Biology subjects

Gramlich, M. W.

Publications and source records attributed to Gramlich, M. W..

6 recordsLinked to original sources

bioRxiv2024

Central Presynapses Regulate Spontaneous Synaptic Vesicle Exocytosis Rate by Constraining Recycling Pool Density

Synapses represent a fundamental unit of information transfer during cognition. They accomplish this via presynaptic vesicle exocytosis, which can occur either spontaneously or by an action potential leading to evoked release. It has been well established that evoked release is probabilistic in nature, but it has been less clear what mechanisms mediate spontaneous release. Understanding spontaneous release is important because it is an essential maintenance mechanism for synaptic connections. We propose a mechanistic framework and model of spontaneous release based on immobile vesicles in the reserve pool geometrically constraining mobile vesicles in the recycling pool, which provides a force leading to a spontaneous release rate. We experimentally support this framework using a combination of Scanning Electron Microscopy (SEM), high-resolution fluorescence microscopy techniques using pHluorin-VGlut1 and a single vesicle SGC5 reporter, and a computational model. We observe that the spontaneous release rate increases linearly with the number of vesicles but is constant in the absence of presynaptic actin. We then use an acute agent, Forskolin, to further constrain the volume of the recycling pool, leading to an increased spontaneous release rate. We show that our framework predicts the increasing spontaneous release rate experimentally observed. These results suggest that synapses constrain the density of the recycling pool to mediate spontaneous release rate via the entropic force.

neuroscience↗

bioRxiv2024

Troriluzole Rescues Glutamatergic Deficits, Amyloid and Tau Pathology, and Synaptic and Memory Impairments in 3xTg-AD Mice

Alzheimers disease (AD) is a neurodegenerative condition in which clinical symptoms are highly correlated with the loss of glutamatergic synapses. While later stages of AD are associated with markedly decreased glutamate levels due to neuronal loss, in the early stages, pathological accumulation of glutamate and hyperactivity contribute to AD pathology and cognitive dysfunction. There is increasing awareness that presynaptic dysfunction, particularly synaptic vesicle (SV) alterations, play a key role in mediating this early-stage hyperactivity. In the current study, we sought to determine whether the 3xTg mouse model of AD that exhibits both beta-amyloid (A{beta}) and tau-related pathology would exhibit similar presynaptic changes as previously observed in amyloid or tau models separately. Hippocampal cultures from 3xTg mice were used to determine whether presynaptic vesicular glutamate transporters (VGlut) and glutamate are increased at the synaptic level while controlling for postsynaptic activity. We observed that 3xTg hippocampal cultures exhibited increased VGlut1 associated with an increase in glutamate release, similar to prior observations in cultures from tau mouse models. However, the SV pool size was also increased in 3xTg cultures, an effect not previously observed in tau mouse models but observed in A{beta} models, suggesting the changes in pool size may be due to A{beta} and not tau. Second, we sought to determine whether treatment with troriluzole, a novel 3rd generation tripeptide prodrug of the glutamate modulator riluzole, could reduce VGlut1 and glutamate release to restore cognitive deficits in 8-month-old 3xTg mice. Treatment with troriluzole reduced VGlut1 expression, decreased basal and evoked glutamate release, and restored cognitive deficits in 3xTg mice. Together, these findings suggest presynaptic alterations are early events in AD that represent potential targets for therapeutic intervention, and these results support the promise of glutamate-modulating drugs such as troriluzole in Alzheimers disease.

pharmacology and toxicology↗

bioRxiv2023

Myofascial Trigger Point (MTrP) Size and Elasticity Properties Can Be Used to Differentiate Active and Latent MTrPs in Lower Back Skeletal Muscle

