Overview of Parkinson’s Disease Genetics and Related Antibody Targets

Parkinson’s disease (PD) is the second most common and progressive neurodegenerative disease that affects around 10 million people globally. Pathologically, PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the accumulation of intracytoplasmic inclusions in the neural cell bodies. Generally, individuals afflicted with PD experience motor symptoms including rigidity, postural instability, bradykinesia, facial paucity (hypomimia), and muscle tremors owing to the depletion of 50% to 80% of dopaminergic neurons. Degeneration of dopamine in the striatum of Parkinson’s disease patients causes elevated activity of the internal segment of the globus pallidus (GPi), the substantia nigra pars reticulata (SNpr) circuits, and subsequent gamma aminobutyric acid (GABA) dysfunction. This elevated activity eventually leads to the inhibition of the thalamus with reduced frontal cortex activation and results in decreased motor activity of PD subjects.

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The identification of causative genes linked to Parkinson’s disease over the past two decades has revealed remarkable heritability of this complex disease. Notably, recent studies suggest 15% of PD patients have a family history of PD and 5-10% of patients follow a Mendelian inheritance pattern. The potential genes responsible for the monogenic forms of PD have been termed as "PARK loci" and named in the chronological order of their identification. Twenty-three PARK genes have been identified so far and mutations in these genes can contribute to either autosomal dominant or autosomal recessive form of the disease.

Extensive research on these causative proteins and mutants is required to completely understand the pathophysiology of PD. Antibodies are an essential tool for Parkinson’s disease protein research because of their high-affinity binding and specificity. Thermo Fisher Scientific provides antibodies validated in multiple applications to study proteins involved in autosomal dominant and recessive Parkinson’s disease.

Alpha-synuclein (SNCA or PARK1) is a cytoplasmic protein encoded by the SNCA gene with six exons. Other types of genetic abnormalities like triplication and duplication of SNCA can also result in PD. SNCA is a presynaptic protein primarily expressed in the substantia nigra, thalamus, hippocampus, cortex, and cerebellum. SNCA consists of three domains: (i) the amino-terminal region, (ii) a central hydrophobic domain, and (iii) an acidic, negatively charged carboxy-terminal domain. Although the native full-length form of SNCA has no secondary structure, it adopts a helical structure once it binds to the phospholipid membrane. The mutants tend to form stable beta sheets that convert to toxic oligomers, protofibrils, and fibrils. Emerging evidence supports the hypothesis that SNCA secreted to the extracellular space of the brain propagates like a prion and potentially damages healthy neurons.

The pathological indicator of PD is the existence of cytoplasmic aggregates within the cell body called Lewy bodies (LBs), primarily made up of filamentous SNCA. In the case of PD and other synucleinopathies, it gains a filamentous structure and develops into an abnormally phosphorylated aggregate. This aberrant deposition of SNCA promotes neuroinflammation by activating the immune cells of the brain known as microglia. The size of a Lewy body varies from 5 to 30 μm in diameter with a halo core (predominantly contains SNCA) surrounded by a granular and fibrillar deposit. Apart from alpha-synuclein, several other molecular components of a Lewy bodies include tau, parkin, heat shock proteins, ubiquitin, oxidized/nitrated proteins, proteasomal and lysosomal elements, and cytoskeletal proteins (such as neurofilaments, MAPs, and tubulin).

Figure 1. Alpha-synuclein antibody in western blot.(A) Western blot was performed using alpha-Synuclein Monoclonal Antibody (Syn 211) (Cat. No. AHB0261) and ~14 kDa band corresponding to alpha-Synuclein was observed in whole cell extracts (30 µg lysate) of SH-SY5Y and HEK-293 cell lines, but not in Hep G2 and A549 cell lines which are reported to be expressing low to no amounts of alpha-Synuclein protein. The blots were probed with alpha-Synuclein Monoclonal Antibody (1µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A28177, 1:10000 dilution). (B) Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein in HeLa cells. A loss of signal was observed for target protein in SNCA (KO) cell line compared to control cell line using alpha-Synuclein Monoclonal Antibody (Syn 211) (Cat. No. AB0261).

