Eukaryon

Introduction

Neurodegenerative Diseases
Most of the cells in our body have regenerative power. Unfortunately, regeneration in the central nervous system is extremely restricted. This lack of regeneration can be particularly devastating, because if these cells are destroyed or damaged, permanent disability ensues.

Our society is afflicted by a variety of human brain disorders. Amongst them are a group of disorders classified as the neurodegenerative diseases. The word neurodegeneration gives us a hint of what these disorders involve: the deterioration or death of neurons in the brain specific to the disease. Neurodegenerative diseases affect millions of people worldwide, and by 2040, they will surpass cancer as the leading cause of death. Unfortunately, no cure currently exists. Understanding the molecular and cellular mechanisms by which these diseases operate might eventually lead to more effective treatments and ultimately a cure (Lozano et al., 2005).

The best-studied neurodegenerative diseases include Alzheimer’s disease, Lou Gehrig’s disease, Creutzfeldt-Jacob disease, Huntington disease, Parkinson’s disease (PD), and prion diseases. They vary in symptoms and onset, but one common characteristic is the presence of an abnormal protein unique to each disease. Each particular protein misfolds, aggregates, usually leading to accumulation as intracellular or extracellular inclusions in the brain of patients (Taylor et al., 2002). In most cases, these proteins are thought to gain a toxic new function leading to pathogenesis, but data is not yet conclusive. My thesis focused on the molecular basis of one such disease, PD.

Parkinson’s Disease
PD afflicts around four million people worldwide, making it amongst the most prevalent neurodegenerative disease (Lozano et al., 2005).  First characterized in the 1800s by physician James Parkinson, classic symptoms include tremors, muscular rigidity, slowness of movement, and impaired balance and coordination. Half of the patients develop symptoms after age 60, suggesting that the disease not only affects the elderly since the other half develop symptoms before then. Currently, no cure for PD exists, but treatment for the symptoms is available. Unfortunately, most patients eventually become resistant to the medication (Lozano et al., 2005).

Similar to other neurodegenerative diseases, PD is linked to the misfolding and accumulation of a particular protein, α-synuclein (Spillantini, et al., 1998). A hallmark symptom of PD is the accumulation and aggregation of α-synuclein into structures called lewy bodies found in the substantia nigra region of the midbrain. These individuals experience loss of dopaminergic neurons in this area of the brain (Giasson et al., 1999; Spillantini et al., 1998, Abeliovich et al., 2000; Lozano et al., 2000). The basal ganglia consists of a group of nuclei that organize motor behavior and the substantia nigra is one component of this pathway. Therefore, the substantia nigra is essential for voluntary movement. Due to this fact, substantia nigra cell death leads to movement disorders since the death of these neurons interferes with the normal signaling to the basal ganglia (Figure 1A; Schmidt and Oertel, 2006). The brain has two pathways through the basal ganglia which function to either initiate volitional movement (direct pathway) or to suppress inappropriate movements (indirect pathway) (Purves, 2008). PD affects the direct pathway to the basal ganglia because it results from the inability to initiate movement, therefore making it a hypokinetic disease (Figure 1B; Purves, 2008). 

PD occurs in two forms: sporadic or familial. An estimated 95 percent of PD cases are sporadic (Schmidt and Oertel, 2006). The misfolding and accumulation of normal wild-type (WT) α-synuclein is linked to the sporadic case of PD. α-Synuclein aggregates and accumulates because of decreasing mitochondrial complex 1 activity, oxidative stress, and environmental factors such as chemicals or toxins (Dawson and Dawson, 2003). For example, the uptake of 1-Methyl-4-phenyl-4-propionoxy-piperidine (MPPP), a byproduct of synthetic heroin, caused PD-like symptoms in patients (Langston et al., 1983). The cause for the misfolding and accumulation of WT α-synuclein is not well understood. Because of the importance of these characteristics in PD pathogenesis, they are essential to examine further.

