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. 2021 Jan 20;41(3):538-554.
doi: 10.1523/JNEUROSCI.0295-20.2020. Epub 2020 Nov 25.

Oxidative Damage and Antioxidant Response in Frontal Cortex of Demented and Nondemented Individuals with Alzheimer's Neuropathology

Anna Fracassi  1 , Michela Marcatti  1 , Olga Zolochevska  1 , Natalie Tabor  2 , Randall Woltjer  3 , Sandra Moreno  4 , Giulio Taglialatela  5
Affiliations

Oxidative Damage and Antioxidant Response in Frontal Cortex of Demented and Nondemented Individuals with Alzheimer's Neuropathology

Anna Fracassi et al. J Neurosci. .

Abstract

Alzheimer's disease (AD) is characterized by progressive neurodegeneration in the cerebral cortex, histopathologically hallmarked by amyloid β (Aβ) extracellular plaques and intracellular neurofibrillary tangles, constituted by hyperphosphorylated tau protein. Correlation between these pathologic features and dementia has been challenged by the emergence of "nondemented with Alzheimer's neuropathology" (NDAN) individuals, cognitively intact despite displaying pathologic features of AD. The existence of these subjects suggests that some unknown mechanisms are triggered to resist Aβ-mediated detrimental events. Aβ accumulation affects mitochondrial redox balance, increasing oxidative stress status, which in turn is proposed as a primary culprit in AD pathogenesis. To clarify the relationship linking Aβ, oxidative stress, and cognitive impairment, we performed a comparative study on AD, NDAN, and aged-matched human postmortem frontal cortices of either sex. We quantitatively analyzed immunofluorescence distribution of oxidative damage markers, and of SOD2 (superoxide dismutase 2), PGC1α [peroxisome proliferator-activated receptor (PPAR) γ-coactivator 1α], PPARα, and catalase as key factors in antioxidant response, as well as the expression of miRNA-485, as a PGC1α upstream regulator. Our results confirm dramatic redox imbalance, associated with impaired antioxidant defenses in AD brain. By contrast, NDAN individuals display low oxidative damage, which is associated with high levels of scavenging systems, possibly resulting from a lack of PGC1α miRNA-485-related inhibition. Comparative analyses in neurons and astrocytes further highlighted cell-specific mechanisms to counteract redox imbalance. Overall, our data emphasize the importance of transcriptional and post-transcriptional regulation of antioxidant response in AD. This suggests that an efficient PGC1α-dependent "safety mechanism" may prevent Aβ-mediated oxidative stress, supporting neuroprotective therapies aimed at ameliorating defects in antioxidant response pathways in AD patients.

