Spyridon Theofilopoulos

Liver X Receptors and Oxysterols Promote Ventral Midbrain Neurogenesis In Vivo and in Human Embryonic Stem Cells

Control over progenitor proliferation and neurogenesis remains a key challenge for stem cell neurobiology and a prerequisite for successful stem cell replacement therapies for neurodegenerative diseases like Parkinsons disease (PD). Here, we examined the function of two nuclear receptors, liver X receptors (Lxralpha and beta) and their ligands, oxysterols, as regulators of cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuron development. Deletion of Lxrs reduced cell cycle progression and VM neurogenesis, resulting in decreased DA neurons at birth. Activation of Lxrs with oxysterol ligands increased the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type but not Lxralphabeta(-/-) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) during DA differentiation increased neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors. Thus, Lxr ligands may improve current hESC replacement strategies for PD by selectively augmenting the generation of DA neurons.

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C27 and C24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C26 sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.

Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis

Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinsons disease.

The neuronal survival effects of rasagiline and deprenyl on fetal human and rat ventral mesencephalic neurones in culture.

The neuronal survival properties of rasagiline (R(+)-N-propargyl-l-aminoindane mesylate or TVP-1012), a novel monoamine oxidase B inhibitor, have been investigated using neuronal cell cultures from fetal rat and human ventral mesencephalon. The ability of rasagiline to reduce the rate of neuronal cell loss in vitro was tested using primary neuronal cell lines and immunohistochemistry to quantify the reduction in cell death. Direct comparison was made with deprenyl, a widely used and long established monoamine oxidase B inhibitor. Rasagiline was shown to act 15–20% more effectively as a neuronal survival agent than deprenyl, increasing both the survival of the total number of neurones and selectively increasing the survival of dopaminergic neurones with no statistically significant increase in survival of GABAergic neurones.

Cholestenoic acids regulate motor neuron survival via liver X receptors

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

Parallel induction of the formation of dopamine and its metabolites with induction of tyrosine hydroxylase expression in foetal rat and human cerebral cortical cells by brain-derived neurotrophic factor and glial-cell derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF; 50 ng/ml), dopamine (DA; 10 microM) and forskolin (Fsk; 10 microM) have previously been shown by this and other laboratories to induce the tyrosine hydroxylase (TH) enzyme in foetal human and rat cerebral cortex during specified sensitive developmental periods. In the present study, these findings were extended for human and rat cells by showing that the induced TH+ cells also produce dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). In addition to this, TH induction and DA plus DOPAC production was observed in foetal human and rat cerebral cortex by using glial-cell derived neurotrophic factor (GDNF) in place of BDNF. The degree of induction by GDNF (1-10 ng/ml) was similar to that produced by BDNF and did not increase further when the two neurotrophic factors were used together. The time-course of induction in human cultures was followed: GDNF was found to cause a more rapid induction process than BDNF during the first 2 weeks. However the degree of induction after 3 weeks was the same for both neurotrophic factors. Inhibitors of transcription (actinomycin D) or of translation (cycloheximide) eliminated all the increase in DA+DOPAC contents elicited by these compounds, indicating that de novo transcription and translation were required for increased expression of the TH and other related enzymes. The intracellular pathways by which these molecules exert this dopaminergic phenotype induction effect are discussed. This study indicates a new source of dopaminergic brain tissue for use as transplants to neurosurgically treat Parkinsons disease patients.

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C21-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time

Tiam1 Regulates the Wnt/Dvl/Rac1 Signaling Pathway and the Differentiation of Midbrain Dopaminergic Neurons

ABSTRACT Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinsons disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.

Dkk1 regulates ventral midbrain dopaminergic differentiation and morphogenesis.

Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1+/− embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1−/− embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.

Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors

Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson disease. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. However, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells after cessation of normal dopamine neurogenesis. We show here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone beyond the major dopamine neurogenic period. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can be stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors as well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis.