A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis.

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A-B56 phosphatase through a coiled-coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. We find that PP2A-B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment.

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

A Consensus Binding Motif for the PP4 Protein Phosphatase.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Retina-specific loss of Ikbkap/Elp1 causes mitochondrial dysfunction that leads to selective retinal ganglion cell degeneration in a mouse model of familial dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive disorder marked by developmental and progressive neuropathies. It is caused by an intronic point-mutation in the IKBKAP/ELP1 gene, which encodes the inhibitor of κB kinase complex-associated protein (IKAP, also called ELP1), a component of the elongator complex. Owing to variation in tissue-specific splicing, the mutation primarily affects the nervous system. One of the most debilitating hallmarks of FD that affects patients' quality of life is progressive blindness. To determine the pathophysiological mechanisms that are triggered by the absence of IKAP in the retina, we generated retina-specific Ikbkap conditional knockout (CKO) mice using Pax6-Cre, which abolished Ikbkap expression in all cell types of the retina. Although sensory and autonomic neuropathies in FD are known to be developmental in origin, the loss of IKAP in the retina did not affect its development, demonstrating that IKAP is not required for retinal development. The loss of IKAP caused progressive degeneration of retinal ganglion cells (RGCs) by 1 month of age. Mitochondrial membrane integrity was breached in RGCs, and later in other retinal neurons. In Ikbkap CKO retinas, mitochondria were depolarized, and complex I function and ATP were significantly reduced. Although mitochondrial impairment was detected in all Ikbkap-deficient retinal neurons, RGCs were the only cell type to degenerate; the survival of other retinal neurons was unaffected. This retina-specific FD model is a useful in vivo model for testing potential therapeutics for mitigating blindness in FD. Moreover, our data indicate that RGCs and mitochondria are promising targets.

Animal and cellular models of familial dysautonomia.

Since Riley and Day first described the clinical phenotype of patients with familial dysautonomia (FD) over 60 years ago, the field has made considerable progress clinically, scientifically, and translationally in treating and understanding the etiology of FD. FD is classified as a hereditary sensory and autonomic neuropathy (HSAN type III) and is both a developmental and a progressive neurodegenerative condition that results from an autosomal recessive mutation in the gene IKBKAP, also known as ELP1. FD primarily impacts the peripheral nervous system but also manifests in central nervous system disruption, especially in the retina and optic nerve. While the disease is rare, the rapid progress being made in elucidating the molecular and cellular mechanisms mediating the demise of neurons in FD should provide insight into degenerative pathways common to many neurological disorders. Interestingly, the protein encoded by IKBKAP/ELP1, IKAP or ELP1, is a key scaffolding subunit of the six-subunit Elongator complex, and variants in other Elongator genes are associated with amyotrophic lateral sclerosis (ALS), intellectual disability, and Rolandic epilepsy. Here we review the recent model systems that are revealing the molecular and cellular pathophysiological mechanisms mediating FD. These powerful model systems can now be used to test targeted therapeutics for mitigating neuronal loss in FD and potentially other disorders.

The familial dysautonomia disease gene IKBKAP is required in the developing and adult mouse central nervous system.