Keywords: Alzheimer's disease; NDAN; PGC1α; PPARα; miRNA-485; oxidative stress.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Correlation analysis between each of the studied parameters and PMI values across all of the assayed specimens. A Pearson's correlation test was performed for each measurement against the PMI. Correlation coefficient (r) and p values are noted in the individual plots showing no significant correlation with PMI values.
Figure 2.
Figure 2.
A, A′, 8-oxo-dG expression and distribution in frontal cortex of control, AD, and NDAN subjects. Immunolocalization of 8-oxo-dG and quantitative analysis of IF images showing increased levels of oxidative damage in brains of AD subjects and low levels in NDAN subjects, compared with control subjects. Original magnification, 20×. Scale bar, 100 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 41.67, p < 0.0001) following Tukey's multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. B–D, 8-oxo-dG expression and distribution in frontal cortex neurons and astrocytes of control, AD, and NDAN subjects. B, B′, Double IF of 8-oxo-dG (green) in combination with NeuN (red) shows high levels of oxidative damage in AD neurons. NDAN neurons demonstrate low levels of oxidative damage marker. Magnification, 60×. Scale bar, 30 µm. The quantitative analysis of IF images shows significantly higher levels of oxidative damage markers in AD neurons. Statistical analyses were made using one-way ANOVA (F(2,15) = 48.47, p < 0.0001) following Tukey's multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. C C′, Double IF of 8-oxo-dG (green) in combination with GFAP (red) showing high levels of oxidative damage to astrocytes in AD subjects compared with control and NDAN subjects, although lower levels than in neurons. Magnification 60×. Scale bar, 30 µm. The quantitative analysis of IF images shows significantly higher levels of the oxidative damage marker in AD astrocytes, while NDAN and control astrocytes displayed comparable levels of damage. Statistical analyses were made using one-way ANOVA (F(2,15) = 37.64, p < 0.0001) following Tukey's multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. D, The analysis demonstrates relatively higher resistance of astrocytes to oxidative damage, compared with neurons, which appear more prone to AD-associated oxidative damage. Statistical analyses were made using two-way ANOVA (F(2,30) = 80, p < 0.0001). Values are expressed as the mean ± SD. ****p < 0.0001. ns, not significant.
Figure 3.
Figure 3.
8-oxo-dG expression and distribution in relation to Aβ accumulation. A, A′, Double IF of 8-oxo-dG (green) and Aβ (red) showing the oxidative damage to nucleic acids around Aβ plaques in AD and NDAN subjects. The quantitative analyses in terms of the intensity of fluorescence (t(10) = 13.06, p < 0.0001, unpaired t test) and number of 8-oxo-dG+ cells (t(10) = 15.02, p < 0.0001, unpaired t test) show increased levels of oxidative damage around amyloid plaques in AD compared with NDAN individuals. Original magnification, 60×. Scale bar, 30 µm. Values are expressed as the mean ± SD. ****p < 0.0001. B, B′, Immunostaining of 8-oxo-dG and Aβ showing significant high levels of oxidative damage in AD subjects compared with control and NDAN subjects even far from Aβ plaques. Statistical analyses were made using one-way ANOVA (IntDens: F(2,15) = 122.1, p < 0.000; count: F(2,15) = 42.34, p < 0.0001) following Tukey's multiple-comparisons test. Original magnification 60×. Scale bar, 30 µm. Values are expressed as the mean ± SD. ****p < 0.0001. ns, not significant.
Figure 4.
Figure 4.
A, A′, 4-HNE expression and distribution in frontal cortex of control, AD, and NDAN subjects. Immunolocalization of 4-HNE and quantitative analysis of IF images showing increased levels of the lipid peroxidation marker in AD brains and low levels in NDAN subjects, compared with control subjects. Original magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 19.44, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ***p < 0.001. B–D, 4-HNE expression and distribution in frontal cortex neurons and astrocytes of control, AD, and NDAN subjects. B, B′, Double IF of 4-HNE (green) in combination with NeuN (red) shows high levels of oxidative damage in AD neurons. NDAN neurons demonstrate low levels of lipid peroxidation marker. Magnification, 60×. Scale bar, 30 µm. The quantitative analysis of IF images shows a significantly higher levels of oxidative damage marker in AD neurons. Statistical analyses were made using one-way ANOVA (F(2,15) = 45.43, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. C, Double IF of 4-HNE (green) in combination with GFAP (red) showing high