revue neurologique 170 (2014) 151–161

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Update in neurosciences

Protein folding and misfolding in the neurodegenerative disorders: A review Le repliement de prote´ine normal et errone´ dans les maladies neurode´ge´ne´ratives : une mise au point N.B. Bolshette a,*, K.K. Thakur a, A.P. Bidkar a, C. Trandafir c, P. Kumar b, R. Gogoi a a Laboratory of Biotechnology, Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Gauhati Medical College, 781032 Bhangagarh, Guwahati, Assam, India b Laboratory of Molecular Pharmacology and Toxicology, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Gauhati Medical College, 781032 Guwahati, Assam, India c Faculty of Medicine, University of Medicine and Pharmacy‘‘Iuliu Hatieganu’’, 400349 Cluj-Napoca, Romania

info article

abstract

Article history:

Protein misfolding is an intrinsic aspect of normal folding within the complex cellular

Received 10 June 2013

environment. Its effects are minimized in living system by the action of a range of protective

Received in revised form

mechanisms including molecular chaperones and quality control systems. According to the

24 November 2013

current growing research, protein misfolding is a recognized key feature of most neurode-

Accepted 26 November 2013

generative diseases. Extensive biochemical, neuropathological, and genetic evidence sug-

Available online 7 March 2014

gest that the cerebral accumulation of amyloid fibrils is the central event in the pathogenesis of neurodegenerative disorders. In the first part of this review we have discussed the general

Keywords:

course of action of folding and misfolding of the proteins. Later part of this review gives an

Protein folding

outline regarding the role of protein misfolding in the molecular and cellular mechanisms in

Molecular chaperone

the pathogenesis of Alzheimer’s and Parkinson along with their treatment possibilities.

Alzheimer

Finally, we have mentioned about the recent findings in neurodegenerative diseases. # 2014 Elsevier Masson SAS. All rights reserved.

Amyloid-b a-synuclein

Mots cle´s :

r e´ s u m e´ Le repliement de prote´ine errone´ est un aspect intrinse`que du processus normal du

Repliement de prote´ine

repliement de prote´ine intervenant au sein de l’environnement complexe des cellules.

Prote´ine chape´rone

Dans l’organisme vivant, les effets du repliement errone´ sont minimise´s par l’action de

Alzheimer

plusieurs me´canismes protecteurs comme les prote´ines chaperons et les syste`mes de

Amyloı¨de-b a-synucle´ine

errone´ apparaıˆt comme l’e´le´ment cle´ de la plupart des maladies neurode´ge´ne´ratives. Les

controˆle de qualite´. Selon les re´sultats de la recherche re´cente, le repliement de prote´ine abondantes donne´es biochimiques, neuropathologiques et ge´ne´tiques sugge`rent que l’accumulation ce´re´brale de fibrilles amyloı¨des est un e´ve´nement central dans la pathoge´ne`se des

* Corresponding author. E-mail addresses: [email protected], [email protected] (N.B. Bolshette). 0035-3787/$ – see front matter # 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.neurol.2013.11.002

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revue neurologique 170 (2014) 151–161

maladies neurode´ge´ne´ratives. Dans la premie`re partie de cette mise au point, nous examinons le processus ge´ne´ral de repliement et de repliement errone´. La deuxie`me partie pre´sente un re´sume´ du roˆle du repliement errone´ dans les me´canismes pathoge´niques mole´culaires et cellulaires des maladies d’Alzheimer et de Parkinson, ainsi que des possibilite´s the´rapeutiques. Enfin, nous mentionnons les re´cents re´sultats avec les maladies neurode´ge´ne´ratives. # 2014 Elsevier Masson SAS. Tous droits re´serve´s.

1.

