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Impaired neural development in a zebrafish model for Lowe syndrome.

Ramirez IB, Pietka G, Jones DR, Divecha N, Alia A, Baraban SC, Hurlstone AF, Lowe M - Hum. Mol. Genet. (2011)

Bottom Line: In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue.Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes.Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.

View Article: PubMed Central - PubMed

Affiliation: University of Manchester, The Michael Smith Building, Manchester M13 9PT, UK.

ABSTRACT
Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.

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Catalytic activity and binding to clathrin are required for OCRL1 function in brain development. (A) Representative bright field images of 26 hpf OCRL1−/− mutant embryos either uninjected (left) or injected with mRNA encoding GFP–OCRL1 D480A (right). The phenotypes were scored as dysmorphic (left) or severely dysmorphic (right), in which the brain size is further reduced when compared with the mutant alone. Quantitation of the morphological phenotypes obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 38–324 embryos from 2 to 18 experiments). (B) Representative AO staining of apoptosis in OCRL1−/− mutant embryos expressing GFP–OCRL1 D480A or GFP–OCRL1 ▵LIDLE. Note the increased apoptosis in the head region (left) and spinal cord (right), indicated by arrows. Quantitation of the AO staining obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 29–136 embryos from 2 to 17 experiments). (C) Quantitation of proliferation as assessed by PH3 staining upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 13–89 embryos from 2 to 12 experiments).
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DDR608F8: Catalytic activity and binding to clathrin are required for OCRL1 function in brain development. (A) Representative bright field images of 26 hpf OCRL1−/− mutant embryos either uninjected (left) or injected with mRNA encoding GFP–OCRL1 D480A (right). The phenotypes were scored as dysmorphic (left) or severely dysmorphic (right), in which the brain size is further reduced when compared with the mutant alone. Quantitation of the morphological phenotypes obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 38–324 embryos from 2 to 18 experiments). (B) Representative AO staining of apoptosis in OCRL1−/− mutant embryos expressing GFP–OCRL1 D480A or GFP–OCRL1 ▵LIDLE. Note the increased apoptosis in the head region (left) and spinal cord (right), indicated by arrows. Quantitation of the AO staining obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 29–136 embryos from 2 to 17 experiments). (C) Quantitation of proliferation as assessed by PH3 staining upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 13–89 embryos from 2 to 12 experiments).

Mentions: To determine the cellular mechanisms underlying the defects, we observe in OCRL1 mutant and morphant embryos, rescue experiments were performed using constructs with point mutations that abrogate catalytic activity (D480A) or binding to the vesicle coat protein clathrin (▵LIDLE). Morphology, apoptosis and proliferation were scored to determine the ability of each construct to rescue these phenotypes. Importantly, expression levels of the various constructs were comparable as assessed by GFP fluorescence. Expression of the catalytically inactive D480A mutant failed to rescue mutant morphology, apoptosis or proliferation, indicating that 5-phosphatase activity is essential for OCRL1 function in vivo (Fig. 8A–C). Interestingly, expression of this construct led to a more severe morphological defect than that seen in the mutant alone, with a greater reduction in the size of the brain (Fig. 8A). Moreover, we observed enhanced apoptosis in embryos expressing the D480A mutant (Fig. 8B). This was particularly striking in the head, suggesting increased apoptosis within the neural tissue. We also observed a greater reduction in cell proliferation in mutant embryos expressing the D480A mutant (Fig. 8C). These results confirm the functional importance of OCRL1 5-phosphatase activity during embryonic development.Figure 8.


Impaired neural development in a zebrafish model for Lowe syndrome.

Ramirez IB, Pietka G, Jones DR, Divecha N, Alia A, Baraban SC, Hurlstone AF, Lowe M - Hum. Mol. Genet. (2011)

Catalytic activity and binding to clathrin are required for OCRL1 function in brain development. (A) Representative bright field images of 26 hpf OCRL1−/− mutant embryos either uninjected (left) or injected with mRNA encoding GFP–OCRL1 D480A (right). The phenotypes were scored as dysmorphic (left) or severely dysmorphic (right), in which the brain size is further reduced when compared with the mutant alone. Quantitation of the morphological phenotypes obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 38–324 embryos from 2 to 18 experiments). (B) Representative AO staining of apoptosis in OCRL1−/− mutant embryos expressing GFP–OCRL1 D480A or GFP–OCRL1 ▵LIDLE. Note the increased apoptosis in the head region (left) and spinal cord (right), indicated by arrows. Quantitation of the AO staining obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 29–136 embryos from 2 to 17 experiments). (C) Quantitation of proliferation as assessed by PH3 staining upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 13–89 embryos from 2 to 12 experiments).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DDR608F8: Catalytic activity and binding to clathrin are required for OCRL1 function in brain development. (A) Representative bright field images of 26 hpf OCRL1−/− mutant embryos either uninjected (left) or injected with mRNA encoding GFP–OCRL1 D480A (right). The phenotypes were scored as dysmorphic (left) or severely dysmorphic (right), in which the brain size is further reduced when compared with the mutant alone. Quantitation of the morphological phenotypes obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 38–324 embryos from 2 to 18 experiments). (B) Representative AO staining of apoptosis in OCRL1−/− mutant embryos expressing GFP–OCRL1 D480A or GFP–OCRL1 ▵LIDLE. Note the increased apoptosis in the head region (left) and spinal cord (right), indicated by arrows. Quantitation of the AO staining obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 29–136 embryos from 2 to 17 experiments). (C) Quantitation of proliferation as assessed by PH3 staining upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 13–89 embryos from 2 to 12 experiments).
Mentions: To determine the cellular mechanisms underlying the defects, we observe in OCRL1 mutant and morphant embryos, rescue experiments were performed using constructs with point mutations that abrogate catalytic activity (D480A) or binding to the vesicle coat protein clathrin (▵LIDLE). Morphology, apoptosis and proliferation were scored to determine the ability of each construct to rescue these phenotypes. Importantly, expression levels of the various constructs were comparable as assessed by GFP fluorescence. Expression of the catalytically inactive D480A mutant failed to rescue mutant morphology, apoptosis or proliferation, indicating that 5-phosphatase activity is essential for OCRL1 function in vivo (Fig. 8A–C). Interestingly, expression of this construct led to a more severe morphological defect than that seen in the mutant alone, with a greater reduction in the size of the brain (Fig. 8A). Moreover, we observed enhanced apoptosis in embryos expressing the D480A mutant (Fig. 8B). This was particularly striking in the head, suggesting increased apoptosis within the neural tissue. We also observed a greater reduction in cell proliferation in mutant embryos expressing the D480A mutant (Fig. 8C). These results confirm the functional importance of OCRL1 5-phosphatase activity during embryonic development.Figure 8.

Bottom Line: In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue.Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes.Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.

View Article: PubMed Central - PubMed

Affiliation: University of Manchester, The Michael Smith Building, Manchester M13 9PT, UK.

ABSTRACT
Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.

Show MeSH
Related in: MedlinePlus