduced retinal vascular density and an increase in the age pigment, lipofuscin. BACE1 expression is highest in the neural retina while BACE2 was greatest in the retinal pigment epithelium (RPE)/choroid. Pigment epithelial-derived factor, a known regulator of γ-secretase, inhibits vascular endothelial growth factor (VEGF)-induced in vitro and in vivo angiogenesis and this is abolished by BACE1 inhibition. Moreover, intravitreal administration of BACE1 inhibitor or BACE1 small interfering RNA (siRNA) increases choroidal neovascularization in mice. BACE1 induces ectodomain shedding of vascular endothelial growth factor receptor 1 (VEGFR1) which is a prerequisite for γ-secretase release of a 100 kDa intracellular domain. The increase in lipofuscin following BACE1 inhibition and RNAI knockdown is associated with lysosomal perturbations. Taken together, our data show that BACE1 plays a critical role in retinal homeostasis and that the use of BACE inhibitors for AD should be viewed with extreme caution as they could lead to retinal pathology and exacerbate conditions such as age-related macular degeneration.

Age-related macular degeneration (AMD) is associated with multiple genetic and cellular defects which lead to a common endpoint, retinal degeneration. Aging and oxidative stress, significant features in the pathogenesis of AMD, are associated with an increase in damaged intracellular organelles and defective autophagy flux in a range of age-related and neurodegenerative diseases. Autophagy is a key process in maintenance of cellular homeostasis that serves to remove dysfunctional organelles and proteins. Autophagy proteins are strongly expressed in the retina and there is now strong evidence that mitochondrial damage and defective autophagy are a feature of the aging retina and that this is further exacerbated in AMD. It is apparent that autophagy makes a significant contribution to lipofuscin accumulation in the RPE. Pharmacological manipulation of autophagy may offer an alternative therapeutic target in AMD.