Leucine-rich repeat kinase 2 (LRRK2 or PARK8) is a large homodimer protein (285 kDa) widely expressed in many tissues such as brain, heart, kidney, and lungs. It is localized in the cytoplasm where it is associated with vesicular structures and intracellular membranes. The LRRK2 gene encodes a multidomain protein consisting of leucine-rich repeat motifs at the N-terminus, a Ras of Complex (Roc), GTPase in the central portion of the protein, WD40 repeats at the C-terminus, Roc (COR) domain in the C-terminus, followed by serine-threonine kinase domains. The catalytic core of LRRK2 is comprised of both the ROC-COR bidomain and kinase region.

LRRK2 is attributed to upregulated kinase activity that has been detected as a risk factor for both sporadic (1–5%) and familial (5–13%) forms of PD. In vitro studies reported that mutant LRRK2 with augmented kinase activity increases phosphorylation of downstream mitogen-activated protein kinase (MAPK) kinase (MKK) that accelerates oxidative damage, dopaminergic neuronal cell death, impaired dopamine neurotransmission, and defective protein degradation. Also, LRRK2 upregulates SNCA transcription through activation of the extracellular signal-regulated kinases (ERK) cascade.

Figure 2. Phospho-LRRK2 (Ser935) antibody in immunofluorescence. Immunofluorescence analysis of LRRK2 (pS935) was done on 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-LRRK2 (Ser935) Recombinant Rabbit Monoclonal Antibody (3H8L19) (Cat. No. 701066) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control.

VPS35 (PARK17) is a core component of a heteropentameric retromer cargo-recognition complex, which is involved in the retrieval of transmembrane cargo proteins from endosomes. VPS35 is the largest subunit of the cargo-selective complex trimer (CSC) that is composed of 796 amino acids. Therefore, the pathogenic mutations associated with Parkinson’s disease suggest VPS35 is a genetic link for the retromer associated neurodegeneration. The missense variants of VPS35, including D620N, are associated with partial loss of function and mitochondrial dysfunction by recycling dynamin-like protein DLP1 complexes. These initial dysfunctions interfere with many downstream cellular events including impaired mitochondrial fusion, altered dendritic sorting of receptors, intriguing connections to other PD-linked gene products (i.e. LRRK2, alpha-synuclein, and parkin), enhanced mitochondrial fission, and defects in macroautophagy.

Figure 3. VPS35 antibody in western blot.(A) Western blot was performed using VPS35 Polyclonal Antibody (Cat. No. PA5-21898) on whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), A549 (Lane 2), U-937 (Lane 3), SH-SY5Y (Lane 4), HeLa (Lane 5), and tissue extracts of mouse lung (Lane 6), rat lung (Lane 7) and mouse spleen (Lane 8). An ~80 kDa band corresponding to VPS35 was observed across the cell lines tested. The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 1:4000 dilution). (B) Western blot was performed in HeLa cells with VPS35 specific siRNAs, Silencer Select (Cat. No. s31375, s31376), cells were transfected with scrambled siRNAs and untransfected control. Densitometric analysis of this western blot is shown in the histogram. A decrease in signal upon siRNA mediated knock down confirms that this antibody is specific to VPS35.

DNAJC13 (PARK21), also known as, receptor-mediated endocytosis 8 (RME-)8 is a DNAJ-domain-bearing protein that is primarily localized in the membranes of the endosomal system. It binds to the molecular chaperone heat shock cognate 70 (Hsc70) through its DNAJ-domain. RME-8/DNAJC13 is required for several endosomal functions such as protein sorting, membrane receptor recycling, generation of endosomal degradative microdomains, retrograde transport from the endosome to the trans-Golgi network, endosomal tubulation, and acts as a scaffold to organize the retromer. The clinicopathological feature of the mutant phenotype is the late onset of Parkinson’s disease. In addition to this, DNAJC6 has recently been identified in juvenile Parkinson’s disease, as well.

