Final Report Summary - FAN (Defining the functions of Elongator in adult brain neurogenesis)
The adult brain retains stem/progenitor cells in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus (DG) that support neurogenesis throughout life. Understanding the molecular pathways that drive neurogenesis in adult brain is important as endogenous neuroblasts may one day be harnessed and manipulated to provide neuronal replacement therapy after brain injury. Recent studies suggest that acetylation of cytoplasmic substrates contributes to brain development and maintenance. Disruption of acetylation is also associated with various progressive neurological disorders. A tight interplay between lysine deacetylases (KDACs) and KATs acts antagonistically to control protein acetylation, but the enzymes that catalyse such modification on non-histone proteins remain mostly unknown. We have focused specifically on Elongator and its acetylation targets. Elongator is a multiprotein complex composed of 6 subunits (Elp1-6), which is expressed both in the nucleus and cytoplasm where it acts as DNA demethylase and KAT (both activities holded by the catalytic subunit Elp3). In the nucleus, it promotes transcriptional elongation and paternal genome demethylation, whereas it contributes to exocytosis and tRNA modifications in the cytoplasm. Molecular analyses showed increased expression of Elp1 after induction of neurogenesis in the DG and suggest that Elongator belongs to the molecular machinery that underlies adult neurogenesis. The goal of our work is to investigate whether Elongator contributes to adult neurogenesis in the SVZ and to focus and characterize, in particular, the acetylation targets of Elongator that contribute to ciliogenesis in ependymal cells .
We showed by western-blot, immunohistochemistry, and qRT-PCR support the expression of Elongator subunits in micro-dissected postnatal mouse SVZ and DG. FoxG1:Cre ;Elp3lox/lox (Elp3 cKO) animals died before postnatal day 23 (P23). The size of the brain and in particular the thickness of the cortical plate are reduced upon Elp3 deletion and knockout animals are hydrocephales. Ependymal cells are multi-ciliated epithelial cells that line the walls of the ventricular system in the adult brain, whose absence often leads to hydrocephalus. Most of them are born prenatally and derived from radial glia cells. Ependymal cells have a remarkable planar polarization that determines orientation of ciliary beating and propulsion of the cerebrospinal fluid flow (CSF). Disruption of ependymal ciliary beating, by injury or disease, results in aberrant CSF circulation and hydrocephalus, a common disorder of the CNS. In addition, the ependymal-generated CSF flow establishes gradients of chemorepellents that guide the migration of newborn neurons in adult mammalian SVZ. Cilia contain highly acetylated microtubule (MT) and Elongator is a tubulin acetyl transferase (TAT). Thus, we will assess whether Elongator controls rostral migration of neuroblasts by regulating ependymal cilia formation and/or beating and thus CSF flow. Our analyses support partial loss of ciliated cells (immunopositive for acetylated-alpha tubulin) at the ventricular surface of P15 Elp3 cKO brains that came together with a reduction of S100 beta staining, a marker of ependymal cells. While Elp3 cKO mutant showed lower number of multi-ciliated cells within the ventricle, they have a larger number of cells presenting a primary cilia, suggesting that many radial glia cells have not differentiated properly into ependymal cells. We will decipher whether ciliogenesis defects is developmental or degenerative. For this purpose we will make whole ventricular wall preparation at P1, P6, P10 and P15 that provide an "en face" view of ventricular surface. We will perform immunohistochemsitry to assess the expression of ependymal (S100beta, CD24, noggin and vimentin) and radial glia markers (GLAST, bLBP, nestin).
Ependymal cells are polarized within the plane of the epithelium through a multi-step process named polar cell polarity (PCP), which is initially organized by primary cilia in radial glia (radial glia planar polarity) and then refined by motile cilia in ependymal cells. Basal bodies in ependymal cells in the lateral ventricle walls of postnatal mice are polarized in two steps: (1) translational polarity, a process initiated in progenitors (radial glia cells) through radial glia polarity and assessed by measuring the deviation angle between cellular vector drawn from the center of the patch of basal bodies relative to the corresponding median vector obtained in multiple cells; (2) rotational polarity, which is assessed by measuring the deviation angle between individual vectors drawn from center of a basal body with respect to its basal foot (basal foot angle) and the cellular median basal foot angle. We showed that both polarities were imparaired upon Elp3 deletion. We further analysed the biological impact of the PCP defects that arise in Elp3 cKO on cilia beating and directionality of CSF flow. For this purpose, we followed the displacement of fluorescent beads added to P15 brain slices (300-μm-thick vibratome sections) in the lateral ventricle by real-time confocal microscopy (25frame/s during 30-60s). In wild-type condition, beads movement should be consistently directed from caudal to rostral pole of lateral ventricles. As we observe defects in cilia beating, we will further analyse rostral migration of neuroblasts in Elp3cKO.
Finally we used a global approach to uncover new protein acetylated by Elp3. We compared acetylome from elp3 cKO and WT cortices. Among the candidates uncovered by our experiments, CEP290 is of particular interest. CEP290 is a centrosomal protein, and mutations in CEP290 have been implicated in many autosomal recessive disorders. In a mouse model, a CEP290 mutation gives rise to early-onset retinal degeneration and olfactory. CEP290 is located at the transition zone and participates strictly in primary cilia formation by regulating ciliary entry of membranes associated proteins (Craige et al, 2010; Tsang et al, 2008). We therefore analysed by immunochemistry the localisation of CEP290 within the lateral wall of the lateral ventricle at P1 and P15 and found that CEP290 seems to be located at the basal foot of the cilia in wild-type mice. To test more precisely the effect of cep290 acetylation on planar polarity, the endogenous candidate proteins will be silenced by injection of lentiviruses shRNA encoding plasmids and replaced by shRNA refractory mutant protein harbouring key acetylated K-to-R mutations. The resulting phenotype will be analysed by immunohistochemistry.
