ΑLPHA SYNUCLEIN MISFOLDING AND NEURODEGENRATIVE DISEASE
Kapoor D*, Vyas RB, Lad C, Patel M
Dr. Dayaram Patel Pharmacy College, Surat, Gujarat, India, Pin-394601
Parkinson’s disease (PD) results primarily from the 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced do death of dopaminergic neurons in the substantia nigra. paminergic neurodegeneration. Exposure of humans to Current PD medications treats symptoms; none halt or MPTP causes a syndrome that mimics the core neuroretard dopaminergic neuron degeneration. Although the etiology for sporadic Parkinson’s disease is unknown, information gleaned fromboth familial forms of the disease and animal models places misfolded _-synuclein at the forefront. The disease is currently without a cure and most therapies target themotoric symptoms relying on increasing dopamine tone. Association of alpha-synuclein with oxidized lipid metabolites can lead to mitochondrial dysfunction in turn leading to dopaminergic neuron death and thus to Parkinson’s disease.
Parkinson’s disease is the second most common neurodegenerative disease, after Alzheimer’s disease, among the aging human population. Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic Parkinson’s disease (PD). α-Synuclein has been identified as themajor component of Lewy bodies and Lewy neurites, the characteristic proteinaceous deposits that are the hallmarks of PD. Alpha-synuclein is a 140 amino acid neuronal protein that has been associated with several neurodegenerative diseases. A point mutation in the gene coding for the a-synuclein protein was the first discovery linking this protein to a rare familial form of Parkinson’s disease (PD).
The role of 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. the different aggregation states of α-synuclein, the molecular mechanism of its aggregation, and the influence of environmental and genetic factors on this process. Abnormal processing of a-synuclein is predicted to lead to pathological changes in its binding properties and function. Various mechanisms for in vitro and in vivo a-synuclein aggregation and neurotoxicity are summarized, and their relevance to neuropathology is explored.
Keywords: Dopamine transporter, Tyrosine hydroxylase, Iron, Muscarinic receptors, Endocytosis, α-Synuclein, Therapeutics Oligomers, Protein misfolding.
Parkinson’s disease is characterized by motor symptoms such as uncontrollable tremor, slowness of movement, muscular rigidity and postural instability. From a neuropathological point of view, the main characteristics are the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of cytoplasmic inclusions known as Lewy bodies 1, 2. Parkinson’s disease (PD) is an age-related progressive neurodegenerative disease with invariant loss of substantia nigra pars compacta dopaminergic neurons (DANs) resulting in a near complete deficit of the striatal presynaptic neurotransmitter dopamine.
Figure 1: Formation of alpha-synuclein fibrils and the loss of dopaminergic neurons in the substantia nigra are observed in patients with Parkinson’s disease. Alpha-synuclein (in grey) is normally a monomeric unstructured protein which undergoes conformational changes upon interaction with lipids and also upon fibrillation.
In addition to the invariable loss of DANs and dystrophic projections to the striatum, postmortem hallmark pathological features include the presence of activated microglia and large intracytoplasmic eosinophilic proteinaceous inclusions called Lewy bodies in the remaining substantia nigra DANs 3, 4, 5.
A strong proof of the involvement of alpha-synuclein in neurodegeneration came from studies showing that three independent mutations in this synaptic protein, alanine to threonine at position 53, alanine to proline at position 30 and glutamate to lysine at position 46, lead to the development of familial Parkinson’s disease 6, 7, 8. Genetic studies led to the discovery of a small percentage of familial PD cases linked directly to genetic mutations, as well as gene duplications and triplications 9, 10, 11. Susceptibility genes involved with protein phosphorylation and degradation, mitochondrial function and oxidative stress responses have been identified including parkin, ubiquitin carboxy-terminalhydrolase- L1, PINK1, DJ-1 and LRRK2 (dardarin) 12-15.
