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Lazarillo-related Lipocalins confer long-term protection against type I Spinocerebellar Ataxia degeneration contributing to optimize selective autophagy.

del Caño-Espinel M, Acebes JR, Sanchez D, Ganfornina MD - Mol Neurodegener (2015)

Bottom Line: GLaz beneficial effects persist throughout aging, and appears when expressed by degenerating neurons or by retinal support and glial cells.GLaz gain-of-function reduces cell death and the extent of ubiquitinated proteins accumulation, and decreases the expression of Atg8a/LC3, p62 mRNA and protein levels, and GstS1 induction.Down-regulation of selective autophagy causes similar and non-additive rescuing effects.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología y Genética Molecular-Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid-CSIC, c/ Sanz y Forés 3, 47003, Valladolid, Spain. manuela@ibgm.uva.es.

ABSTRACT

Background: A diverse set of neurodegenerative disorders are caused by abnormal extensions of polyglutamine (poly-Q) stretches in various, functionally unrelated proteins. A common feature of these diseases is altered proteostasis. Autophagy induction is part of the endogenous response to poly-Q protein expression. However, if autophagy is not resolved properly, clearance of toxic proteins or aggregates cannot occur effectively. Likewise, excessive autophagy induction can cause autophagic stress and neurodegeneration. The Lipocalins ApoD, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz) are neuroprotectors upon oxidative stress or aging. In this work we test whether these Lipocalins also protect against poly-Q-triggered deterioration of protein quality control systems.

Results: Using a Drosophila retinal degeneration model of Type-1 Spinocerebellar Ataxia (SCA1) combined with genetic manipulation of NLaz and GLaz expression, we demonstrate that both Lipocalins protect against SCA1 neurodegeneration. They are part of the endogenous transcriptional response to SCA1, and their effect is non-additive, suggesting participation in a similar mechanism. GLaz beneficial effects persist throughout aging, and appears when expressed by degenerating neurons or by retinal support and glial cells. GLaz gain-of-function reduces cell death and the extent of ubiquitinated proteins accumulation, and decreases the expression of Atg8a/LC3, p62 mRNA and protein levels, and GstS1 induction. Over-expression of GLaz is able to reduce p62 and ubiquitinated proteins levels when rapamycin-dependent and SCA1-dependent inductions of autophagy are combined. In the absence of neurodegeneration, GLaz loss-of-function increases Atg8a/LC3 mRNA and p62 protein levels without altering p62 mRNA levels. Knocking-down autophagy, by interfering with Atg8a or p62 expression or by expressing dominant-negative Atg1/ULK1 or Atg4a transgenes, rescues SCA1-dependent neurodegeneration in a similar extent to the protective effect of GLaz. Further GLaz-dependent improvement is concealed.

Conclusions: This work shows for the first time that a Lipocalin rescues neurons from pathogenic SCA1 degeneration by optimizing clearance of aggregation-prone proteins. GLaz modulates key autophagy genes and lipid-peroxide clearance responsive genes. Down-regulation of selective autophagy causes similar and non-additive rescuing effects. These data suggest that SCA1 neurodegeneration concurs with autophagic stress, and places Lazarillo-related Lipocalins as valuable players in the endogenous protection against the two major contributors to aging and neurodegeneration: ROS-dependent damage and proteostasis deterioration.

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Effects on Atg8a and p62 expression of GLaz loss- and gain-of-function in control conditions. A-B, Immunofluorescence of control (either WTG10 or gmr > +), GLaz loss-of-function (GLazΔ2/Δ2) and GLaz gain-of-function (gmr > GLaz2) retinas showing p62 protein expression. C, mRNA levels of p62, measured by RT-qPCR in control and GLaz loss- and gain-of-function fly heads. D, mRNA levels of Atg8a, measured by RT-qPCR in control, GLaz loss- and gain-of-function fly heads. Statistical differences were assayed by Student’s t-test (immunofluorescence) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.
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Fig8: Effects on Atg8a and p62 expression of GLaz loss- and gain-of-function in control conditions. A-B, Immunofluorescence of control (either WTG10 or gmr > +), GLaz loss-of-function (GLazΔ2/Δ2) and GLaz gain-of-function (gmr > GLaz2) retinas showing p62 protein expression. C, mRNA levels of p62, measured by RT-qPCR in control and GLaz loss- and gain-of-function fly heads. D, mRNA levels of Atg8a, measured by RT-qPCR in control, GLaz loss- and gain-of-function fly heads. Statistical differences were assayed by Student’s t-test (immunofluorescence) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.

Mentions: In basal conditions, when no neurodegeneration-triggered autophagy activity is present, GLaz mutant flies show higher levels of p62 protein (Figure 8A), while over-expressing GLaz under basal conditions does not change p62 protein levels (Figure 8B). Transcript levels of p62 slightly decrease in GLaz mutants, but do not change significantly by GLaz gain-of-function in the absence of neurodegeneration (Figure 8C). Atg8a transcript levels increase in GLaz mutant flies, but no changes in Atg8a mRNA levels are observed in flies over-expressing GLaz (Figure 8D). These results suggest, on the one hand, the existence of a GLaz-dependent effect on basal autophagy progression, since the absence of GLaz expression results in increased Atg8a expression and accumulation of p62 without a parallel increase in p62 transcription. On the other hand, increasing GLaz expression levels does not affect, by itself, the expression of genes monitoring autophagic activity or the accumulation of p62 protein in the absence of a deteriorating cellular context.Figure 8