Hereditary sensory and autonomic neuropathies (HSANs) are a genetically and clinically diverse group of disorders defined by peripheral nervous system (PNS) dysfunction. HSAN type III, known as familial dysautonomia (FD), results from a single base mutation in the gene IKBKAP that encodes a scaffolding unit (ELP1) for a multi-subunit complex known as Elongator. Since mutations in other Elongator subunits (ELP2 to ELP4) are associated with central nervous system (CNS) disorders, the goal of this study was to investigate a potential requirement for Ikbkap in the CNS of mice. The sensory and autonomic pathophysiology of FD is fatal, with the majority of patients dying by age 40. While signs and pathology of FD have been noted in the CNS, the clinical and research focus has been on the sensory and autonomic dysfunction, and no genetic model studies have investigated the requirement for Ikbkap in the CNS. Here, we report, using a novel mouse line in which Ikbkap is deleted solely in the nervous system, that not only is Ikbkap widely expressed in the embryonic and adult CNS, but its deletion perturbs both the development of cortical neurons and their survival in adulthood. Primary cilia in embryonic cortical apical progenitors and motile cilia in adult ependymal cells are reduced in number and disorganized. Furthermore, we report that, in the adult CNS, both autonomic and non-autonomic neuronal populations require Ikbkap for survival, including spinal motor and cortical neurons. In addition, the mice developed kyphoscoliosis, an FD hallmark, indicating its neuropathic etiology. Ultimately, these perturbations manifest in a developmental and progressive neurodegenerative condition that includes impairments in learning and memory. Collectively, these data reveal an essential function for Ikbkap that extends beyond the peripheral nervous system to CNS development and function. With the identification of discrete CNS cell types and structures that depend on Ikbkap, novel strategies to thwart the progressive demise of CNS neurons in FD can be developed.

Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein (IKBKAP). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre (Tα1-Cre). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients.

Transgenic expression of the proneural transcription factor Ascl1 in Müller glia stimulates retinal regeneration in young mice.

Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.

A transient wave of BMP signaling in the retina is necessary for Müller glial differentiation.

The primary glial cells in the retina, the Müller glia, differentiate from retinal progenitors in the first postnatal week. CNTF/LIF/STAT3 signaling has been shown to promote their differentiation; however, another key glial differentiation signal, BMP, has not been examined during this period of Müller glial differentiation. In the course of our analysis of the BMP signaling pathway, we observed a transient wave of Smad1/5/8 signaling in the inner nuclear layer at the end of the first postnatal week, from postnatal day (P) 5 to P9, after the end of neurogenesis. To determine the function of this transient wave, we blocked BMP signaling during this period in vitro or in vivo, using either a BMP receptor antagonist or noggin (Nog). Either treatment leads to a reduction in expression of the Müller glia-specific genes Rlbp1 and Glul, and the failure of many of the Müller glia to repress the bipolar/photoreceptor gene Otx2. These changes in normal Müller glial differentiation result in permanent disruption of the retina, including defects in the outer limiting membrane, rosette formation and a reduction in functional acuity. Our results thus show that Müller glia require a transient BMP signal at the end of neurogenesis to fully repress the neural gene expression program and to promote glial gene expression.

ASCL1 reprograms mouse Muller glia into neurogenic retinal progenitors.

Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.

EGF stimulates Müller glial proliferation via a BMP-dependent mechanism.

Müller glia, the major type of glia in the retina, are mitotically quiescent under normal conditions, though they can be stimulated to proliferate in some pathological states. Among these stimuli, EGF is known to be a potent mitogen for Müller glia. However, the signaling pathways required for EGF-mediated proliferation of Müller glia are not clearly understood. In this study, postnatal day 12 (P12) or adult trp53(-/-) mouse retinas were explanted and cultured in the presence of EGF to stimulate Müller glial proliferation. Treatment with signaling inhibitors showed that activation of both MEK/ERK1/2 and PI3K/AKT pathways is required for EGF-induced proliferation of Müller glia. Interestingly, BMP/Smad1/5/8 activation downstream of PI3K/AKT signaling was also necessary for robust Müller glial proliferation, though activation of BMP/Smad1/5/8 signaling alone failed to stimulate their proliferation. In dissociated Müller glial culture, treatment with EGF induced the upregulation of Bmp7, and this upregulation was blocked significantly by co-treatment with the BMP inhibitor dorsomorphin, suggesting that BMP/Smad1/5/8 activation is mediated at least in part by an autocrine mechanism in Müller glia. A better understanding of how BMP/Smad1/5/8 signaling is involved in glial proliferation may have important implications for proliferative disorders, as well as for retinal regeneration in mammalian retinas.

P53 is required for the developmental restriction in Müller glial proliferation in mouse retina.