α-Synuclein is a 140-amino acid protein, which is encoded by a single gene consisting of seven exons located in chromosome 4. α-Synuclein is an abundant brain protein of 140 residues, lacking both cysteine and tryptophan residues. This protein is present in high concentration at presynaptic terminals and is found in both soluble and membrane-associated fractions of the brain. α-Synuclein was estimated to account for as much as 1% of the total protein in soluble cytosolic brain fractions. The involvement of α-synuclein in the control of the neuronal apoptotic response and in the protection of neurons from various apoptotic stimuli was demonstrated. Knockout of all synucleins (there are three members of the synuclein family in the vertebrata: α-, β-, and γ- synucleins) in mice leads to age-dependent neuronal dysfunction indicating that are important contributors to long-term operation of the nervous system. α-Synuclein was shown to physically interact with at least 30 proteins, underlying its important role in cell signaling 16-20.
Structures of α-synuclein oligomers
Oligomers ofα-synuclein, similar to those of other amyloidogenic proteins, are highly structurally diverse. Some of themare β-sheet rich,while others are primarily disordered. Recent studies identified several distinct populations of α-synuclein oligomers and obtained their structural information. For example, Giehm and coworkers identified wreath-like oligomers with a diameter of approximately 18 nm 21. These oligomerswere able to disrupt the membranes and easily assembled into fibrils. Hong et al. identified the oligomers that formed in parallel with fibril formation 22.
Propagation of α-synuclein aggregates in animal models of PD and in-vitro:
The seeding and propagating properties of α-synuclein aggregates have been elegantly demonstrated by recent cell culture studies. In human embryonic kidney (QBI-293) cells that are
overexpressing α-synuclein, exogenous α-synuclein fibrils promoted and induced endogenous α-synuclein to form LB-like intracellular inclusions. Intracellular propagation of α-synuclein fibrils was dependent on the presence of a fibrilforming core, further confirming in vitro experiments that demonstrated nucleation-dependent fibrillation of α-synuclein 23, 24. Seeding of endogenous α-synuclein is not specific to the fibrils, but was also previously described for α-synuclein oligomers, in which three distinct types of oligomers promoted intracellular oligomer seeding of endogenous α-synuclein in cultured human neuroblastoma (SH-SY5Y) cells 25. Lastly, the propagation and spreading of misfolded synuclein is reported in cases where human PD patients that received embryonic nigral transplants. In these cases, the embryonic dopaminergic neurons grafted into PD patients developed α-synuclein inclusions and have reduced the dopamine transporter over a period of 14 years 26.
α-Synuclein aggregation and cell death:
The mechanisms proposed to describe the neurotoxicity of α- synuclein and its aggregates can be grouped into three major classes — mechanical disruption of cellular compartments/processes, toxic gain of function, and toxic loss of function. One of the most commonly accepted examples of the former is permeation of cellular membranes by amyloid aggregates. α-Synuclein oligomers can bind to lipid membranes and disrupt membrane bilayers 27. Certain oligomeric forms of α-synuclein were shown to penetrate membranes, forming pore-like channels 28, 29.Membrane permeation by amyloid oligomers without pore formation has also been proposed. It is believed that this is one of the main mechanisms of toxicity for protein aggregates.
Interaction of α-synuclein with membranes:
α-Synuclein contains several class A2 lipid-binding helices, distinguished by clustered basic residues at the polar–apolar interface, positioned ±100° from the center of apolar face; a predominance of lysines relative to arginines among these basic residues; and several glutamate residues at the polar surface 30. These structural features allow α-synuclein to bind to synthetic vesicles containing acidic phospholipids and to cellular membranes 31. In presynaptic termini, monomeric α-synuclein exists in equilibrium between free and membrane- or vesicle-bound states. The equilibrium is tightly regulated, and it has been estimated that approximately 15% of α-synuclein is membrane-bound within the synaptic termini 32.
Current Parkinson’s disease therapies
The first line therapy for the last 40 years has been levodopa (l-dopa) a DA precursor capable of crossing the blood brain barrier and efficacious in a large number of PD patients relieving akinesia/ bradykinesia 33. Commonly given in conjunction with an inhibitor of extracerebral dopa decarboxylase such as carbidopa this pharmaceutical combination is themost effective symptomatic treatment available. Levodopa therapy results in a “honeymoon period” when patients respond extremelywell to therapy followed in 5–15 years by a “wearing-off” effect 34. During this phase, levodopa-induced dyskinesias develop and the patient is forced to halt treatment. In addition, for levodopa to be effective a portion of SN DANs and striatal projections must be present and in fact as the neuropathology progresses, this therapy ultimately fails.
