rTg(TauP301L)4510 mice exhibit increased VGlut1 in hippocampal presynaptic glutamatergic vesicles and increased extracellular glutamate release

Abstract

The molecular pathways that contribute to the onset of symptoms in tauopathy models, including Alzheimer’s disease (AD), are difficult to distinguish because multiple changes can happen simultaneously at different stages of disease progression. Understanding early synaptic alterations and their supporting molecular pathways is essential to develop better pharmacological targets to treat AD. Here, we focus on an early onset rTg(TauP301L)4510 tauopathy mouse model that exhibits hyperexcitability in hippocampal neurons of adult mice that is correlated with presynaptic changes and increased extracellular glutamate levels. However, it is not clear if increased extracellular glutamate is caused by presynaptic changes alone, or if presynaptic changes are a contributing factor among other factors. To determine whether pathogenic tau alters presynaptic function and glutamate release, we studied cultured hippocampal neurons at 14–18 days in vitro (DIV) from animals of both sexes to measure presynaptic changes in tauP301L positive mice. We observed that presynaptic vesicles exhibit increased vesicular glutamate transporter 1 (VGlut1) using immunohistochemistry of fixed cells and an established pH-sensitive green fluorescent protein approach. We show that tauP301L positive neurons exhibit a 40% increase in VGlut1 per vesicle compared to tauP301L negative littermates. Further, we use the extracellular glutamate reporter iGluSnFR to show that increased VGlut1 per vesicle directly translates into a 40% increase in extracellular glutamate. Together, these results show that increased extracellular glutamate levels observed in tauP301L mice are not caused by increased vesicle exocytosis probability but rather are directly related to increased VGlut1 transporters per synaptic vesicle.

Mitochondria Exhibit Changes in Morphology/Function to Support Increased Glutamate Release in TauP301LNeurons Prior to Reduction in Presynaptic Vesicle Release

“…We use our previously established cell culturing approach of hippocampal neurons cultured at PND5 that showed increased VGlut1 and glutamate release. 8 Here, we show that mitochondria morphology and structure change in P301L hippocampal neurons compared to hippocampal neurons from normal, non-transgenic litter mates (hereafter called tauP301L neg). We show that altered structure correlates with decreased basal P301L membrane potential.…”

“…34 We then counted intensity spikes following our previously established approach of at least 1 standard deviation above noise level. 8 The number of identified release events were then aggregated and divided by the observation time to estimate release frequency. The intensity per vesicle was determined as the three frame average of the peak minus the three-frame average of baseline before release.…”

“…2 We then showed that increased presynaptic glutamate release is directly caused by a tau-mediated increase in the number of vesicular glutamate transporter 1 (VGlut1) per glutamatergic vesicle. 8 Recent studies have shown that tau mediates an increase in VGlut1 via altering nuclear epigenetic transcription factors. 9 Increased VGlut1 per vesicle then increases the concentration of glutamate per vesicle, 8 resulting in an increase in glutamate release per vesicle during activity.…”

“…8 Recent studies have shown that tau mediates an increase in VGlut1 via altering nuclear epigenetic transcription factors. 9 Increased VGlut1 per vesicle then increases the concentration of glutamate per vesicle, 8 resulting in an increase in glutamate release per vesicle during activity. 2,8,10 Understanding the consequences of this molecular pathway is important because we separately showed that reducing VGlut1 levels and glutamate release rescued cognitive deficits in tauP301L pos mice, 10 suggesting the increased VGlut1 and glutamate release may represent potential therapeutic targets for the treatment of early-stage tauopathies.…”

Tau-dependent HDAC1 nuclear reduction is associated with altered VGluT1 expression

“…We recently demonstrated that Tau destabilization from MTs and its increased expression as a soluble MT-unbound protein, occurring at early stages of the disease, cause a significant increase in Tau nuclear levels, changing the expression of genes involved in synaptic transmission and leading to toxic hyperexcitability ( Siano et al, 2019b ). Accordingly, neuronal hyperexcitability, associated also with VGluT1 increase, has been identified as a hallmark of early AD pathology ( Ghatak et al, 2019 ; Siano et al, 2020 ; Taipala et al, 2022 ). The molecular mechanisms mediating this effect are still elusive.…”