In contrast, familial PD is caused by genetic factors. Studies of families demonstrate that point mutations in the α-synuclein gene cause PD. These mutations include the A30P (Polymeropoulos et al., 1997), A53T (Kruger et al., 1998) or E46K (Zarranz et al., 2004) changes. Six other genes have been identified in the onset of familial PD: parkin (Kitada et al., 1998), UCH-L1 (Liu et al., 2002), DJ-1 (Bonifati et al., 2003), PINK1 (Valente et al., 2004), LRRK2 (Funayama et al., 2002; Paisan-Ruiz et al., 2004), and PARK9 (Ramirez et al., 2006). The parkin E3 ubiquitin ligase and UCH-L1 ubiquitin hydrolase gene mutations are involved in interfering with the ubiquitin proteasome system (Kitada et al., 1998; Liu et al., 2002).

Figure 1: Parkinson’s disease Pathway. A. The motor cortex controls movement of our muscles, while the basal ganglia controls timing and coordination of that movement. The substantia nigra is part of the basal ganglia. This pathway is disrupted in PD by the loss of the dopaminergic neurons in the substantia nigra. This leads to less/shaky voluntary movement because the basal ganglia cannot send signals to the motor cortex, which would send instructions for movement. B. Direct pathway through the basal ganglia. In PD (red), the inputs by the substantia nigra are diminished, making it more difficult for a transient inhibition of the caudaute and putamen. This results in less inhibition of the globus pallidus and in turn more inhibition of the VA/VL complex of the thalamus, which results in less thalamic excitation of the motor cortex (Adapted from Purves, 2008). 

DJ-1 gene mutations are implicated in the pathogenesis of PD because of its possible role in response to oxidative stress (Bonifati et al., 2003). PINK1 is involved in the phosphorylation of mitochondrial proteins, as a response mechanism for mitochondrial dysfunction, and its mutation has been linked to PD progression (Valente et al., 2004). A mutation in protein LRRK2 has similarly been linked to pathogenesis due to possible phosphorylation of α-synuclein protein or causing accumulation due to kinase activity (Paisan-Ruiz et al., 2004; Zimprich et al., 2004). Studies demonstrate that the LRRK2 mutation causes a significant increase in kinase activity, suggesting a gain in PD pathology (Gloeckner et al. 2006). The most recently identified gene in familial PD has been PARK9, since mutations in this gene cause loss of function. Loss of PARK9 might be involved in the accumulation of proteins because of degradation dysfunction through either the proteasome or lysosome (Ramirez et al., 2006). Most importantly PARK9 protects cells from manganese toxicity, since manganese is an environmental factor in PD (Gitler et al., 2009). The mechanisms involved in progression to familial PD are a large area of study that still needs to be fully uncovered. Studying α-synuclein properties is important in order to understand PD pathogenesis, since α-synuclein misfolds in both sporadic and familial forms of the disease. 

α-Synuclein
α-Synuclein has been implicated in a variety of other neurodegenerative diseases including AD (Lucking and Brice, 2000). As mentioned previously, the misfolding and aggregation of α-synuclein leads to Lewy body formation in the brains of patients. α-Synuclein is part of a larger family of proteins including α-, β-, and γ-synuclein (George, 2009). The protein has a molecular mass of 19 kDA, is 140 amino acids long (Jakes et al., 1994), and is composed of the N-terminal amphiphathic domain, the hydrophobic middle domain, and the acidic C-terminal domain (Lucking and Brice, 2000). α-Synuclein’s flexible nature is due to its unfolded C-terminal region (Eliezer et al., 2001).