Lazarillo-related Lipocalins confer long-term protection against type I Spinocerebellar Ataxia degeneration contributing to optimize selective autophagy.

del Caño-Espinel M, Acebes JR, Sanchez D, Ganfornina MD - Mol Neurodegener (2015)

Effects on Atg8a and p62 expression of GLaz loss- and gain-of-function in control conditions. A-B, Immunofluorescence of control (either WTG10 or gmr > +), GLaz loss-of-function (GLazΔ2/Δ2) and GLaz gain-of-function (gmr > GLaz2) retinas showing p62 protein expression. C, mRNA levels of p62, measured by RT-qPCR in control and GLaz loss- and gain-of-function fly heads. D, mRNA levels of Atg8a, measured by RT-qPCR in control, GLaz loss- and gain-of-function fly heads. Statistical differences were assayed by Student’s t-test (immunofluorescence) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374295&req=5

Fig8: Effects on Atg8a and p62 expression of GLaz loss- and gain-of-function in control conditions. A-B, Immunofluorescence of control (either WTG10 or gmr > +), GLaz loss-of-function (GLazΔ2/Δ2) and GLaz gain-of-function (gmr > GLaz2) retinas showing p62 protein expression. C, mRNA levels of p62, measured by RT-qPCR in control and GLaz loss- and gain-of-function fly heads. D, mRNA levels of Atg8a, measured by RT-qPCR in control, GLaz loss- and gain-of-function fly heads. Statistical differences were assayed by Student’s t-test (immunofluorescence) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.
Mentions: In basal conditions, when no neurodegeneration-triggered autophagy activity is present, GLaz mutant flies show higher levels of p62 protein (Figure 8A), while over-expressing GLaz under basal conditions does not change p62 protein levels (Figure 8B). Transcript levels of p62 slightly decrease in GLaz mutants, but do not change significantly by GLaz gain-of-function in the absence of neurodegeneration (Figure 8C). Atg8a transcript levels increase in GLaz mutant flies, but no changes in Atg8a mRNA levels are observed in flies over-expressing GLaz (Figure 8D). These results suggest, on the one hand, the existence of a GLaz-dependent effect on basal autophagy progression, since the absence of GLaz expression results in increased Atg8a expression and accumulation of p62 without a parallel increase in p62 transcription. On the other hand, increasing GLaz expression levels does not affect, by itself, the expression of genes monitoring autophagic activity or the accumulation of p62 protein in the absence of a deteriorating cellular context.Figure 8

Bottom Line: GLaz beneficial effects persist throughout aging, and appears when expressed by degenerating neurons or by retinal support and glial cells.GLaz gain-of-function reduces cell death and the extent of ubiquitinated proteins accumulation, and decreases the expression of Atg8a/LC3, p62 mRNA and protein levels, and GstS1 induction.Down-regulation of selective autophagy causes similar and non-additive rescuing effects.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología y Genética Molecular-Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid-CSIC, c/ Sanz y Forés 3, 47003, Valladolid, Spain. manuela@ibgm.uva.es.

ABSTRACT

Background: A diverse set of neurodegenerative disorders are caused by abnormal extensions of polyglutamine (poly-Q) stretches in various, functionally unrelated proteins. A common feature of these diseases is altered proteostasis. Autophagy induction is part of the endogenous response to poly-Q protein expression. However, if autophagy is not resolved properly, clearance of toxic proteins or aggregates cannot occur effectively. Likewise, excessive autophagy induction can cause autophagic stress and neurodegeneration. The Lipocalins ApoD, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz) are neuroprotectors upon oxidative stress or aging. In this work we test whether these Lipocalins also protect against poly-Q-triggered deterioration of protein quality control systems.

Results: Using a Drosophila retinal degeneration model of Type-1 Spinocerebellar Ataxia (SCA1) combined with genetic manipulation of NLaz and GLaz expression, we demonstrate that both Lipocalins protect against SCA1 neurodegeneration. They are part of the endogenous transcriptional response to SCA1, and their effect is non-additive, suggesting participation in a similar mechanism. GLaz beneficial effects persist throughout aging, and appears when expressed by degenerating neurons or by retinal support and glial cells. GLaz gain-of-function reduces cell death and the extent of ubiquitinated proteins accumulation, and decreases the expression of Atg8a/LC3, p62 mRNA and protein levels, and GstS1 induction. Over-expression of GLaz is able to reduce p62 and ubiquitinated proteins levels when rapamycin-dependent and SCA1-dependent inductions of autophagy are combined. In the absence of neurodegeneration, GLaz loss-of-function increases Atg8a/LC3 mRNA and p62 protein levels without altering p62 mRNA levels. Knocking-down autophagy, by interfering with Atg8a or p62 expression or by expressing dominant-negative Atg1/ULK1 or Atg4a transgenes, rescues SCA1-dependent neurodegeneration in a similar extent to the protective effect of GLaz. Further GLaz-dependent improvement is concealed.

Conclusions: This work shows for the first time that a Lipocalin rescues neurons from pathogenic SCA1 degeneration by optimizing clearance of aggregation-prone proteins. GLaz modulates key autophagy genes and lipid-peroxide clearance responsive genes. Down-regulation of selective autophagy causes similar and non-additive rescuing effects. These data suggest that SCA1 neurodegeneration concurs with autophagic stress, and places Lazarillo-related Lipocalins as valuable players in the endogenous protection against the two major contributors to aging and neurodegeneration: ROS-dependent damage and proteostasis deterioration.

Show MeSH
Related in: MedlinePlus