Introduction

Alzheimer disease (AD) and Parkinson disease (PD) are the most frequent neurological disorders and causes of dementia in elderly people (over 60 years old) [1]. They lead to progressive disability and decreased quality of life and are associated mainly with age-related changes, such as the high production of free oxygen radicals which damage the cell components including the DNA, or a decreased enzymatic activity which lead to accumulation of abnormal proteins in the brain cells [2,3]. Both of these disorders are protein misfolding diseases, which are caused by the presence and accumulation of abnormal proteins, and are associated with cell dysfunctions [4]. Protein folding is an intrinsic feature of normal folding within the complex cellular environment, and its effects are minimized in living systems by the action of a range of protective mechanisms, including molecular chaperons and quality control system of cell (Fig. 1) [5]. Cellular protein quality control relies on three separate yet interrelated strategies whereby misfolded proteins can either be refolded, degraded, or delivered to distinct quality control compartments that sequester potentially harmful misfolded species [6]. In all the tissues, the majority of intracellular proteins are degraded by ubiquitin–proteosome pathway (UPP). The protein degradation by UPP involves two sucessive steps:  tagging of substrate by covalent attachment of ubiquitin molecules;

Fig. 1 – Protein folding funnel. The protein folding funnel is based on the concept of minimizing free energy and gives an explanation for how proteins fold in to their native structure.

 degradation of tagged proteins in to small peptide by 26s proteosome complex with release of free and reusable ubiquitin [7,8]. Fig. 2 illustrates about the fate of misfolded proteins toward the pathogenicity of neurodegenerative diseases [9]. Along with the ubiquitin degradation pathway misfolded aggregated proteins can also be degraded by a seprate autophagy pathway that involves an ultimate delivery to the lysosome [10]. Autophagy is a nonspecific bulk degradation pathway that was initially described for long lived cytoplasmic proteins and demanded organelles. Additionally, protein inclusions may enhance the efficiency of aggregate clearance, presumably by facilitating interactions with lysosomal and autophagic pathways [11]. When a protein is unable to fold correctly upon synthesis or misfolds at a later stage in its cellular life time, it can no longer fulfill its biological functions [12]. The folding of most newly synthesized proteins in the cell requires the interaction of a variety of protein co-factors known as molecular chaperones. These are set of proteins that link with unfolded polypeptides thereby preventing aggregation and prolific folding in an ATP-dependent manner [13]. Unfolded and misfolded polypeptides have a tendency to form a variety of aggregate, including the highly ordered and kinetically stable amyloid fibrils. These aggregates signify a generic form of structure resulting from the innate polymer properties of polypeptide chain, and their formation is associated with a wide range of debilitating human diseases [14]. Current knowledge interplay between different forms of protein structure and their generic distinctiveness provides a platform for rational therapeutic intervention designed to prevent and to treat this whole family of diseases [5]. It is well recognized that protein misfolding diseases (PMD’s) also known as ‘conformational diseases’ are caused by the misfolding of proteins into intermolecular b sheet aggregation [15]. Such conformation is stabilized by intermolecular interactions, leads to formation of oligomers, protofibrils and fibrils, which then accumulate as amyloid deposits in affected tissues. Aggregates of prion protein (PrPSc) in prion diseases, amyloidbeta (Ab) in the Alzheimer’s disease (AD), islet amyloid polypeptide (IAPP) in type 2 diabetes (T2D) or serum amyloid A (SAA) in secondary amyloidosis accumulate extracellularly [13,15]. Further, misfolded aggregates that accumulate intracellularly, are alpha-synuclein (a-syn) in Parkinson disease (PD), superoxide dismutase (SOD) in amyotrophic lateral sclerosis (ALS), tau in Tauropathies and huntingtin (Htt) in Huntington disease (HD) [13]. In AD, a small protein fragment called Ab accumulates initially in the hippocampus, disturbing the complex neural networks of this brain region, resulting in cell death and loss of memory function [16]. They are generated from smaller, less ordered protein clumps called

