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Expression profiling in APP23 mouse brain: inhibition of Abeta amyloidosis and inflammation in response to LXR agonist treatment.

Lefterov I, Bookout A, Wang Z, Staufenbiel M, Mangelsdorf D, Koldamova R - Mol Neurodegener (2007)

Bottom Line: Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Abeta.The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation.An increased understanding of the LXR controlled regulation of Abeta aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15219, USA. iliyal@pitt.edu.

ABSTRACT

Background: Recent studies demonstrate that in addition to its modulatory effect on APP processing, in vivo application of Liver X Receptor agonist T0901317 (T0) to APP transgenic and non-transgenic mice decreases the level of Abeta42. Moreover, in young Tg2576 mice T0 completely reversed contextual memory deficits. Compared to other tissues, the regulatory functions of LXRs in brain remain largely unexplored and our knowledge so far is limited to the cholesterol transporters and apoE. In this study we applied T0 to APP23 mice for various times and examined gene and protein expression. We also performed a series of experiments with primary brain cells derived from wild type and LXR knockout mice subjected to various LXR agonist treatments and inflammatory stimuli.

Results: We demonstrate an upregulation of genes related to lipid metabolism/transport, metabolism of xenobiotics and detoxification. Downregulated genes are involved in immune response and inflammation, cell death and apoptosis. Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Abeta. In primary LXRwt but not in LXRalpha-/-beta-/- microglia and astrocytes LXR agonists suppressed the inflammatory response induced by LPS or fibrillar Abeta.

Conclusion: The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation. An increased understanding of the LXR controlled regulation of Abeta aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.

No MeSH data available.


Related in: MedlinePlus

Extended T0 treatment increases the levels of soluble apoE and apoA-I proteins and decreases insoluble Aβ in APP23 mice. APP23 mice (n = 5) were treated by gastric gavage with T0 for 4 weeks at a dose of 20 mg/kg/day and age-matched control mice (n = 5) received vehicle. At the end of the treatment one hemisphere was used for total RNA isolation and the other was used for protein extraction. Soluble brain proteins were extracted with DEA followed by the extraction of the insoluble proteins from the pellet using formic acid. A: Expression level of ABCA1, apoE and apoA-I mRNA as determined by RT-QPCR. B: Insoluble Aβtotal in aliquots from the insoluble brain fraction was determined by WB using by 6E10 antibody which recognizes both Aβ40 and Aβ42. The level of Aβtotal was normalized to the level of APP full length (APPfl). C: Amounts of soluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenates. The bands were quantified and the level of apoE normalized to the level of GAPDH. D: Insoluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenate as in C. Values (A, B, C and D) are means ± SEM and represent fold of vehicle (two-tailed Student's t test). E. The level of insoluble Aβtotal correlates negatively to the level of soluble apoE (Spearman Nonparametric correlation analysis).
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Figure 2: Extended T0 treatment increases the levels of soluble apoE and apoA-I proteins and decreases insoluble Aβ in APP23 mice. APP23 mice (n = 5) were treated by gastric gavage with T0 for 4 weeks at a dose of 20 mg/kg/day and age-matched control mice (n = 5) received vehicle. At the end of the treatment one hemisphere was used for total RNA isolation and the other was used for protein extraction. Soluble brain proteins were extracted with DEA followed by the extraction of the insoluble proteins from the pellet using formic acid. A: Expression level of ABCA1, apoE and apoA-I mRNA as determined by RT-QPCR. B: Insoluble Aβtotal in aliquots from the insoluble brain fraction was determined by WB using by 6E10 antibody which recognizes both Aβ40 and Aβ42. The level of Aβtotal was normalized to the level of APP full length (APPfl). C: Amounts of soluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenates. The bands were quantified and the level of apoE normalized to the level of GAPDH. D: Insoluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenate as in C. Values (A, B, C and D) are means ± SEM and represent fold of vehicle (two-tailed Student's t test). E. The level of insoluble Aβtotal correlates negatively to the level of soluble apoE (Spearman Nonparametric correlation analysis).

