Therapeutics potentiating microglial p21-Nrf2 axis can rescue neurodegeneration caused by neuroinflammation

Abstract

Neurodegenerative disorders are caused by progressive neuronal loss, and there is no complete treatment available yet. Neuroinflammation is a common feature across neurodegenerative disorders and implicated in the progression of neurodegeneration. Dysregulated activation of microglia causes neuroinflammation and has been highlighted as a treatment target in therapeutic strategies. Here, we identified novel therapeutic candidate ALGERNON2 (altered generation of neurons 2) and demonstrate that ALGERNON2 suppressed the production of proinflammatory cytokines and rescued neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease model. ALGERNON2 stabilized cyclinD1/p21 complex, leading to up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which contributes to antioxidative and anti-inflammatory responses. Notably, ALGERNON2 enhanced neuronal survival in other neuroinflammatory conditions such as the transplantation of induced pluripotent stem cell-derived dopaminergic neurons into murine brains. In conclusion, we present that the microglial potentiation of the p21-Nrf2 pathway can contribute to neuronal survival and provide novel therapeutic potential for neuroinflammation-triggered neurodegeneration.

Fig. 1. ALGERNON2 rescues DA neuronal loss upon MPTP injection.

(A₁) Experimental scheme. (A₂) Western blotting of the striatal tissue of animals treated as indicated. TH, tyrosine hydroxylase; DAT, dopamine transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (A₃) Quantification of Western blotting analysis. *P < 0.05. (B₁) Experimental scheme. (B₂) Western blotting analyses from the striatal tissue of animals with indicated treatment. (B₃) Quantification of Western blotting analysis. The expression level of TH was normalized to that of sham control. *P < 0.05. (C₁) Experimental scheme. (C₂) Representative images of the substantia nigra (SN) and striatum (Cpu) of sham control or MPTP-treated animals administered with vehicle (Veh) or ALGERNON2. Tissue was stained with anti-TH antibody. Scale bars, 200 μm (SN) and 100 μm (Cpu). (D) Quantification of the number of TH-positive cells in SN. *P < 0.05. n = 3 each. (E) Measurement of DA amount in striatal tissues of animals with indicated treatment. *P < 0.05. n = 6 each. (F) Average time taken to make a turn (T-turn) and of locomotor activity (T-LA) of animals with indicated treatment during a pole test. **P < 0.01. n = 6, 7, and 6 for each group.

Fig. 2. ALGERNONs mediate neuroprotection in the presence of glial cells.

(A) Representative images of ventral midbrain–derived neurons at 7 days in vitro. DA neurons and glial cells were visualized with TH (green) and S100β (magenta), respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm. (B) Viability was assessed by the number of TH-positive neurons and normalized to that of control conditions at 0 μM MPP⁺. Treatment with ALGERNONs improved neuronal survival upon oxidative stress in the presence of glial cells. *P < 0.05. DMSO, dimethyl sulfoxide. (C) Representative images of human induced pluripotent stem cell (iPSC)–derived DA progenitors cultured with murine glial cells. Human iPSC-derived DA neurons were visualized with anti–human nuclei (hNuclei; blue), Nurr1 (magenta), and TH (green) antibodies. Scale bar, 50 μm. (D) Quantitative analyses of the number of hNuclei, and the level of Nurr1 and TH mRNA. Nurr1 and TH were used as markers of DA neurons in early differentiation and maturation, respectively. ALGERNON2 rescued neuronal loss in the presence of glial cells. Data were normalized to the control condition without H₂O₂ treatment. *P < 0.05.

Fig. 3. Nrf2 and p21 enhancement by ALGERNONs.

(A) Representative images of glial cultures isolated from the murine brain. Scale bar, 10 μm. (B). Experimental scheme for the automated evaluation of the expression of cyclin D1 and p21. The image acquisition and analyses were performed in an automated manner. (C₁) Representative images of glial cells treated with indicated compounds. Scale bar, 25 μm. (C₂) An example of quantification of the signal intensity of Nrf2 in nuclei of Cd11b-positive cells. Glial cells were treated with ALGERNON2 (2.5 μM) for indicated periods. The full set of experimental data is available in fig. S3F. (D₁) Representative examples of the histogram of Nrf2 intensity in nuclei of Cd11b-positive cells treated as indicated. ALGERNON2 treatment shifted the histogram rightward; this shift was abolished in the presence of p21 small interfering RNA (siRNA). (D₂) Dot plots from the same experiments shown in (D₁). ***P < 0.001. Note that the increase in nuclear Nrf2 signal upon lipopolysaccharide (LPS) and LPS/ALGERNON2 treatment was not observed in cells treated with p21 siRNA compared with those that were treated with control siRNA. (E) Experimental scheme (top) and Western blotting of striatal tissue from animals treated as indicated (bottom). Quantitative analyses of Western blotting (right). *P < 0.05.

