VGLUT2 in Parkinson’s disease: an emerging therapeutic target

Abstract

Introduction:

Parkinson’s disease is among the most common age-related neurodegenerative disorders today and characterized by midbrain dopamine neuron degeneration. However, this cell loss is not uniform. Though most dopamine neuron loss occurs in the substantia nigra, some ventral tegmental area and dorsal-tier substantia nigra dopamine neurons are relatively resistant to degeneration in Parkinson’s disease. Yet, the mechanisms underlying this dopamine neuron resilience remain unclear. Novel insights into neuronal resilience are provided by evidence of a midbrain dopamine neuron subpopulation that expresses the vesicular glutamate transporter 2, VGLUT2, and is more resistant to cell loss in both preclinical models and clinically in Parkinson’s disease. Thus, VGLUT2 expression has emerged as a defining signature difference between vulnerable versus resilient dopamine neuron subpopulations.

Areas covered:

Here, we review recent developments in the potential mechanisms of dopamine neuron resilience and the therapeutic potential associated with boosting cellular resilience via VGLUT2.

Expert opinion:

We discuss approaches to maximize VGLUT2-mediated dopamine neuron resiliency. This includes increasing the efficiency of vesicular sequestration of dopamine to decrease generation of cytotoxic reactive oxygen species and increasing levels of neuroprotective antioxidant glutathione. Taken together, VGLUT2-mediated pathways represent original directions to treat Parkinson’s disease symptoms and modify disease progression.

1. Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disease and fastest growing neurological disorder today [1]. Dopamine (DA) neurodegeneration is a pathological hallmark of PD, but it is not uniform. Most DA neuron loss occurs in the substantia nigra pars compacta (SNc). Yet, some ventral tegmental area (VTA) and dorsal-tier SNc DA neurons are relatively resistant to degeneration in PD [2]. Nevertheless, the transcriptomic/proteomic signatures that make these DA neurons resilient are not clear [3–5].

Mechanistic clues underlying DA neuron resiliency come from sex differences in PD. PD occurs ~50% more frequently in men than women and has an earlier age of onset in men [6–8]. Though rodent PD models historically focused on males, recent studies reveal sex differences resembling those in humans. Females exhibit less SNc DA neurodegeneration and milder motor symptoms versus males [9]. These gaps in knowledge raise important longstanding questions: What distinguishes resilient DA neuron subtypes from more vulnerable cells in the face of exposure to the same genetic and environmental factors? Are the molecular modulators of midbrain DA neuron resilience regulated differently in men versus women? Increased understanding of these resilience mechanisms may offer innovative new approaches to better treat PD, as current therapies are geared towards symptom relief rather than modifying or preventing the disease course [10,11].

Additional insights into DA neuron resilience are provided by evidence that the midbrain is composed of heterogeneous DA neuron subtypes that possess unique transcriptomic, molecular, and functional properties [12–15]. This includes relatively resilient subpopulations of midbrain DA neurons that are enriched in calbindin, a calcium buffering protein and/or vesicular glutamate transporter 2 (VGLUT2) [12,13,16,17]. Indeed, VGLUT2+ DA neurons are relatively spared both in animal models and clinical PD. Therefore, VGLUT2 has emerged as a defining signature difference between the vulnerable versus resilient DA neuron populations [3,18,19]. Here, we will focus on VGLUT2-associated DA neuroprotection and the potential of VGLUT2 as a therapeutic target to promote DA neuron survival in PD.

2. Physiological roles of VGLUT2 in dopamine neurons

2.1. Localization and properties of VGLUT2-expressing midbrain dopamine neurons

VGLUT2, encoded by SLC17A6, was originally characterized as a phosphate transporter [20]. Yet, VGLUT2 also functions as a vesicular H⁺/glutamate antiporter, transporting glutamate into the synaptic vesicle (SV) lumen in exchange for H⁺ [21–23]. VGLUT2 also functions as a Cl⁻ channel which helps maintain the vesicular membrane potential [24].