Other dopaminergic agents currently utilized effectively increase the amount of DA by extending the half-life and/or decreasing the metabolism. For example, the monoamine oxidase inhibitor deprenyl increases the half-life of DA and in one study delayed the need for levodopa therapy. Surgical intervention with the goal of suppressing abnormal neural activity resulting from the loss of dopaminergic input is utilized in conjunction with pharmacotherapy. The most common surgical approach is deep brain stimulation (DBS) which entails MRI guided positioning of an electrode most often into the subthalamic nucleus and implantation of an impulse generator under the patient’s collarbone to produce high frequency electrical impulses which dampen the increased glutamate output from this nuclei 35,36. This technology is most effective in patients that respond to levodopa. As with other therapies, as disease progresses DBS fails to be effective Tissue implantation and gene therapeutic approaches to PD are still in the investigative stages.
Synuclein: a novel therapeutic target for PD
A novel method to interfere with misfolded SYN utilizes single chain fragment variable antibodies (ScFvs) directed at specific SYN protein sequences and/or conformations 37. ScFvs are composed of the minimal antigen binding site, the VH and VL variable domains, joined by a flexible polypeptide linker 38. These antibodies are powerful since they are encoded on one contiguous gene sequence and therefore amenable to renewable large-scale production from bacterial and mammalian gene expression systems. In addition, ScFvs can be expressed intracellularly (intra bodies) or designed for secretion making them compatible with gene therapeutic delivery approaches and a broad range of applications. ScFvs have been identified which bind to specific regions of SYN 39.
Other potential classes of OPSyn inhibitors are currently under investigation. Statins reduce neuronal detergent insoluble SYN suggesting that the regulation of cholesterol synthesis may be a novel therapeutic locus for PD. Prolyl oligopeptidase (PO) cleaves proteins at the carboxyl side of l-proline and indeed the C-terminal fragment of SYN is a PO substrate. However, full-length SYN is not cleaved by PO yet SYN aggregation is enhanced 40. Antioxidants also represent a large class of potential pharmaceuticals for neurodegenerative diseases.
These compounds are intended to prevent oxidation of other molecules reducing overall free radical levels and cellular oxidative stress. Antioxidant compounds also inhibit the formation of amyloid and SYN fibrils and importantly can destabilize preformed fibrils which bodes well for their usefulness in established disease . In addition, ScFvs can be expressed intracellularly (intrabodies) or designed for secretion making them compatible with gene therapeutic delivery approaches and a broad range of applications. ScFvs have been identified which bind to specific regions of SYN. Recently ScFv directed against the non-amyloid component of SYN (NAC), a region important for aggregation, reduced the amount of intracellular SYN aggregates and associated toxicity in a cell culture model 41. Furthermore, ScFv that specifically recognizes DA-modified proteins has been identified which may prove to be important in PD since SYN misfolding has been linked to direct interaction with DA.
The focus of studies on the molecular mechanisms of PD pathology became strongly α-synuclein-centric due to the two important discoveries made in 1997, the demonstration that a specific mutation in the α- synuclein gene is related to familial cases of early-onset PD , and the demonstration that α-synuclein is highly abundant in LBs. Much has been learned about α-synuclein structure, function, and aggregation properties after these discoveries. This multifactorial nature of PD and other synucleinopathies, and a limited understanding of the key molecular events provoking neurodegeneration, are among the major reasons determining the lack of drugs for the successful inhibition and cure of these diseases.
Another factor is the lack of the precise knowledge of the nature of the neurotoxic species that accumulate during α-synuclein misfolding and aggregation, and eventually lead to cell death. As a result, the current arsenal of anti-Parkinsonian drugs is not able to halt or retard neuron degeneration, and all drugs developed so far treat disease symptoms. There are several potential solutions for these problems. One of them is a search for small molecules stabilizing the intrinsically disordered conformation of α-synuclein or completely blocking its aggregation, or resulting in the complete disaggregation of the preformed aggregates down to the monomeric state. Alternatively, one can search for chemical compounds that can either clear toxic misfolded proteins or protect neurons from their impact. Finally, a very promising approach relies on compounds that promote protein aggregation, accelerate formation of large inclusions, and eliminate the toxic effects of misfolded protein conformations and small oligomers.
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