The following key α-synuclein findings may help us better understand PD: 1) The protein’s normal function is not well understood, but data indicates it is highly expressed in the brain, particularly in presynaptic nerve terminals (Kaplan et al., 2003; Jakes et al., 1994). 2) α-Synuclein deletion mice exhibit a reduction in striatal dopamine neurons, supporting the finding that the protein is an essential presynaptic regulator of dopamine neurotransmission (Abeliovich et al., 2000). 3) Lewy bodies have an increased accumulation of fibrillar α-synuclein, which point to their role in disease pathogenesis (Goldberg and Lansbury, 2000; Kaplan et al., 2003). 4) The presence of a weak transient or residual secondary structure in the protein is what may be playing a role in amyloid fibril formation, therefore resulting in aggregation (Eliezer et al., 2001). 5) Cytoplasmic concentrations of dopamine have been demonstrated to promote and stabilize protofibrillar intermediates, linking the dopaminergic selectivity of α-synuclein (Conway et al. 2001). 6) Data on the cytotoxicity of α-synuclein protofibrils has been inconclusive, but research demonstrates that α-synuclein protofibrils are the ones that tightly bind to vesicles and cause membrane permeabilization and destruction, a toxic effect (Volles et al. 2001). 7) To further support α-synuclein’s gain of function characteristics in PD, experiments in animal models show PD-like symptoms with α-synuclein over-expression. This was demonstrated with wild-type α-synuclein and familial mutants A30P and A53T in mice (Masilah et al., 2000), flies (Feany and Bender, 2000), and worms (Lakso et al., 2003). 8) In yeast models, WT and E46K α-synuclein enhanced cell toxicity (Dixon et al, 2005). WT and the other familial mutant, E46K localize to the plasma membrane, confirming α-synuclein’s membrane binding affinity. The familial mutant A30P localizes to the cytoplasm (Ouitero and Lindquist, 2003; Dixon et al., 2006; Sharma et al., 2006). However, the role of α-synuclein membrane binding in PD pathogenesis remains unknown. 9) Since it is found associated in diverse cellular locations, α-synuclein degradation is likely complex.

Major α-Synuclein Questions
The past decade provided tremendous insight into the PD field. Questions regarding the molecular basis of PD still remain unanswered. Is the accumulation and inclusion formation of α-synuclein causing the progression of PD? If this is true, what is the causative agent? From the studies with the familial causing PD genes, the role of oxidative stress in PD pathogenesis needs to be fully understood. Studying the effect of mitochondrial dysfunction’s role in the disease is a priority. A major gap in the field is whether α-synuclein accumulation causes PD. If so, a better understanding of the different degradation routes α-synuclein takes is essential for developing treatments. My thesis specifically focused on this last question.

The Degradation Problem
α-Synuclein is found in many cell locations (Figure 2A). α-Synuclein is mostly found in the cytoplasm, while studies demonstrate an affinity to phospholipids along with vesicle binding (Kahle, 2000; Davidson et al., 1998; Eliezer et al., 2001). Recently, data indicate that α-synuclein is delivered to the plasma membrane through its interactions with the ER-Golgi secretory pathway (Dixon et al., 2005). Lastly, smallamounts of α-synuclein are secreted from cells and are present in human body fluids, such as the blood plasma and cerebrospinal fluid (Lee et al., 2008).

Figure 2: α-Synuclein location and possible degradation routes. A. α-Synuclein is found in several places throughout a cell: 1. In cytoplasm, 2. Membrane-bound, 3. In secretory pathway, and 4. Extracellularly. B. Old, misfolded or damaged proteins need to be degraded. Extracellular and membrane-bound proteins use the lysosome as a route, while nuclear cytoplasmic proteins use the proteasome.

α-Synuclein degradation is likely complex due to its localization (Figure 2A). The cell has mechanisms to degrade old, damaged, or misfolded proteins, as eliminating such proteins is critical for the health of an organism. These mechanisms break proteins down to their constituent amino acids through one of two different degradation routes: the ubiquitin proteasome system (UPS) or the lysosome (Figure 2B). Given α-synuclein’s localization, its degradation is likely complex. Data reveals that prion proteins, another set of proteins linked to neurodegeneration, can use both pathways. In mice brains, the prion protein is deposited in the lysosome (Laszlo et al., 1992), while in yeast it uses the proteasome for degradation (Ma and Lindquist, 2001). Depending on where α-synuclein is localized, it might use one or both routes. I first examine the evidence for the proteasome as a site for α-synuclein degradation, followed by the lysosome.

Route One: The Proteasome
The ubiquitin proteasome system (UPS) is one of two recycling mechanisms in the cell. The proteasome usually degrades proteins from the cytoplasm or the nucleus. Exceptions to this rule exist, however (Figure 2B; Alberts, 2004). The UPS uses ubiquitin, a small protein that is attached to molecules that need to be degraded, as a marker for the molecules’ destruction. The enzymes that add the ubiquitin protein recognize signals for misfolding or chemical damage in the proteins.

