This study revealed multifaceted regulation of ALDH1A1 by Cdk5 in Alzheimer’s disease (AD) pathogenesis. ALDH1A1 is a multifunctional enzyme with dehydrogenase, esterase, and anti-oxidant activities. ALDH1A1 is also a major regulator of retinoic acid (RA) signaling, which is critical for normal brain homeostasis. We identified ALDH1A1 as both physiological and pathological target of Cdk5. First, under neurotoxic conditions, Cdk5-induced oxidative stress upregulates ALDH1A1 transcription. Second, Cdk5 increases ALDH1A1 levels by preventing its ubiquitylation via direct phosphorylation. Third, ALDH1A1 phosphorylation increases its dehydrogenase activity by altering its tetrameric state to a highly active monomeric state. Fourth, persistent oxidative stress triggered by deregulated Cdk5 inactivates ALDH1A1. Thus, initially, the good Cdk5 attempts to mitigate ensuing oxidative stress by upregulating ALDH1A1 via phosphorylation and paradoxically by increasing oxidative stress. Later, sustained oxidative stress generated by Cdk5 inhibits ALDH1A1 activity, leading to neurotoxicity. ALDH1A1 upregulation is highly neuroprotective. In human AD tissues, ALDH1A1 levels increase with disease severity. However, ALDH1A1 activity was highest at mild and moderate stages, but declines significantly at severe stage. These findings confirm that during the initial stages, neurons attempt to upregulate and activate ALDH1A1 to protect from accruing oxidative stress-induced damage; however, persistently deleterious conditions inactivate ALDH1A1, further contributing to neurotoxicity. This study thus revealed two faces of Cdk5, good and bad in neuronal function and survival, with a single substrate, ALDH1A1. The bad Cdk5 prevails in the end, overriding the good Cdk5 act, suggesting that Cdk5 is an effective therapeutic target for AD.
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Aldehyde dehydrogenase A1 isoform
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This work was supported by the National Institute on Aging (R21-AG 47447 to KS.). We thank Dr. David Schubert for the HT22 cells. pLKO.1 TRC vector was a gift from David Root (Addgene plasmid #10878) . We thank New York Brain Bank for the cohort 1 clinical specimens (NIH P50 AG008702) and Michigan Brain Bank (University of Michigan Alzheimer’s Disease Core Center supported by 5P30 AG053760) for providing the cohort 2 clinical specimens (Table 1).
This work was supported by grant from National Institute on Aging, National Institutes of Health (R21-AG 47447).
The authors declare that they have no competing interests.
Electronic Supplementary Material
Validation of Antibodies and siRNA knockdown (A) Validation of ALDH1A1 shRNA. HT22 cells were transfected with ALDH1A1 shRNA and cell lysates were analyzed 30 h later by immunoblot analysis (B) Validation of Cdk5 shRNA. HT22 cells were transfected with Cdk5 shRNA and cell lysates were analyzed 30 h later by immunoblot analysis (C) ALDH1A1 localization in HT22 cells. Immunofluorescent analysis of ADH1A1 in paraformaldehyde-fixed HT22 cells using ALDH1A1 antibody (D) Cdk5 localization in HT22 cells. Immunofluorescent analysis of Cdk5 in formaldehyde-fixed HT22 cells using a Cdk5 antibody. Supplementary Fig. 2. Cdk5-mediated phosphorylation regulates ALDH1A1 levels. (A) HT22 cells were treated with glutamate for 12 and 24 h, in the presence and absence Cdk5 shRNA and then the total levels of ALDH1A1 mRNA were analyzed using semi quantitative RT-PCR. (B) ALDH1A1 mRNA levels in HT22 cells in response to glutamate treatment with or without Cdk5 shRNA. Graphical results are mean ± SEM of three independent experiments. *P < 0.05, compared with untreated HT22 cells. #P < 0.05, compared with only glutamate treated HT22 cells. (C) ALDH1A1-HT22 cells were treated similarly as described for A and total levels of ALDH1A1 analyzed. (D) Average relative ratios of HA (ALDH1A1) band intensities to alpha-tubulin band intensities upon glutamate treatment as obtained from three independent experiments. *P < 0.05, compared with untreated ALDH1A1-HT22 cells. Supplementary Fig. 3. Cdk5-mediated phosphorylation of ALDH1A1 increases its dehydrogenase activity. (A) Cdk5 increases ALDH1A1 enzymatic activity. Comparative spectrophotometric analysis of ALDH1A1 activity upon phosphorylation by Cdk5 after 150 min. *P < 0.05, compared with ALDH1A1. (B) ALDH1A1-phosphorylation-resistant mutant have minimal enzymatic activity. ALDH1A1 activity was measured after 150 min. *P < 0.05, compared with ALDH1A1. Supplementary Fig. 4. ALDH1A1 activity initially increases, but later decreases in glutamate-treated HT22 cells. (A) Glutamate treatment increased ALDH1A1 activity in HT22 cells while the phosphorylation-resistant mutant cells have diminished enzymatic activity. ALDH1A1 activity was measured after 105 min. *P < 0.05, compared with control group. (B) Cdk5 depletion partially prevents the increase in ALDH1A1 activity in 12 h glutamate-treated cells, but rescues~50% of the decrease in ALDH1A1 activity in 24 h glutamate-treated cells. ALDH1A1 activity was measured after 105 min. *P < 0.05, compared with control group. (C) Elimination of oxidative stress restores ALDH1A1 activity to a significant extent. ALDH1A1 dehydrogenase activity determined after 105 min. *P < 0.05, compared with control group. (D) DTT treatment is beneficial for ALDH1A1 enzymatic activity initially. HT22 cells were treated with glutamate for 12 h and 24 h. ALDH1A1 was immunoprecipitated using ALDH1A1 antibody, and enzyme activity was performed with or without 10 mM DTT in reaction buffers as described in material and methods. ALDH1A1 activity was measured after 105 min. *P < 0.05, compared with control group. Supplementary Fig. 5. Cdk5 increases ALDH1A1 activity in primary neurons. (A) Cdk5 depletion partially prevents the increase in ALDH1A1 activity in 12 h glutamate-treated cells, but rescues~50% of the decrease in ALDH1A1 activity in 24 h glutamate-treated cells. ALDH1A1 activity was measured after 105 min. *P < 0.05, compared with control group. (B) Elimination of oxidative stress restores ALDH1A1 activity to a significant extent. ALDH1A1 dehydrogenase activity determined after 105 min. *P < 0.05, compared with control group. (C) DTT treatment is beneficial for ALDH1A1 enzymatic activity initially. Primary neurons were treated with glutamate for 12 h and 24 h. ALDH1A1 was immunoprecipitated using ALDH1A1 antibody, and enzyme activity was performed with or without 10 mM DTT in reaction buffers as described in material and methods. ALDH1A1 activity was measured after 105 min. *P < 0.05, compared with control group. (PDF 443 kb)
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Nikhil, K., Viccaro, K. & Shah, K. Multifaceted Regulation of ALDH1A1 by Cdk5 in Alzheimer’s Disease Pathogenesis. Mol Neurobiol 56, 1366–1390 (2019). https://doi.org/10.1007/s12035-018-1114-9