Identification of -A2 and Annexin-A5 Interactions with Synaptic Vesicles and the

Parkinson’s Disease Associated α-Synuclein

Michaela Peterson

Abstract

Parkinson’s Disease (PD) is a neurodegenerative disorder characterized by a loss of motor control neurons. Normally, neurons communicate with one another via neurotransmitter released at the synapse. In PD patients, trafficking of these neurotransmitter-containing synaptic vesicles is disrupted due to mutations in or overexpression of the protein α-synuclein. Due to the lack of understanding of the targets of α-synuclein, the development of PD treatments has not progressed. One way excess α-synuclein may be altering the exo- and endocytic processes is by inhibiting the function of other synaptic vesicle trafficking regulatory such as members of the Annexin family. The Annexin family is evolutionarily conserved, and can be examined more closely using sea lamprey as a model organism. Binding interactions have been observed between aggregated α-synuclein and specific Annexin family members, Annexin-A2 and

Annexin-A5, indicating that these proteins may also have a role in the progression of PD. The expression patterns of the and α-synuclein genes were analyzed in a mammalian brain.

Annexin expression was also examined using immunofluorescence in the sea lamprey nervous system. An increase in the understanding of the expression of the Annexins and α-synuclein combined with their binding interactions may lead to the development of future treatments and an improved ability to diagnose patients with PD.

Introduction

Parkinson’s Disease (PD) is a neurodegenerative disorder that affects approximately

0.6% of adults aged 45 or older (Marras et al., 2018). Globally, nearly 6.1 million individuals have been diagnosed with PD (Rocca, 2018). PD is characterized by a loss of motor control due to the reduction in the number of neurons in the nigrostriatal pathway. This pathway is responsible for fine motor movement (Chinta & Andersen, 2005). The nigrostriatal pathway runs from the substantia nigra pars compacta located in the midbrain, to the dorsal striatum located in the forebrain. In this pathway, neurons are connected to one another at a point called the synapse, through which they can communicate with one another via chemicals called neurotransmitters

(Pang & Südhof, 2010). As degeneration progresses in this pathway, the neurons that are affected can result in a loss of the neurotransmitter dopamine and reduced communication between these neurons. If these neurotransmitter signals are not sent between neurons that are synapsed onto one another, then the connection between them may be lost, ultimately leading to the death of the neuron (Miraglia et al., 2018). While the mechanism behind the neurodegeneration seen in the nigrostriatal pathway of PD patients is not completely understood, the mutation in or overexpression of the protein α-synuclein has been linked to its progression in

PD (Krüger et al., 1998; Polymeropoulos, 1997; Tu et al., 1998). The brains of individuals who have PD have been found to contain abnormal protein aggregates called Lewy bodies. These

Lewy bodies are primarily composed of the protein α-synuclein (Spillantini et al., 1998).

However, not all of the overexpressed α-synuclein results in the formation of Lewy bodies.

Small aggregates of α-synuclein have also been observed at the presynapse of PD patients (Bridi

& Hirth, 2018). From the α-synuclein aggregates that are observed in PD patients, it can be inferred that one of the mechanisms behind the development of PD involves the regulation of the expression of α-synuclein in the brain. Expression of α-synuclein is controlled by the SNCA gene. More specifically, familial PD can be the result of a mutation in the α-synuclein gene affecting the promoter region, the area of DNA that helps to control where and when the gene is expressed in an organism. Multiple missense mutations, changes in a single component of the genetic code leading to a change in a gene’s expression, can also lead to PD (Farrer et al., 2001).

Cases of sporadic PD are also linked to an increase in the expression of α-synuclein (Chiba-Falek et al., 2006; Dumitriu et al., 2012). These resulting genetic abnormalities can lead to the formation of a dysfunctional protein (Singleton et al., 2003). Overall, these mutations affecting

SNCA have been found to lead to the overexpression of α-synuclein that characterizes PD

(Farrer et al., 2001).

While the mechanism behind the aggregation of α-synuclein and the resulting impact of this aggregation on PD patients is unknown, the structure of α-synuclein can provide more information about its potential function. α-Synuclein is a small acidic protein that is normally located at the presynapse (Bridi & Hirth, 2018, Emamzadeh, 2016). α-Synuclein consists of three main components: a basic NTD that folds into an α-helix (Suzuki et al, 2018; Busch et al., 2014), an amyloid-binding central domain, and an acidic C-terminal (Lautenschlager et al., 2018;

