Journal of Young Investigators RESEARCH REVIEW

The Role of Mutant and α-Synuclein Oligomerization in Neurodegeneration: Linking Gaucher’s Disease and Parkinson’s Disease Brendan Scot Tanner1* and John Porter 2

Gaucher’s disease is a pathology associated with intracellular accumulation of glucosylceramide due to glucocerebrosidase dysfuntion. Gaucher’s disease, type I in particular, does not usually present with neurologic components; however, researchers and physicians have recently noted an increased incidence of Parkinsonism in patients with type I Gaucher’s. Parkinson’s disease is a pathology affecting dopaminergic nerve tracts in motor cortices, most notably the substantia nigra. The disease is microscopically characterized by the presence of α-synuclein inclusions (Lewy bodies) in dopaminergic neurons. In an attempt to link the pathologies at a biomolecular level, researchers utilized numerous strategies, all culminating in several common observations: mutations of glucocerebrosidase, mutations of α-synuclein, and lysosomal dysfunction. Accumulated mutations and lysosomal dysfunction result in a complex feed-forward mechanism leading to aberrant ER-Golgi function. This review aims to highlight the previously unknown mechanism of Gaucher-linked Parkinsonism and to shed light on the future direction of linking and treating similar pathologies.

INTRODUCTION 1q22 (Cullen et al. 2011). Each variant of the Observing an increased incidence of Parkinsonism in patients disease manifests in a slightly different manner. This paper will with type I Gaucher’s disease focus on type I Gaucher's disease, common among the Ashkenazi Jews. Type I Gaucher’s disease is the non-neuropathic variant Researchers have spent considerable amounts of energy in an that results in hepatomegaly, splenomegaly, thrombocytopenia, attempt to link two pathophysiological events, α-synuclein and leukopenia, although most patients remain asymptomatic aggregation and glucocerebrosidase dysfunction. α-Synuclein is a (Rosenbloom et al. 2011). Current research is focused on highly acidic, ubiquitous protein that serves numerous functions elucidating a common biomolecular link between Parkinson's in the central nervous system, including acting as a chaperone disease and type I Gaucher's disease. Patients with the type I protein in the synthesis of soluble NSF attachment protein variant of Gaucher's disease preferentially develop an atypical, complexes and mediating vesicle trafficking in the neuronal severe form of early onset Parkinson's disease (Rosenbloom et al. Golgi apparati (Burré et al. 2010; Cooper et al. 2006). Due to the 2011). prevalence of α-synuclein, any pathology affecting α-synuclein The focus has been placed on linking mutations in the metabolism or distribution could have dire pathophysiological glucocerebrosidase GBA1 with accumulation of α-synuclein consequences. The intracellular accumulation of normal and in neuronal tissue, particularly dopaminergic neurons in the misfolded α-synuclein is implicated in Parkinson's disease, substantia nigra pars compactica, in hippocampal layers CA2-4, dementia with Lewy bodies, and other synucleinopathies (Auluck in the locus ceruleus, in glutaminergic neurons, and in other et al. 2010; Brockmann et al. 2011; Choi et al. 2011). motor cortices (Auluck et al. 2010; Goker-Alpan et al. 2010). Hypoglucocerebrosidemia or absence of functional Prevailing hypotheses put forward by the leading research groups glucocerebrosidase is implicated in the development of Gaucher's are that an etiological toxic gain-of-function mutation in GBA1 disease (Mazzulli et al. 2011). Gaucher's disease is the most results in a complex feed-forward mechanism in which common autosomal recessive lysosomal storage disorder accumulation of α-synuclein and complex lipids block ER-Golgi (Dawson and Dawson 2011). It is pathologically characterized by glucocerebrosidase transport and contributes to lysosomal intralysosomal and leukocytic accumulation of glucosylceramide dysfunction (Dawson and Dawson 2011; Gasser 2009; Parnetti et and other glycosphingolipids (Guschina et al. 2011; Latsoudis al. 2009; Sardi 2011; Velayati et