Müller glia are normally mitotically quiescent cells, but in certain pathological states they can re-enter the mitotic cell cycle. While several cell cycle regulators have been shown to be important in this process, a role for the tumor suppressor, p53, has not been demonstrated. Here, we investigated a role for p53 in limiting the ability of Müller glia to proliferate in the mature mouse retina. Our data demonstrate that Müller glia undergo a developmental restriction in their potential to proliferate. Retinal explants or dissociated cultures treated with EGF become mitotically quiescent by the end of the second postnatal week. In contrast, Müller glia from adult trp53-/+ or trp53-/- mice displayed a greater ability to proliferate in response to EGF stimulation in vitro. The enhanced proliferative ability of trp53 deficient mice correlates with a decreased expression of the mitotic inhibitor Cdkn1a/p21(cip) and an increase in c-myc, a transcription factor that promotes cell cycle progression. These data show that p53 plays an essential role in limiting the potential of Müller glia to re-enter the mitotic cycle as the retina matures during postnatal development.

Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo.

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.

Genome-wide analysis of Müller glial differentiation reveals a requirement for Notch signaling in postmitotic cells to maintain the glial fate.

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10-14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.

Unexpected transcriptional activity of the human VMD2 promoter in retinal development.

Vitelliform macular dystrophy (VMD) is associated with mutations in the VMD2 gene, which encodes a chloride channel protein and is thought to be preferentially expressed in the retinal pigmented epithelium (RPE). In an effort to establish an inducible gene knockout system for the RPE, we recently used a 3.0-kb human VMD2 promoter to direct the expression of a reverse tetracycline-inducible system controlled Cre recombinase in transgenic mice. Although Cre function was localized to the RPE in most VMD2-cre mouse lines, Cre activity was also identified in neural retina in approximately half of the transgenic lines. In two VMD2-cre mouse lines, Cre activity was predominantly localized to retinal Müller cells. This surprising expression pattern is likely caused by the transcriptional activity of our transgene system during retinal development. Therefore, our results suggest that transcription of VMD2 gene may occur in progenitors of Müller cells. The two VMD2-cre mouse lines that demonstrated Cre activity specifically in the RPE or predominantly in the Müller cells were fully characterized. These VMD2-cre mice are potentially useful for dissecting cellular mechanisms of age-related macular degeneration or diabetic retinopathy, two leading causes of blindness with high relevance to gene expression in the RPE or Müller cells.

gp130 activation in Müller cells is not essential for photoreceptor protection from light damage.

Members of IL-6 family cytokines, such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), activate the common signal-transducing receptor gp130. We and others have previously shown that application of exogenous gp130 ligands promotes photoreceptor survival in light-induced and inherited retinal degeneration in animal models. While there is strong evidence that gp130 plays an essential role in photoreceptor protection, it is not clear whether protection is cell-autonomous in photoreceptors or an effect of Müller cell activation. To investigate the role of Müller cells in gp130-mediated photoreceptor protection, we have generated conditional gp130 knockout (KO) mice in retinal Müller cells using the Cre/lox system. Western blot and immunohistochemical analyses show that in our conditional gp130 KO mice, approximately 50% Müller cells no longer respond to LIF with activation of known downstream signaling proteins, STAT3 and ERK1/2. Despite the loss of gp130 activity in many Müller cells, intravitreal injection of LIF still induced significant degree of photoreceptor protection that was comparable to normal littermates. These data suggest that Müller cell activation of gp130 is not essential for photoreceptor protection, and support the hypothesis that the protection is mediated by cell-autonomous mechanisms in photoreceptors.

Preconditioning-induced protection of photoreceptors requires activation of the signal-transducing receptor gp130 in photoreceptors.