Suzuki et al., 2018). The NTD of α-synuclein is both water-loving and water-hating, or amphipathic, with polar and nonpolar areas that are similar to the polar and nonpolar regions that make up the lipid membrane of a cell. The amphipathic properties of the NTD of α-synuclein promote its binding to lipids. When calcium binds to α-synuclein’s C-terminal, the lipid-binding ability of α-synuclein’s NTD is increased (Lautenschlager et al., 2018). The calcium-binding ability of α-synuclein is noteworthy as an influx of calcium ions at the presynapse is one of the first steps in the neurotransmission process (Lautenschlager et al., 2018; Suzuki et al, 2018). In addition, α-synuclein folds into a characteristic α-helix shape when it is associated with phospholipids. In contrast, it is predicted that α-synuclein may exist in an unfolded state in the cytosol of the cell (Selkoe et al., 2014). At a synapse where α-synuclein is not aggregated, neurotransmission is able to occur as normal. In order for neurons to communicate with one another via the neurotransmission process, signals are transmitted from neuron to neuron by neurotransmitter released from synaptic vesicles. These synaptic vesicles have a phospholipid membrane with many integral and associated proteins. One example of an integral protein is SV2. α-Synuclein and the members of the Annexin family are examples of synaptic vesicle-associated proteins. The synaptic vesicles, along with their associated proteins, are clustered at the presynaptic active zone. In this zone, they are ready to fuse with the plasma membrane and be released from the presynaptic neuron through a multi-step process known as exocytosis. In this process, a stimulus, or action potential, is first applied to the presynaptic neuron, and calcium ions enter the cell through voltage-gated calcium channels (Pang & Südhof, 2010). As the concentration of calcium increases in the cell, the SNARE protein complex assembles. This complex has been observed to bind with α- synuclein, and aids in the fusion of synaptic vesicles with the (Bridi & Hirth,

2018). The calcium influx from the action potential induces the synaptic vesicles to fuse with the presynaptic membrane and release neurotransmitter into the synaptic cleft. The neurotransmitter then binds to receptors located on the neuron receiving the signal (Pang & Südhof, 2010).

Endocytosis occurs after the presynaptic neuron has released the neurotransmitter. In this process, the presynaptic membrane is recycled to be used in future neurotransmission events.

This process can be carried out by a protein called clathrin. Clathrin-mediated endocytosis functions at synapses to internalize membrane. The clathrin protein is recruited to the presynaptic membrane by adaptor and accessory proteins, where a clathrin-coated pit forms. Many synaptic proteins are involved in clathrin coat formation and synaptic vesicle maturation. The clathrin coat proteins are then removed from the internalized vesicle by a protein complex, the vesicle is filled with neurotransmitter, and clustered with other vesicles at the synaptic active zone for future neurotransmission events (Busch et al., 2014). Clathrin-mediated endocytosis is especially important to examine in relation to α-synuclein because interactions have been observed between

α-synuclein and Hsc70, a protein that removes the clathrin coat from the synaptic vesicle. This interaction may be a possible mechanism behind the progression of the neurodegeneration characteristic of PD (Banks et al., 2020). However this interaction cannot completely explain the synaptic defects observed in the presence of excess α-synuclein, because the specific targets of α-synuclein are not known. For example, when an excess of α-synuclein is introduced at the presynapse, observed synaptic vesicle defects include the extension of the plasma membrane, as well as the formation of cisternae (Busch et al., 2014). This data indicates that excess α-synuclein may be playing a role in the inhibition of the trafficking of neurotransmitter-containing synaptic vesicles at the presynapse (Miraglia et al., 2018). However,

α-synuclein is only able to have this inhibitory effect on the trafficking of neurotransmitter- containing synaptic vesicles when its NTD is folded into an α-helix, and a stimulus is applied at the presynapse. The lack of synaptic defects observed when a stimulus is not applied to the presynapse indicates that the synaptic vesicle-associated proteins such as α-synuclein and the members of the Annexin family are also normally functioning under unstimulated conditions

(Busch et al., 2014). While some proteins at the presynapse have been well-studied, a large amount of information regarding their function at the synapse in relation to PD is still unknown.

In order to gain more information about the mechanisms behind PD, the interactions of well-studied proteins, along with their regulatory mechanisms at the synapse, should be explored. In addition, their interaction with the PD-associated protein α-synuclein should be examined. One group of proteins that can be examined in this way is the Annexin family. In past studies, binding interactions have been observed between α-synuclein and members of the

Annexin family (Banks et al., unpublished data; Bedrood et al., 2009). The members of the

Annexins family of proteins may have a regulatory role at the synapse. These Annexins are calcium and anionic phospholipid binding proteins that can exist as monomers or heterotetrameric complexes. These proteins are able to organize membrane proteins and lipids, and regulate endocytosis and exocytosis (Hakobyan et al., 2017). Research suggests that one member of the Annexin family, Annexin-A2, plays a role in endocytosis, exocytosis, the conductance of ion channels, and the organization of the cell membrane (Bharadwaj, 2013). In addition to this, Annexin-A2 has a binding interaction with phospholipids that are negatively charged when calcium is introduced As both Annexin-A2 and α-synuclein exhibit binding to phospholipids and play a role in endocytosis and exocytosis, examining their interactions may have a biological relevance in relation to the inhibition of synaptic vesicle recycling that is observed with an excess of α-synuclein (Hakobyan et al., 2017). Because of the biological relevance of Annexin-A2, it is important to learn more about the other members of the Annexin family. Direct binding between aggregated α-synuclein and Annexin-A5 has been observed, indicating that this protein may also play a role in the impairment of synaptic vesicle recycling that is observed with excess α-synuclein (Bedrood et al., 2009). An interaction between the amyloidogenic protein, h-IAPP, has also been observed, with Annexin-A5 reducing the toxicity levels for both proteins (Bedrood et al., 2009). Annexin-A5 is also known to play a role in the inhibition of the of blood, and has multiple radiopharmaceutical, pharmacologic, and biodistribution applications such as probing for cell-death related proteins (Kietselaer et al.,