Until 2004, the PD field believed α-synuclein was degraded solely by the proteasome, because α-synuclein is found in the cytoplasm. Several lines of genetic evidence support the proteasome as an α-synuclein degradation route.

Figure 3: Three routes to the lysosome. There are three major routes to the lysosome: phagocytosis, autophagy, and endocytosis. In phagocytosis, the cell engulfs bacteria or viruses for degradation. In autophagy, a membrane forms and eventually surrounds the organelle, then transporting it to the lysosome. Lastly, during endocytosis, the plasma membrane invaginates and material from outside of the cell or from the plasma membrane is tagged to the lysosome.

Patients with two mutant genes involved in the ubiquitin-proteasome system caused familial PD (UCH-L1 and parkin). UCH-L1 is a neuronal enzyme that hydrolyses ubiqutin, and studies show that α-synuclein aggregates when mutated UCH-L1’s concentration exceeds a certain threshold (Liu et al., 2002). Parkin is an E3 ligase that ubiquitylates α-synuclein among other proteins and tags them for degradation (Liu et al., 2002; Kitada et al., 1998). Several lines of genetic and pharmacological evidence further provide a link to the importance of the proteasome in α-synuclein degradation. A 26S proteasome subunit mouse knockout model resulted in mice with intra-neuronal Lewy-like inclusions and neurodegeneration in the nigrostriatal pathway (Bedfort et al., 2008). Pharmacological studies have shown that proteasome inhibition, by lactacystin, for example, leads to α-synuclein inclusion formation (Rideout et al., 2002; Sawada et al., 2004). Proteasome inhibition in mice increases cell death through activation of cytoplasmic p53. Findings suggest that p53 abnormalities may play a role in dopaminergic cell death (Nair et al., 2006). These results highlight the proteasome as a key organelle in α-synuclein degradation.

Route Two: The Lysosome
The lysosomal system is the second recycling mechanism in the cell which degrades proteins from the plasma membrane or from outside of the cell. The cell has three pathways to the lysosome: phagocytosis, autophagy and endocytosis (Figure 3). Extracellular particles, such as bacteria, are taken up through phagocytosis. Once the particle is bound to the cell, special receptors trigger invagination of the membrane. The vesicle then pinches off and this vacuole is now a phagosome. Then enzymes begin breaking the bacteria down and transport it to the lysosome; once it fuses with the lysosome, it becomes a phagolysosome (Alberts, 2004). Since α-synuclein is not a bacterium, phagocytosis is not studied as a degradation route. 

The second pathway to the lysosome is autophagy and a method that the cell uses to get rid of unwanted or damaged organelles and proteins found in the cytoplasm. Autophagy is a stimulus-induced (self-cannibalism) mechanism: for example, if the cell is experiencing amino acid starvation the cell will degrade nonessential cellular components.

Figure 4: MVB/Endocytosis Pathway. A. The endocytic pathway brings in proteins from outside or from the plasma membrane into the cell and to the lysosome. It also brings in proteins that the lysosome needs to break down substances. These proteins are made in the ER. It does this using multivesicular bodies (MVBs), which are vesicles that form inside the late endosome. B. MVBs are formed when the endosome membrane invaginates into the lumen. The invagination is made possible by the endosomal-sorting complex required for transport (ESCRT). The ESCRT complex helps form MVBs which then make it possible for those membrane-bound proteins to be degraded once the endosome and lysosome fuse.

The process begins with the formation of a membrane around an organelle, a process called nucleation. The membrane expands until the organelle is completely enclosed by a double membrane, creating an autophagosome (expansion). Lastly, this vesicle is then transported to the lysosome for degradation and the union of an autophagosome and a lysosome is called fusion (Alberts, 2004).

In endocytosis, the third route to the lysosome, fluid or small molecules are taken from the outside of the cell, from the plasma membrane, or from the ER-Golgi secretory pathway to the lysosome. This happens when the plasma membrane buds in and then pinches off to form an endosome. This material is eventually taken to the lysosome for digestion (Alberts, 2004).