Mentions: Next we examined if the T0-mediated up-regulation of ABCA1 and apolipoproteins had an effect on the level of insoluble Aβ. T0 was administered to 6-month old APP23 mice (n = 5), 5 times per week for 4 weeks at a daily dose of 20 mg/kg body weight; the control group (n = 5) received vehicle. We decreased the dose of T0 in an attempt to minimize the liver steatosis which is a well known side effect of LXR ligands. At the end of the treatment, mRNA was isolated from the cortex and hippocampus of one hemisphere of each mouse and the activation of LXRs was confirmed by the increased level of ABCA1 mRNA in T0 versus vehicle treated mice (Fig. 2A). Cortices and hippocampi from the other hemisphere were homogenized and soluble and insoluble proteins were extracted, as before [14]. We were interested in the levels of apoE mRNA and protein because of its effect on Aβ aggregation and clearance [29,30] and we also examined the effect of LXR on apoA-I which was shown previously to affect Aβ aggregation in vitro [31]. ApoE and apoA-I in the soluble brain fraction (solApoE and solApo-A-I as determined by WB) of T0 treated mice were substantially increased: apoE more than 3 times and apoA-I more than 13-fold compared to their level in the same fraction of vehicle treated animals (Fig. 2C). In contrast, T0 treatment decreased significantly the level of total Aβ (comprising of Aβ40 and Aβ42) as measured by WB in the insoluble brain fraction using 6E10 antibody (Fig. 2B), while the level of soluble Aβ in the same set of samples was not changed (not shown). The increase in apoE protein level in T0 treated mice was accompanied by a small but statistically significant up-regulation of apoE mRNA (Fig. 1A). In contrast, whereas apoA-I protein level was increased by 13-fold, the increase of its mRNA was not statistically significant (1.6-fold, p < 0.2, Fig. 2A). It should be noted that we found similar results for ApoA-I on the microarrays: a 1.5-fold increase in apoA-I mRNA which was not statistically significant (p < 0.19, data not shown). In the insoluble fraction of brain homogenates of T0 treated mice there was no change in the level of apoE and the increase of apoA-I was not statistically significant (Fig. 2D). Finally, we found that there was a negative correlation between the levels of soluble apoE level and insoluble Aβ (Fig. 2E). This is in accordance with our studies using APP23/ABCA1-/- mice, where the lack of ABCA1 caused an increase in amyloid load accompanied by a substantial decrease in soluble apoE level, with no change in the level of insoluble apoE [14]. We conclude that LXR activation increases the level of soluble apolipoproteins in the brain which correlates negatively to the level of insoluble Aβ.


Expression profiling in APP23 mouse brain: inhibition of Abeta amyloidosis and inflammation in response to LXR agonist treatment.

Lefterov I, Bookout A, Wang Z, Staufenbiel M, Mangelsdorf D, Koldamova R - Mol Neurodegener (2007)

Extended T0 treatment increases the levels of soluble apoE and apoA-I proteins and decreases insoluble Aβ in APP23 mice. APP23 mice (n = 5) were treated by gastric gavage with T0 for 4 weeks at a dose of 20 mg/kg/day and age-matched control mice (n = 5) received vehicle. At the end of the treatment one hemisphere was used for total RNA isolation and the other was used for protein extraction. Soluble brain proteins were extracted with DEA followed by the extraction of the insoluble proteins from the pellet using formic acid. A: Expression level of ABCA1, apoE and apoA-I mRNA as determined by RT-QPCR. B: Insoluble Aβtotal in aliquots from the insoluble brain fraction was determined by WB using by 6E10 antibody which recognizes both Aβ40 and Aβ42. The level of Aβtotal was normalized to the level of APP full length (APPfl). C: Amounts of soluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenates. The bands were quantified and the level of apoE normalized to the level of GAPDH. D: Insoluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenate as in C. Values (A, B, C and D) are means ± SEM and represent fold of vehicle (two-tailed Student's t test). E. The level of insoluble Aβtotal correlates negatively to the level of soluble apoE (Spearman Nonparametric correlation analysis).
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Related In: Results  -  Collection