2004). Although the interaction of Annexin-A5 with aggregated α-synuclein has been observed in past studies, the specific region of this interaction is uncertain. More information about the binding interactions of Annexin-A5 and Annexin-A2 at stimulated and unstimulated synapses will lead to a better understanding of the mechanisms behind PD. While the interactions of α-synuclein and the Annexins in vivo is unclear, both types of proteins may play a role in endocytosis and exocytosis. Because synaptic vesicle recycling is impaired with the introduction of excess α-synuclein at the presynapse, the interactions of these proteins may take place on the synaptic vesicle itself. Excess α-synuclein may be binding to the

Annexins and moving them away from the membrane, or the excess α-synuclein could be binding to the membrane itself, blocking the ability of the Annexins to interact with it. The interaction between α-synuclein and the Annexins at the synapse may lead to Annexin dysfunction and an impairment of synaptic vesicle recycling, suggesting that the interaction of the Annexins with α-synuclein may play a role in the development of PD.

One goal of this research is to identify the specific interaction that takes place between the Annexins and α-synuclein with synaptic vesicles under conditions that mimic PD. Because the Annexins have a binding interaction with negatively-charged phospholipids when calcium is introduced, they may also play a role in the development of PD through their interactions with α- synuclein at the presynapse. Another goal of this research is to characterize the interactions of the Annexins with α-synuclein at unstimulated and stimulated synapses. It is predicted Annexins will interact with α-synuclein and/or synaptic vesicles under conditions that simulate PD because of their role as regulatory proteins at the presynapse. However, to better understand the interaction of α-synuclein and the Annexin family, the behavior of the Annexin family with synaptic vesicles at the presynapse must first be understood on its own. This will be examined through Annexin-A2. Its interaction can take the form of direct binding with the synaptic vesicle itself. As the recycling of the synaptic membrane occurs with a when a stimulus is applied at the presynapse, the Annexins may also differ in their distribution in the area where the synaptic vesicles are located with and without the application of this stimulus. This may be the result of their role in the regulation of endocytosis and exocytosis. data for Annexin-A5 and α-synuclein can also be used to determine the possible regions of co-expression, thus indicating potential for interaction and dysfunction in patients with PD.

Methods

Synaptic Vesicle Purification

Synaptic vesicles were isolated from a rat brain with all of the integral and associated proteins using the subcellular fractionation process. This process allowed the different parts of a cell to be separated from the cell’s other components. A modified version of the standard synaptic vesicle purification process was used (Huttner et al., 1983; Baumert et al., 1989).

Briefly, a frozen rat brain (BioChem Services, cat# 031-ATBR) was broken down into a homogenate. To break down the homogenate, the brain sample was placed into a test tube, then drilled with a power drill until the sample was a milky pink color. Then, the sample was spun, with each centrifugation step varying in speed and length of spin. After each centrifugation round was completed, the components of the sample were separated into two layers based on their respective densities, with the most dense component in a pellet at the bottom of the tube, and the least dense portion at the top in a liquid state as the supernatant. The first round of centrifugation of the homogenate yielded pellet 1 (P1) and supernatant 1 (S1). P1 contained nuclei and other large membrane structures. The second round yielded pellet 2 (P2) and supernatant 2 (S2), and the remaining sample was washed. P2 contained mitochondria. The next round of centrifugation yielded pellets 2’ (P2’), 2’’ (P’’), and 2’’’ (P’’’). Supernatants 2’ (S2’),

2’’ (S’’), and 2’’’ (S’’’) were also obtained from this round of centrifugation. The fourth round of centrifugation yielded lysed pellet 1 (LP1) and lysed supernatant 1 (LS1) after hypotonic lysis.

LP1 contained lysed mitochondria, as well as the cell membrane. The last round of centrifugation yielded lysed pellet 2 (LP2), which contained the low-density synaptic vesicles. Lysed supernatant 2 (LS2) was also obtained in this round of centrifugation. For each centrifugation round of the sample, the speed was increased, and the sample was spun for a longer time period, as the remaining components became progressively less dense. As a result of this separation process, the low-density synaptic vesicles were able to be isolated for experimental use after many rounds of centrifugation (Huttner et al., 1983).

After each round of centrifugation, samples from the pellet and liquid supernatant were collected. The synaptic vesicles were collected during the last round of centrifugation because they were the lightest component that was present in the original sample. Proteins that were in each of these collected samples were then separated based on their respective sizes by Sodium

Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and run through a standard

Western blot to be visualized. This was accomplished by transferring separated proteins to a nitrocellulose membrane, and exposing them to to detect proteins. All antibodies were diluted 1:1000 in 1% milk in TBS + 0.1% Tween 20. The enrichment of synaptic vesicles at the end of the protocol was then be detected with a mouse monoclonal against SV2

(DSHB, cat# SV2-c), a protein located on the membranes of synaptic vesicles. α-Synuclein was detected using a rabbit polyclonal primary antibody (Abcam, cat# ab53726). Annexin A5 was detected using a rabbit polyclonal primary antibody ( Technology, cat# 8555S), that was previously validated in the laboratory using rat brain lysate and lamprey central nervous system lysates (Hendrick, unpublished data). All primary antibodies were detected using horseradish peroxidase (HRP) goat anti-mouse (Thermo Scientific Pierce, cat# 31430) or goat anti-rabbit (Thermo Scientific Pierce, cat# 31460). The areas in which binding occurred between the target protein and antibody resulted in a band on the membrane of the Western blot after a reaction with a chemiluminescence developing substrate solution (Thermo Scientific Pierce, cat#

32106). Bands were then detected using chemiluminescence and the Azure Biosystems c600 Gel

Imaging System. Synaptic vesicle purification was also repeated using buffers that were spiked with 1 mM of calcium because of the calcium-binding characteristics of Annexin-A5.