al. 2010; Yap et al. 2011). The and Papapetropoulos 2010; Leverenz et al. 2009; Lopez and pathophysiological mechanism eventually results in neuronal Sidransky 2010; Manning-Boğ et al. 2009; Neumann et al. 2009; death with Parkinson-like sequelae (Mazzulli et al. 2011). The Nishioka et al. 2011). Three variants of Gaucher's disease exist goal of this review is to highlight the progress of research linking as a result of over 200 characterized mutations in GBA1 on Gaucher's disease. Novel methodologies coupled with commonly utilized technologies have shed new light on linking Gaucher’s 1Misher College of Arts and Science, University of the Sciences, 600 disease and Parkinson’s disease in such a way that other S. 43rd St., Philadelphia, PA 19104.2 Philadelphia College of researchers examining similar pathologies could benefit. After Pharmacy 600 S. 43rd St. Philadelphia, PA 19104 understanding the complex biochemical aetiology, researchers can redirect their focus to developing novel therapeutics targeting *To whom correspondence should be addressed: [email protected]

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the various pathways leading to Parkinsonism secondary to of α-synuclein oligomers. Research groups have demonstrated that Gaucher's disease. under Gaucher's disease conditions, α-synuclein monomers oligomerize and form fibrils. The fibrils eventually form Lewy bodies in neurons. Glucocerebrosidase The right side of the figure shows the ramifications of α-synuclein oligomerization. Adapted from Cookson 2009.

Glucocerebrosidase is an important present at different sites throughout the body. There are three glucocerebrosidase α-Synuclein isozymes that are found within Homo sapiens: GBA1 is located in , GBA2 is an isoform found in bile, and GBA3 in α-Synuclein is an acidic 140 amino acid protein encoded by the the cytosol (Nagase et al. 2000). Due to the glucocerebrosidase SNCA gene (Auluck et al. 2010). α-Synuclein is typically found gene's proximity to two pseudogenes and the metataxin gene on as a membrane-bound protein primarily in presynaptic junctions chromosome 1q22, recombinant alleles occur (Díaz-Font et al. in an α-helical form (Auluck et al. 2010; Cookson 2009). α- 2003). Genetic studies have demonstrated the polymorphisms Synuclein, when not bound to a membrane, exists in an unfolded causing Gaucher's disease arise as a result of gene conversion or state (Cookson 2009). Interactions with the negatively-charged irregular crossing-over between GBA and GBAP, the phospholipid bilayer occur due to the numerous cationic lysine glucocerebrosidase gene and pseudogene respectively (Díaz-Font residues (Auluck et al. 2010). α-Synuclein serves a myriad of et al. 2003). The normal function of glucocerebrosidase in functions−dopamine release and transport, fibril formation of lysosomes is to catabolize the glycolipid glucocerebroside to microtubule-associated protein tau, and inhibition of caspase 3, glucose and ceramide as a final step in glycoshingolipid one of the canonical executioner caspases (Goers et al. 2003; catabolism (Goker-Alpan et al. 2010) Glucocerebrosidase Goker-Alpan et al. 2010). α-Synuclein contains three domains, dysfunction is implicated in lysosomal storage disorders, most each with a specific function (Waxman et al. 2009). The non A-β notably Gaucher's disease (Aerts et al. 2011). Patients with type I component of Alzheimer disease amyloid plaque domain (NAC Gaucher's disease are more likely to develop Parkinson's disease domain) is responsible for fibril formation in various with the presence of Lewy bodies (Mazzulli et al. 2011). Several synucleinopathies (Waxman et al. 2009). The next proximal glucocerebrosidase mutations have been identified and linked to region is the hydrophobic domain (Waxman et al. 2009). The premature development of Parkinson's disease. Mutations hydrophobic domain of α-synuclein (Δ71-82) is responsible for commonly occur in the Ashkenazi Jewish population and membrane binding (Waxman et al. 2009). The hydrophobic typically manifest as N370S and L444P (Brockmann et al. 2011). domain plays a role in fibrillization and toxicity in vitro and in vivo (Auluck et al. 2010). The C-terminal domain regulates oligomerization and filamentous diameter (Waxman et al. 2009). Post-translational modification of α-synuclein is altered in synucleinopathies and in Gaucher-linked Parkinson's disease (Figure 1); phosphorylation at Ser129, tyrosine residue nitration, Dysfunctional and dityrosine cross-linking are implicated in formation of glucosylcerami insoluble α-synuclein fibers (Okochi et al. 2000). Approximately de 90% of α-synuclein involved in proteinaceous pathologies are phosphorylated at Ser129 (Okochi et al. 2000). Pathophysiologically, α-synuclein fibrils are found in β- amyloid plaques, Lewy bodies, glial inclusions, and axonal spheroids (Auluck et al. 2010). Accumulation of α-synuclein leads to the development of a synucleinopathy (e.g., Alzheimer's disease and Gaucher's-linked Parkinson's disease) but are not directly responsible for cytotoxicity. α-Synuclein oligomerization has also been implicated in inhibiting ER-Golgi transport, impaired synaptic vesicle release, mitochondrial dysfunction, and inhibition of chaperone-mediated autophagy (Figure 1) (Cookson 2009). Studies have shown that misfolded forms of α-synuclein are implicated in neuronal cytotoxicity (Auluck et al. 2010). In Gaucher-linked Parkinson's disease, α-synuclein preferentially Figure 1. The left side of the figure shows key players in the process of accumulates in the dopaminergic neurons of the substantia nigra Lewy body formation in neurons. The central panel illustrates the pars compacta (Figure 2) (Sidhu et al. 2004). Irreversible theorized pathway leading to α-synuclein oligomerization and Lewy cytotoxicity in neurons eventually results in progressive body formation. α-synuclein exists in a helical form when bound to neurodegeneration (Sardi 2011). membranes and in an unfolded state when unbound. An equilibrium is There are several keystone mutations in α-synuclein that proposed to exist between the two forms of α-synuclein. The red arrow have been shown to abrogate or enhance cytotoxicity. demonstrates the effect of glucocerebrosidase dysfunction on formation

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Biochemical analysis of the A53T mutation show that the glucocerebrosidase results in a buildup of glucosylceramide. hydrophobic core (Δ71-82) is expanded from eleven amino acid Glucosylceramide preferentially accumulates in macrophages residues to thirty amino acid residues (Auluck et al. 2010). and is internalized after receptor-mediated phagocytosis (Kok et Expanding the hydrophobic core destroys the normal al. 1989). A histological description of a "crinkled paper" thermodynamic stability of the α helical domain in amino acids morphology is commonly used to describe Gaucher cells (Zhang 51-66; β-sheets are subsequently adopted as the most stable et al. 2011). The presence of glucosylceramide has been conformation (Auluck et al. 2010). An expanded hydrophobic demonstrated to stabilize the formation of α-synuclein oligomers core mediates oligomer formation due to α-synuclein's (Choi et al. 2011). Glucosylceramide accumulation in neurons propensity to form β-sheets (Auluck et al. 2010). The A30P and macrophages in Gaucher’s patients leads to neuronal death, mutation in α-synuclein results in decreased β-amyloid plaque particularly in dopaminergic neurons (Hisaki et al. 2004). formation (Yonetani et al. 2009). The prevailing hypothesis concerning the ability of A30P to reduce plaque formation lies Principal Approaches within an equilibrium shift from free α-synuclein monomers to Protein/Lipid Identification and Confirmation: Non- oligomers (Yonetani et al. 2009). E46K mutations in α-synuclein fluorescent Methods are thought to increase the ability of charged residues in α- synuclein to interact with the negatively-charged phosphate Most research groups utilized sodium dodecyl sulfate heads of the phospholipid bilayer (Rospigliosi 2009). Increased polyacrylamide gel electrophoresis to identify and quantitatively membrane interaction is thought to facilitate the formation of size target proteins−α-synuclein, glucocerebrosidase, ubiquitin, dityrosine cross-linked dimers (Rospigliosi 