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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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Rescue of hATXN182Q-dependent photoreceptor degeneration by GLaz over-expressed with a native spatiotemporal pattern. A, Representative examples of scanning electron microscopy and magnified images of retinas degenerated (gmr > hATXN182Q), and co-expressing different GLaz expression transgenes (glaz:GLaz-GFP[FX] and glaz:GLaz-GFP[F2]). B, Quantitative estimate of degeneration by computing a regularity index based on the variance (σ) of the local intensity maxima distances. A percent recovery of the degeneration is indicated for each genotype. 20–35 eyes/genotype were used to compute regularity indexes. Average ± S.E.M. are represented. Asterisk represents statistically significant differences (Student’s t-test, *P < 0.05) with respect to the degenerated gmr > hATXN182Q genotype. C, a,b: GLaz expression revealed by GFP fluorescence immunohistochemistry of paraffin sections of retinas of flies expressing hATXN182Q and the transgene glaz:GLaz-GFP[FX]. Nuclei are shown with DAPI staining. c,d: Double fluorescence immunohistochemistry revealing GLaz and the photoreceptor marker rhodopsin (4C5). DAPI stains nuclei in C.
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Fig2: Rescue of hATXN182Q-dependent photoreceptor degeneration by GLaz over-expressed with a native spatiotemporal pattern. A, Representative examples of scanning electron microscopy and magnified images of retinas degenerated (gmr > hATXN182Q), and co-expressing different GLaz expression transgenes (glaz:GLaz-GFP[FX] and glaz:GLaz-GFP[F2]). B, Quantitative estimate of degeneration by computing a regularity index based on the variance (σ) of the local intensity maxima distances. A percent recovery of the degeneration is indicated for each genotype. 20–35 eyes/genotype were used to compute regularity indexes. Average ± S.E.M. are represented. Asterisk represents statistically significant differences (Student’s t-test, *P < 0.05) with respect to the degenerated gmr > hATXN182Q genotype. C, a,b: GLaz expression revealed by GFP fluorescence immunohistochemistry of paraffin sections of retinas of flies expressing hATXN182Q and the transgene glaz:GLaz-GFP[FX]. Nuclei are shown with DAPI staining. c,d: Double fluorescence immunohistochemistry revealing GLaz and the photoreceptor marker rhodopsin (4C5). DAPI stains nuclei in C.

Mentions: GLaz produces a significant rescue of photoreceptor degeneration when co-expressed with hATXN182Q (Figure 1C-F, Additional file 2). Two UAS:GLaz and two EP insertions upstream of GLaz were used to drive the expression of the Lipocalin under the control of gmr:GAL4. The adult eye external morphology was inspected (Figure 1C; Additional file 2A) and the degree of surface regularity analyzed (Figure 1D) and quantified (Figure 2B) by estimating a regularity index according to the variance of intensity maxima (See Methods section; [31]). Histochemistry of paraffin sections (Figure 1E) shows severe atrophy of retinal cells in the hATXN182Q-expressing flies, while the retinal epithelial pattern is reasonably preserved in the presence of gain-of-function constructs of GLaz. The degeneration observed in hATXN182Q-expressing photoreceptors of 3 days old flies is already extensive, and no further deleterious effect is observed when native GLaz expression is eliminated in the mutant background (Additional file 3A). The expression of an unrelated control protein (LacZ) does not rescue the degeneration phenotype of the SCA1 model (Additional file 2C, Figure 2B). Specificity was further tested by simultaneously over-expressing GLaz in photoreceptors (with the UAS:GLaz2) and decreasing the expression of GLaz with a UAS:GLazRNAi construct. The rescue is abolished in this situation (Additional file 3B, Figure 2B). Moreover, the rescue observed with GLaz gain-of-function is dependent on temperature (due to the temperature sensitivity of the GAL4/UAS system) and the transgene expression levels. The GLaz-triggered rescue at 25°C shows a similar extent when comparing flies with either one or two copies of the UAS:hATXN182Q transgene located in the 1st chromosome (Additional file 2D, Figure 2B). However, a lesser rescue was obtained either at 29°C or with a 3rd chromosome UAS:hATXN182Q insertion line that shows a higher expression level (results not shown).Figure 2


