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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 of GLaz over-expression on Atg8a expression and under pharmacological induction of autophagy. A, Pathogenic expression of hATXN182Q and starvation increase autophagic activity, monitored by Atg8a mRNA levels. B, Atg8a expression is decreased by co-expression of GLaz and the hATXN182Q transgene. Statistical differences were assayed by Mann–Whitney U-test. *P < 0.05. C, Levels of ubiquitinated proteins in fly heads, measured by immunoblot after treatment with the autophagy inducer rapamycin. An increase in ubiquitin load is observed when SCA1 model flies are exposed to rapamycin, while a decrease in ubiquitin signal is clear upon GLaz over-expression. Color code applies for blot and graphs in B and C. Statistical differences were assayed by Student’s t-test. *P < 0.05.
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Fig6: Effects of GLaz over-expression on Atg8a expression and under pharmacological induction of autophagy. A, Pathogenic expression of hATXN182Q and starvation increase autophagic activity, monitored by Atg8a mRNA levels. B, Atg8a expression is decreased by co-expression of GLaz and the hATXN182Q transgene. Statistical differences were assayed by Mann–Whitney U-test. *P < 0.05. C, Levels of ubiquitinated proteins in fly heads, measured by immunoblot after treatment with the autophagy inducer rapamycin. An increase in ubiquitin load is observed when SCA1 model flies are exposed to rapamycin, while a decrease in ubiquitin signal is clear upon GLaz over-expression. Color code applies for blot and graphs in B and C. Statistical differences were assayed by Student’s t-test. *P < 0.05.

Mentions: In order to understand the protective mechanism of Lipocalins we studied the endogenous expression of the Drosophila Atg8a gene, a homologue of LC3 in vertebrates [38], as a first approximation to the levels of autophagy activity. Transcription of Atg8a is known to be strongly up-regulated upon starvation [39] and to show a strong correlation with autophagy activity. Although autophagy initiation might not be dependent on Atg8a up-regulation, this transcriptional response contributes to replenish the protein consumed by autophagic activity [5]. By measuring the transcription levels of the Atg8a gene we can therefore assess whether autophagic activity is increased in the SCA1 model of photoreceptor degeneration. A twofold Atg8a induction is observed (Figure 6A). This indication of increased autophagic activity is in agreement with the observed induction of GstS1 (Figure 4C), which upon oxidation would contribute to JNK-based autophagy induction, and supports the view that inducing autophagy might be a general response in poly-Q based pathologies of different etiologies [3]. Neurodegeneration-triggered Atg8a induction in the fly head is similar in extent to the induction obtained upon starvation in this tissue (Figure 6A), although quantitatively smaller than the changes observed in larval and fat body tissues [39]. If flies expressing hATXN182Q are starved, Atg8a transcription is further increased, suggesting that different mechanisms of autophagy induction are acting in an additive manner.Figure 6


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 of GLaz over-expression on Atg8a expression and under pharmacological induction of autophagy. A, Pathogenic expression of hATXN182Q and starvation increase autophagic activity, monitored by Atg8a mRNA levels. B, Atg8a expression is decreased by co-expression of GLaz and the hATXN182Q transgene. Statistical differences were assayed by Mann–Whitney U-test. *P < 0.05. C, Levels of ubiquitinated proteins in fly heads, measured by immunoblot after treatment with the autophagy inducer rapamycin. An increase in ubiquitin load is observed when SCA1 model flies are exposed to rapamycin, while a decrease in ubiquitin signal is clear upon GLaz over-expression. Color code applies for blot and graphs in B and C. Statistical differences were assayed by Student’s t-test. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Effects of GLaz over-expression on Atg8a expression and under pharmacological induction of autophagy. A, Pathogenic expression of hATXN182Q and starvation increase autophagic activity, monitored by Atg8a mRNA levels. B, Atg8a expression is decreased by co-expression of GLaz and the hATXN182Q transgene. Statistical differences were assayed by Mann–Whitney U-test. *P < 0.05. C, Levels of ubiquitinated proteins in fly heads, measured by immunoblot after treatment with the autophagy inducer rapamycin. An increase in ubiquitin load is observed when SCA1 model flies are exposed to rapamycin, while a decrease in ubiquitin signal is clear upon GLaz over-expression. Color code applies for blot and graphs in B and C. Statistical differences were assayed by Student’s t-test. *P < 0.05.
Mentions: In order to understand the protective mechanism of Lipocalins we studied the endogenous expression of the Drosophila Atg8a gene, a homologue of LC3 in vertebrates [38], as a first approximation to the levels of autophagy activity. Transcription of Atg8a is known to be strongly up-regulated upon starvation [39] and to show a strong correlation with autophagy activity. Although autophagy initiation might not be dependent on Atg8a up-regulation, this transcriptional response contributes to replenish the protein consumed by autophagic activity [5]. By measuring the transcription levels of the Atg8a gene we can therefore assess whether autophagic activity is increased in the SCA1 model of photoreceptor degeneration. A twofold Atg8a induction is observed (Figure 6A). This indication of increased autophagic activity is in agreement with the observed induction of GstS1 (Figure 4C), which upon oxidation would contribute to JNK-based autophagy induction, and supports the view that inducing autophagy might be a general response in poly-Q based pathologies of different etiologies [3]. Neurodegeneration-triggered Atg8a induction in the fly head is similar in extent to the induction obtained upon starvation in this tissue (Figure 6A), although quantitatively smaller than the changes observed in larval and fat body tissues [39]. If flies expressing hATXN182Q are starved, Atg8a transcription is further increased, suggesting that different mechanisms of autophagy induction are acting in an additive manner.Figure 6

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