Protein changes in synaptosomes of Huntington's disease knock-in mice are dependent on age and brain region

Disposition of Proteins and Lipids in Synaptic Membrane Compartments Is Altered in Q175/Q7 Huntington’s Disease Mouse Striatum

“…We also looked at the distribution of the neuronal specific protein GLUT3, presynaptic proteins such as VGLUT1 and VGLUT2, and post synaptic proteins. For PSD95 and VGLUT2 we have reported significant reductions in levels in synaptosomes of Q175/Q7 versus Q7/Q7 striatum; no change in protein levels was observed for Na + /K + ATPase, SNAP25, and VGLUT1 (Sapp et al, 2020). We observed that several membrane-associated proteins that distribute to fractions 1-4 appeared altered from their normal distribution of peaking in fraction 2 in Q7/Q7 mice to peaking in fraction 1 in Q175/Q7 HD mice, ( Figure 1B, shown for GLUT3 and Supplementary Figure 1B) similar to SNAP25.…”

“…Mutant Huntingtin expression altered functions of Synapsin 1 when targeted to the synapse in transgenic mice (Xu et al, 2013), reduces transcription of Synaptic Vesicle Protein 2C in N171-82Q transgenic mice (Peng et al, 2018) and affected interactions with Syntaxin 1A (Kaltenbach et al, 2007). Differences were found in levels of pre-and postsynaptic proteins in Q50/Q7, Q140/Q7, Q175/Q7, and Q140/Q140 HD mouse brain synaptosomes compared to wild-type including SCN4B, GABAa1, calmodulin, VGlut1, VGlut2, SNAP25, and PSD95 (Valencia et al, 2013;Sapp et al, 2020). Significant loss of the postsynaptic signaling proteins, PDE10a and DARPP32 was reported by us and others (Beaumont et al, 2016;Hosp et al, 2017;Skotte et al, 2018;Sapp et al, 2020).…”

“…our previous results with n = 3 mice showed that SNAP25 had a trend to shift from fraction 2 in Q7/Q7 to fraction 1 in Q175/Q7 HD (see also Figure 1B), the change was not significant with a larger sample size (Figure 2; p = 0.0527, unpaired t-test, N = 9). We also analyzed distribution of proteins wellknown to have reduced levels in HD mouse models including DARPP32, PDE10A, and SCN4B (Sapp et al, 2020). Although the levels of these proteins were all reduced in total signal on the western blots of fractions from Q175 compared to wildtype, their peak distribution was in lower fractions (3-7) and no shift in their percent distribution in the gradient occurred.…”

“…Previous study has shown successful isolation of synaptosome fractions from postmortem fresh brain tissue of different species using sucrose for separation (Shinagawa et al, 1963;Whittaker et al, 1964;Garey et al, 1974;Sapp et al, 2020). To determine whether frozen postmortem tissue could be assessed using iodixanol gradient fractionation and if HD mutation affected the subcellular distribution of proteins in the synaptic compartment, we examined samples of human putamen from 3 controls to 3 HD postmortem brains (Supplementary Table 9).…”

“…Based on studies in human postmortem brain and Q140 HD mice there are stages of synaptic loss with reduced inputs from cortex and thalamus to the striatum occurring early and output pathways of the striatum affected later (Reiner and Deng, 2018). In neurons, mutant Huntingtin collects in aggregates including at synapses (Li et al, 2003;Sapp et al, 2020), and distributes to the same sites as wild-type Huntingtin including on membranes and in synapses (Aronin et al, 1995). Wild-type Huntingtin has been shown to be associated with the presynaptic cytomatrix (Yao et al, 2014) and mutant Huntingtin can sequester Bassoon, a polyglutamine containing structural protein, and Piccolo away from the cytoskeletal matrix of the active zone (CAZ) in R6/1 mice and human HD brain (Huang et al, 2020).…”