Beyond Dopamine: The Role of Glutamate in Parkinson’s Disease
Having defended my PhD via Zoom earlier this month, this is an opportune time to reflect on how the findings from 6 years of experiments in a genetic mouse model of Parkinson’s disease (PD) fit into the larger context of basic science research, and contribute to the endeavor of developing earlier disease biomarkers, therapeutic targets, and disease-modifying treatments.
My research has focused on identifying early changes within the basal ganglia circuitry that may contribute to PD onset and progression. With increasing acknowledgement of non-motor symptoms that often precede motor dysfunction, PD is now recognized as a multi-system disorder, involving multiple neurotransmitters within the brain. As the majority of symptoms arise before the complete loss of dopamine neurons in the substantia nigra (SN), the latter is now considered a consequence—rather than a cause—of PD. As in other neurodegenerative disorders, it is highly probable that neuronal dysfunction precedes degeneration, and that other disease mechanisms drive the vulnerability of dopamine neurons. The fact that current dopamine replacement therapies neither improve most non-motor symptoms nor slow disease progression highlights the importance of studying the involvement of non-dopaminergic systems. The glutamatergic system is of particular interest: not only do complex functional interactions between glutamate and dopamine underlie L-DOPA-induced dyskinesia, but they may also contribute to disease onset. Thus, studying early changes in glutamate can lead to identifying biomarkers for earlier and more precise diagnosis, but also informs us of what systems or circuitry we need to target in order to modify—and eventually prevent—disease progression.
One way to identify changes that precede clinical diagnosis is to study how PD risk factors alter neurobiology in animals. PD arises from an interaction of environmental and genetic factors, most of which can be modeled in animals to investigate their pathological mechanisms. Whereas toxin-based models successfully mirror many clinical and pathological features of late-stage PD, genetic models—while exhibiting more subtle disease phenotypes—are instrumental for studying earlier pathophysiology. The most common cause of familial PD is mutations in the LRRK2 gene, leading to altered LRRK2 protein function. Mutant LRRK2 has additionally been implicated in sporadic PD, can worsen the pathological effects of toxins, and interacts on a cellular level with other PD-associated proteins; thus, investigating its function has important implications beyond understanding familial PD. LRRK2 is highly expressed in the basal ganglia circuitry, particularly within the striatum, and numerous groups have demonstrated its involvement in multiple aspects of neurotransmission (the communication between neurons at synapses).
As neurotransmission is a crucial element of neural functioning and circuitry, we are interested in how it may be disrupted by the most common LRRK2 mutation, p.G2019S—and whether this contributes to the vulnerability of dopamine neurons. We use LRRK2-G2019S knock-in (GKI) mice, which carry the mutation in their LRRK2 gene, and look at early time points to study phenotypes that arise before the typical manifestation of PD.
My research has focused on glutamate transmission within the striatum—not only due to its high LRRK2 expression, but also given its role as the gateway to the basal ganglia. The striatum receives glutamate from the cortex and thalamus and is modulated by dopamine from the SN; this convergence of inputs makes it particularly interesting for investigating how perturbed circuitry could drive neuronal degeneration. Indeed, the “axonal dying-back” theory posits that SN neurons are first impacted at their axons within the striatum, partly due to aberrant activity at synapses. This is further supported by structural changes in striatal neurons, abnormal cortico-striatal neurotransmission, and degeneration of thalamic inputs in PD. In line with this, our group and others previously reported increased striatal dopamine and glutamate activity in young GKI mice. However, the question remained if both cortical and thalamic inputs were affected, and what modes of neurotransmission are altered by mutant LRRK2—details that are consequential for determining the underlying mechanism. Most importantly, we wanted to test if abnormal neurotransmission can be reversed by targeting LRRK2 protein function, which is emerging as a potential treatment strategy.
To this end, we used a technique called optogenetics to selectively stimulate glutamate release from cortical or thalamic inputs in brain slices, while recording the resulting activity in striatal neurons. This technique provides some advantage over intrastriatal electrical stimulation, as the latter non-selectively activates multiple axons within the striatum, and may consequently mask changes at glutamate synapses. It also allows us to distinguish between evoked versus spontaneous (non-stimulated) neurotransmission, which could provide important mechanistic clues. These experiments revealed that glutamate release is altered in both cortical and thalamic inputs from 2- to 3-month-old GKI mice, as compared to their wild-type (WT) littermates. While we had previously observed that spontaneous release is increased in young GKI mice, these findings indicate that evoked release is disrupted, likely due to impaired trafficking and recycling of glutamate.
Given this newly identified phenotype, we tested whether it could be rescued by targeting LRRK2 function. G2019S—as well as several other pathogenic mutations in LRRK2—increases LRRK2’s kinase activity, thereby increasing the phosphorylation (a functional modification) of its substrates. This accounts for many of the pathophysiological effects of mutant LRRK2, and previous reports have shown that acute application of LRRK2 kinase inhibitors can reverse some cellular phenotypes. As LRRK2 inhibitors are currently in early clinical trials, determining its effect on our synaptic phenotype was not only for proof of mechanism, but also of therapeutic relevance. We found that injecting animals with the inhibitor MLi-2 rapidly reversed the disrupted evoked release in GKI mice, while activity in WT mice was not altered. Surprisingly, reduced phosphorylation of Rab10, a LRRK2 substrate and standard readout of kinase inhibition, was only observed in MLi-2-treated WT, but not GKI, mice. This suggests that LRRK2 inhibition may produce therapeutic benefit through a Rab10-independent mechanism, prompting us to examine other proteins that may interact with LRRK2 in regulating trafficking and recycling. While the preliminary results are intriguing, a more thorough investigation will be conducted once lab work can resume, and we our focusing on publishing our findings in the meantime.
This brings me to the end of my PhD, but the story will evolve. In the course of my research, we detected early glutamate dysfunction resulting from the highest genetic risk factor of PD, and, in doing so, have filled a gap in our knowledge of LRRK2 neurobiology. This is vital to identifying which molecular mechanisms contribute to PD onset, and how these may be targeted to modify disease progression. Our ability to rapidly reverse the observed phenotype with a LRRK2 inhibitor provides hope that this clinically-relevant drug has neuroprotective potential, and raises questions as to what readouts we should use to accurately measure its efficacy. We have laid the groundwork for further investigating how glutamate dysfunction impacts dopaminergic neurons in animal models, but also for determining how this may manifest as non-motor symptoms—or at least biomarkers—in people with PD. I look forward to having more answers in time for the next WPC.
Naila Kuhlmann, PhD just received her doctoral degree from the University of British Columbia, and is currently wrapping up some work in Dr. Austen Milnerwood’s lab at the Montreal Neurological Institute. She plans to pursue a postdoctoral fellowship at McGill University this September, leading a knowledge dissemination project that will bring together neuroscientists, performing artists, and people with Parkinson’s disease for creative collaboration.
This research was first shared as an abstract at the WPC 2019 in Kyoto. WPC is pleased to support abstract authors by sharing their ongoing work. Digital files of WPC abstract books can be downloaded from the past three Congresses HERE.
Ideas and opinions expressed in this post reflect that of the author(s) solely. They do not necessarily reflect the opinions of the World Parkinson Coalition®