Allen Brain Atlas

The Allen Brain Atlas was used to examine the expression patterns of the α-synuclein

(SNCA) and Annexin-A5 (ANXA5) genes in a mouse brain for α-synuclein and Annexin-A5 respectively. Expression patterns were generated using in situ hybridization in a C57B1/6J mouse brain (Lau et al., 2008). Each gene was searched on the software in the Allen Mouse

Brain Atlas expression library, and a corresponding data set of a brain section was selected. Data from a sagittal section of a mouse brain was then used for analysis with a sagittal Nissl overlay. This Nissl stain is commonly used in the identification of different different types of nervous tissue, as each type contains varying amounts of Nissl substance. The sagittal section was selected from the gene expression data set as it allowed for a clearer view of all of the mouse brain’s structures (Paul et al., 2008). The density and intensity of each data set was then adjusted to eliminate background noise, while still showing overall expression patterns. The density was set to a minimum of 0.0145 and a maximum of 0.0979, while the intensity was set to a minimum of 102 and a maximum of 210. Expression data was examined specifically in the nigrostriatal pathway of the brain, the pathway that corresponds to an individual's ability to regulate motor control. Images were captured for both SNCA and ANXA5. These images were used to compare the location of expression and the amount of expression that occurred for these genes in the mouse brain. A table containing the major sections of the brain was used to compare and contrast these things.

Immunofluorescence and Imaging

All procedures involving lampreys were performed following the guidelines of the National

Institutes of Health and approved by the Marine Biological Laboratory Institutional Animal Care and Use Committee (Protocol # 19-15). Following standard protocols, the animals were anesthetized and the spinal cord was dissected from the animal (Banks et al., 2020). Spinal cords were either immediately fixed in 4% Paraformaldehyde in 0.1 M PBS pH 7.4 or were treated with high potassium lamprey ringer (61 mM NaCl, 50 mM KCl, 2 mM MgCl2, 4 mM glucose, 2 mM HEPES, 0.5 mM L-glutamate, 2.6 mM CaCl2) followed by paraformaldehyde fixation. The fixed spinal cords were embedded in Optimal Cutting Temperature Medium (Tissue Tek, Cat #

4583), frozen, and sectioned on a cryostat (Leica CM1860). Sections were collected on slides

(Superfrost Plus, Fisher Scientific, Cat# 12-550-15) and stored at -20°C. Sections to be imaged were blocked (10% Normal Goat Serum, 10% Triton-X 100), washed (240 mM sodium phosphate buffer pH 7.4, 10% Triton-X 100, 4M NaCl), and incubated in primary antibody

(1:100; rabbit anti-, Cell Signaling Technology, Cat# 8235S and 1:100 mouse anti-

SV2, DSHB) diluted in Antibody Signal Enhancer Solution (10 mM Glycine, 0.05% Tween-20,

0.1% Triton-X 100, 0.1 M PBS pH 7.4, 0.1% hydrogen peroxide). The sections were washed and incubated in secondary antibody (1:300 488 Goat anti-rabbit, Thermo Fisher, Cat# A11070 and

1:300 594 Goat anti-mouse, Thermo Fisher, Cat# A-11005). The sections were washed, mounted (ProLong Gold Mounting Medium with DAPI, Thermo Fisher, Cat# P36931), and sealed with nail polish. Samples were imaged on a Zeiss LSM 710 scanning confocal microscope using a Zeiss 40X, 0.8 NA Achroplan objective. Analysis of the immunofluorescence images was conducted using FIJI ImageJ 2.0.0 software. Composite images were made of each z-stack, then a scale of a pixel distance of 14.6, known distance of 1, pixel aspect ratio of 1, and a µm unit of length was set. For an individual z- stack, axons were outlined using the region of interest (ROI) tool. Next, an individual synapse with SV2 labeling was selected using a 3µm x 3µm ROI square marker. Synapses were selected independent of any Annexin staining that was present. These measurements allowed for enough room for the synapse and its surrounding area to be analyzed, and reduced the possibility of analyzing a synaptic area more than once. The synapse was located in the center of this square.

The frame that was utilized in this step was the frame that was judged to be most representative of the center of that synaptic cluster. Each synapse is represented throughout multiple frames, but the specific frames that contain these synapse components varies from synapse to synapse. Each individual synapse was cropped and the frames where the synapse was present were selected to make a substack. This substack was then used to make a max Z projection. An ROI circle was then used to fully surround the synaptic vesicle cluster in the middle of the image. Next, lines 0.2

µm in length were drawn outwards from the top, bottom, left, and right of this circle. This length of the lines took into account some shrinkage that occurred during preparation of the sample.