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)

Rescue of hATXN182Q-dependent photoreceptor degeneration by GLaz over-expressed with a native spatiotemporal pattern. A, Representative examples of scanning electron microscopy and magnified images of retinas degenerated (gmr > hATXN182Q), and co-expressing different GLaz expression transgenes (glaz:GLaz-GFP[FX] and glaz:GLaz-GFP[F2]). B, Quantitative estimate of degeneration by computing a regularity index based on the variance (σ) of the local intensity maxima distances. A percent recovery of the degeneration is indicated for each genotype. 20–35 eyes/genotype were used to compute regularity indexes. Average ± S.E.M. are represented. Asterisk represents statistically significant differences (Student’s t-test, *P < 0.05) with respect to the degenerated gmr > hATXN182Q genotype. C, a,b: GLaz expression revealed by GFP fluorescence immunohistochemistry of paraffin sections of retinas of flies expressing hATXN182Q and the transgene glaz:GLaz-GFP[FX]. Nuclei are shown with DAPI staining. c,d: Double fluorescence immunohistochemistry revealing GLaz and the photoreceptor marker rhodopsin (4C5). DAPI stains nuclei in C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Rescue of hATXN182Q-dependent photoreceptor degeneration by GLaz over-expressed with a native spatiotemporal pattern. A, Representative examples of scanning electron microscopy and magnified images of retinas degenerated (gmr > hATXN182Q), and co-expressing different GLaz expression transgenes (glaz:GLaz-GFP[FX] and glaz:GLaz-GFP[F2]). B, Quantitative estimate of degeneration by computing a regularity index based on the variance (σ) of the local intensity maxima distances. A percent recovery of the degeneration is indicated for each genotype. 20–35 eyes/genotype were used to compute regularity indexes. Average ± S.E.M. are represented. Asterisk represents statistically significant differences (Student’s t-test, *P < 0.05) with respect to the degenerated gmr > hATXN182Q genotype. C, a,b: GLaz expression revealed by GFP fluorescence immunohistochemistry of paraffin sections of retinas of flies expressing hATXN182Q and the transgene glaz:GLaz-GFP[FX]. Nuclei are shown with DAPI staining. c,d: Double fluorescence immunohistochemistry revealing GLaz and the photoreceptor marker rhodopsin (4C5). DAPI stains nuclei in C.
Mentions: GLaz produces a significant rescue of photoreceptor degeneration when co-expressed with hATXN182Q (Figure 1C-F, Additional file 2). Two UAS:GLaz and two EP insertions upstream of GLaz were used to drive the expression of the Lipocalin under the control of gmr:GAL4. The adult eye external morphology was inspected (Figure 1C; Additional file 2A) and the degree of surface regularity analyzed (Figure 1D) and quantified (Figure 2B) by estimating a regularity index according to the variance of intensity maxima (See Methods section; [31]). Histochemistry of paraffin sections (Figure 1E) shows severe atrophy of retinal cells in the hATXN182Q-expressing flies, while the retinal epithelial pattern is reasonably preserved in the presence of gain-of-function constructs of GLaz. The degeneration observed in hATXN182Q-expressing photoreceptors of 3 days old flies is already extensive, and no further deleterious effect is observed when native GLaz expression is eliminated in the mutant background (Additional file 3A). The expression of an unrelated control protein (LacZ) does not rescue the degeneration phenotype of the SCA1 model (Additional file 2C, Figure 2B). Specificity was further tested by simultaneously over-expressing GLaz in photoreceptors (with the UAS:GLaz2) and decreasing the expression of GLaz with a UAS:GLazRNAi construct. The rescue is abolished in this situation (Additional file 3B, Figure 2B). Moreover, the rescue observed with GLaz gain-of-function is dependent on temperature (due to the temperature sensitivity of the GAL4/UAS system) and the transgene expression levels. The GLaz-triggered rescue at 25°C shows a similar extent when comparing flies with either one or two copies of the UAS:hATXN182Q transgene located in the 1st chromosome (Additional file 2D, Figure 2B). However, a lesser rescue was obtained either at 29°C or with a 3rd chromosome UAS:hATXN182Q insertion line that shows a higher expression level (results not shown).Figure 2

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