Parkinson’s Disease (PD) is one of the most devastating medical conditions the human population faces, as more than 10 million people live with PD and 60,000 people are diagnosed yearly in the United States–with this number rising each year . It is the second most common neurodegenerative disease, only following Alzheimer’s Disease in terms of prevalence. PD is typically a condition of the elderly, and the hallmark symptoms include motor function deterioration, tremors, bradykinesia, hypokinesia, postural instability, and cognitive decline . Symptoms often begin on one side, with stiffness in the limbs when walking. These symptoms eventually transition into the ones listed above as the disease progresses. However, Parkinson’s Disease typically progresses in proportion to normal aging, so it does not significantly reduce the life span of patients .
In terms of pathology, Parkinson’s Disease is characterized by the loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) . The SNpc is one of two parts in the Substantia nigra, a nucleus of neurons in the midbrain. The Substantia nigra is critical in managing motor and reward function using the basal ganglia circuitry. The basal ganglia are responsible for several essential functions such as voluntary movement, planning, emotions, and basic cognitive function . This loss of dopaminergic neurons is likely what leads to the traditional symptoms of Parkinson’s Disease, which include the motor function and cognitive decline discussed.
In addition, the presence of Lewy bodies (a form of neurofibrillary tangle) is a major hallmark of PD . Like other neurodegenerative diseases, the Lewy body is an aggregation of a misfolded protein and is thought to lead to the progression of the disease. In PD, this protein is α-synuclein, which is ordinarily involved in synaptic function, as it serves for synaptic vesicle trafficking and neurotransmitter release . However, when there is a mutation in the SNCA gene, the gene which codes for the α-synuclein protein, the protein misfolds and becomes insoluble. This insoluble α-synuclein begins to aggregate and forms dense inclusions (Lewy bodies) within the cell. These Lewy bodies are not restricted to the brain, as they can be found throughout the nervous system. They can even extend as far as the spinal cord and the peripheral nervous system.
Oxidative stress is thought to play a major role in Parkinson’s Disease pathogenesis, and it is hypothesized that Lewy bodies are one potential avenue through which Reactive Oxygen Species (ROS) form. The ubiquitin-proteasome system (UPS) is the primary pathway in which cells discard waste and damaged proteins. The UPS is used as a defense mechanism during oxidative stress to prevent oxidized proteins from inflicting harm. However, in PD, it is believed that dysfunction in the UPS leads to improper maintenance of ROS and thus, unregulated oxidative stress, as seen in Figure 1. In fact, α-synuclein, is a substrate of UPS, indicating that there is a link between α-synuclein/Lewy Bodies and the oxidative stress that forms during Parkinson’s Disease .
Along with the UPS, excessive levels of dopamine are a major source of ROS formation in the Substantia nigra of PD brains. When dopamine is present in excessive amounts, which is the case for SNpc neurons , there is saturation, meaning the excess dopamine cannot be efficiently transported by the vesicular monoamine transporters. Free dopamine is left remaining, and this excess dopamine is readily oxidized to form dopamine quinones, superoxides, and hydrogen peroxide, which contribute to oxidative stress . In various studies, it was found that dopamine quinones formed eumelanin after reacting with neuromelanin . There are several other proposed mechanisms for ROS formation in Parkinson’s Disease. For example iron, calcium, and lipids are all molecules that play a role in oxidative stress in the PD brain .
Although there are various intracellular antioxidant defense mechanisms, the glutathione (GSH) system is considered to be the most important antioxidant defense system for cellular viability and function maintenance . GSH is a ubiquitous thiol tripeptide which interacts with various ROS, such as hydroxyl radicals, peroxynitrite, and superoxide radicals, and reduces them. GSH is also responsible for reducing any dopamine quinone in the cell that was oxidized from free dopamine. In addition, GSH is important in iron metabolism, as it forms various iron complexes including iron(II)glutathione and the diglutathionyl- dinitrosyl‐ iron‐ complexes . It has been shown across multiple studies that diminished levels of GSH leads to elevated levels of oxidative stress in cells. Multiple processes are affected by low levels of GSH, such as increased oxidative stress in mitochondrial fractions, lipid peroxidation, intracellular calcium, and γ-glutamyl transpeptidase (γGT) activity .
