1) Identification of sleep-dependent phosphorylation sites of tau in the mouse
I combined an analysis on published databases on sleep-wake evoked changes in phosphoproteomics (whole brain homogenate), with the analysis of our in-house optimized phosphoproteomics pipeline in both cortex and hippocampus. Specifically, we investigated what the impact of 6 hours ad lib sleep at the beginning of the light phase (the habitual rest phase of mice) is on pTau levels, compared to mice that were sleep deprived for 6 hours at the same time of the day. In our data, we found in both cortex and hippocampus significant sleep-dependent increases in several pTau sites that partially overlapped with known pTau sites present in Alzheimer’s Disease.
Implementation: Excellent. Brain specific differences in pTau induced by sleep could be assessed by measuring pTau in cortex and hippocampus separately. With our proposed technique (immunohistofluorescence) to assess subcellular relocalization of pTau, we did not identify sleep-evoked changes. We are currently establishing a different technique to image pTau at higher resolution to understand whether there is a global increase in pTau during sleep, or whether it relocates to specific subcellular locations. Quantification of pTau in cerebrospinal fluid has not been completed and will be assessed in future work.
2) Temperature-dependence of p-tau sites, using primary cortical neurons
Using primary cortical neurons from the mouse, I confirmed that a decrease in temperature of 2°C for 6 hours also induces a significant phosphorylation in some but not all phosphosites evoked by sleep (aim 1), phenocopying partly our observation in sleep. Combined with the reanalysis of available literature and published databases, we have determined a group of pTau sites that are evoked by temperature and sleep. Intriguingly, most of these sites are associated with tauopathies including Alzheimer’s Disease.
Implementation: Good. There is confirmation that a sleep-like reduction in temperature can evoke an increase in pTau using mass spectrometry. Assessment on whether this finding holds in human induced neurons is pending.
3) Identify the contribution of sleep-evoked pTau sites to sleep-dependent synaptic plasticity
If Tau and its phosphorylation contribute to sleep-dependent changes in neuronal functioning, removal of the complete Tau protein will impact brain functioning during sleep. To address this question, I used an antisense-oligonucleotide method to reduce Tau protein levels in mice. This method allows to knock down Tau specifically in adulthood, thereby avoiding confounding factors such as developmental compensation via other proteins. As expected from the literature, sleep deprivation impaired synaptic plasticity. In the absence of the Tau protein in the sleep condition, the synaptic plasticity resembles that of the animals that are sleep deprived, pointing to a sleep-dependent role of the Tau protein in the synapse. Our sleep-wake phenotyping, based on brain activity and muscle tone, did not point to an overt sleep phenotype in mice lacking Tau protein under baseline conditions. Brain activity during a specific sleep stage was altered though, pointing to a role of Tau in regulating neuronal activity during specific sleep stages. However, the limited number of animals (n=4 per group) does not allow to draw solid conclusions, and we are currently expanding this study. These results suggest that Tau contributes to sleep-dependent changes in synaptic plasticity.
We have successfully established a drosophila screen method for sleep-dependent synaptic plasticity. Like in the mouse, synaptic plasticity is impaired following sleep deprivation in flies, and this impairment is attenuated in Tau knock-out flies. We are currently assessing whether reinstating mouse-Tau in a dTau knock-out fly rescues the ERG phenotype. Our next step is then to use different phosphomimetic (i.e. as in sleep) and phosphodead (as in sleep deprived conditions) forms of Tau and assess their impact on neuronal transmission under respectively sleep deprived and sleep conditions. Once we have found a combination of Tau phosphosites in the phoshomimetic mutant that rescues the sleep deprived phenotype and/or in the phosphodead mutant that imitates the sleep deprived phenotype under sleep conditions in the flies, we will assess the function of these Tau phosho sites in sleep-dependent neuronal transmission in mice.
Implementation: Good. I established that the Tau protein by itself contributes to sleep-dependent synaptic plasticity, and generated preliminary data suggesting that it contributes to brain activity. Currently we are screening for the function of the established pTau sites in neuronal transmission in the fly, and pending these results, the role of these sites will be assessed in the mouse.
