Templated α-Synuclein Inclusion Formation Is Independent of Endogenous Tau

Abstract

for her help with purifying recombinant α-synuclein and Dr. Karen Gamble for advice on statistics. We also are thankful for UAB's High Resolution Imaging Core which provides the confocal microscope for the expansion microscopy images.

Neuronopathic GBA1 L444P mutation accelerates Glucosylsphingosine levels and formation of hippocampal alpha-synuclein inclusions

“…To test whether GBA1 +/L444P mice show enhanced dopaminergic loss, immunohistochemistry and unbiased stereology were performed using antibodies to TH to quantify SNc dopamine neurons. Both GBA1 +/+ mice and GBA1 +/L444P mice injected with fibrils had an approximately 50% reduction of dopaminergic neurons in the SNc compared to GBA +/+ mice injected with α-syn monomer ( Figure 6a and 6c ) as previously shown (Froula et al, 2019; Stoyka et al, 2020, 2021). Similarly, GBA1 +/L444P fibril-injected mice treated with venglustat showed a 50% decrease in TH + neurons compared to GBA1 +/+ monomer injected mice fed venglustat.…”

“…To study the effects of GBA1 L444P expression on α-syn inclusion formation, we first used primary hippocampal neurons from GBA1 +/+ mice and GBA1 +/L444P knock-in mice exposed to sonicated α-syn fibrils on DIV7 and analyzed 14 days later (DIV21) (Volpicelli-Daley et al, 2011). Primary hippocampal neurons from mice were chosen as our model culture system because we achieve reliable, robust formation of α-syn inclusions in response to exposure to fibrils (Volpicelli-Daley et al, 2014; Froula et al, 2018; Brzozowski et al, 2021; Stoyka et al, 2021). Because the formation of α-syn inclusions depend on neuronal outgrowth (Murphy et al, 2000), we quantified axonal density using neurofilament-H (NFH; heavy polypeptide).…”

“…GBA1 +/L444P mice showed a faster total descent time compared to GBA1 +/+ mice ( Figure 3b, left ), but there was no difference in turnaround time or the time descending the pole ( Figure 3b, middle and right ). Lastly, fear conditioning was performed to measure associative learning, previously shown to be impaired in mice with fibril-induced inclusions (Stoyka et al, 2020, 2021). During the training phase, both groups of mice showed increased freezing over time in response to a tone paired with a shock with approximately 73.6% and 73.8%% time freezing after foot shock for GBA1 +/+ and GBA1 +/L444P mice, respectively ( Figure 3c, left ).…”

“…Ten months post injection, mice were perfused, and immunofluorescence was performed on fixed brain sections for p-α-syn positive inclusions. The dmPFC, hippocampus, and SNc were analyzed as previously described (Stoyka et al, 2020, 2021). Mice injected with monomer showed no p-α-syn inclusions, regardless of GBA1 L444P expression as previously shown in our publications (Froula et al, 2019; Stoyka et al, 2020).…”

“…Brains were then frozen in methylbutane solution (−50 to −60 °C) and sectioned into 40 μm serial sections with a freezing microtome (Leica SM 2010R). Serial sections spaced 240 μm apart representing the entire forebrain were collected in a 6-well tray using a freezing microtome (Stoyka et al, 2020, 2021). Sections were stored at −20 °C in a solution of 50% glycerol, and 0.01% sodium azide in Tris-buffered saline (TBS).…”