Retinal degenerations are a class of neurodegenerative disorders that ultimately lead to blindness due to the death of retinal photoreceptors. In most cases, death is the result of long-term exposure to environmental, inflammatory, and genetic insults. In age-related macular degeneration, significant vision loss may take up to 70-80 years to develop. The protracted time to develop blindness suggests that retinal neurons have an endogenous mechanism for protection from chronic injury. Previous studies have shown that endogenous protective mechanisms can be induced by preconditioning animals with sublethal bright cyclic light. Such preconditioning can protect photoreceptors from a subsequent damaging insult and is thought to be accomplished through induced expression of protective factors. Some of the factors shown to be associated with protection bind and activate the signal transducing receptor gp130. To determine whether stress-induced endogenous protection of photoreceptors requires gp130, we generated conditional gp130 knockout (KO) mice with the Cre/lox system and used light-preconditioning to induce neuroprotection in these mice. Functional and morphological analyses demonstrated that the retina-specific gp130 KO impaired preconditioning-induced endogenous protection. Photoreceptor-specific gp130 KO mice had reduced protection, although the Müller cell KO mice did not, thus gp130-induced protection was restricted to photoreceptors. Using an animal model of retinitis pigmentosa, we found that the photoreceptor-specific gp130 KO increased sensitivity to genetically induced photoreceptor cell death, demonstrating that gp130 activation in photoreceptors had a general protective role independent of whether stress was caused by light or genetic mutations.

Expression of Cre recombinase in retinal Müller cells.

PURPOSE: In an effort to generate inducible RPE-specific Cre mice using a 3.0-kb human vitelliform macular dystrophy-2 (VMD2) promoter, we identified a mouse line with unanticipated Cre activity in the neural retina, including Müller glial cells. Müller cells play important roles in the function and maintenance of the retina, and this mouse line would be potentially useful for conditional gene targeting in Müller glia. We therefore characterized the timing, inducibility, and cell specificity of Cre expression, as well as Müller cell-specific efficiency of Cre-mediated recombination in this mouse line. METHODS: Transgenic mice carrying cassettes of human P(VMD2)-rtTA and TRE-cre were generated. Cre expression was characterized using a Cre-activatable lacZ reporter mouse line (R26R) and a floxed interleukin six signal transducing receptor (gp130) mouse line. RESULTS: beta-Galactosidase (beta-gal) assay and immunohistochemical analysis of VMD2-cre/R26R double transgenic mice indicated that Cre activity was detected in cells located in the inner nuclear layer, with prominent expression of beta-gal in Müller cells. Cre activity was also detected in photoreceptors in the outer nuclear layer. PCR analysis demonstrated that Cre-mediated recombination initiated by embryonic day 15. Immunohistochemical analysis indicated that Cre-mediated deletion of floxed gp130 gene occurred in 52% of the retinal Müller cells. Retinal function and morphology were normal in 10-month-old VMD2-cre mice. CONCLUSION: We generated a transgenic cre mouse that is useful to study gene activation and inactivation in retinal Müller cells.

STAT3 activation in photoreceptors by leukemia inhibitory factor is associated with protection from light damage.

Members of the interleukin-6 cytokine family, including leukemia inhibitory factor (LIF), signal through gp130. The neuroprotective role of gp130 activation has been widely demonstrated in both CNS and PNS, but the mechanism by which this is accomplished is not well established. We investigated temporal and cell-specific activation of signaling pathways induced by LIF in the mature mouse retina. Intravitreal injection of LIF preserved photoreceptor function and prevented photoreceptor cell death from light-induced oxidative damage in a dose-dependent manner (2 days post-injection). A therapeutic dose of LIF induced rapid and sustained activation of signal transducer and activator of transcription (STAT) 3. Activated STAT3 was localized to all the retinal neurons and glial cells, including photoreceptors. Activation of extracellular signal-regulated kinase 1 and 2 was robust but transient in Müller glial cells, and undetectable at the time of light exposure. Akt was not activated by LIF. We also show that at the time of neuroprotection, STAT3 but not extracellular signal-regulated kinase 1 and 2 or the Akt pathways was active in LIF-treated retinas, and activated STAT3 was clearly localized in transcriptionally active areas of photoreceptor nuclei. Our data suggest that photoreceptor protection in response to LIF can be directly mediated by activation of STAT3 in photoreceptors.