These lines were evenly spaced around the circumference of the circle. Another ROI circle was then drawn touching each of these lines, enveloping the inner synapse area in another circle divided into four quadrants. This process was repeated for each synapse in each z-stack image. A subset of synapses were selected for further analysis resulting in a sample size of 10 for each condition. All of the selected synapse images were then adjusted for brightness in the Annexin channel to a minimum of 0 and a maximum 42. For each max Z projection image, the pixels of the highest intensity in the green channel were selected with ROI circles for each quadrant surrounding the inner circle, as well as from the inner circle itself. A second ROI of the same size as the previous ROI was selected immediately adjacent to the previous ROI to provide data about background level intensity. The mean intensity of each ROI was measured. Next, the background level intensity was subtracted from this value, and the resulting data was then normalized for analysis. Normalizing of the data was done to ensure that a comparison was possible between the results for the unstimulated and stimulated conditions. A parametric t-test of this normalized data was performed.

Results

Synaptic Vesicle Purification

Because members of the Annexin family have exhibited a binding interaction with negatively-charged phospholipids when calcium is introduced in past studies, they might also interact with α-synuclein to produce synaptic defects that can lead to the development of PD. To identify the specific interaction that takes place between the Annexins and α-synuclein that can lead to the inhibition of synaptic vesicle recycling, each protein’s individual interactions with synaptic vesicles must first be understood. Western blot analysis of Annexin-A5 from a synaptic vesicle purification without the addition of Ca2+ led to the generation of the Western blot transfer membrane below (Fig. 1). On the blot, the homogenate (H) and supernatant (S1) lanes exhibited bands at the known molecular weight for Annexin-A5 (35 kD). Purified synaptic vesicles are expected to be present in the LP2 fraction. However, the rest of the blot did not exhibit any bands, suggesting that there is not any Annexin-A5 present on the purified synaptic vesicles (Fig.

1).

Figure 1. Western Blot of Annexin-A5 from a Synaptic Vesicle Purification without Ca2+. A

Western blot transfer membrane for Annexin-A5 from a synaptic vesicle purification without

Ca2+ is displayed above. Bands are present in H and S1 at the molecular weight of Annexin-A5

(35kD). A lack of bands is also observed in the rest of the blot, suggesting that Annexin-A5 is not present on the purified synaptic vesicles.

Allen Brain Atlas

Gene expression data can also aid in increasing the understanding of the individual roles that the Annexins and α-synuclein may each play in synaptic vesicle recycling. Expression in different regions of the brain and the corresponding functions of these sections may indicate what may occur in the presence of excess α-synuclein as observed in PD. In situ hybridization in a C57B1/6J mouse brain generated a gene expression pattern for the SNCA gene for α-synuclein.

The data was obtained from a sagittal mouse brain section overlaying a sagittal Nissl. The presence of green coloring and its intensity in the image corresponds to SNCA expression levels and intensity (Fig. 2). SNCA is expressed in several regions throughout the brain. The highest levels and intensity of expression is observed in the cerebrum, nucleus of the solitary tract in the medulla, motor-related area of the midbrain, olfactory area, striatum, and thalamus. Overall, these proteins seem to be expressed at relatively high levels through the entire mouse brain, with exception to the cerebellum area (Fig. 2).

Figure 2. Gene expression pattern of SNCA in a C57B1/6J mouse brain. The image shows an expression pattern search for SNCA generated by in situ hybridization in a C57B1/6J mouse brain. The data set is from a sagittal mouse brain section overlaying a sagittal Nissl. Expression of SNCA is represented by green coloring, with greater amounts of green representing higher levels of expression. SNCA is expressed in several regions throughout the brain, with the highest levels of expression observed in the cerebrum, nucleus of the solitary tract in the medulla, motor-related area of the midbrain, olfactory area, striatum, and thalamus. In situ hybridization in a C57B1/6J mouse brain also generated a gene expression pattern for the ANXA5 gene for Annexin-A5. The data was obtained from a sagittal mouse brain section overlaying a sagittal Nissl. The presence of pink coloring and its intensity in the image corresponds to Annexin-A5 expression levels and intensity (Fig. 3). The highest levels and intensity of ANXA5 expression occurs in the cerebellum, hypothalamus, medulla, olfactory area, superior olivary complex and motor-related areas of the pons, striatum, and thalamus. This data indicates that Annexin-A5 is also present in relatively high levels through the mouse brain (Fig. 3).

Figure 3. Gene expression pattern of ANXA5 in a C57B1/6J mouse brain. The image shows an expression pattern search for ANXA5 generated by in situ hybridization in a C57B1/6J mouse brain. The data set is from a sagittal mouse brain section overlaying a sagittal Nissl. Expression of ANXA5 is represented by pink coloring, with greater amounts of pink representing higher levels of expression. The highest levels of ANXA5 expression occur in the cerebellum, hypothalamus, medulla, olfactory area, superior olivary complex and motor-related areas of the pons, striatum, and thalamus.