revue neurologique 170 (2014) 151–161

153

Fig. 2 – Fate of cellular misfolded protein. Sometimes, due to specific mutations or cellular stress, the biosynthesized polypeptides are converted after translation to folded or misfolded forms. Initially, Hsp40/70/90 assisted by E1, E2, and E3, direct them to the proteosomal pathway through ubiquitination by activating, conjugating, and ligating of misfolded proteins. If the ubiquiting enzymes are damaged, misfolded proteins are directed to the aggregation pathway. The resulting ubiquitin complex with the misfolded forms enters to proteosome system, which degrades the protein to small peptides and ubiquitin molecules. Damage of the proteosomal system causes failure in the protein degradation. Cellular Hsp104 and Hsp70 divide the compact aggregates and form partially folded monomers. They are further converted to native polypeptides with the help of Hsp60 chaperones. However, aggregates of fibrillar amyloid may interact with each other to form amyloid plaques which accumulate in different cellular spaces or become toxic. Furthermore, the toxicity leads to cell death and causes several neurodegenerative diseases. Also, the accumulation of nontoxic matured amyloid causes amyloidosis.

soluble oligomers[17]. These aggregates are highly ordered fibrils that are known as amyloid [18]. Recent work in model organisms such as C. elegans suggests that capacity of protein quality control declines during aging thus as we age our body gradually loses the ability to prevent accumulation of misfolded proteins [14]. At some point, this may result in a catastrophic breakdown of protein homeostasis and in the manifestation of disease. Not only protein folding but also misfolding and aggregation, in the biological environment can be differentially affected by conditions such as the presence of high molecular concentrations or highly reactive molecules including reactive oxygen species and sugars that promote destabilizing chemical modification of proteins [19]. Continuous stream of misfolded proteins can arise the cell because of, for example insufficient protein synthesis, mutant protein expression, excess unassembled subunits of oligomeric complexes and insufficiently translocated secretory and mitochondrial precursors [20]. Along with these physiological sources of misfolded proteins, a number of pathological, environmental and metabolic factors such as stress, aging and cancer can also enhance the production of misfolded proteins [21]. Accordingly, protein aggregation can be privileged by impairment or overwhelming of any of these molecular machineries. Currently, there is no cure for any of these diseases. However, it is believed that the concerted research effort in the areas of protein folding, combined with system biology analysis of networks of protein quality control hopefully will provide the knowledge base for the development of new therapeutic strategies [19]. This review will

present some of the most-recent findings and ideas on some of the basic features of protein folding, misfolding and aggregation with a special focus on the role in Alzheimer’s and Parkinson’s. It will also emphasize the role of protein misfolding and chaperone proteins in therapeutic approaches that might be used to successfully treat these disorders.

2.

Physics of protein folding

Specific protein conformation is largely determined by the flexibility of polypeptide backbone and specific consistent intermolecular interactions of the side chains [22]. In globular proteins, internal core is mostly formed by hydrophobic amino acid residues held together by van der waals forces and surface formed by charged and polar side chains. However, restricted flexibility of a polypeptide chain is a major factor among those determining protein structures and folding [23]. According to thermodynamic concept, the free energy of protein molecules affected by the hydrophobic forces, hydrogen bonding, electrostatic interactions and conformational entropy due to inhibited motion of main and side chains. Stability of protein depends on the free energy difference between folded and unfolded states, which is expressed by Gibb’s free energy equation, RTlnK ¼ DG ¼ DH  TDS Where, R stands for Avogadro’s number, which is 6.023  1023; T is the absolute temperature; K represents equilibrium constant between folded and unfolded forms of