VGLUT2 is expressed in several brain regions, including medulla, midbrain, hippocampus, subthalamic nucleus, and thalamus in neurons that primarily transmit glutamate [20,22,25,26]. Importantly, there is a unique subpopulation of midbrain DA-transmitting neurons that also express VGLUT2 [27–29]. These cells are mainly localized to the medial VTA and primarily project to the medial shell of the nucleus accumbens [27,30,31]; however, a smaller population of VGLUT2+ DA neurons are also found in the lateral SNc and project to the tail of the striatum [14,32–34].

VTA neurons possess electrophysiological and functional properties distinct from SN neurons [27,29,35–38]. For example, some medial VTA DA neurons exhibit higher frequency firing over extended periods of time relative to SN neurons [35,38,39], raising the possibility that VGLUT2 may contribute to these cells’ distinct activities.

2.2. Physiological roles of VGLUT2 in midbrain dopamine neurons

VGLUT2 modulates SV acidification within DA/glutamate neurons in response to depolarization [40]. Since the vesicular pH gradient is the main driving force for DA loading into SVs, this activity-dependent SV hyperacidification mediated by VGLUT2 enables tuning of vesicular DA content and release in response to changes in firing frequency [40–42]. VGLUT2-dependent modulation of DA release permits active neurons to reliably meet the demands of high-frequency burst firing. Just as importantly, VGLUT2 expression is highly dynamic in DA neurons. In flies and mice, expression of either VGLUT2 or the Drosophila VGLUT2 ortholog, dVGLUT, in DA neurons increases in response to synaptic DA depletion following either longer-term exposures to drugs of abuse like amphetamine or in response to depletion of SV DA stores by reserpine, an inhibitor of the vesicular monoamine transporter 2 (VMAT2) [43,44]. These data suggest that VGLUT2 expression is tied to changes in DA homeostasis. We posit that dynamic regulation of VGLUT2 expression is a homeostatic mechanism to maintain adequate synaptic DA in response to progressive DA loss in PD. Relatedly, an intriguing hypothesis is that nigrostriatal DA loss in PD can be reflected by corresponding increases in DA neuron VGLUT2, making VGLUT2 a potential early diagnostic marker of DA neuron dysfunction.

3. Dopamine neuron VGLUT2 and its relevance to PD

3.1. Dynamic VGLUT2 upregulation in response to SN DA neuron injury

Dynamic changes in DA neuron VGLUT2 expression are not limited to the adult midbrain. Though most adult SN DA neurons do not express VGLUT2 [45], almost all SN DA neurons express VGLUT2 in development [3]. By adulthood, most SN DA neurons repress or ‘turn off’ VGLUT2 expression in contrast to the limited subpopulations of DA neurons that maintain VGLUT2 expression throughout life. VGLUT2 expression can also re-emerge in adult SNc DA neurons in response to injury including toxicant exposures in rodent PD models and in clinical PD [3,18,34,46]. Moreover, interfering with VGLUT2 upregulation after insults or during healthy aging can increase SNc DA neuron vulnerability and even result in neurodegeneration [3,18,43,47]. These findings suggest that VGLUT2 upregulation may represent an adaptive compensatory response by DA neurons to maintain cell function and prevent cell death. This phenomenon is consistent across species including flies, rodents, and humans [3,18,19,34,43,46]. Nevertheless, the precise molecular mechanisms responsible for VGLUT2 re-emergence in adult SNc neurons remain unclear. Collectively, these data suggest that DA neurons that endogenously express or upregulate VGLUT2 expression exhibit the greatest resilience. Therefore, if VGLUT2 expression can confer resilience, why is VGLUT2 differentially expressed across DA neurons within the adult midbrain, and why is VGLUT2 expression reduced over the course of development? While age-related changes in VGLUT2 expression are well-documented, VGLUT2’s role(s) in healthy DA neuron development is unknown. It may be that VGLUT2 expression is high in early postnatal periods to protect developing neurons from environmental insults. Additionally, we speculate that there may be transient periods of heightened activity during DA neuron development that necessitate VGLUT2-mediated enhancement of vesicular DA loading and release to maintain neurotransmission. Yet, during later periods of brain maturation and thereafter, there may be less need for these VGLUT2-mediated protective mechanisms. This raises the related question: how does expression of VGLUT2 re-emerge in adult SNc DA neurons? Increased research into both the developmental mechanisms limiting DA neuron VGLUT2 expression and post-developmental mechanisms that re-activate VGLUT2 expression in adult cells and the functional implications of these events is clearly warranted.