Figure 4. DNAJC13 antibody in immunofluorescence. Knockdown of DNAJC13 was achieved by transfecting A-431 cells with specific Silencer Select siRNA (Cat. No. s23548, s23550). Immunofluorescence analysis was performed on A-431cells (untransfected, panel a), transfected with DNAJC13 specific siRNA (panel c), or non-specific scrambled siRNA (panels b). Cells were fixed, permeabilized, and labelled with DNAJC13 Recombinant Rabbit Monoclonal Antibody (11H13L5) (Cat. No. 702773, 1:100 dilution), followed by Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2000 dilution). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962) and Rhodamine Phalloidin (Cat. No. R415, 1:300 dilution) was used for cytoskeletal F-actin (red) staining. Significant reduction of signal was observed upon siRNA mediated knockdown (panel i-l) confirming specificity of the antibody to DNAJC13 (green).

The ubiquitin carboxyl-terminal esterase L1 (UCH-L1/PGP9.5/PARK5) protein expression is limited to neurons, cells of the diffuse neuroendocrine system (DES), testes, and ovaries. UCH-L1 is involved in the degradation of misfolded and damaged proteins via the ubiquitin-proteasome system. UCH-L1 is a deubiquitinating enzyme that belongs to the C-terminal hydrolase family and hydrolyzes the polymeric ubiquitin chains. UCH-L1 has a role in dimerization-dependent ubiquitin ligase activity. The missense mutation that replaces the isoleucine with methionine at the position 93 (Ile93Met/I93M) and another variant that replaces serine with tyrosine (Ser18Tyr or S18Y) affects hydrolase and ligase activity, respectively, thereby emerging as a pathological hallmark of Parkinson’s disease.

Figure 5. PGP9.5 (UCH-L1) antibody in immunofluorescence. Immunofluorescence analysis of PGP9.5 was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PGP9.5 Monoclonal Antibody (BH7) (Cat. No. 480012) at 1:500 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Cat. No. A32766, 1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300 dilution). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e represents Hep G2 cells having no expression of PGP9.5 Panel f represents control cells with no primary antibody to assess background.

Parkin (PRKN or PARK2) is an E3 ubiquitin ligase that contains 12 exons composed of an amino-terminal ubiquitin-like (Ubl) domain and a carboxyl-terminal ubiquitin ligase domain with two ring finger domains flanking a cysteine-rich domain, termed In Between RING fingers (IBR). Parkin plays an essential role in protein degradation with the help of a ubiquitin proteasome system and maintains the healthy mitochondrial network. Interestingly, there have been close to a hundred different autosomal recessive mutants of the PRKN gene identified to date, including insertion, deletion, and nonsense/missense mutations. Genetic linkage analysis shows that a mutational change in chromosome 6q25.2-27 is the typical cause of autosomal recessive juvenile Parkinson (AR-JP) disease and early onset of Parkinson’s disease.

Figure 6. Parkin recombinant rabbit monoclonal antibody in western blot.(A) Western blot analysis was performed on whole cell extracts (60 µg lysate) of SH-SY5Y (Lane 1) and tissue extracts of mouse brain (Lane 2) and rat brain (Lane 3). The blots were probed with Parkin Recombinant Rabbit Monoclonal Antibody (21H24L9) (Cat. No. 702785, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP (Cat. No. A27036, 0.25 µg/mL, 1:4000 dilution). A 48 kDa band corresponding to Parkin was observed across the cell line and tissues tested. (B) Western blot analysis of Parkin was performed on cell extracts (100 µg of lysate) of HEK-293 wild type (Lane 1) and Parkin knockout (Lane 2). Densitometric analysis of this western blot is shown in the histogram. Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Parkin.

PINK1 (PARK6) was the second gene identified in early-onset recessive Parkinson’s disease caused by the mutation in the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1). It consists of 8 exons that encode the 581 amino acid protein PINK1, a highly conserved serine/threonine kinase domain, C-terminal auto-regulatory domain, and a mitochondrial targeting sequence at the N-terminus. PINK1 is predominantly localized in the mitochondria and has been hypothesized to have a neuroprotective role against mitochondrial dysfunction by the removal of dysfunctional mitochondria. Mutations (truncating mutations, point mutations, and frameshift mutations) of the PINK1 gene results in functional aberration that contributes to disease vulnerability later in life and is responsible for 7-9% of early-onset autosomal recessive PD.