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protein; G is the Gibbs-free energy. DH is the enthalpy change and DS is the change in entropy between folded and unfolded forms of protein [24]. The Gibb’s free energy difference between biologically active form and unfolded conformation is small and it is up to 10 Kcal/mol [25]. The theory of protein folding funnel emanates in reality when discrete biosynthesized protein renovate from an unstructured state to the final equilibrium state [26]. The unfolded protein had both high entropy (S) and free energy. The presence of high entropy in misfolded proteins allow the main and side chain to gain the possible number of conformational states in order to orient itself in a stable conformation. The high free energy means that the molecule is unstable and flops easily in different conformational states. The walls of the funnel are not supposed to be smooth and Follow-on bumps and valleys explain the kinetic traps where the protein show non equilibrium structures for a short period of time. These fleeting structures are disordered molten globules and are partially ordered intermediates. This physical picture of protein folding in which one population of protein portion promptly reaches the folded state while the remaining fraction gets trapped in metastable states was stated as kinetic portioning mechanism [27]. At the bottom of the funnel, the free energy is minimum and there is only one conformational state available for protein molecule called native state. It is plausible that the native state is not unique, there can be several states with nearly equal free energy at the bottom of the funnel [28]. In conclusion, the current view pronounces the energy landscape theory, or ‘‘new view’’ of protein folding. It provides a conceptual framework to describe the general mechanism of protein folding and the justification to design protein folding experiments and to interpret their results.

3.

Protein folding models

Ever since Anfinsen’s experiment in the 1960s, it has been known that the complex three-dimensional structure of protein molecules is encoded in their amino acid sequences, and the chain autonomously folds under proper conditions [29]. A linear polypeptide chain is autonomously organized into a space

filling, compact and well defined three-dimensional structures. The law which states how the protein orients itself in a specific conformational space is called ‘Levinthal’s paradox’. It states that a protein cannot fold by random search and there must be specific folding pathways under which folding occurs [30]. For example, in the nucleation/growth model it is assumed that nucleation is the rate limiting step in folding process, once nucleation occurs the nuclei grow fast and folding complete [31]. In Diffusion-collision-adhesion model microdomains diffuse and randomly collide with each other that leads to aggregation of domains in larger unit, and diffusion is the rate limiting step in this model [32]. Framework model states that folding process is hierarchical, it starts with secondary structure formation and performed structure docking is the rate limiting step [9]. Chaperons will aid in correct folding by the help of energy from ATP. Moreover, they play a role in avoiding b sheet formation thus halting protein aggregation finally resulting in avoidance of protein misfolding [33]. In spite of chaperone actions, some proteins may undergo misfolding under normal condition and in this case the quality control system within the cell will detect and them send for their degradation [34].

4.

Role of different chaperones in folding

Under high-stress conditions, such as extreme temperature or oxidative stress, cells produce a special type of proteins called heat shock protein (HSPs) or molecular chaperones [5]. Molecular chaperones bind reversibly to the folding intermediates, prevent aggregation and drive their passage down to a productive folding path [35]. These chaperones include GroEL from bacteria, Hsp60 from mitochondria and chloroplast and TRiC from the eukaryotic cytoplasm. Table 1 shows the list of chaperone family with their specific roles [9]. Chaperones from Hsp70 class and their co-factor inhibit the premature folding and aggregations by binding to the nascent polypeptide chain from a ribosome until the domain forms stable structure [36]. GroEL seems to be the best studied chaperonin (a group of chaperones with molecular weight of about 60 kDa) with regard to folding mechanism [9]. GroEL works with GroES, a co-factor of HSp10 family. Inside the ring

Table 1 – Different subgroup of chaperonin with function. Chaperone family

ATP

Eukaryotic

Hsp100

+

Hsp104

Hsp90

+

Hsp90

Hop, p23, CDC37

Hsp70

+

Hsp70,Hsc70

Hsp40, Bag1, Hip, Chip, Hop, HspBP1

Hsp60 sHsp

+

CCT/TRiC Hsp25(Crystalline)

prefoldin

Co-chaperone

Function Thermo tolerance Disaggregation together with Hsp70 Stress tolerance Control of folding and activity of steroid hormone receptors, protein kinases, etc. De novo protein folding, prevention of aggregation of heat-denatured proteins Solubilization of protein aggregates together with Hsp104, regulation of the heat shock response, regulation of the activity of folded regulatory proteins (such as transcription factors and kinases) De novo folding of actin and tublin Prevention of aggregation of heat-denatured proteins Component of the lens of the vertebrate eye