2009). prosaposin, cathepsin D, lysosomal associated membrane protein 2 (LAMP2), hsc70, glucosylceramide, and glucosylsphingosine (Alvarez-Erviti et al. 2010; Choi et al. 2011; Guschina et al. 2011; Manning-Boğ et al. 2009; Mazzulli et al. 2011; Neumann et al. 2009; Nishioka et al. 2011; Sardi 2011; Segarane 2009). α- Synuclein, glucocerebrosidase, ubiquitin, prosaposin, glucosylceramide, glucosylsphingosine, and cathepsin D are proteins involved in glycolipid catabolism, membrane structure, and protein degradation (Guschina et al. 2011; Manning-Boğ et al. 2009; Nishioka et al. 2011; Segarane 2009) LAMP2A and Hsc70 are autophagy-related proteins (Alvarez-Erviti et al. 2010). Several research groups used slightly different methods to achieve protein and glycolipid separation. These include native size exclusion chromatography, liquid chromatography, anion- exchange chromatography (due to the acidic nature of α- synuclein), NMR, 2D-TLC, electrospray ionization mass spectrometry, and gas chromatography (Guschina et al. 2011; Mazzulli et al. 2011; Sardi 2011; Xu et al. 2011; Yap et al. Figure 2: The formation of α-synuclein oligomers forms a self- 2011). Although the methods among research groups differed, maintained neurotoxic cycle through downregulation of tyrosine the results were consistent: defective glucocerebrosidase and hydroxylase (TH) activity, vesicular monoamine transporter 2 (VMAT2) aberrant lysosomal degradation pathways resulted in an activity, and dopamine transporter (DAT) activity. Subsequently, free accumulation of complex lipids and α-synuclein. Furthermore, dopamine (DA) is oxidized, forming reactive oxygen species (ROS). the researchers noted the lack of self-limiting kinetics in the The genesis of ROS propagates the formation of α-synuclein fibrils. Retrieved from Sidhu et al. 2004 accumulation of the lipids and α-synuclein. Taken together, these results are consistent with the existence of a feed-forward mechanism of lipid and protein accumulation in patients with Glucosylceramide Gaucher-linked Parkinsonism. While many research groups studied well-classified Glucosylceramide is an amphipathic glycolipid composed of proteins and pathways, Tarantino et al. (2011) took a different glucose and ceramide (Jennemann et al. 2005). approach. The group examined a protein, LRRK2, that was Glucosylceramide, as well as other glycosphingolipids, are an hypothesized to play a role in phosphorylation of α-synuclein at integral component of neuronal membranes in the central Ser129. Past research has shown that phosphorylated α-synuclein nervous system and are essential for proper brain development in Lewy bodies is a hallmark histological finding in patients with (Jennemann et al. 2005). Normal degradation of Parkinson's disease (Tarantino et al. 2011). Reverse transcriptase glucosylceramide is mediated by the enzyme glucocerebrosidase. PCR was used to identify four polymorphisms (rs871196, Glucocerebrosidase hydrolyzes the β-glucosidic bond between rs2420616, rs7069375, rs4752293) in LRRK2 (Tarantino et al. the fatty and sugar moieties of glucosylceramide. Dysfunctional 2011). Mutated full-length Lrrk2 and Lrrk2 fragments maintaining kinase activity were shown to have a greater

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capacity to phosphorylate α-synuclein compared to wild-type 2010; Cullen et al. 2011; Goker-Alpan et al. 2010; Mazzulli et al. Lrrk2 (Tarantino et al. 2011). The results suggested mutations in 2011; Nishioka et al. 2011; Parkkinen et al. 2011; Sardi 2011; Xu LRRK2 can lead to pathologic levels of phosphorylated α- et al. 2011; Yakunin et al. 2010; Yap et al. 2011). The synuclein. With this in mind, researchers used this finding as a predominant method of labeling proteins and histopathological building block to look for overlapping mechanisms in patients structures was with fluorescently-tagged antibodies or GFP with Gaucher-linked Parkinsonism. recombinant proteins; however, Mazzulli et al. (2011) also utilized thioflavin T binding to quantify amyloid Molecule(s) of Observation Physiological Reference plaques in cortical cells. Interest consequence An additional method used