Hello, my name is Patrick Keefe, and I am a second-year medical student at Midwestern University Chicago College of Osteopathic Medicine, and here I present our study of the effects of the leucine-rich repeat kinase 2, or LRRK2, gene mutation in Parkinson's disease. Parkinson's, I'm sure I don't have to tell you, stems from a loss of dopaminergic neurons within the basal ganglia and causes symptoms of bradykinesia, rigidity, tremor, gait disturbance, and others. The impetus for this study was our belief that the schema of Parkinson's solely as a central disease is perhaps blinding to peripheral manifestations worthy of study, and namely the neuromuscular junction, which provides a synaptic connection between the peripheral nervous system and the musculature. In other movement disorders, degradation of the nicotinic acetylcholine receptor stability is well articulated and an accepted contributor to the disease progression. So here we hypothesize that changes in the neuromuscular junction, and specifically alterations in the stability of the nicotinic acetylcholine receptors at the synapse may occur during or even precede disease manifestation. For this project, we chose animals at 3, 6, and 9 months of age because the purported phenotypic presentation of Parkinson's in the animal is not said to be until 18 months of age, and we wish to establish the current state of the neuromuscular junction at this age. So for this project, we focused on muscles of respiration diglottation, the hind limb and the forelimb for their clinical significance and functional relevance. Muscles were removed and they were washed in an alpha-bangarotoxin, that's a snake venom that binds quasi-irreversibly to the nicotinic receptors at the neuromuscular junction and allows for their visualization. But to begin, we performed a battery of behavioral tests. We performed a gait analysis, grip strength, and a cylinder test. The grip strength demonstrated a reduction in grip strength by 6 months of age. The gait analysis showed a reduction in stride length, an increase in stride width, and a decrease in overall initiations of movement. And so we conclude that LRRK2 transgenic mice exhibit a significant decline in motor performance compared to controls. As mentioned, we focused our study on the musculature of respiration diglottation, the hind limb, and the forelimb. So to begin, I'd like to discuss our findings in the sternomastoid muscle, a muscle here at the anterior neck, which is a secondary or tertiary muscle of respiration or diglottation because of its function to maintain the patency of the airway. So you're seeing the neuromuscular junction at 3, 6, and 9 months of age with wild type on the left and Parkinson's on the right. The normal morphology of the neuromuscular junction has these uniform fluorescence to its branches or gutters that you're seeing here. The shape is sometimes said to look roughly like a pretzel. When we compare to Parkinson's disease at the various ages, we would frequently see a finding such as this. These white arrows indicate these black areas. This is known as a perforation or a hole. This is an area in the neuromuscular junction where the nicotinic receptors are failing to insert into the muscle cell membrane. We would also frequently see morphology such as this. This is known as fragmentation. A normal neuromuscular junction, as it begins to age, these gutters or these troughs begin to break apart and form these clusters or these islands. But to see such a presentation at 3, 6, or 9 months of age would not be typical as the presentation of an aging neuromuscular junction would not be said to be normal until roughly 24 months of age. The quantification of the receptors you can see here with a significant reduction in the nicotinic receptor density. Other muscles of respiration and diglutition that we assessed was the intercostal, genioglossus, and diaphragm where we saw a similar presentation of reductions in the nicotinic receptor densities. Muscles of the limbs such as pectoralis, tibialis anterior, soleus, and extensor digitorum longus were explored. And you saw again a similar presentation of a reduction in the nicotinic acetylcholine receptor densities. And so we concluded that in LRRK2 mice, the postsynaptic nicotinic acetylcholine receptor density was significantly reduced in the muscles assessed independently of muscle fiber type when compared to age-matched controls. Because we were seeing, you can see my dog just decided to join us, that was Walter. Because we were seeing these demonstrations of an aging neuromuscular junction or a senescent neuromuscular junction, we wished to explore other presentations that are accepted forms of an aging neuromuscular junction, and namely the number of myonuclei at neuromuscular junction. So to do so, we would quantify the number of myonuclei which are seen here. So these figures, this is the composite, and then it's broken apart to demonstrate the neuromuscular junction and the myonuclei respectively. We would count the number of myonuclei per neuromuscular junction and normalize to area. And so you can see that in Parkinson's disease, there was a significant increase in myonuclei per unit area at the neuromuscular junction, which is again an accepted presentation of an aging neuromuscular junction. Because we were seeing such demonstrations, we wish to understand the current state of the muscle fiber type fibers currently. And so we performed a variety of histological stainings, including hematoxylin and eosin, H&E staining, which you can see here, modified Gamori here, and NADH staining here. Within the H&E staining, we would frequently see figures such as this, where the muscle fiber type has begun to hypertrophy with a central nucleization. Muscle fibers, as I'm sure you know, have a nuclei at the periphery. And so when the myonuclei begin to aggregate into the center of the muscle cell, that is a sign of a muscle cell under stress. We were also frequently seeing demonstrations like this, where clusterings of these muscle cells that had a different shape to them, they would frequently look like diamonds or triangles in these clusters, which we believe represents atrophying fibers. We quantified the area of the muscle fiber cells areas with a slight increase in area. You can see here in the distribution curve with the light gray being Parkinson's, that there is a general increase in area for Parkinson's. But we were interested in this spike here. There's a single size, which is at the small range, where there was a significantly more increase in fiber sizes at that size. And so we did the NADH staining, which allows you to see type 1 versus type 2 fibers. In the NADH staining, type 1 is dark and type 2 is light. And so you saw here that in Parkinson's disease, there's a significant increase in type 1 fibers. This is consistent with other publications where Parkinson's frequently demonstrates what's known as type 1 fiber grouping. Here in the modified Gomory staining, the muscle cells would typically stain this bluish greenish color. And it's typically used to look for what's known as ragged red fibers, which you're actually seeing here. This accumulation of magenta or red underneath the sarcolemma, this non-uniform sort of bordering of red indicates an accumulation of defective mitochondria underneath the sarcolemma. And that is a non-specific but frequently seen finding in mitochondrial myopathies. With all that said, we conclude that mutations in the leucine-rich repeat kinase 2 gene impact the integrity of the neuromuscular junction in Parkinson's disease, and that a pattern of changes may exist with clinical relevance. Thank you.

LRRK2 gene mutation

Parkinson's disease

neuromuscular junctions

nicotinic acetylcholine receptor

transgenic mice