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structure of GroEL, a central cavity is formed in which an incompletely folded polypeptide is sequestered via hydrophobic interaction. Hsp70 is a leader chaperone and forms the central component of chaperone family. It is the first chaperone, which binds to newly synthesized polypeptide and involves in translocation of the unfolded polypeptide across the intracellular membranes [37]. The Hsp40 cochaperone accelerates the ATPase cycle to physiological level [5]. Substrate binding and association of Hsp40 stimulate rapid hydrolysis of ATP leading to peptide capture and retention. Hsp40 proteins, such as bacterial DnaJ, which transiently interact with substrates and deliver them to Hsp70 in ATPbound state. Hsp70 and Hsp40 play a main role during the process of protein folding. TRiC constituted the different subgroup of chaperonin because its function is independent of Hsp10 co-factor [9]. The folding in ER of eukaryotic cell has a compartment, where folding occurs [37]. Only those proteins that fold and mature to properly pass a stringent selection process and go to their own ways to target compartments. Whenever proper maturation fails, complex degradation mechanism eliminates misfolded or unassembled secretary proteins from the ER. Chaperones of the Hsp70 (Bip) and Hsp90 (GRP94) families recognize structural features of unfolded polypeptides and assist in their folding and assembly. The enzyme oxidoreductase of protein disulfide isomerase (PDI) family is required for correct formation of disulfide bonds [38]. Calnexin and calreticulin are involved in addition and deletion of carbohydrates in proteins. These lectin chaperones and associated co-chaperones ERp57, act as sensors to monitor and promote efficient folding of wide range of glycoproteins. Some of the secondary factors, as Hsp47, are also involved in folding and are transiently associated with procollagen [39]. Furthermore, they are involved in collagen processing and prevent the secretion of procollagen with abnormal conformation and receptor associated protein (RAP). This exerts chaperone action on certain LDL-receptor family members. Also, cellular mechanism plays an important role in regulating synthesis and translocation. Folding and degradation of proteins seem to operate in a very stringent manner to ensure that protein aggregation is minimized [38].

5.

Alzheimer’s disease

Neurodegenerative diseases were the first described protein misfolding diseases [5]. Alzheimer’s disease (AD) was first described by Alois Alzheimer in 1907. It is one of the most common neurodegenerative disorder and its symptoms evolve gradually from memory loss to thoughtful dementia, leading to death after an average of eight years [17]. Numerous biochemical and neuropathological evidence suggests that the cerebral accumulation of misfolded Ab proteins is the vital event in pathogenesis [40]. Some of the neuropathological features representing a significant step in AD pathogenesis, are extracellular depositions of amyloid b (Ab), which is over expressed in AD [40,41]. Ab is produced from its membrane rooted precursor, the amyloid precursor protein (APP) through chronological cleavage by the enzymes b and g secretases. APP comprises a group of ubiquitously expressed polypeptides, whose heterogeneity arises from both alternative axon splicing