3.2. Precise regulation of VGLUT2 expression determines dopamine neuron resilience

While disruption of endogenous VGLUT2 upregulation increases SNc DA neuron vulnerability, the converse is also true: low levels of ectopic VGLUT2 overexpression boost DA neuron survival [18,43]. Nevertheless, it should be noted that in both fly and mouse experiments using VGLUT2 overexpression systems that produce persistently high levels of VGLUT2, DA neuron survival is diminished [3]. Therefore, it is possible that tight regulation of VGLUT2 expression by DA neurons is necessary for VGLUT2’s neuroprotective properties. Indeed, systems that circumvent endogenous mechanisms of VGLUT2 expression at both extremes (e.g., knockdown, strong overexpression), may profoundly affect cell resilience [43].

3.2. Sex differences in VGLUT2 expression in midbrain dopamine neurons

There are species-conserved sex differences in DA neuron VGLUT2 expression in flies, rats, and humans, with females expressing more VGLUT2 than males [43]. In flies, DA neuron-specific knockdown of dVGLUT renders female DA neurons less resilient to age-related DA cell loss and motor deficits [43]. Conversely, DA neuron dVGLUT knockdown increases cytotoxic mitochondrial reactive oxygen species (ROS) specifically in males in the fly paraquat model of PD [47]. These data offer a potential VGLUT2-mediated mechanism for greater vulnerability of male DA neurons and may explain why females are more protected in toxicant PD models [47].

In humans, while PD prevalence is lower in women, upon developing PD, female patients exhibit a faster rate for time to require caregivers compared to men. This suggests a potential faster clinical progression of the disease in women [7,8,48]. Such a dichotomy may be related to sex differences in DA neuron VGLUT2 expression. We hypothesize that in later disease stages, ongoing DA neuron loss may induce progressively increasing compensatory VGLUT2 upregulation as a means of facilitating vesicular DA stores in remaining dopaminergic terminals. However, after a certain point, higher VGLUT2 expression in female midbrain neurons may become cytotoxic akin to prior ectopic overexpression studies, hastening clinical disease progression [43]. Further work exploring the role of sex hormones in DA neuron VGLUT2 expression and its implications for sex differences in DA neuron resilience is therefore needed.

4. Expert opinion

The evidence above points to VGLUT2 as a modulator of selective DA neuron resilience. Nevertheless, we also acknowledge important caveats. Foremost, work in flies and rodents shows that ectopically raising VGLUT2 levels in DA neurons beyond endogenous thresholds may increase cell vulnerability via mechanisms yet to be elucidated [3,43]. These data suggest that directly manipulating VGLUT2 expression may not be an ideal approach to boost DA neuron resilience. Instead, targeting VGLUT2- and glutamate-mediated pathways may offer promising therapeutic approaches.