In Parkinson’s Disease, it has been established that lower levels of GSH are present in patients. In a study from 1994, researchers found upon postmortem analysis of the CNS that glutathione levels in the substantia nigra of PD patients were reduced by 40% compared to the substantia nigra of similar-age controls. However, this was unique to PD, as patients with other basal ganglia-based neurodegenerative diseases did not experience this drop in GSH levels in the substantia nigra .
Glutathione peroxidases (GPXs) are a set of enzymes that reduce hydrogen peroxide, a toxic ROS, into water–utilizing GSH as a reducing agent. It has been found that upregulation of GPX proteins leads to less neuronal loss . In dopaminergic neurons in the SNpc, there are overall fewer levels of both GSH and GPX proteins, which provides an explanation as to why this region of the brain is particularly vulnerable to oxidative stress and neurodegeneration.
Glutathione S-transferases (GSTs) are a class of proteins that catalyze glutathione conjugation and are responsible for much of the ROS reduction in the cell. Of the various subtypes of GSTs, the cytosolic forms are predominant in the brain. Because GSTs are vital to the management of intracellular oxidative stress and their levels have been known to be diminished in PD brains, GSTs are a reliable biomarker to track the progression of PD.
GSH is a tripeptide, and its amino acid constituents include cysteine, glutamic acid, and glycine. Glutamine, through the Glutamine/Glutamate cycle, forms glutamate via the enzyme Glutaminase. The glutamate then is eventually synthesized to GSH. Therefore, the levels of glutathione are proportional to the intracellular levels of glutamine, as glutamine is a precursor to glutathione . In one study, it was shown that glutamine deprivation increases oxidative stress and decreases GSH levels in a neuroblastoma cell line. Several studies have demonstrated similar results in that reduction of glutamine levels has led to excessive oxidative stress due to not enough GSH being synthesized . Therefore, it follows that an increase/decrease in glutamine would lead to modulation of oxidative stress in Parkinson’s Disease . In fact, certain cancers have been shown to exhibit a “glutamine addiction” in which they significantly increase intracellular glutamine levels as one of their primary means of avoiding apoptosis and preserving cell viability .
As seen in Figure 2, glutamine uptake into the cell is mediated by transport protein ASCT2 (also known as SLC1A5). ASCT2 is a 541 amino acid-long membrane transporter protein which belongs to the protein family of Solute Carrier Family 1. Although ASCT2 is responsible for the transport of several amino acids such as asparagine, it primarily functions as a glutamine transporter. The levels of ASCT2 are directly proportional to glutamine uptake and intracellular glutamine levels. In recent years, ASCT2 has become an increasingly popular target for cancer treatment. For numerous in vitro cancerous cell lines and even murine in vivo models, ASCT2 inhibition and downregulation has led to intrinsic oxidative stress and apoptosis. The cancer cells were actively using glutamine and converting it into GSH to neutralize the ROS produced from the rapid proliferation and mitochondrial function .
Since PD cells (along with other neurodegenerative diseases) have lower levels of GSH–which results in increased oxidative stress–modulating the glutamine levels of the cells could possibly change the levels of oxidative stress accordingly. Similarly, since the amount of ASCT2 is proportional to glutamine uptake, if ASCT2 concentration is increased, oxidative stress theoretically might decrease and if ASCT2 concentration is dropped, oxidative stress should increase. The goal of this research is to potentially identify a functional relationship between the glutamine to glutathione conversion process and PD progression through modulation of the ASCT2 transporter.
To understand the glutamine-glutathione conversion process’ role in PD progression, V-9302, a chemical designed to act as a competitive ASCT2 inhibitor , was used to limit glutamine uptake into SH-SY5Y cells, transfected to overexpress α-synuclein. This should in turn worsen PD “progression” in these cells. To assess PD progression in this model, various assays will be performed including cell viability, cell proliferation, Reactive Oxygen Species production, and α-synuclein quantification. In addition, various hallmarks in the glutamine-glutathione conversion process will be tested, such as glutamine uptake, glutamate levels, and glutathione levels within the cell.