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and post translation modification [42]. Gene mutations leading to early onset of familial AD have been identified in the APP gene on chromosome 21q, in the presenilin 1 gene (PSEN1) on chromosome 14q and the presenilin 2 gene (PSEN2) located on chromosome 1q. The identification of causative mutation within the APP and presenilin genes (PSEN1 and PSEN2) has most likely overinflated the public’s perception of genes in causing the sporadic form of AD [16]. Sporadic AD, which is far more prevalent present the same clinical and pathological characteristics, suggests that the factors that affect the APP to Ab pathway play a significant role in this disease [43]. The accumulation of the product of interaction between Ab and tau protein leads to mitochondrial dysfunction. A fundamental characteristic of AD is the presence of neurofibrilary tangles (NFTs) inside the neurons, which are composed of Tau proteins. Tau binds to microtubules, supporting them and maintaining the cell’s cytoskeleton as well as promoting transport across microtubules. However, in Alzheimer disease, tau protein is aberrantly hyperphosphorylated, forming insoluble fibrils, which commence inside the cell [44]. Regarding the tau phosphorylation, the sequential phosphorylation by cyclindependent kinase (Cdk5) and glycogen synthase kinase-3b (Gsk-b) is firmly implicated in the pathogenic hyperphosphorylation of tau [45]. Also, microtubules stabilize the growing axons, which are necessary for the development and growth of neuritis [5]. Another group reported that the tau mediates Ab neurotoxicity via the disturbance of axonal transport of mitochondria. Thus, Ab and tau may amplify their neurotoxic effects through the inhibition of mitochondrial function and its axonal transport [45]. Ab aggregation occurs first in the neuron’s cytosol and then at the exterior. Soluble forms of Ab in the extracellular compartment facilitate the flow of calcium through calcium-conducting ion channels in the plasma membrane, leading to neuron death by excitotoxicity [46]. Besides its neurotoxicity, Ab is also acts an antigen mediating chronic inflammation. It stimulates the astrocytes and microglia to produce inflammatory agents and activates the cascade complement [47]. In the neuron cytosol, chronic accumulation of Ab aggregates decreases the respiratory capacity of the mitochondrial electron transport chain, disturbing the energy homeostasis in mitochondria. This leads to accumulation of reactive oxygen species inside the mitochondria and its destruction [48,49] activating caspase-3 and the production of caspase-3-truncated tau species in neurons. The activation of caspase-3 leads to apoptosis mediated cell death [39,50].

6.

Mechanism of APP cleavage

The mechanism of aggregation involves a seeding-nucleation process (Fig. 3) [42], in which the significant step is the production of a misfolded seed that acts as a nucleus to catalyze supplementary aggregation for development of oligomeric and fibrillar species [40]. The mechanism of aggregate formation begins when the APP that is attached onto the surface of neuron membranes is cleaved by a sequence of enzymes called a-secretase, b-secretase and gsecretase. However, the APP undergo cleavage by two pathways: a benign cleavage by first b-secretase and then gsecretase, which releases the Ab fragment of APP in to the

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Fig. 3 – Mechanism of Ab aggregation. The seeding- polymerization is the decisive process in the pathogenesis of Alzheimer’s disease (AD). The amyloid precursor protein (APP) is cleaved by the b-secretase and g-secretase complexes, and releases the amyloid-beta (Ab) peptide. Sometimes, segments of Ab peptides are can be polymerized to oligomers in the cytoplasm and then secreted into the interstitial fluid of brain. Further, these Ab oligomers are polymerized to insoluble amyloid fibrils, which then aggregate into spherical plaques and cause dysfunction of neighboring dendrites. The activation of kinases in the neuronal cytoplasm causes the hyperphosphorylation of tau protein. This protein further polymerizes into insoluble fibrils that aggregate as neurofibrillary tangles. These tangles induce local inflammation, which may lead to neurotoxicity.

space outside the cell. However, these cleaved Ab fragments clump together if there is an excess amount [17]. In the benign cleavage pathway, APP is cut by a-secretase resulting in an 83amino acid C-terminal peptide which is additionally cleaved by g-secretase into short P3 peptide [39]. In the harmful cleavage pathway, the cleavage occurs at 99 instead of 83 amino acids from the C-terminus and is interceded by the bsecretase. It results in 99-amino acid peptide that contains an undamaged hydrophobic region termed the Ab region. Subsequent cleavage by g-secretase releases this peptide region forming amyloid peptides of normally 40-amino acids in length (Ab-40) [42]. An alternate cleavage by the g-secretase results in a less abundant form of peptide 42 residue in length. The small, 40- and 42-amino acid long amyloid-b peptide aggregates accumulate and form ‘‘senile plaques’’ in the brain of a patient with Alzheimer’s disease [5]. However, in the healthy brain these b amyloid proteins are broken down and eliminated [5]. These senile plaques are highly insoluble and proteolysis resistant fibrils. Thus, the processing of APP through cleavage by g-secretase is considered as a key to the Alzheimer disease process. Ab misfolding occurs when the soluble, monomeric, extracellular assembly of extended conformations and Ab oligomers is transformed first into a spherical assembly. Then, a number of intermediates finally convert it to a fibrillar cross b-sheet quaternary structures known as amyloid. Amyloid fibrils and related structures recruit soluble Ab into aggregates by a seeded polymerization mechanism [39].

