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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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GLaz over-expression decreases p62 protein accumulation in response to ATXN1 degeneration and autophagy induction, and modifies p62 mRNA levels in neurodegenerative conditions. A, p62 accumulation, measured by immunoblot, decreases in SCA1-affected retinas that over-express GLaz. B, p62 protein expression increases upon rapamycin treatment in wild type flies. C, Treatment of SCA1 flies with rapamycin, combined with GLaz over-expression, further reduces p62 accumulation. D, A decrease in p62 mRNA expression is observed in SCA1 flies that over-express GLaz. Statistical differences were assayed by Student’s t-test (immunoblots) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.
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Fig7: GLaz over-expression decreases p62 protein accumulation in response to ATXN1 degeneration and autophagy induction, and modifies p62 mRNA levels in neurodegenerative conditions. A, p62 accumulation, measured by immunoblot, decreases in SCA1-affected retinas that over-express GLaz. B, p62 protein expression increases upon rapamycin treatment in wild type flies. C, Treatment of SCA1 flies with rapamycin, combined with GLaz over-expression, further reduces p62 accumulation. D, A decrease in p62 mRNA expression is observed in SCA1 flies that over-express GLaz. Statistical differences were assayed by Student’s t-test (immunoblots) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.

Mentions: To discern between the alternatives stated above we monitored the accumulation of p62, a protein acting as a decision point for the degradation of aggregasomes (aggregates of misfolded proteins too large to be degraded by the proteasome system). p62 docks aggregasomes to newly formed autophagic membranes, thus controlling their entrance in the autophagosome [41-43]. If autophagy is working properly, p62 should also be degraded in the process. Therefore, measurement of endogenous p62 can be used to quantitatively assess the flow of autophagy of protein aggregates [44,45] serving as a readout of autophagic degradation efficiency [5]. The hypothesis that GLaz might be promoting the resolution of autophagy predicts a reduction in p62 accumulation. Levels of p62 show a 30-40% decrease upon GLaz over-expression (Figure 7A). Moreover, p62 protein levels increase upon rapamycin-induced autophagy in both, wild type control flies (Figure 7B) and in flies undergoing photoreceptor neurodegeneration (Figure 7C), as expected by the known transcriptional up-regulation of p62 in autophagy-inducing conditions [39]. Over-expression of GLaz reduces p62 levels in the SCA1 model with or without further induction of autophagy by rapamycin (Figure 7A,C). The effect of GLaz over-expression on autophagy markers does not support its role in clearance of aggregate-prone proteins by an autophagy-independent proteasome mechanism.Figure 7


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)

GLaz over-expression decreases p62 protein accumulation in response to ATXN1 degeneration and autophagy induction, and modifies p62 mRNA levels in neurodegenerative conditions. A, p62 accumulation, measured by immunoblot, decreases in SCA1-affected retinas that over-express GLaz. B, p62 protein expression increases upon rapamycin treatment in wild type flies. C, Treatment of SCA1 flies with rapamycin, combined with GLaz over-expression, further reduces p62 accumulation. D, A decrease in p62 mRNA expression is observed in SCA1 flies that over-express GLaz. Statistical differences were assayed by Student’s t-test (immunoblots) 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

Fig7: GLaz over-expression decreases p62 protein accumulation in response to ATXN1 degeneration and autophagy induction, and modifies p62 mRNA levels in neurodegenerative conditions. A, p62 accumulation, measured by immunoblot, decreases in SCA1-affected retinas that over-express GLaz. B, p62 protein expression increases upon rapamycin treatment in wild type flies. C, Treatment of SCA1 flies with rapamycin, combined with GLaz over-expression, further reduces p62 accumulation. D, A decrease in p62 mRNA expression is observed in SCA1 flies that over-express GLaz. Statistical differences were assayed by Student’s t-test (immunoblots) and Mann–Whitney U-test (RT-qPCR). *P < 0.05.
Mentions: To discern between the alternatives stated above we monitored the accumulation of p62, a protein acting as a decision point for the degradation of aggregasomes (aggregates of misfolded proteins too large to be degraded by the proteasome system). p62 docks aggregasomes to newly formed autophagic membranes, thus controlling their entrance in the autophagosome [41-43]. If autophagy is working properly, p62 should also be degraded in the process. Therefore, measurement of endogenous p62 can be used to quantitatively assess the flow of autophagy of protein aggregates [44,45] serving as a readout of autophagic degradation efficiency [5]. The hypothesis that GLaz might be promoting the resolution of autophagy predicts a reduction in p62 accumulation. Levels of p62 show a 30-40% decrease upon GLaz over-expression (Figure 7A). Moreover, p62 protein levels increase upon rapamycin-induced autophagy in both, wild type control flies (Figure 7B) and in flies undergoing photoreceptor neurodegeneration (Figure 7C), as expected by the known transcriptional up-regulation of p62 in autophagy-inducing conditions [39]. Over-expression of GLaz reduces p62 levels in the SCA1 model with or without further induction of autophagy by rapamycin (Figure 7A,C). The effect of GLaz over-expression on autophagy markers does not support its role in clearance of aggregate-prone proteins by an autophagy-independent proteasome mechanism.Figure 7

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