Identification of human α-synuclein specific single chain antibodies

doi:10.1016/j.bbrc.2006.08.127

Biochemical and Biophysical Research Communications

Volume 349, Issue 4, 3 November 2006, Pages 1198-1205

https://doi.org/10.1016/j.bbrc.2006.08.127 Get rights and content

Abstract

Parkinson’s disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, α-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between α-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of α-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of α-synuclein’s role in PD.

Section snippets

Antibodies

Commercially available antibodies were utilized for synuclein and scFv detection (mouse anti-α-synuclein; BD Biosciences, San Jose, CA; mouse monoclonal anti-M2 Flag™; Sigma–Aldrich, St. Louis, MO). Horseradish peroxidase-conjugated (HRP) secondary antibodies were from GE Healthcare. In some cases primary antibodies were HRP-conjugated and used for direct detection (anti-M2 Flag™-HRP, Sigma; anti-HA-HRP, Roche Diagnostics, Indianapolis, IN).

Bacterial expression and purification of α-synuclein

The bacterial expression vector pRK172 containing

Identification of anti-synuclein single-chain antibodies

In its native form human wild-type α-synuclein (SYN) exists as a random coil but can misfold into oligomers and large molecular weight aggregates. Since misfolded SYN is toxic in both cell culture and animal models, we bacterially produced and biochemically purified human SYN which was subsequently experimentally induced to misfold. We then utilized both monomeric and misfolded SYN to pan against a human phage library harboring single-chain antibodies (scFv). Specifically, monomeric SYN was

Discussion

Since SYN is a likely important contributor to Parkinson’s disease pathogenesis and given that its tendency to misfold may be integral in its toxicity we sought to identify scFvs that recognize both native and misfolded forms of SYN. Here, we describe three classes of scFvs that have specificity for: (1) monomeric and aggregated SYN; (2) monomeric, aggregated, and DA-modified SYN; (3) DA-modified proteins exclusively. Of those scFvs that interact with monomeric SYN we have identified specific

