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  • Additionally we observed an increase in global O GlcNAcylati


    Additionally, we observed an increase in global O-GlcNAcylation levels in KRN 7000 structure tissue from mature mice, an effect more pronounced in brain cortex from WT mice (Fig. 2). In fact, it is known that age alters brain glucose metabolism [39] modulating O-GlcNAcylation levels. In accordance with our observations, Fülöp et al. [40] observed an increase in the levels of this posttranslational modification in the brain of aged Brown-Norway rats. However, other studies revealed a decrease in O-GlcNAcylation levels in aged mice [41,42] rendering the brain more prone to dysfunction under stress conditions such as brain ischemia. However, those studies used 22–24-month-old WT mice while in our study we used younger (11–12-month old) mice. In our study, 11–12-month-old 3xTg-AD mice show a reduction in brain O-GlcNAcylation levels, an effect more pronounced in brain cortex (Fig. 2). Accordingly, a recent study performed in 12-month-old 3xTg-AD mice shows a decrease of total O-GlcNAcylation levels associated with altered OGT and OGA activation [43]. Proteomics analysis identified several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the AD progression such as neuronal structure, protein degradation and glucose metabolism [43]. In accordance the proteomic quantitative analysis performed by Wang and colleagues [44] revealed that post mortem AD brain tissue presents altered O-GlcNAcylated proteins belonging to several structural and functional categories such as synaptic, cytoskeleton and memory-associated proteins. Besides that, altered O-GlcNAcylation cycling might result in abnormal O-GlcNAcylated tau and APP contributing to the accumulation of toxic species in the brain supporting the idea that impaired O-GlcNAcylation levels contribute to the progression of AD [34]. The exposure of differentiated SH-SY5Y cells to Aβ1–42 (Fig. 3C), OA (Fig. 4C) or STZ (Fig. 5C) induced a significant decrease in global O-GlcNAcylation levels. Evidence from the literature supports our observations since both Aβ and tau can be modified by O-GlcNAcylation [34]. It was shown that the increase in O-GlcNAcylated APP, particularly at threonine 576 residue [45], promotes its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased Aβ production [[45], [46], [47]]. It was also reported that genetic and pharmacological tools that increase the levels of O-GlcNAcylation increase non-amyloidogenic α-secretase processing resulting in increased levels of the neuroprotective sAPPα fragment and decreased Aβ secretion [48]. A reciprocal relationship between O-GlcNAcylation levels and phosphorylation was also documented during the pathological course of AD. Using starved mice to mimic AD-related hypometabolism Liu and collaborators [38] reported that decreased O-GlcNAcylation levels are associated with increased tau hyperphosphorylation. Interestingly, hyperphosphorylated tau contains 4-fold less O-GlcNAcylation than non-hyperphosphorylated tau, which fosters the relationship between O-GlcNAcylation and phosphorylation of tau in the human brain [35]. The present study also establishes a strong correlation between global O-GlcNAcylation levels and cellular viability and ΔΨm (Figs. 3D, 4D, 5D) in three different KRN 7000 structure in vitro models of AD. The loss of cell viability that accompanies O-GlcNAcylation reduction in these in vitro models of AD is not surprising taking into account that this posttranslational modification is particularly enriched in neuronal synapses [19,49] and modifies several post-synaptic density proteins [50]. Regarding ΔΨm decay, it was recently found that mitochondrial components are modified by O-GlcNAcylation. An elegant study from Cha and collaborators revealed that ATP synthase subunit α (ATP5A) is a substrate of O-GlcNAcylation [51]. Remarkably, these authors observed that O-GlcNAcylation of ATP5A is decreased in AD pathology since Aβ blocks the direct interaction between ATP5A and mitochondrial OGT leading to impaired ATPase activity and ATP depletion [51]. Another study showed that disruption of posttranslational modification of proteins with O-GlcNAcylation via overexpression of OGT or OGA impairs mitochondrial function [52]. More recently, it was reported that sustained O-GlcNAcylation elevation in SH-SY5Y neuroblastoma cells increase OGA expression and reduced cellular respiration and ROS generation and cells with elevated O-GlcNAcylation levels had elongated mitochondria and increased ΔΨm [53]. The same study shows that OGT knockdown in the liver of mice increases ROS levels and the nuclear respiratory factor (NRF) 2 antioxidant response and impairs respiration [53]. Using siRNA in HeLa cells, Sacoman and colleagues [54] found that reducing endogenous mitochondrial OGT (mOGT) expression leads to alterations in mitochondrial structure and function, including dynamin-related protein (Drp)1-dependent mitochondrial fragmentation, reduction in ΔΨm, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. We also evaluated the impact of reduced O-GlcNAcylation on mitochondrial morphology and distribution in in vitro models of AD. In our study the exposure of differentiated SH-SY5Y to OA and STZ promoted the collapse of mitochondrial network as evidenced by the perinuclear accumulation of smaller and rounder mitochondria, these alterations being more pronounced in the OA-induced model of AD (Fig. 6). It has been reported that the mitochondrial motor-adaptor Milton that tethers mitochondria to the kinesin motors facilitating the anterograde movement of mitochondria is targeted by O-GlcNAcylation, being a substrate for OGT. Briefly, during synaptic activity Milton's O-GlcNAcylation arrests the motile pool of mitochondria in pre- and post-synaptic areas in order to assure ATP production and Ca2+ buffering essential for synapse maintenance, acting as a stop signal [20]. In this sense, it is tempting to speculate that loss of Milton O-GlcNAcylation underlies mitochondrial mislocalization in AD, leading to a drastic reduction in the stationary mitochondrial pool at the synapses, which in turn contributes to an energetic catastrophe that culminates in synaptic “starvation” and neuronal loss. However, several components of the mitochondrial fusion-fission and trafficking machinery should be evaluated in future experiments to gain further insights on the relation between O-GlcNAcylation and mitochondrial dynamics in AD pathology.