Neurotoxic Microglial Activation via IFNγ‐Induced Nrf2 Reduction Exacerbating Alzheimer's Disease

Abstract Microglial neuroinflammation appears to be neuroprotective in the early pathological stage, yet neurotoxic, which often precedes neurodegeneration in Alzheimer's disease (AD). However, it remains unclear how the microglial activities transit to the neurotoxic state during AD progression, due to complex neuron‐glia interactions. Here, the mechanism of detrimental microgliosis in AD by employing 3D human AD mini‐brains, brain tissues of AD patients, and 5XFAD mice is explored. In the human and animal AD models, amyloid‐beta (Aβ)‐overexpressing neurons and reactive astrocytes produce interferon‐gamma (IFNγ) and excessive oxidative stress. IFNγ results in the downregulation of mitogen‐activated protein kinase (MAPK) and the upregulation of Kelch‐like ECH‐associated Protein 1 (Keap1) in microglia, which inactivate nuclear factor erythroid‐2‐related factor 2 (Nrf2) and sensitize microglia to the oxidative stress and induces a proinflammatory microglia via nuclear factor kappa B (NFκB)‐axis. The proinflammatory microglia in turn produce neurotoxic nitric oxide and proinflammatory mediators exacerbating synaptic impairment, phosphorylated‐tau accumulation, and discernable neuronal loss. Interestingly, recovering Nrf2 in the microglia prevents the activation of proinflammatory microglia and significantly blocks the tauopathy in AD minibrains. Taken together, it is envisioned that IFNγ‐driven Nrf2 downregulation in microglia as a key target to ameliorate AD pathology.

Normal vs sAD 40 Two-tailed t test <0.001Y (1) One-way ANOVA was performed with Tukey post-hoc correction for multiple comparisons.
Two-tailed unpaired Student's t-test was performed for two variances.

Figure S4 .
Figure S4.Production of IFNg by reactive astrocytes in AD mini-brains.Immunostaining results show that the co-localization of reactive astrocytes (GFAP high , green) and IFNg (red) in 9 wks AD models.Scale bars, 20 μm.

Figure S5 .
Figure S5.Investigation of soluble factors from human AD mini-brains affecting neurodegenerative phenotype transition.a-b.iMGs were treated with each soluble factor from 9 wks AD models and stained with neurodegenerative markers as (a) CD86 and (b) iNOS.All data represent mean ± SD (one-way ANOVA with Tukey's multiple-comparisons test, n=20).

Figure S7 .
Figure S7.Confirmation of Nrf2 downregulation by siRNA.a-b.(a) Immunostaining and (b) western-blotting data confirmed the Nrf2 downregulation by the treatment of siNrf2 encapsulated in polymeric-based carriers.Scale bars, 20 μm.

Figure S8 .
Figure S8.Investigation of neuroprotective phenotype transition by the treatment of AD CM. a-b.(a) Florescent images and (b) quantitative results showing the expression of CD206, a marker for M2 type microglia, in MG or Nrf2 OE MG treated with Con CM or AD CM (oneway ANOVA with Tukey's multiple-comparisons test, n=11).Scale bars, 50 μm.All data represent means ± SD.

Figure S9 .
Figure S9.Effects of H2O2 scavenger on the production levels of H2O2 and IFNγ in AD mini-brains.a-b.(a) H2O2 and (b) IFNγ presented in the conditioned media of Con and AD mini-brains were measured (one-way ANOVA with Tukey's multiple-comparisons test, n=4).All data represent means ± SD. **, P<0.01.

Figure S11 .
Figure S11.Downregulation of Nrf2 in neurodegenerative microglia correlates with neurodegeneration in AD animal models.a. Preparation of 5XFAD mouse models as described previously. [1]We assigned female and male mice (40-weeks old) to each group equally.Aβ plaques were formed in the cerebral cortex region of 5XFAD mice confirmed by immunostaining.b.Significant increase of IFNγ level was confirmed by qPCR (two-tailed unpaired t-test, n=11).c-d.(c) Western-blotting data and (d) quantitative analysis showing the expression level of Nrf2 in microglia extracted from Normal and 5XFAD mouse brains (twotailed unpaired t-test, n=4).e. Immunofluorescent images of mouse brain tissues indicating the presence of Nrf2 low CD86 high microglia (marked with arrows).e-f.(e) Fluorescent images and (f) quantitative analysis showing the significant increase of Nrf2 low CD86 high population among the neurodegenerative microglia in microglia in 5XFAD mice (two-tailed unpaired t-test, n=4).g.Passive avoidance test confirming the recovery of fear conditioning memory by the overexpression of microglial Nrf2 in 5XFAD mice (one-way ANOVA with Tukey's multiplecomparisons test, n=6).Scale bars, 50 μm.All data represent means ± SD. *, P<0.05; **, P<0.01.

Figure S12 .
Figure S12.Induction of microglial recruitment by soluble factors from AD CM. a-b.(a) Fluorescent images and (b) quantitative analysis showing the microglial recruitment from the annular chamber (a.c.) to the central chamber (c.c.) by soluble factors found in AD CM (oneway ANOVA with Tukey's multiple-comparisons test, n=5).Scale bars, 500 μm.All data represents means ± SD. **, P<0.01.