Mutation in the protein DJ-1 (PARK7) gene is associated with 1-2% of autosomal recessive early onset PD with dyskinesia, rigidity, and tremors. It can also lead to psychiatric symptoms, such as psychotic disturbance, anxiety, and cognitive decline. DJ-1 has seven coding exons, has ubiquitous expression, and functions as a scavenger of reactive oxygen species through self-oxidation. DJ-1 protects the neuronal cells from oxidative stress. This protein is conserved with a cysteine (Cys) residue at position 106 which is the preferential site for oxidative protein modification. The reduced form of DJ-1 (DJ-1 Cys106-SH) can be oxidized to sulfinic acid forms which are more stable, as compared to, the oxidized sulfonic acid form (DJ-1 Cys106-SO3H) which tends to form aggregates. Recent findings reveal the significance of DJ-1 as a promising biomarker and therapeutic target for Parkinson’s disease.

SOD1 is a Cu/Zn metalloenzyme that generates hydrogen peroxide by the dismutation of superoxide, a mechanism of mitigating cytoplasmic oxidative stress in substantia nigra (SN) dopaminergic neurons. The copper/zinc-dependent SOD1 is located largely in the cytosol. Conversion of the apo-SOD1 to the catalytically active holo-SOD1 dimer results from Zn binding, followed by the insertion of a copper cofactor and dimerization. Increased oxidative stress in dopaminergic neurons stimulates expression of apo-SOD1, however, maturation of active SOD1 is hindered by the copper deficiency specific to PD. Without adequate neuronal copper to enable an oxidative modification of free cysteine, arginine, lysine, and histidine residues there is stimulation of the oligomerization and aggregation of the copper- or zinc-deficient form or apo-SOD1. SOD1 has also been shown to work with oligomerized alpha-synuclein leading to the accumulation of both proteins. These accumulated proteins contribute to oxidative stress and impaired axonal transport in dopaminergic neurons in Parkinson’s disease.

OPTN (Optineurin) is composed of 577 amino acids and contains several domains, including 2 leucine zippers, an LC3 interacting domain, ubiquitin-binding domain, bZIP motif, a C-terminal C2H2 type of zinc finger, and multiple coiled-coil motifs. Expression of OPTN is enriched in dopamine neurons, SNpc pyramidal neurons of the hippocampus, and cortical layers 1 and 2. OPTN’s expression is regulated in a neurotoxic PD model. The structural motifs of OPTN are linked to its cellular functions including structural maintenance of the Golgi, vesicle trafficking, and the innate immune response. Point mutations in OPTN are linked to impaired autophagy and mitochondrial dysfunction in amyotrophic lateral sclerosis (ALS), which also occur in the earlier stages of Parkinson’s disease. Genome-wide association studies show that a single nucleotide polymorphism in OPTN (M98K), may affect OPTN-mediated autophagy and be identified as a PD risk modulator.

Figure 9. Antibodies specific to SOD1 and OPTN.(A) Immunofluorescence analysis was performed on HeLa cells (untransfected, panel a), transfected with SOD1 specific siRNA (panel c) or non-specific scrambled siRNA (panel b). Cells were fixed, permeabilized, and labelled with SOD1 Recombinant Rabbit Monoclonal Antibody (11H3L1) (Cat. No. 702783, 1:100 dilution), followed by Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2000). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962), and Rhodamine Phalloidin (Cat. No. R415, 1:300) was used for cytoskeletal F-actin (red) staining. Significant reduction of signal was observed upon siRNA mediated knockdown (panel i-l) confirming specificity of the antibody to SOD1 (green). (B) Immunofluorescence analysis was performed on mouse retina tissue. Transverse sections of mouse retina were incubated with Optineurin Recombinant Polyclonal Antibody (Cat. No. 711879, 1:250 dilution) overnight at 4°C, followed by Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2000, 45 minutes). Nuclei (blue) were stained using SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938), Panel a) represents staining with the matched isotype control. Panel b) shows a representative mouse retinal section stained for optineurin (green).