to assay α-Synuclein Increased Formation of insoluble Tarantino et al. 2011 monomeric and oligomeric α-synuclein was phosphorylation oligomers leading to cell death sandwich ELISA (Cullen et al. 2011). Sandwich Mutation in Protein Decreased clearance of Mazulli et al. 2011 ELISA is useful in confirming the aggregating malformed protein protein in brain samples was truly α-synuclein and resulting in intracellular Yakunin et al. 2010 not an additional synuclein protein. The specificity accumulation of protein, culminating in cell; of the antibodies for α-synuclein specifically ERAD malfunction expelled any notion of cross-reacting with other Glucocerebrosid Dysfunctional Enzyme Accumulation of complex Mazulli et al. 2011 synuclein and synuclein-like proteins. -ase lipids and perpetuation of formation of insoluble a- synclein oligomers Induction of WT and Mutant α-Synuclein and resulting in cell death Glucocerebrosidase: In Vitro Models Adeno-like virus- Reversal of memory Sardi 2011 mediated exogenous deficits and motor Several cell lines and types were used in order to expression in dysfunction caused by express and examine abnormal α-synuclein hippocampus Gba1 mutation glucocerebrosidase interaction−MES23.5, PC12, Prosaposin Decreased expression Accumulation of Mazzulli et al. 2001 complex lipids dude to SH-SY5Y, HEK293, and induced dopaminergic lysosomal dysfunction, neurons (Alvarez-Erviti et al. 2010; Cullen et al. eventually leading to cell 2011; Manning-Boğ et al. 2009; Mazzulli et al. death Lysosomal Mutation in protein Gaucher model Parnetti et al. 2009 2011; Yakunin et al. 2010). The MES23.5, PC12, demonstrating decreased and SH-SY5Y cell lines were used because the catabolism of complex cells were of a neural cancer origin and are able to lipids resulting in cell produce large amounts of protein as a result death (Biedler et al. 1978; Liu 1999). HEK cells were

Table 1: Summary of key experiments demonstrating complex mechanisms used due to the ease to transfect the cells using an associated with Gaucher-associated Parkinsonism. adenovirus vector (Yakunin et al. 2010). Yap et al. (2011) took a slightly different approach to produce α-synuclein by using engineered Escherichia coli BL21(DE3)pLysS. Greater control of α-synuclein production led to the decision of Yap et al. (2011) Protein/Lipid Identification and Confirmation: Fluorescent to use the genetically-engineered E. coli system; Methods BL21(DE3)pLysS contains an IPTG-inducible gene for T7 RNA polymerase. Immunofluorescence is extremely common in identifying key To express mutant α-synuclein and dysfunctional proteins implicated in Gaucher's disease, Parkinson's disease, glucocerebrosidase using an in vitro model, some research dementia with Lewy bodies, and other lysosomal storage groups introduced the mutations through PCR-directed disorders and synucleinopathies. Research groups favored using mutagenesis (Alvarez-Erviti et al. 2010; Cullen et al. 2011; in vitro methods and tissue samples obtained from international Mazzulli et al. 2011; Saha et al. 2004; Xu et al. 2011; Yap et al. histology banks (Alvarez-Erviti et al. 2010; Cullen et al. 2011; 2011). Some of the common mutations introduced into α- Goker-Alpan et al. 2010; Mazzulli et al. 2011; Nishioka et al. synuclein were G7C, A30P, A53T, S87D, S87A, Y125F, 2011; Parkkinen et al. 2011; Sardi 2011; Xu et al. 2011; Yakunin Y125D, S129D, S129A, and Y136C (Alvarez-Erviti et al. 2010; et al. 2010; Yap et al. 2011). Cullen et al. 2011; Saha et al. 2004; Yap et al. 2011). The Fluorescent microscopy allowed research groups to mutations induced in α-synuclein were chosen based on past locate and identify specific structures implicated in reviews of medical records and computerized molecular models. synucleinopathies and in lysosomal storage disorders. Structures The most common mutations introduced into glucocerebrosidase of interest included ubiquitin-tagged proteins, τ-tangles, β- were N370S, V394L, D409V, and L444P (Cullen et al. 2011; amyloid plaques, classical and cortical Lewy bodies, and other Mazzulli et al. 2011; Sardi 2011). abnormal lysosomal and neuron inclusions (Alvarez-Erviti et al.