Several lines of pharmacological evidence now point to the lysosome as another α-synuclein degradation route: 1) Evidence demonstrates no changes in levels of α-synuclein with proteasome inhibition (Rideout et al., 2004; Ancolio et al., 2000). 2) Studies performed with human neuroblastoma and rat embryonic cortical neuron cell cultures with inhibited lysosome show rapid accumulation of oligomeric α-synuclein (Lee et al., 2004). Cuervo et al.(2004) conducted experiments demonstrating WT α-synuclein is internalized and degraded by the lysosome through chaperone-mediated autophagy (CMA). The A30P and A53T mutants, on the other hand, were not degraded using this mechanism. 3) Results were confirmed in similar experiments with inhibited CMA and macroautophagy, resulting in WT α-synuclein accumulation (Vogliatzi et al, 2008). 4) Other experiments with autophagy found evidence of the presence of α-synuclein in autophagy-like vesicles. Data also showed that autophagy inducers, such as rapamycin, increased the clearance of α-synuclein in PC12 cell lines (Webb et al., 2003).

The studies above focus mostly on autophagy and less on endocytosis; almost all evidence is pharmacological. My thesis, therefore, focused on the endocytosis route.

Multivesicular body/Endosome Pathway
The main function of the endocytosis pathway is to import proteins located extracellularly or proteins on the membrane to the lysosome. Endocytosis also brings in proteins from the ER that the lysosome needs to break down substances (Figure 4A).

Transmembrane proteins are delivered to the endosome lumen in multivesicular bodies (MVBs), which are vesicles that form inside the late endosome. The sorting of proteins into the MVB pathway is a complex, multistep process. Monoubiquitination of these transmembrane proteins signals their sorting into an MVB (Katzmann et al., 2001). A common substrate that uses the MVB/endosome pathway is the epidermal growth factor receptor (EGF-R). The signal for this protein to become degraded by MVBs is tyrosine phosphorylation, and mutations impair its degradation (Fedler et al. 1990). Experiments with growth hormone receptor (GHR) demonstrate that ubiquitin needs to be present for GHR degradation by endocytosis (Strous et al. 1996). In accordance, other proteins such as Ste3, Gap1, Tat2t in yeast and GHR, MHCII, E-Cadherin in mammals also uses ubiquitin as a signal for degradation by the MVB/endocytosis pathway (Katzmann et al., 2002).

The invagination of the endosome membrane is made possible by subcomplexes, the endosomal-sorting complex required for transport (pre-ESCRT, ESCRT I, II, III and post-ESCRT). The ESCRT complexes helps form MVBs which then make it possible for those membrane-bound proteins to be degraded once the endosome and lysosome fuse (Figure 4B; Katzmann et al., 2001; Katzman et al., 2002).

The ESCRT complexes are composed of less than 20 proteins, known as class E Vacuolar Protein Sorting (vps) proteins, since the vacuole is the yeast counterpart of the lysosome (Katzmann et al., 2002). Mutations in these genes cause defective sorting of transmembrane proteins from the plasma membrane to the vacuolar lumen by means of the MVB pathway (Hierro et. al. 2004). vps34, a protein kinase, is involved at the pre-ESCRT step with initiation and cargo recognition. vps34 is also responsible for the synthesis of a specific phospholipid, phosphatidylinositol 3-phosphate, which then forms a complex at the membrane with vps15 to regulate protein sorting (Herman & Erm, 1990; Stack et al. 1993). Moreover, pre-ESCRT protein vps27 is recruited to the early endosome by its interaction with the ubiquitinated cargo. vps27 then recruits ESCRT-I complex (composed of mvb12, vps23, vps28, and vps37) into the membrane, these proteins are responsible for the cargo sorting. ESCRT-I recruits ESCRT-II (composed of vps22, vps25, and vps36), and ESCRT-II recruits ESCRT-III (composed of vps2, vps20, vps24,and vps32) into the membrane (Babst et al., 2002). ESCRT-III is responsible for cargo sequestration and finally MVB vesicle formation. Post-ESCRT steps include vps4, vps60 and vta1. These are involved in disassembly and membrane release (Lee and Gao, 2008).