To maximize resiliency, ideal translational entry points include strategies that enhance VGLUT2-mediated regulation of the SV pH gradient to increase vesicular DA sequestration of DA. This could include pharmacological levers that employ VGLUT2 to increase the efficiency of vesicular pH/VMAT2 coupling. It is established that non-sequestered DA SVs is oxidized to form a highly reactive quinone, which increases cytoplasmic ROS [49]. Therefore, facilitating vesicular DA sequestration by regulating VGLUT2 may lower cytoplasmic ROS in DA neurons (Figure 1A). Given the high bioenergetic demands of midbrain DA neurons, these cells are under great metabolic and mitochondrial strain, thus heightening their antioxidant requirements. This is further exacerbated during prolonged periods of PD-related neuron stress, making VGLUT2-mediated reductions in ROS especially neuroprotective. In flies, there is also recent evidence showing that dVGLUT is a modulator of depolarization-induced increases in intracellular ATP within DA neurons. Moreover, these dVGLUT-mediated ATP increases are strongly correlated with activity-dependent mitochondrial ROS generation [47]. Together, these findings raise the intriguing possibility that VGLUT2’s ability to modulate activity-dependent energetic demands via intracellular ATP availability may further contribute to its neuroprotective properties, in concert with its roles in modulating cellular ROS levels.

Figure 1. Targeting VGLUT2- and glutamate-mediated pathways to boost dopamine neuron resilience.

(A) Enhancing VGLUT2-mediated sequestration of dopamine (DA) into synaptic vesicles via transport through the vesicular monoamine transporter 2 (VMAT2) will reduce cytoplasmic DA levels that contribute to the formation of cytotoxic reactive oxygen species (ROS) and preserve mitochondrial health within DA neurons. (B) Increased incorporation of glutamate into the glutathione biosynthetic pathway will increase glutathione levels to more efficiently lower intracellular ROS. Created with BioRender.com and adapted from Buck et al. (2022) ACS Chem Neurosci.

Another translational entry point would rely on VGLUT2 to boost synthesis of glutathione, a key antioxidant. Since VGLUT2+ DA neurons are adapted to handle glutamate, we also posit that these cells may be better suited to detoxify ROS via enhanced glutamate incorporation into glutathione. Thus, strategies that enhance DA neuron glutathione biosynthesis can further contribute to DA neuron resilience (Figure 1B).

Besides VGLUT2, a small literature has explored the roles of other VGLUT subtypes including VGLUT1 and VGLUT3 in PD therapeutics. VGLUT1 is primarily expressed in cortical and hippocampal glutamatergic neurons while VGLUT3 is mostly expressed in non-glutamatergic cells including striatal cholinergic interneurons and raphe serotonergic neurons [28,41,50]. Electroacupuncture can reverse reductions in VGLUT1 within the subthalamic nucleus alongside motor improvements in a rat model of PD [51]. Considerably less is known about VGLUT3 in the contexts of PD pathophysiology and treatment. VGLUT3 was shown to be upregulated in the soma of GABAergic neurons in the substantia nigra pars reticulata in the 6-hydroxydopamine (6-OHDA) model of PD in rats [52]. Conversely, VGLUT3 deletion in mice prevented motor deficits following DA depletion by 6-OHDA and attenuated L-DOPA-mediated dyskinesias, suggesting that VGLUT3 may be a promising therapeutic target in PD [53].

Overall, optimizing the ability of DA neurons to diminish ROS-induced stress via VGLUT2-mediated pathways represent original directions to treat PD symptoms and potentially modify disease progression. These approaches will require preclinical and clinical development, including identification of specific drugs and effective targeting strategies.

Article highlights.

• VGLUT2-expressing dopamine neurons are more resilient to neurodegeneration in preclinical models and clinically in Parkinson’s disease.

• VGLUT2-expressing midbrain dopamine neurons are primarily localized in the medial ventral tegmental area and the lateral substantia nigra pars compacta (SNc).

• A key physiological function of VGLUT2 is to modulate synaptic vesicle acidification in dopamine neurons following depolarization to tune vesicular loading and release of dopamine.

• VGLUT2 expression is dynamic in dopamine neurons and re-emerges in adult SNc dopamine neurons as a potential adaptive response to maintain cell function and prevent cell death.

• Therapeutic targeting of VGLUT2 to optimize the ability of dopamine neurons to diminish cytotoxic reactive oxygen species may represent an original direction to treat symptoms of Parkinson’s disease and potentially modify disease progression.