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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 cell death due to hATXN182Q-dependent degeneration, and on SCA1-modifiers expression. A, TUNEL analysis of apoptotic cell death in paraffin sections of degenerated (gmr > hATXN182Q) and GLaz rescued (gmr > hATXN182Q + GLaz2) retinas. Representative examples of retina sections showing a decreased number of apoptotic nuclei (white arrows) in flies that over-express GLaz. B, hATXN1 mRNA levels in fly heads of the SCA1 model and lines expressing a double dose of GLaz. C, RT-qPCR expression levels of the SCA1 neurodegeneration modifiers GstS1 and Vib. Statistical differences of RT-qPCR data were assayed by Mann–Whitney U-test. *P < 0.05.
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Fig4: Effects of GLaz over-expression on cell death due to hATXN182Q-dependent degeneration, and on SCA1-modifiers expression. A, TUNEL analysis of apoptotic cell death in paraffin sections of degenerated (gmr > hATXN182Q) and GLaz rescued (gmr > hATXN182Q + GLaz2) retinas. Representative examples of retina sections showing a decreased number of apoptotic nuclei (white arrows) in flies that over-express GLaz. B, hATXN1 mRNA levels in fly heads of the SCA1 model and lines expressing a double dose of GLaz. C, RT-qPCR expression levels of the SCA1 neurodegeneration modifiers GstS1 and Vib. Statistical differences of RT-qPCR data were assayed by Mann–Whitney U-test. *P < 0.05.

Mentions: Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis reveals that photoreceptor cells die by apoptosis upon hATXN182Q expression (Figure 4A). GLaz over-expression causes a reduction in the number of apoptotic cells detected in the fly retina (Figure 4A). To have a quantitative estimate of the cell death rescue, we measured the levels of hATXN1 mRNA by qRT-PCR under two assumptions: (i) at pupal stage, hATXN1 mRNA levels are similar when degeneration is not yet present (hATXN1 protein levels show no difference, Additional file 1), and (ii) only intact photoreceptors would contribute significantly to the pool of detectable mRNA transcribed from the human transgene once the degeneration has started. GLaz does increase the levels of human Ataxin 1, both when co-expressed in photoreceptor neurons and when expressed by the nearby support cells and basal glial retinal cells (Figure 4B), in agreement with the decrease observed in the number of TUNEL-positive cells.Figure 4


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 cell death due to hATXN182Q-dependent degeneration, and on SCA1-modifiers expression. A, TUNEL analysis of apoptotic cell death in paraffin sections of degenerated (gmr > hATXN182Q) and GLaz rescued (gmr > hATXN182Q + GLaz2) retinas. Representative examples of retina sections showing a decreased number of apoptotic nuclei (white arrows) in flies that over-express GLaz. B, hATXN1 mRNA levels in fly heads of the SCA1 model and lines expressing a double dose of GLaz. C, RT-qPCR expression levels of the SCA1 neurodegeneration modifiers GstS1 and Vib. Statistical differences of RT-qPCR data were assayed by Mann–Whitney U-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

Fig4: Effects of GLaz over-expression on cell death due to hATXN182Q-dependent degeneration, and on SCA1-modifiers expression. A, TUNEL analysis of apoptotic cell death in paraffin sections of degenerated (gmr > hATXN182Q) and GLaz rescued (gmr > hATXN182Q + GLaz2) retinas. Representative examples of retina sections showing a decreased number of apoptotic nuclei (white arrows) in flies that over-express GLaz. B, hATXN1 mRNA levels in fly heads of the SCA1 model and lines expressing a double dose of GLaz. C, RT-qPCR expression levels of the SCA1 neurodegeneration modifiers GstS1 and Vib. Statistical differences of RT-qPCR data were assayed by Mann–Whitney U-test. *P < 0.05.
Mentions: Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis reveals that photoreceptor cells die by apoptosis upon hATXN182Q expression (Figure 4A). GLaz over-expression causes a reduction in the number of apoptotic cells detected in the fly retina (Figure 4A). To have a quantitative estimate of the cell death rescue, we measured the levels of hATXN1 mRNA by qRT-PCR under two assumptions: (i) at pupal stage, hATXN1 mRNA levels are similar when degeneration is not yet present (hATXN1 protein levels show no difference, Additional file 1), and (ii) only intact photoreceptors would contribute significantly to the pool of detectable mRNA transcribed from the human transgene once the degeneration has started. GLaz does increase the levels of human Ataxin 1, both when co-expressed in photoreceptor neurons and when expressed by the nearby support cells and basal glial retinal cells (Figure 4B), in agreement with the decrease observed in the number of TUNEL-positive cells.Figure 4

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