Combined, the gene expression data for α-synuclein and Annexin-A5 can be examined to see if there are any similar patterns in their expression. This can give more information about their relationship to one another. Both Annexin-A5 and α-synuclein were expressed at low levels in the substantia nigra pars compacta, one of the parts of the nigrostriatal pathway that is lost in individuals with PD (Table 1). α-Synuclein is also expressed at low levels in the cerebellum and in the motor-related area of the pons (Table 1).

Location Function SNCA ANXA5 Expression Expression

Cerebellum Coordination of Low High voluntary muscle movement, balance Cerebrum Movement High Medium initiation/coordination, sensory regulation, reasoning, learning

Hypothalamus Connects nervous and Low High endocrine systems

Medulla Control of autonomic Low High nervous system

Medulla Relay of visceral High Medium (nucleus of sensory information solitary tract)

Midbrain Regulation of motor High Medium (motor-related movement area)

Olfactory bulb Odor detection High High

Pons (superior Part of the afferent and Low High olivary efferent auditory complex, pathways, motos areas motor-related regulate fine motor areas) movements

Striatum Regulation of High High voluntary motor movement

Substantia Regulation of motor Low Low nigra pars movement, contains compacta dopaminergic neurons

Thalamus Relay of sensory/motor High High information to cerebral cortex

Table 1. Expression pattern comparison of SNCA and ANXA5. The table shows the expression levels of SNCA and ANXA5 in the sagittal mouse brain sections above (Figures 1 and 2 respectively). Both SNCA and ANXA5 are expressed at low levels in the substantia nigra pars compacta, one part of the nigrostriatal pathway that is lost in PD. Low levels of SNCA are also observed in the cerebellum and motor-related area of the pons.

Immunofluorescence and Imaging

The behavior of the Annexins at the presynapse on their own must also be understood to better-characterize their interactions with α-synuclein. To examine this, immunofluorescence images of the spinal cord were analyzed (Fig. 4). In Figure 4, individual axons are outlined in yellow for each cross section. FIJI ImageJ software then generated composite images for each of the stimulated and unstimulated synapses. In these multi-channel images, green represents

Annexin-A2, and red represents SV2, an integral synaptic vesicle membrane protein. The red and green channels were also combined for analysis purposes. From these cross-section images, a more in-depth analysis of individual synapses was able to be conducted (Fig. 4).

Figure 4. Immunofluorescence Cross-Section of a Lamprey Spinal Cord. The image above shows two immunofluorescence cross-sections of a lamprey spinal cord. Axons are outlined in yellow for each image. The image on the left is from unstimulated z-stack 1, and the image on the right is from stimulated z-stack 1. Unstimualted z-stack 1 contains axons 4, 3, 1, and 2 from top to bottom. Stimulated z-stack 1 contains axons 5, 3, 1, 2, and 4 from top to bottom. The red coloration represents SV2, green represents Annexin-A2, and blue represents nuclei.

After synapses were selected from each z-stack image, individual synapses were then analyzed to better understand the behavior of the Annexin family at the synapse. Images of the individual synapses were made into composites with a green channel representing Annexin-A2, and a red channel representing SV2. The full compote can be viewed in the rightmost portion of the image (Fig. 5). In the image where the color channels are combined, an inner circle surrounding the synaptic vesicle cluster is shown, surrounded by a circle containing the periactive zone. Smaller ROIs circles inside of these large circles were used to examine the intensity of the green pixels within that circle (Fig. 5).

Figure 5. Unstimulated and Stimulated Lamprey Spinal Cord Synapse Channels. In the image above, synapse 1 from axon 2 unstimulated z-stack 1 is shown in the top row. In the bottom row, synapse 3 from axon 2 stimulated z-stack 1 is shown. From left to right, the images show their corresponding green Annexin-A2 channels, red SV2 channels, and combined red and green channels containing ROI analysis circles. The inner ROI circle surrounds the synaptic vesicle cluster, while the outer ROI circle surrounds the peractive zone. The small circles within these zones were used for the intensity analysis mentioned below.

Data from the immunofluorescence images analysis for the synaptic vesicle cluster was analyzed using a parametric t-test (p = 0.0125, mean D = 16.23, mean E = 29.38, (E-D) ± SEM =

13.15 ± 4.737). In the graph, the normalized intensity of Annexin-A2 inside the periactive zone and synaptic vesicle cluster can be observed. The data was obtained from both stimulated and unstimulated synapses. A lower intensity of Annexin-A2 was observed in the synaptic vesicle cluster of the unstimulated synapses. This indicates that more Annexin-A2 can be found in the periactive zone for this condition (Fig.4). A higher intensity of Annexin-A2 was observed in the synaptic vesicle cluster of the stimulated synapses. This information indicates that less Annexin- A2 can be found in the periactive zone for this condition (Fig. 6).

Figure 6. Normalized Intensity of Annexin-A2 in the Periactive Zone and Synaptic Vesicle

Cluster. The data in the graph was obtained from a parametric t-test (p = 0.0125). The graph shows the normalized intensity of Annexin-A2 in the periactive zone and synaptic vesicle cluster of stimulated and unstimulated synapses. The synaptic vesicle cluster had a lower intensity of

Annexin-A2 in the unstimulated synapses, suggesting that more Annexin-A2 was present in the periactive zone under this condition. The synaptic vesicle cluster had a higher intensity of

Annexin-A2 in the stimulated synapses, suggesting that more Annexin-A2 was present in the synaptic vesicle cluster under this condition.