Myofascial Trigger Points (MTrPs) are localized contraction knots that develop after muscle overuse or an acute trauma. Significant work has been done to understand, diagnose, and treat MTrPs in order to improve patients suffering from their effects. However, effective non-invasive diagnostic tools are still a missing gap in both understanding and treating MTrPs. Effective treatments for patients suffering from MTrP mediated pain require a means to measure MTrP properties quantitatively and diagnostically both prior to and during intervention. Further, quantitative measurements of MTrPs are often limited by the availability of equipment and training. Here we develop ultrasound (US) based diagnostic metrics that clinicians can use during patient diagnosis and treatment. We highlight the advantages and limitations of previous US-based approaches that utilize elasticity theory. We show how US-based measurements can distinguish Active from Latent MTrPs. We demonstrate that Active MTrPs tend to be smaller, stiffer, and deeper in the muscle tissue. We provide evidence that more than one MTrP within a single US-image field increases the stiffness of neighboring MTrPs. Finally, we highlight a combination of metrics (depth, thickness, and stiffness) that can be used to assess individual MTrPs in combination with standard clinical assessments.

biophysics↗

bioRxiv2023

Mitochondria Exhibit Changes in Morphology/Function to Support Increased Glutamate Release in TauP301L Neurons Prior to Reduction in Presynaptic Vesicle Release

We have shown that tauopathy models display early-stage hyperexcitability due to increased presynaptic glutamate release that is mediated by an increase in vesicular glutamate transporter-1 (VGlut1). This hyperexcitability increases energy demand which in turn would increase demand on mitochondria. It is unclear, however, how early-stage presynaptic changes in glutamate release are supported by or influence the function of mitochondria. Using Large Area Scanning Electron Microscopy (LA-SEM) and fluorescence microscopy, we demonstrate that mitochondrial changes in morphology, structure, and function in CA1/CA3 hippocampal neurons decrease resting mitochondrial membrane potential in P301L mice. However, P301L mitochondria maintain a high membrane potential during levels of high activity, suggesting that they can support increased energy demand during hyperexcitability. These activity-dependent differences in membrane potential can be rescued by inhibiting ATP-dependent VGlut1 vesicle refilling. This indicates that the increased VGlut1 per vesicle observed in P301L mice contributes to the differences in mitochondria membrane potential. Notably, the mitochondrial dysfunction in P301L mice occurs before any observable alterations in presynaptic release mechanics, suggesting these changes may represent early therapeutic targets. Finally, we propose a model of increased glutamate-mediated changes in mitochondrial morphology and function in P301L neurons that represents a potentially targetable pathway to reduce or arrest neurodegeneration.

neuroscience↗

bioRxiv2022

rTg(TauP301L)4510 mice exhibit increased VGLUT1 in hippocampal presynaptic glutamatergic vesicles and increased extracellular glutamate release

The molecular pathways that contribute to the onset of symptoms in tauopathy models, including Alzheimers 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 in order 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 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.

neuroscience↗

bioRxiv2020

Vesicle Navigation of Microtubule Ends Distinguished by A Single Rate-Constant Model

Axonal motor driven cargo utilizes the microtubule cytoskeleton in order to direct cargo, such as presynaptic vesicle precursors, to where they are needed. This transport requires vesicles to travel up to microns in distance. It has recently been observed that finite microtubule lengths can act as roadblocks inhibiting vesicles and increasing the time required for transport. Vesicles reach the end of a microtubule and pause until they can navigate to a neighboring microtubule in order to continue transport. The mechanism by which axonal vesicles navigate the end of a microtubule in order to continue mobility is unknown. In this manuscript we model experimentally observed vesicle pausing at microtubule ends in C. elegans. We show that a single rate-constant model reproduces the time vesicles pause at MT-ends. This model is based on the time a vesicle must detach from its current microtubule and re-attach to a neighboring microtubule. We show that vesicle pause times are different for anterograde and retrograde motion, suggesting that vesicles utilize different proteins at plus and minus end sites. Last, we show that vesicles do not likely utilize a tug-of-war like mechanism and reverse direction in order to navigate microtubule ends.

biophysics↗