7.

Treatment possibilities

Ab seems to play a decisive role during the initiation and aggravation of other pathological changes. However, Ab reduction or elimination in brain is a key therapeutic target for AD [45]. Treatments targeting on APP processing involve the cleavage towards non-amyloidogenic a-secretase pathway, a-secretase pathway enhancing and inhibition of b and g amyloidogenic pathway. The g-secretase inhibitors are designed in such a way that they lower the APP by 20–40% without interfering in a quantitatively important way with the indentation processing [45]. These agents which increase asecretase activities are NSAIDs, statins and estrogens through activation of protein kinase C [17]. Many AD-related genes are involved in multiple aspects of the disease: generation of neurofibrilary tangles, amyloid aggregation, amyloid clearance, oxidative stress, hypoxia, inflammation, mitochondrial dysfunction, permanent alteration of cytoplasmic membrane, abnormal calcium concentration and apoptotic cell death. Most of the above processes were previously targeted by therapeutic agents directly or through the effect of drugs chosen to target another disease mechanism [17]. Recent approaches concern use of small molecules which bind Ab monomers in order to avoid their assembly into potentially cytotoxic oligomers or coating small amyloid oligomers to mask their toxicity [42]. Recently, Ozawa D et al. reported that the shuttling-protein nucleolin possesses scavenger receptor

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activity. This receptor interacts specifically with Ab type 1-42 and mediates its phagocytosis by microglia. Their results indicate that nucleolin is a receptor that permits microglia to distinguish monomeric and fibril Ab42 [51].

8.

Parkinson’s disease

After Alzheimer’s disease, the second most common neurodegenerative disorder is Parkinson’s disease (PD), which causes loss of motor control and cognitive function. PD has higher incidence in men and affects about 1% of people beyond 65 years of age [52]. PD has different pathological characteristics, such as clumps of proteins named Lewy bodies (LBs) and degeneration of dopaminergic neurons within substantia nigra and pars compacta, which may lead to the decrease of dopamine levels. This will cause resting tremor, postural instability and muscular rigidity. However, a number of genetic loci associated to PD have been identified and one of them is a-synuclein (a-syn), which forms intracellular aggregates [53]. Current studies on infrequent genetic forms of PD have discovered that mutations in the genes encoding parkin (PARK2), PINK1 (PARK6), a-syn (PARK1/ 4), ubiquitin C-terminal hydrolase L1 (UCH-L1), leucineopulent repeat kinase-2 (LRRK2) (PARK8), are associated with PD pathology [37]. Detection of mutations in these genes can influence patients with very unusual familial forms of PD. It allowed us to start studying the mechanisms of protein aggregation and neuronal loss in the more common sporadic forms of PD. The process of oligomerization, fibrillization and aggregation of a-syn are the felons behind the neurodegeneration seen in PD (Fig. 4) [54]. For illustration, the identification

157

of a-syn as a familial PD gene led to the recognition that one of the key constituents LBs in sporadic PD brains is a-syn [37].

9.

Structure and role of a-synuclein in PD

a-syn belongs within the synuclein family that consists of band g-syn [55]. a-syn and b-syn are mostly expressed in the thalamus, hippocampus, and cerebellum [56]. g-syn is found in different regions throughout the brain, mostly in the substantia nigra [57]. The synucleins are encoded by a gene located on chromosome 4q21, 5q35, and 10q23 respectively. asyn gene on chromosome 4q21 encodes a 140- amino acid protein which is subdivided into three domains. The highly conserved N-terminal domain consists of an amphipathic ahelical that associates with membrane microdomains [58]. The central region of a-syn contains a highly hydrophobic motif that comprises 65-90 amino acid residues and is known as the non-amyloid-b component of AD amyloid plaques (NAC). The NAC region is crucial for a-syn aggregation; the deletion of large segments within this motif greatly diminished a-syn oligomerization and fibrillogenesis in vitro and in a cell-based assay [59]. Although the normal function of a-syn remains unknown, in its localization at presynaptic terminals, and association with distal reserve pool of synaptic vesicles. The deficiencies in synaptic transmission observed in response to knockdown or over-expression of a-syn unravelled its role in the regulation of neurotransmitter release, synaptic function and in plasticity [60]. a-syn is expressed throughout the brain and is enhanced in presynaptic nerve terminals. The protein can form various folded structures with monomeric and oligomeric a-helix and b-sheet conformations, as well as