Combined, the data above gives a better understanding of the individual roles of the

Annexin family and α-synuclein. Based on this better understanding of their individual interactions near synapses and at synaptic vesicles specifically, more information can also be learned about their combined interaction with one another that may lead to the impairment of synaptic vesicle recycling.

Discussion

The Western blot transfer membrane for Annexin-A5 from the synaptic vesicle purification without Ca2+ exhibits bands in the H and S1 zones at 35 kD, the molecular weight of Annexin-A5. No other bands are observed on the blot, indicating that Annexin-A5 is not present on the purified synaptic vesicles (Fig. 1). As stated earlier, the Annexin family is known to play a role in regulation at the synapse, and are known to be synaptic vesicle-associated proteins (Banks et al., 2020; Bedrood et al., 2009). As a synaptic vesicle-associated protein, it is expected that Annexin-A5 would be able to be purified with synaptic vesicles in the purification process mentioned above, yet it is not. In past research, the protein α-synuclein has been able to be purified with synaptic vesicles, and was observed in the LP2 fraction based on the presence of a band in this area of its corresponding Western blot (Cantor, unpublished data). As α-synuclein is an associated protein like Annexin-A2, and is actually able to be purified out with synaptic vesicles, this information could have implications for the ability of these proteins to interact with one another, as α-synuclein may be localized to synaptic vesicles, while Annexin-A2 (and by extension the rest of the Annexin family) may not be. Overall, the specific interaction that takes place between Annexin-A5 and α-synuclein is still not clear from these results, but the direct binding between Annexin-A5 and aggregated α-synuclein that was observed in the past indicates that Annexin-A5 may still be playing a role in the impairment of synaptic vesicle recycling at the presynapse (Bedrood et al., 2009). However, the observed absence of Annexin-A5 on the purified synaptic vesicles indicates that the interaction between Annexin-A5 and α-synuclein may not be taking place directly on the synaptic vesicle itself. Instead, another type of interaction between these proteins could be occurring away from the synaptic vesicle. Excess α-synuclein may be binding to Annexin-A5 elsewhere, resulting in its movement away from the presynapse, and leading to less synaptic regulation. The excess α-synuclein could also be binding to the synaptic vesicle membrane, blocking the ability of Annexin-A5 to interact with it under conditions similar to PD. This synaptic vesicle purification and binding should be repeated under the same conditions to verify its results. To examine the interactions of the Annexin family under conditions similar to PD, experiments should be done using excess α-synuclein. Under these conditions, the interaction between these two proteins may follow a similar pattern to what was observed in the data above, indicating that the excess α-synuclein characteristic of PD is interacting with members of the Annexin family away from the membrane of the synaptic vesicle. Because the exact mechanisms of this interaction still need to be characterized, it is important to examine the location of Annexin and α-synuclein expression in the brain itself. If these proteins are expressed in a given region of the brain, then the corresponding function of that area may tell us more about what happens when the proteins are overexpressed. Data from the Allen Brain Atlas maps the expression of the SNCA gene for α-synuclein, and the ANXA5 gene for Annexin-A5 in a C57B1/6J mouse brain (Lau et al., 2008). The α- synuclein expression data shows high SNCA expression in the cerebrum, nucleus of the solitary tract in the medulla, motor-related area of the midbrain, olfactory area, striatum, and thalamus

(Fig. 2). The Annexin-A5 expression data shows high ANXA5 expression in the cerebellum, hypothalamus, medulla, olfactory area, superior olivary complex and motor-related areas of the pons, striatum, and thalamus (Fig. 3). Combined, this data indicates that these proteins normally have high expression in the majority of the brain’s structures. In contrast to this data however, both SNCA and ANXA5 are expressed at low levels in the substantia nigra pars compacta, one part of the nigrostriatal pathway that is lost in PD (Table 1). Because the amount of α-synuclein in this pathway is typically so low for normally functioning individuals, it can be inferred that even a slight increase in the amount of α-synuclein can have a large impact on the brain’s function. This data supports the fact that the increased levels of α-synuclein observed in PD patients in an aggregated form can contribute to synaptic defects (Busch et al., 2014). These defects can lead to neurodegeneration, and eventually to a loss of motor control (Miraglia et al.,

2018). Annexin expression in the nigrostriatal pathway may simply be low because that is the amount that is needed for regulatory purposes, as the Annexin family is known to play a role in synaptic regulation. Annexin-A5 may also be playing an unknown role in the mouse brain in this case (Bedrood et al., 2009; Hakobyan et al., 2017) Low levels of SNCA expression is also observed in the cerebellum and motor-related area of the pons (Table 1). The expression patterns of the other members of the Annexin family should also be examined. If the pattern between this family of proteins is consistent with the data above, then more concrete conclusions about their roles in the brain can be drawn. From the information gathered about the Annexins, it is more clear where their potential interactions with excess α-synuclein may be taking place. As observed in past studies, aggregated α-synuclein in the brain can lead to synaptic defects, which can ultimately lead to the neurodegeneration that is characteristic of diseases like