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Figure 2: Extended T0 treatment increases the levels of soluble apoE and apoA-I proteins and decreases insoluble Aβ in APP23 mice. APP23 mice (n = 5) were treated by gastric gavage with T0 for 4 weeks at a dose of 20 mg/kg/day and age-matched control mice (n = 5) received vehicle. At the end of the treatment one hemisphere was used for total RNA isolation and the other was used for protein extraction. Soluble brain proteins were extracted with DEA followed by the extraction of the insoluble proteins from the pellet using formic acid. A: Expression level of ABCA1, apoE and apoA-I mRNA as determined by RT-QPCR. B: Insoluble Aβtotal in aliquots from the insoluble brain fraction was determined by WB using by 6E10 antibody which recognizes both Aβ40 and Aβ42. The level of Aβtotal was normalized to the level of APP full length (APPfl). C: Amounts of soluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenates. The bands were quantified and the level of apoE normalized to the level of GAPDH. D: Insoluble apoE and apoA-I were determined by WB of formic acid extracted brain homogenate as in C. Values (A, B, C and D) are means ± SEM and represent fold of vehicle (two-tailed Student's t test). E. The level of insoluble Aβtotal correlates negatively to the level of soluble apoE (Spearman Nonparametric correlation analysis).
Mentions: Next we examined if the T0-mediated up-regulation of ABCA1 and apolipoproteins had an effect on the level of insoluble Aβ. T0 was administered to 6-month old APP23 mice (n = 5), 5 times per week for 4 weeks at a daily dose of 20 mg/kg body weight; the control group (n = 5) received vehicle. We decreased the dose of T0 in an attempt to minimize the liver steatosis which is a well known side effect of LXR ligands. At the end of the treatment, mRNA was isolated from the cortex and hippocampus of one hemisphere of each mouse and the activation of LXRs was confirmed by the increased level of ABCA1 mRNA in T0 versus vehicle treated mice (Fig. 2A). Cortices and hippocampi from the other hemisphere were homogenized and soluble and insoluble proteins were extracted, as before [14]. We were interested in the levels of apoE mRNA and protein because of its effect on Aβ aggregation and clearance [29,30] and we also examined the effect of LXR on apoA-I which was shown previously to affect Aβ aggregation in vitro [31]. ApoE and apoA-I in the soluble brain fraction (solApoE and solApo-A-I as determined by WB) of T0 treated mice were substantially increased: apoE more than 3 times and apoA-I more than 13-fold compared to their level in the same fraction of vehicle treated animals (Fig. 2C). In contrast, T0 treatment decreased significantly the level of total Aβ (comprising of Aβ40 and Aβ42) as measured by WB in the insoluble brain fraction using 6E10 antibody (Fig. 2B), while the level of soluble Aβ in the same set of samples was not changed (not shown). The increase in apoE protein level in T0 treated mice was accompanied by a small but statistically significant up-regulation of apoE mRNA (Fig. 1A). In contrast, whereas apoA-I protein level was increased by 13-fold, the increase of its mRNA was not statistically significant (1.6-fold, p < 0.2, Fig. 2A). It should be noted that we found similar results for ApoA-I on the microarrays: a 1.5-fold increase in apoA-I mRNA which was not statistically significant (p < 0.19, data not shown). In the insoluble fraction of brain homogenates of T0 treated mice there was no change in the level of apoE and the increase of apoA-I was not statistically significant (Fig. 2D). Finally, we found that there was a negative correlation between the levels of soluble apoE level and insoluble Aβ (Fig. 2E). This is in accordance with our studies using APP23/ABCA1-/- mice, where the lack of ABCA1 caused an increase in amyloid load accompanied by a substantial decrease in soluble apoE level, with no change in the level of insoluble apoE [14]. We conclude that LXR activation increases the level of soluble apolipoproteins in the brain which correlates negatively to the level of insoluble Aβ.

Bottom Line: Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Abeta.The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation.An increased understanding of the LXR controlled regulation of Abeta aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15219, USA. iliyal@pitt.edu.

ABSTRACT

Background: Recent studies demonstrate that in addition to its modulatory effect on APP processing, in vivo application of Liver X Receptor agonist T0901317 (T0) to APP transgenic and non-transgenic mice decreases the level of Abeta42. Moreover, in young Tg2576 mice T0 completely reversed contextual memory deficits. Compared to other tissues, the regulatory functions of LXRs in brain remain largely unexplored and our knowledge so far is limited to the cholesterol transporters and apoE. In this study we applied T0 to APP23 mice for various times and examined gene and protein expression. We also performed a series of experiments with primary brain cells derived from wild type and LXR knockout mice subjected to various LXR agonist treatments and inflammatory stimuli.

Results: We demonstrate an upregulation of genes related to lipid metabolism/transport, metabolism of xenobiotics and detoxification. Downregulated genes are involved in immune response and inflammation, cell death and apoptosis. Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Abeta. In primary LXRwt but not in LXRalpha-/-beta-/- microglia and astrocytes LXR agonists suppressed the inflammatory response induced by LPS or fibrillar Abeta.

Conclusion: The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation. An increased understanding of the LXR controlled regulation of Abeta aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.

No MeSH data available.


Related in: MedlinePlus