levels of oxidative damage to astrocytes in AD subjects compared with control and NDAN subjects, although less than in neurons. Magnification, 60×. Scale bar, 30 µm. C′, The quantitative analysis of IF images shows a significantly higher levels of the oxidative damage marker in AD, while NDAN and control astrocytes displayed comparable levels of damage. Statistical analyses were made using one-way ANOVA (F(2,15) = 407.9, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. D, The analysis demonstrates the significant slightly higher distribution of lipid peroxidation end product in AD neurons versus astrocytes, and the relatively higher resistance of astrocytes to oxidative damage in NDAN. Statistical analyses were made using two-way ANOVA (F(2,30) = 172.5, p < 0.0001). Values are expressed as the mean ± SD. *p < 0.05; ** p < 0.01. ns, not significant.
Figure 5.
Figure 5.
A, A′, SOD2 expression in frontal cortex of control, AD, and NDAN subjects. IF images and quantitative analyses showing a significant downregulation of SOD2 in AD patients and preserved levels of SOD2 in NDAN individuals, compared with control subjects. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 30.82, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. B–D, SOD2 expression and distribution in frontal cortex neurons and astrocytes of control, AD, and NDAN subjects. B, B′, Double IF of SOD2 (green) in combination with NeuN (red) and quantitative analysis showing significant low levels of the antioxidant enzyme in AD neurons and significantly higher levels in NDAN neurons, compared with control. Magnification 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 151.8, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. *p < 0.05; ****p < 0.0001. C, C′, Double IF of SOD2 (green) in combination with GFAP (red), and quantitative analysis of images showing the downregulation of the antioxidant enzyme in AD and NDAN, while in AD brains SOD2 mainly localizes to astrocytes. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 68.34, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. **p < 0.01; ****p < 0.0001. D, The diagram shows an impairment of the antioxidant response in AD subjects and a preserved scavenging system in NDAN. Significantly higher levels of SOD2 in neurons and astrocytes of NDAN and AD, respectively, are highlighted. Statistical analyses were made using two-way ANOVA (F(2,30) = 39, p < 0.0001). Values are expressed as the mean ± SD. ****p < 0.0001. ns, not significant.
Figure 6.
Figure 6.
A, A′, PGC1α expression in frontal cortex of control, AD, and NDAN subjects. The quantitative analyses of the IF images showing a downregulation of PGC1α in AD and preserved levels in NDAN subjects. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 18.4, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ***p < 0.001. B–D, PGC1α expression and distribution in frontal cortex neurons and astrocytes of control, AD, and NDAN subjects. B, B′, Double IF of PGC1α (green) in combination with NeuN (red) showing significantly lower levels of the transcription factor in AD neurons and preserved levels in NDAN neurons. Magnification, 60×. Scale bar, 30 µm. Quantitative analysis of IF images shows significantly higher levels of PGC1α in NDAN neurons compared with AD neurons. Statistical analyses were made using one-way ANOVA (F(2,15) = 134, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. C, C′, Double IF of PGC1α (green) in combination with GFAP (red) and quantitative analysis showing the downregulation of the transcription factor in AD and NDAN astrocytes compared with controls. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 54.69, p < 0.0001) following Tukey's multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. D, The analysis shows a downregulation of PGC1α in AD frontal cortex although with a prevalent localization in astrocytes compared with neurons. Conversely, NDAN and control astrocytes display comparable levels of PGC1α, and a significant increase in neurons. Statistical analyses were made using two-way ANOVA (F(2,30) = 134.8, p < 0.0001). Values are expressed as the mean ± SD. **p < 0.01; ***p < 0.001. ns, not significant.
Figure 7.
Figure 7.