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Other reagents and chemicals were of analytical grade. Recombinant human α-Syn and α-Syn(A53T) were expressed in Escherichia coli and purified as described previously (41). Briefly, α-Syn or α-Syn(A53T) cDNAs subcloned into pET3a were transformed in E. coli BL21(DE3) and protein expression was induced by 0.1 mm IPTG for 3 h. Bacterial pellets were resuspended in TE buffer (10 mm Tris-HCl, pH 7.5, and 1 mm EDTA) containing 750 mm NaCl (TE, 750 mm NaCl) with protease inhibitors, heated at 100 °C for 10 min, and centrifuged at 70,000 × g for 30 min.
α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein–protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P₁R) from the lipid raft fractions. S1P₁R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P₁R became refractory to S1P stimulation required for activating inhibitory G-protein (G_i) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P₁R on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn–treated cells. Furthermore, cholesterol-depleting agent–induced S1P₁R expulsion from the rafts also resulted in S1P₁R uncoupling. Taken together, these results suggest that extracellular α-Syn–induced expulsion of S1P₁R from lipid rafts promotes the uncoupling of S1P₁R from G_i, thereby blocking subsequent G_i signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.
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scFvs have been generated against a number of misfolded proteins that have been implicated in neurodegenerative diseases, with the goal of using them for treatment. For example, scFvs have been generated against alpha-synuclein in Parkinson's disease (Lynch et al., 2008; Maguire-Zeiss et al., 2006; Yuan and Sierks, 2009), huntingtin in Huntington's disease (reviewed in (Butler et al., 2012)), amyloid precursor protein and beta-amyloid in Alzheimer's disease (reviewed in (Robert and Wark, 2012)), and prion protein in prion diseases (reviewed in (Sakaguchi et al., 2009)). In the present study, we isolated a number of scFvs directed against SOD1 by affinity selection using phage display libraries.
Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~ 25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity.
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The advantage of these molecules is their versatile molecular manipulation for the expression from a variety of viral vectors [71]. ScFv that bind to specific regions of α-syn have been isolated, with the aim of inhibiting the rate of protein aggregation and therefore limiting its neurotoxic effect [32,70,69]. Modulation of the microglial response and cytokines may also provide a valid strategy to control progression of neurodegeneration.
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Intrabody and Parkinson's disease
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The intrabody technology has become a promising therapeutic avenue for a variety of incurable diseases. This technology is an intracellular application of gene-engineered antibodies, aimed at ablating the abnormal function of intracellular molecules. Parkinson's disease (PD) is a common neurodegenerative disease with no cure. Recent studies have explored possible intrabody applications against alpha-synuclein (α-syn), whose misfolding is believed to cause a familial form of PD. Here, we review the origin, production, and therapeutic mechanisms of intrabodies and the potential of intrabody protection against α-syn toxicity. Furthermore, we propose possible intrabody applications against leucine-rich repeat kinase 2 (LRRK2), whose mutations are the most frequent known cause of familial and sporadic PD.
Immunolocalization of alpha-synuclein in the rat spinal cord by two novel monoclonal antibodies
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This study provides the first immunohistochemical evidence of the presence and distribution patterns in the rat spinal cord of α-synuclein (α-Syn), a soluble acidic protein, widely expressed in the CNS and closely associated to the pathogenesis of neurodegenerative conditions such as Parkinson's and Alzheimer's diseases. We used two novel homemade monoclonal antibodies (2E3 and 3D5) recognizing two different epitopes of α-Syn. Both antibodies localized α-Syn within the nerve terminals, whereas 3D5 alone also localized it within the neuronal nuclei. α-Syn-immunoreactive nervous elements were widely recognized throughout rat spinal cord and in almost all the gray matter laminae. However, they appeared particularly concentrated within laminae I, II, VII and X and more scattered in the others. Double immunofluorescent labeling showed that α-Syn colocalized with synaptophysin in the presynaptic nerve terminals, with neuropeptide Y (NPY) in lamina I, II, IX and X, and had close relationships with tyrosine hydroxylase (TH) immunoreactive neurons in laminae VII and X. Interestingly, the α-Syn-immunoreactive nerve elements, in lamina X, contained little of calbindin-28KD and calretinin-31KD. Our findings could help in understanding the genesis of some early clinical symptoms of Parkinson's disease (PD), such as pain and dysautonomic disorders, and indicate the spinal cord as their probable starting point, according to the ascending theory of PD, proposed by Braak.
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Parkinson’s disease is a progressive age-related neurodegenerative disease with invariant loss of substantia nigra dopamine neurons and striatal projections. This disorder is well known for the associated motoric symptoms including resting tremor and the inability to initiate movement. However, it is now apparent that Parkinson’s disease is a multisystem disorder with patients exhibiting symptoms derived from peripheral nervous system and extra-nigral dysfunctions in addition to the prototypical nigrostriatal damage. Although the etiology for sporadic Parkinson’s disease is unknown, information gleaned from both familial forms of the disease and animal models places misfolded α-synuclein at the forefront. The disease is currently without a cure and most therapies target the motoric symptoms relying on increasing dopamine tone. In this review, the role of α-synuclein in disease pathogenesis and as a potential therapeutic target focusing on toxic conformers of this protein is considered. The addition of protofibrillar/oligomer-directed neurotherapeutics to the existing armamentarium may extend the symptom-free stage of Parkinson’s disease as well as alleviate pathogenesis.

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^☆: We thank Dr. Semyon Papernov and the Laboratory for Laser Energetics at the University of Rochester for help with atomic force microscopy and Andrew Tompkins and Karthik Venkatesh for technical assistance. This work was supported by DAMD17-03-1-0009 to H.J.F.

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Identification of human α-synuclein specific single chain antibodies☆

Abstract

Section snippets

Antibodies

Bacterial expression and purification of α-synuclein

Identification of anti-synuclein single-chain antibodies

Discussion

Trends Neurosci.

J. Neurol. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Immunol. Methods

J. Biol. Chem.

J. Immunol. Methods

J. Biol. Chem.

Brain Res. Mol. Brain Res.

Trends Biotechnol.

Expression pattern of synucleins (non-Abeta component of Alzheimer’s disease amyloid precursor protein/alpha-synuclein) during murine brain development

J. Neurochem.

Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal

J. Neurosci.

Subcellular localization of wild-type and Parkinson’s disease-associated mutant alpha-synuclein in human and transgenic mouse brain

J. Neurosci.

Intravesicular localization and exocytosis of alpha-synuclein and its aggregates

J. Neurosci.

Neural activity controls the synaptic accumulation of alpha-synuclein

J. Neurosci.

Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy

Acta Neuropathol. (Berl.)

Aggregation of neurofilament and alpha-synuclein proteins in Lewy bodies: implications for the pathogenesis of Parkinson disease and Lewy body dementia

Arch. Neurol.

Alpha-synuclein in Lewy bodies [letter]

Nature

Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies

Proc. Natl. Acad. Sci. USA

Alpha synuclein is present in Lewy bodies in sporadic Parkinson’s disease

Mol. Psychiatry

alpha-Synuclein locus triplication causes Parkinson’s disease

Science

Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease

Science