Analysis of protein SUMOylation and its role in Alzheimer's disease using mouse models

Post-translational modifications serve as a cellular mechanism for the regulation of the activity, stability and localization of proteins. SUMOylation is a dynamic and reversible post-translational modification, which entails the attachment of a SUMO protein to a lysine residue of the target protein. SUMOylation is involved in the regulation of numerous cellular processes including transcription, nucleocytoplasmic trafficking, and DNA repair. Three or four SUMO paralogs are present in mammals – SUMO1, SUMO2, SUMO3 and SUMO4. SUMO2 and SUMO3 exhibit extremely high sequence homology and therefore cannot be distinguished by antibodies. Interestingly, SUMO2/3 conjugation has been shown to change dramatically in response to aberrant cellular conditions. The identification of endogenous SUMO substrates has long been hindered by the transient nature of SUMOylation, the lack of reliable antibodies for affinity purification, and the modification of only a small percentage of a given SUMO substrate at a given time. Thus, in a first project, analogous to a His6-HA-SUMO1 knock-in mouse model generated in our lab, we generated a Strep-Myc-SUMO3 knock-in mouse model expressing Strep-Myc-tagged SUMO3 instead of wild type SUMO3 from the endogenous SUMO3 locus. Importantly, a main advantage of this model is the possibility to distinguish specifically SU-MO3 from SUMO2. Strep-Myc-SUMO3 knock-in and wild type mice brain homogenates were used to perform anti-Myc affinity purification, which resulted in the enrichment of free SUMO3 and SUMO3 conjugates in the eluate from the knock-in mice. Thus, we proved that the newly generated mouse model can be used as a tool for the identification of SUMO3 sub-strates. However, despite the utilization of several anti-Myc and one anti-Strep antibody, we were not able to clearly localize Strep-Myc-SUMO3 in brain sections of SUMO3 knock-in mice as the antibodies showed different staining patterns. This mouse model will be further used to study SUMO3 conjugation profiles under physiological and non-physiological condi-tions. A constantly increasing number of studies have suggested a link between SUMOylation and Alzheimer's disease. Thus, in a second project, we crossbred His6-HA-SUMO1 knock-in mice with 5xFAD, a mouse model of Alzheimer's disease, in order to assess SUMO1 conjugation profile in the context of Alzheimer's disease pathology. Using mice at different stages of dis-ease progression, we intended to identify specific changes in the localization of SUMO1 and in the global SUMO1 conjugation levels. Anti-HA immunostaining of brain sections showed that in subiculum and cortical layer V SUMO1 exhibited nuclear presence in both His6-HA-SUMO1 and His6-HA-SUMO1;5xFAD mice at any of the ages examined. Furthermore, two different anti-HA antibodies produced two different types of non-nuclear anti-HA signal in His6-HA-SUMO1;5xFAD mice. While one of the antibodies produced anti-HA signal localiz-ing to amyloid plaques, the other resulted in line-shaped signals or signals with the shape of amorphous mass, with some of the line-shaped signal surrounding amyloid plaques. Im-portantly, both anti-HA antibodies produced similar signals in the 5xFAD non-knock-in mice which strongly speaks against specificity of the signal. The predominantly nuclear localiza-tion of His6-HA-SUMO1 in both 5xFAD and non-5xFAD mice was confirmed by subcellular fractionation followed by Western blot. Regarding SUMO1 conjugation levels upon Alzhei-mer's disease pathology, anti-HA Western blot did not reveal any significant differences be-tween His6-HA-SUMO1 and His6-HA-SUMO1;5xFAD mice in both cortex and hippocampus at any of the examined ages. Furthermore, a quantitative comparison of the anti-HA signal in the neuronal nuclei of His6-HA-SUMO1 and His6-HA-SUMO1;5xFAD in both subiculum and cortical layer V did not reveal substantial differences between the two genotypes. A mi-nor increase of 25.8% was observed in the pyramidal neurons of cortical layer V of 8-week-old His6-HA-SUMO1;5xFAD mice when compared to age-matched His6-HA-SUMO1 mice. In summary, we did not discover substantial changes in SUMO1 localization and SUMO1 conjugation levels in the context of increased amyloid burden. However, we cannot conclude that the SUMO1 profile is undisturbed upon Alzheimer's disease pathology as changes in the SUMOylation pattern of individual proteins may not be detected by the techniques utilized in this study. Thus, the next step will be the investigation of differentially SUMOylated sub-strates by anti-HA affinity purification of brain homogenates from His6-HA-SUMO1, His6-HA-SUMO1;5xFAD, 5xFAD and wild type mice followed by mass spectrometry analysis.