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An additional model used to simulate dysfunctional defects in the ERAD pathway, as modeled by Yakunin et al. glucocerebrosidase involved the use of a potent, irreversible (2010), showed abnormal accumulation of lipids and α-synuclein antagonist of glucocerebrosidase, conduritol B epoxide (Cullen et resulting in non-specific neurodegeneration. al. 2011; Manning-Boğ et al. 2009; Xu et al. 2011). Conduritol B epoxide binds with high affinity to the acidic residues of Gene Analysis and Quantitative Transcript Analysis glucocerebrosidase (Rempel and Withers 2008). Irreversibly inhibiting glucocerebrosidase provides an accurate and efficient Numerous research groups relied on the ability to analyze in vitro model of Gaucher's disease. in order to determine specific mutations in many types of samples and to confirm the success or failure of genetic Induction of WT and Mutant α-Synuclein and modification in bacterial, cellular, and animal models (Cullen et Glucocerebrosidase: In Vivo Models al. 2011; Goker-Alpan 2010; Manning-Boğ et al. 2009; Neumann et al. 2009; Nishioka et al. 2011; Segarane 2009; Tarantino et al. In vivo methods to examine the role of normal and abnormal α- 2011; Yakunin et al. 2010). Transcriptional analysis was synuclein and glucocerebrosidase typically rely on genetically- performed to measure the amount of glucocerebrosidase mRNA modified animals, particularly mice. A common approach to and whether the mRNA was affected by α-synuclein aggregation mimicking dysfunctional glucocerebrosidase is to use knock-in or whether it was the source of α-synuclein oligomerization. If α- or knock-out methods. Heterozygous mice (Gba1+/-) and synuclein inhibited translation of glucocerebrosidase transcript, homozygous recessive mice were used in behavioral experiments one would observe normal to increased amounts of such as object recognition, fear conditioning, and field glucocerebrosidase transcript and α-synuclein. In the case of exploration (Sardi 2011). Sardi (2011) demonstrated decreased concentrations of glucocerebrosidase transcript, one Gba1D409V/D409V mice exhibited α-synuclein and ubiquitin would also observe α-synuclein accumulation. The preferred aggregates in hippocampal neurons. Consistent with methods to measure glucocerebrosidase transcript were qPCR hippocampal degradation, Gaucher mice exhibited memory and long range PCR (Cullen et al. 2011; Goker-Alpan et al. deficit, consistent with what would be expected in a human with 2010; Manning-Boğ et al. 2009; Neumann et al. 2009; Nishioka Gaucher-linked Parkinsonism; however, Sardi (2011) probed the et al. 2011; Segarane 2009; Tarantino et al. 2011; Yakunin et al. effects of adeno-associated virus-mediated expression of 2011). exogenous glucocerebrosidase. Gaucher mice treated with Alvarez-Erviti et al. (2010) analyzed and validated exogenous glucocerebrosidase in the hippocampus showed a siRNA inhibition of LAMP2A by using PCR amplification. regain of motor function and memory. Consistent with the data LAMP2A encodes a lysosomal receptor that binds certain and what would be expected in humans, mutations in GBA1 proteins associated with chaperone-mediated autophagy resulted in a synucleinopathy resembling Parkinson’s disease. (Alvarez-Erviti et al. 2010). The authors investigated if there was The effects of the mutation can be ameliorated by resuscitating an alteration in autophagy due to the degradation of neurons normal glucocerebrosidase function. observed in patients with Gaucher-related Parkinson's disease Expanding on the experiments performed by Sardi (Alvarez-Erviti et al. 2010). α-Synuclein oligomerization leads to (2011), an additional research method utilized mice with point- reactive oxygen species production, which subsequently leads to mutated GBA1 in addition to hypomorphic prosaposin (Mazzulli impaired mitochondrial function. If mitochondrial function was et al. 2011; Xu et al. 2011). Prosaposin is a precursor to saposin impaired, one would expect to observe an increase in autophagy A, saposin B, saposin C, and saposin D (Morimoto et al. 1990). as