Altogether this study brings the first evidence for a role of the Elongator complex in ciliogenesis. Interestingly, a project that we have initiated with a local team, show interesting phenotype after depletion of Elp3 in kidney, suggesting a broader function of Elongator in different ciliated organs.
We showed by western-blot, immunohistochemistry, and qRT-PCR support the expression of Elongator subunits in micro-dissected postnatal mouse SVZ and DG. FoxG1:Cre ;Elp3lox/lox (Elp3 cKO) animals died before postnatal day 23 (P23). The size of the brain and in particular the thickness of the cortical plate are reduced upon Elp3 deletion and knockout animals are hydrocephales. Ependymal cells are multi-ciliated epithelial cells that line the walls of the ventricular system in the adult brain, whose absence often leads to hydrocephalus. Most of them are born prenatally and derived from radial glia cells. Ependymal cells have a remarkable planar polarization that determines orientation of ciliary beating and propulsion of the cerebrospinal fluid flow (CSF). Disruption of ependymal ciliary beating, by injury or disease, results in aberrant CSF circulation and hydrocephalus, a common disorder of the CNS. In addition, the ependymal-generated CSF flow establishes gradients of chemorepellents that guide the migration of newborn neurons in adult mammalian SVZ. Cilia contain highly acetylated microtubule (MT) and Elongator is a tubulin acetyl transferase (TAT). Thus, we will assess whether Elongator controls rostral migration of neuroblasts by regulating ependymal cilia formation and/or beating and thus CSF flow. Our analyses support partial loss of ciliated cells (immunopositive for acetylated-alpha tubulin) at the ventricular surface of P15 Elp3 cKO brains that came together with a reduction of S100 beta staining, a marker of ependymal cells. While Elp3 cKO mutant showed lower number of multi-ciliated cells within the ventricle, they have a larger number of cells presenting a primary cilia, suggesting that many radial glia cells have not differentiated properly into ependymal cells. We will decipher whether ciliogenesis defects is developmental or degenerative. For this purpose we will make whole ventricular wall preparation at P1, P6, P10 and P15 that provide an "en face" view of ventricular surface. We will perform immunohistochemsitry to assess the expression of ependymal (S100beta, CD24, noggin and vimentin) and radial glia markers (GLAST, bLBP, nestin).
Ependymal cells are polarized within the plane of the epithelium through a multi-step process named polar cell polarity (PCP), which is initially organized by primary cilia in radial glia (radial glia planar polarity) and then refined by motile cilia in ependymal cells. Basal bodies in ependymal cells in the lateral ventricle walls of postnatal mice are polarized in two steps: (1) translational polarity, a process initiated in progenitors (radial glia cells) through radial glia polarity and assessed by measuring the deviation angle between cellular vector drawn from the center of the patch of basal bodies relative to the corresponding median vector obtained in multiple cells; (2) rotational polarity, which is assessed by measuring the deviation angle between individual vectors drawn from center of a basal body with respect to its basal foot (basal foot angle) and the cellular median basal foot angle. We showed that both polarities were imparaired upon Elp3 deletion. We further analysed the biological impact of the PCP defects that arise in Elp3 cKO on cilia beating and directionality of CSF flow. For this purpose, we followed the displacement of fluorescent beads added to P15 brain slices (300-μm-thick vibratome sections) in the lateral ventricle by real-time confocal microscopy (25frame/s during 30-60s). In wild-type condition, beads movement should be consistently directed from caudal to rostral pole of lateral ventricles. As we observe defects in cilia beating, we will further analyse rostral migration of neuroblasts in Elp3cKO.
Finally we used a global approach to uncover new protein acetylated by Elp3. We compared acetylome from elp3 cKO and WT cortices. Among the candidates uncovered by our experiments, CEP290 is of particular interest. CEP290 is a centrosomal protein, and mutations in CEP290 have been implicated in many autosomal recessive disorders. In a mouse model, a CEP290 mutation gives rise to early-onset retinal degeneration and olfactory. CEP290 is located at the transition zone and participates strictly in primary cilia formation by regulating ciliary entry of membranes associated proteins (Craige et al, 2010; Tsang et al, 2008). We therefore analysed by immunochemistry the localisation of CEP290 within the lateral wall of the lateral ventricle at P1 and P15 and found that CEP290 seems to be located at the basal foot of the cilia in wild-type mice. To test more precisely the effect of cep290 acetylation on planar polarity, the endogenous candidate proteins will be silenced by injection of lentiviruses shRNA encoding plasmids and replaced by shRNA refractory mutant protein harbouring key acetylated K-to-R mutations. The resulting phenotype will be analysed by immunohistochemistry.
Altogether this study brings the first evidence for a role of the Elongator complex in ciliogenesis. Interestingly, a project that we have initiated with a local team, show interesting phenotype after depletion of Elp3 in kidney, suggesting a broader function of Elongator in different ciliated organs.