A, A′, PPARα expression in frontal cortex of control, AD, and NDAN subjects. The quantitative analyses of the IF images showing upregulation of PPARα in AD compared with control subjects. NDAN and control subjects show comparable levels of the nuclear receptor. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 52.78, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. B–D, PPARα expression and distribution in frontal cortex neurons and astrocytes of control, AD, and NDAN subjects. B, B′, Double IF of PPARα (green) in combination with NeuN (red) showing significant downregulation of the nuclear receptor in AD neurons. Magnification, 60×. Scale bar, 30 µm. Quantitative analysis of IF images showing a similar neuronal localization of PPARα in NDAN compared with AD subjects. Statistical analyses were made using one-way ANOVA (F(2,15) = 31.94, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. ****p < 0.0001. C, C′, Double IF of PPARα (green) in combination with GFAP (red) and quantitative analysis showing a predominant localization of the nuclear receptor in AD astrocytes, while NDAN and control astrocytes display comparable levels of PPARα. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 19.85, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. **p < 0.01; ****p < 0.0001. D, The analysis shows the significant upregulation of PPARα in AD astrocytes. NDAN and control subjects display comparable levels of PPARα in both neurons and astrocytes. Statistical analyses were made using two-way ANOVA (F(2,30) = 42.54, p < 0.0001). Values are expressed as the mean ± SD. *p < 0.05; ***p < 0.001; ****p < 0.0001. ns, not significant.
Figure 8.
Figure 8.
A, A′, CAT expression in frontal cortex of control, AD, and NDAN subjects. A, The quantitative analyses of the IF images showing upregulation of CAT in AD compared with control subjects. NDAN and control subjects show comparable levels of the antioxidant enzyme. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 6.387, p = 0.0099) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. *p < 0.05. B–D, CAT expression and distribution in frontal cortex neurons and astrocytes of control, AD, and NDAN subjects. B, B′, Double IF of CAT (green) in combination with NeuN (red) and quantitative analysis showing significant lower levels of the antioxidant enzyme in AD neurons compared with control neurons, and significantly higher levels in NDAN neurons compared with AD neurons. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 50.94, p < 0.0001) following Tukey's test multiple-comparisons test. Values are expressed as the mean ± SD. **p < 0.01; ****p < 0.0001. C, C′, Double IF of CAT (green) in combination with GFAP (red) showing predominant nuclear localization of the H2O2-scavenging enzymes in NDAN patients. Quantitative analysis of images showing no significant changes of CAT expression in astrocytes. Magnification, 60×. Scale bar, 30 µm. Statistical analyses were made using one-way ANOVA (F(2,15) = 6.752, **p = 0.0081) following Tukey's multiple-comparisons test. Values are expressed as the mean ± SD. **p < 0.01. D, The diagram shows a significantly predominant localization of CAT in astrocytes rather than in neurons in all the three considered conditions. Statistical analyses were made using two-way ANOVA (F(2,30) = 28, p < 0.0001). Values are expressed as the mean ± SD. **p < 0.01; ****p < 0.0001. ns, not significant.
Figure 9.
Figure 9.
Regulation of PGC1α via miRNA-485. Assessment of miRNA-485 levels in frontal cortices of control, AD, and NDAN subjects by real-time PCR shows an increase in AD, whereas a significant decrease in NDAN versus control is observed. Statistical analyses were made using one-way ANOVA (F(2,9) = 21.46, p = 0.0004) following Tukey's multiple-comparisons test. Values are expressed as the mean ± SD. **p < 0.01; ***p < 0.001. ns, not significant.
Figure 10.
Figure 10.
Antioxidant response and oxidative stress in AD and NDAN frontal cortices. Left, Aβ plays a critical role in AD pathogenesis leading to mitochondrial alterations in terms of biogenesis and functions. Downregulation of PGC1α, possibly inhibited by high levels of miRNA-485, and its target gene SOD2 contribute to energy dysmetabolism. Mitochondrial dysfunction and antioxidant response impairment lead to ROS increase and oxidative stress, affecting both mitochondria and peroxisomes (yellow lightning). PPARα increase, in response to redox imbalance, may activate a peroxisomal-based energy metabolism, as well an ROS-detoxifying mechanism (dotted lines), compensating for mitochondrial dysfunction. Right, In the frontal cortex of NDAN subjects, the lack of PGC1α miRNA-485-related inhibition results in unchanged levels of PGC1α and SOD2, and thus preserved antioxidant response and mitochondrial integrity, blunting oxidative damage. This suggests that the activation of a PGC1α-dependent response, to cope with the redox imbalance, is crucial to prevent Aβ-mediated toxicity. The unchanged levels of PPARα keep peroxisomes at a physiological level. Based on this, both mitochondria and peroxisomes cooperate in ROS and energy metabolism.
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