Fig. 4. ALGERNONs suppress cytokine production following LPS stimulation through Nrf2 stabilization in microglia.

(A) Representative images of SN tissues of sham control or MPTP-treated animals administered with vehicle or ALGERNON2 at day 3. Tissues were stained with anti-TH (green), Iba1 (magenta), and GFAP (gray scale) antibodies. Scale bar, 50 μm (left panels). Amoeboid-shaped activated microglia were observed in MPTP-treated animals with vehicle administration. Scale bar, 20 μm (right panels). (B and D) The production of cytokines (B), chemokines, and iNOS mRNA (D) upon LPS treatment was assessed by qPCR. The values were normalized to those of nontreated conditions. *P < 0.05. (C) Cytokine production upon LPS stimulation was quantified with enzyme-linked immunosorbent assay (ELISA). *P < 0.05. IL, interleukin. (E and F) Real-time qPCR analyses of the production of indicated genes in the presence or absence of Nrf2 (E) or upon treatment of ALG, SLF, or combined (F). *P < 0.05, **P < 0.01, and ***P < 0.001. (G) Experimental scheme (top). Animals were administered with ALGERNONs 1 hour before MPTP injection. Tissues were collected 24 hours after the last MPTP injection. Quantitative analyses of striatal tissue by qPCR (left) and ELISA (right) (lower graphs). *P < 0.05. n = 6 each.

Fig. 5. ALGERNON2 rescues the neurodegeneration caused by neuroinflammation.

(A) Experimental scheme. LPS was intraperitoneally injected at 1 mg/kg for four consecutive days. Brains were collected for analyses on indicated days. (B) Representative images of the SN on indicated days. Scale bar, 200 μm. (C) Quantitative analysis of mRNA expression in the striatum. n = 10 each. *P < 0.05 and **P < 0.01. (D) Representative images of the SN from animals treated as indicated. TH (green), Iba1 (gray scale), and GFAP (magenta) were used as markers of DA neurons, microglia, and astrocytes, respectively. Scale bar, 200 μm. (E) Quantification of the number of TH-positive cells in the SN pars compacta (SNpc). n = 5 to 6 animals analyzed for each condition. Error bars represent SEM. *P < 0.05. (F) Quantitative analyses of glial activation by qPCR in striatal tissues at D1 from last LPS injection. Error bars represent SEM. *P < 0.05. +P = 0.11. (G and H) The levels of indicated cytokines and chemokines from striatal tissues on D1 were analyzed by qPCR (G) and ELISA (H). Error bars represent SEM. *P < 0.05. TNF-α, tumor necrosis factor–α.

Fig. 6. ALGERNON2 enhances the efficacy of transplantation of iPSC-DA neurons.

(A) Experimental scheme. Recipients were administered ALGERNON2 1 hour before transplantation of human iPSC-derived DA neurons. Drug administration was performed for four consecutive days after the operation. Animals were left untreated for 4 weeks. (B) Representative images of transplanted cells in striatal tissues. Arrowheads indicate iPSC-derived cells colabeled with TH or Nurr1. Scale bar, 50 μm. (C) Quantitative analyses of transplanted cells. The number of hNuclei/TH or Nurr1 double-positive cells was normalized to the number of hNuclei-positive cells. n = 7 and 5 for each condition, respectively. *P < 0.05 and **P < 0.01. (D) Graphic abstract: Upon injury or exposure to neuroinflammatory pathogens (1), microglia are activated and release cytokines and ROS (2), which triggers neuronal degeneration (3) (“neuroinflammation”). Following treatment with Dyrk1A inhibitors (4), the cyclin D1/p21 complex becomes stabilized (5). Stabilized p21 halts the degradation of Nrf2 by interrupting Nrf2-Keap1 binding (6). Stabilized Nrf2 suppresses the production of proinflammatory cytokine genes (7), which contributes to neuronal survival under neuroinflammatory conditions (8).