an energy compensation mechanism. As expected in vitro, Saposins are required cofactors in the metabolism of certain siRNA-mediated inhibition of LAMP2A resulted in increased glycosphingolipids in the by action of autophagy levels in a dopaminergic cell line. In conjunction with glucocerebrosidase (Mazzulli et al. 2011; Xu et al. 2011). the in vitro findings, examination of brain samples from seven Paralleling the findings of Sardi (2011), Mazzulli et al. (2011) Parkinsonism patients showed reduced chaperone-mediated showed hypomorphic prosaposin mice exhibit characteristics, autophagy and an increased half-life of α-synuclein in the both at the macroscopic and microscopic level, consistent with substantia nigra pars compacta and amygdala, as measured by the existence of a synucleinopathy such as Parkinson’s disease. LAMP2A and hsc70 concentrations, when compared with age- Contrary to most research groups, Yakunin et al. (2010) matched brain samples (Alvarez-Erviti et al. 2010). examined the effects of α-synuclein aggregation independent of Kono et al. (2010) examined GBA1 genetic variations glucocerebrosidase dysfunction. The group used genetically- by examining restriction fragment length polymorphisms engineered Pex2-/-, Pex5-/-, and Pex13-/- mice. Due to the (RFLPs). Although RFLP analysis is considered to be an archaic homology between the peroxisomal proteome and the practice by some due to the abundance of other, efficient, DNA- endoplasmic-reticulum-associated protein degradation (ERAD) sequencing technology, observing RFLPs in homologous DNA pathway, a Pex knock-out system models defective ubiquination molecules allows for a practical method to localize genes that are and proteasomal catabolism (Schlüter 2006). A hypothesis implicated in genetic disorders. This is important as it allows proposed by research groups is that defects in the ERAD researchers to examine and characterize all of the mutations pathway perpetuate the neurodegenerative effects of α-synuclein associated with similar pathologies. fibril formation (Sardi 2011). Consistent with the hypothesis,

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11 Assaying Enzyme Activity Raclopride is a C radiolabeled D2 dopamine receptor antagonist (Antonini et al. 1997). Raclopride is useful for Two principal approaches were taken to assay the activity of WT examining dopaminergic neuron function and useful in and mutant glucocerebrosidase. One approach involved simply differentiating Parkinson's disease from multiple system atrophy incubating purified glucocerebrosidase with a proper substrate; (Antonini et al. 1997). The use of raclopride-PET was important the primary substrate used in incubations was β-D-glucoside in confirming Parkinson's disease as well as assaying the (Choi et al. 2011; Xu et al. 2011). An additional method used to functionality of dopaminergic neurons in a Gaucher-Parkinson measure the amount of glucocerebrosidase activity involved the model. use of glucocerebrosidase substrates coupled to a fluorescent The experiments performed by Kono et al. (2010) molecule, 4-methylumbelliferone (4-MU) (Parnetti et al. 2009; radiographically confirmed a loss-of-function in dopaminergic Sardi 2011; Xu et al. 2011). The substrates that were coupled neurons, consistent with other experiments with Parkinsonism- with 4-MU included β-D-glucocerebroside-N-acetate, β-D- affected Gaucher’s patients. glucocerebroside, and β-D-galactoside (Parnetti et al. 2009; Sardi 2011; Xu et al. 2011). When bound to the substrate, 4-MU is Discussion non-fluorescent; however, when cleaved by , 4-MU Much time and effort has been dedicated to the comprehension of fluoresces. In essence, greater enzyme activity will result in a the understanding of Gaucher-linked Parkinsonism higher observed fluorescence and vice-versa for lower enzyme etiopathogenesis. Although a great deal is already known activity. regarding the link between dysfunctional glucocerebrosidase and Parnetti et al. (2009) examined an additional enzyme synuclein oligomerization, there are still many gaps to fill with class, lysosomal hydrolases. Lysosomal