PD (Busch et al., 2014). As a result, it is important to examine the activity of the Annexins at the synapse itself more closely. As this specific behavior had not been observed before this research took place, finding a way to effectively view activity of proteins such as α-synuclein, members of the Annexin family, and SV2 was of the utmost importance. As a result, immunofluorescence images of Annexin-A2 were used to give more information on this subject (Fig. 4, Fig. 5). Based on the images that were generated, immunofluorescence imaging effectively accomplishes this goal, as the various axons, synapses, and immunofluorescence tagging of proteins is fully visible and clear (Fig. 4, Fig. 5). This technique can be utilized in future analysis of Annexin-A2, as well as other members of the Anneixn family. Analysis was then conducted using data from the small ROI circles (Fig. 5). After analysis of the immunofluorescence images above, a normalized intensity graph was generated to show the intensity of the Annexin-A2 in the periactive zones and synaptic vesicle clusters of stimulated and unstimulated synapses. As a p-value of 0.0125 was obtained from this analysis, it can be inferred that the results were significant. Unstimulated synapses exhibited a lower synaptic vesicle cluster intensity of Annexin-A2, while the stimulated synapses exhibited a higher intensity of Annexin-A2 in the synaptic vesicle cluster (Fig. 6).

Based on this information, more Annexin-A2 seems to be present in the periactive zone when the axons corresponding to these synapses are not stimulated. When axons are stimulated, their corresponding synapses have more Annexin-A2 present in the synaptic vesicle cluster. This information fits with prior knowledge of the role of Annexins at the synapse. More specifically, the Annexins are able to organize membrane proteins and lipids, and regulate endocytosis and exocytosis (Hakobyan et al., 2017). Before a stimulus has occurred, it can be inferred that the

Annexin-A2 from the results was inactive in the periactive zone. Upon stimulation, the Annexin-

A2 was then recruited to the synaptic vesicles to help in the release neurotransmitter from the synaptic membrane, explaining the higher levels that were observed in the synaptic vesicle cluster area after stimulation of the sample (Fig. 6). Immunofluorescence studies should be done with the other members of the Annexin family, as well as α-synuclein. Doing these supplemental studies will allow for more more concrete conclusions to be made about the synaptic vesicle purification data above (Fig. 1). For example, as Annexin-A5 did not exhibit purification with synaptic vesicles (Fig. 1), it may not be expected to be localized to the synaptic vesicle membrane without the application of a stimulus, in a fashion similar to the movement of

Annexin-A2 towards the synthetic vesicle cluster with the application of a stimulus. If this behavior of the Annexins proves to be true for multiple members of the Anneixn family, then more concrete conclusions can be drawn about their interactions with α-synuclein at the presynapse.

Although the results of the immunofluorescence experiment were significant, error for the analysis was larger than expected (Fig 6). This relatively large amount of error is likely the result of the method by which synapses were picked. More specifically, synapses were blindly picked based on the SV2 coloration that was observed. This blind method of picking synapses allowed bias towards synapses with a large amount of Annexin-A2 coloration to be eliminated.

Because of this, we can also infer that the results are unbiased. However, the large amount of error between synapses may have been generated due to the fact that stimulation was unequal between synapses. More specifically, when a stimulus is applied to an axon/synapse, they may not all fire at the exact same time. It is also possible the spinal cord preparation could generate a low level of stimulation in the unstimulated sample.

Overall, this research can take multiple directions in the future. Western blot analysis with Annexin-A5 can be done with synuclein as a comparison to the data above. This data will act as a simulation of conditions that are similar to PD. Western blot analysis of synaptic vesicle purifications with and without synuclein can take place for other members of the Annexin family, allowing for more knowledge to be gained about how the Annexin family may impact the development of PD as a whole. The Allen Brain Atlas software can be used to analyze the expression patterns of other members of the Annexin family in the mouse brain in relation to α- synuclein expression patterns. Based on the areas where the other members of the Annexin family are expressed in the brain, inferences can be made about their impact on the development of PD. The IF analysis of Annexin-A2 should also be conducted in the same way with a larger sample size, to examine if the results remain the same. Lastly, IF analysis can also be conducted in the same way as above, with excess synuclein. This will allow for the generation of conditions similar to PD.

While future studies like the ones mentioned above are needed to fully understand the mechanisms behind the interaction between the Annexins and α-synuclein, the data does provide more insight into this interaction overall. First, it can be concluded that Annexin-A5 did not behave as expected as a synaptic vesicle-associated protein in the synaptic vesicle purification process. Because it did not purify out with the synaptic vesicles like α-synuclein, it can be concluded that more research must be done to determine how this result can apply to the interaction between the Annexin family and α-synuclein. It is also evident that both Annexins and α-synuclein expression corresponds to past knowledge about the development of PD, specifically in relation to motor control area and the nigrostriatal pathway. Annexin-A2 also clearly demonstrates movements to the synaptic vesicle cluster upon the receipt of a stimulus, indicating that the Anneixn family as a whole may also exhibit this behavior. Because of this information, Annexins may be playing an active role in the inhibition of synaptic vesicle recycling with an excess of α-synuclein. From this information, progress towards learning the specific mechanisms behind PD is made. Through this progress, PD can be better-treated and diagnosed.

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