A few studies have focused on the endocytosis/MVB pathway as a mechanism for α-synuclein degradation. First, genome-wide yeast deletions identified two endocytosis genes, vps24 and vps28, that demonstrated α-synuclein dependent toxicity (Willingham et al., 2003).

Figure 5: Predictions and genes examined. A. Predictions for endocytotic regulation of a-synuclein. We induced a-synuclein expression in both the endocytosis intact and the endocytosis deficient yeast. The endocytosis intact is expected to degrade a-synuclein via the endosome-lysosome pathway, leading to healthy cells. On the other hand, we predict some, but not all endocytosis deficient yeast will increase a-synuclein accumulation, toxicity and change localization. B. Table showing the genes that make ESCRT-I, II and III. The genes examined in this study have a checkmark. 

Also, phosphorylated α-synuclein accumulated in C. elegans with endocytosis gene knockouts (Kuwahara et al., 2008). Even though α-synuclein is mostly a cytoplasmic protein, small amounts of its aggregated form are found outside the cells in PD patients, in particular in the blood plasma and cerebrospinal fluid. Lee et al. (2008) showed that accumulated extra cellular α-synuclein was internalized using the endocytosis pathway and eventually degraded by the lysosome (Lee et al., 2008). Rab5, a GTPase, seems to be an important factor in neuronal endocytosis. One study showed that Rab5 is critical for the degradation of exogenous α-synuclein in cells (Sung et al., 2001). In a yeast model, the familial mutant A30P binds to endocytic protein YPP1 at the plasma membrane, causing the budding of endocytic vesicles in receptor mediated endocytosis, and eventually targeted to the vacuole for degradation, and thus again linking endocytosis in α-synuclein degradation by the lysosome (Flower et al., 2007). Therefore, if α-synuclein uses this pathway, it should interact with all or some of the other ESCRT pathway proteins involved.

Gap in Knowledge: What we still do not know

Proteasome degradation of α-synuclein is supported by pharmacological, genetic and biochemical evidence. In order for the lysosome to receive a similarly wide level of support, additional genetic and biochemical studies need to be done. Past evidence for α-synuclein degradation by the lysosome mostly focused on pharmacological studies (Cuervo et al., 2004; Lee et al., 2003). Alex Ayala ‘09 undertook the first study of the MVB/endocytosis protein complexes, and it was one of the few times where several PD-linked α-synuclein properties, accumulation, localization, and toxicity, were studied. She found that all six strains examined altered at least one of the three α-synuclein PD-related properties (Table 1; Ayala Thesis, 2009). She reported three significant findings: 1) The MVB/endocytosis pathway is implicated in α-synuclein degradation, 2) Some ESCRT genes subtly regulate α-synuclein properties, and 3) An overall absence of toxicity and how we can understand those differences. The rest of the MVB/endocytosis genes still need to be analyzed (Figure 5B). My thesis filled this gap in knowledge using the   same budding yeast model.

Table 1: Past evaluation of α-synuclein degradation by endocytosis. Ayala Thesis (2009) evaluated α-Synuclein degradation toxicity, localization, and accumulation for six total ESCRT protein knockout strains. The changes were assessed as a strong, weak or none when compared to BY4741.

Budding Yeast
S. cerevisiae, budding yeast, was used as a model organism to evaluate the role of the endocytosis/MVB pathway in α-synuclein degradation. The MVB/endosome pathway is best studied in budding yeast and because of its importance, the MVB pathway to the lysosome has been conserved from yeasts to humans (Katzman et al., 2001). Budding yeast are extremely powerful molecular and biological tools because their genome is known and the entire gene deletion library is available (OpenBiosystems). This eukaryotic model system is effective for the study of different neurodegenerative diseases. Budding yeast are used in the study of Huntington’s disease and PD among others (Willingham et al., 2003; Outeiro and Lindqust, 2004, Dixon et al., 2005, Sharma et al., 2006).