hydrolases (e.g., regard to the effect of β-amyloid formation and non-functional cathepsins and nucleases) are involved in the proteolysis of glucocerebrosidase on the ubiquitination-proteasome mechanism amyloid-precursor proteins. Defects in lysosomal hydrolases to catabolize malformed proteins.. Misfolded α-synuclein is have been shown to cause formation of β-amyloid plaques in involved in the onset and perpetuation of Gaucher-linked various neurodegenerative disorders (Mazzulli et al. 2011). Parkinsonism (Choi et al. 2011). A typical cellular response to Reactions consisted of 4-MU-coupled substrates (α-mannose, β- prevent incorporation of malformed proteins is to tag the protein mannose, and β-glucose) and isolated lysosomal with ubiquitin for subsequent degradation by proteasomes enzymes from the cerebrospinal fluid of patients with (Pickart 2001). If possible, manipulating normal physiological pathologically-confirmed cases of dementia with Lewy bodies, ubiquitination to tag aberrant α-synuclein and glucocerebrosidase frontotemporal degeneration, and Alzheimer's disease (Parnetti et could become a pharmacological targeting strategy against al. 2009). neurodegeneration. Related to ubiquitination, the unfolded protein response PET Scans (UPR) should also be the subject of further study in Gaucher- Few groups chose functional medical imaging to analyze linked Parkinsonism. Concerning the brain, there is only one Gaucher's disease’effect on α-synuclein aggregation. Kono et al. favorable alternative for the UPR, refolding of the defective (2010) decided to analyze subjects with clinically diagnosed protein. In order to refold the protein, certain chaperone proteins Gaucher-associated Parkinson's disease. Three types of PET must be mobilized (Ron and Walter 2007). Manipulating the scans were used: fluorodeoxyglucose-PET (18F; FDG), (–)-2-β- UPR to refold proteins should be investigated as a potential carbomethoxy-3-β-(4-fluorophenyl)tropane-PET (11C; CFT), and therapeutic target. One possible method to alter the UPR includes raclopride-PET (11C) (Kono et al. 2010). The purpose of the examining pharmacophores of key proteins involved in the UPR study was to demonstrate the presence of dopaminergic neuron and designing small-molecule therapeutics to promote cell deficit in patients with Parkinsonism secondary to Gaucher’s survival, sense malformed proteins, and trigger cell death when disease. appropriate. The one fault in targeting the UPR is that it is an The FDG molecule is rapidly taken up by neurons and is essential cell response perfected through evolution. Tilting the metabolized to 18F-FDG-6-phosphate. Due to the absence of a 2'- UPR one way or the other can have profound, unforeseen hydroxyl group, the phosphorylated metabolite is trapped inside impacts on the organism. This is not to say the UPR should not the cell. Metabolically active neurons will display a higher be explored as a possible therapeutic target, but rather it must be concentration of 18F-FDG-6-phosphate intracellularly (Ewers et extensively studied in in vitro and in vivo models before al. 2011). Concentration of 18F-FDG is a good indicator of beginning human studies. healthy cells versus damaged or dying cells. The development of novel therapeutics to treat Gaucher- CFT is a derivative of cocaine and is a functionally linked Parkinsonism should also be an area of exploration. useful molecule to assess the location and distribution of Although Parkinson-like symptoms may be treated with L- dopamine transporters in neurons (Tang et al. 2011). The use of DOPA, the eventual depletion of dopaminergic neurons due to α- CFT in patients with Parkinsonism would most likely synuclein oligomerization renders L-DOPA treatment demonstrate a lack of dopamine transporters in the brain, ineffective. Enzyme replacement therapies and glucosylceramide particularly in motor cortices and in the substantia nigra pars inhibitors for Gaucher's disease have received much attention in compacta (Kono et al. 2010). the past few years. The use of recombinant glucocerebrosidase or

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