Fig. 4 – Mechanism for a-syn aggregation. Alpha-synuclein (a-syn) can aggregate either in the cytoplasm or within the cell membrane. In the cytosol, this generates spherical and ring-like oligomers that are converted into fibrils. Sometimes, intermediates are formed during the aggregation process which affects the mitochondrial function, Golgi trafficking, protein degradation pathway or the synaptic transmission, leading to neurodegeneration. However, when it has a higher concentration, fibril is polymerized into aggregated b sheets forms. These aggregates form inclusions called Lewy bodies (LBs) and lead to cell death.

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aggregates that are amorphous to amyloid-like fibrils, These formed folded structures are origin of LBs in familial and idiopathic PD [61]. In the process of fibril formation various intermediates forms of a-syn generates. These are primarily soluble oligomeric forms of a-syn that have spherical, ring and string-like features when seen under the electron microscope. These structures collectively referred to as protofibrils, slowly turn into insoluble and unite into fibrils [60].

10.

Mechanism of a-syn aggregation

The level of a-syn in the CNS depends upon the balance between the rates of a-syn synthesis, aggregation and clearance. The formation of fibrillar moieties which are components of LBs or the oligomeric protofibrils is augmented by an increase in SNCA expression [62]. a-syn is a synaptic protein in which four mutations (A53 T, A30P, E46 K and gene triplication) have been found [54]. The a-helix forming domain hosts shows two independent missense mutations at position 53, changing an Alanine to Threonine (A53 T), and at location 30, changing an Alanine to Proline (A30P); which have a key role in autosomal PD. A30P and A53 T mutations cause a-syn to be in aggregated stage, which causes toxicity to the cells [63,64]. Further, these mutated peptides have increased potential for self-aggregation and oligomerization into protofibrils. Although only A53 T promotes the formation of fibrils, the effect of A30P is to disrupt the interaction between a-syn and the lipid membrane, which drives fibrils away from synapses [65]. The aggregation and accumulation of these abnormal a-syn proteins in dopaminergic neurons have been suggested to be responsible for the consequence neurodegeneration [66]. Normal a-syn and its mutated forms (A30P and A53 T) have licentious coil conformation and do not from considerable secondary structure in aqueous solution at low concentration, though at higher concentration, they are prone to self-aggregate, and form amyloid fibrils. However, a-syn can also form protofibrillar intermediates that may contribute to its toxicity [67]. Recent studies show that the intracellular accumulation of a-syn leads to oxidative stress, mitochondrial dysfunction, and caspase degradation emphasized by mutations associated with familial PD [68]. An additional mechanism for a-syn aggregation is associated with protein degradation system and ubiquitin–proteosome system (UPS), which is a degradation pathway for intracellular proteins [69]. An ubiquitin molecule attaches to protein to tag it for degradation, and the tagged protein is degraded by the 26S proteosome. In case of LBs, ubiquitin forms polyubiquitin chains which cannot be degraded by proteosome. Inactivation of UPS leads to neurodegeneration in PD characterized by LB formation [70]. A further mechanism for a-syn aggregation is related to the toxic intermediates formed due to dopamine and its metabolites. They inhibit the formation of major fibrils from protofibrils, probably through the formation of dopamine-a-syn adducts [63]. However, a comprehensive mechanism of a-syn aggregation and the specific role of toxic oligomeric species formed through the amyloid development course are still unknown [71]. However, in familial PD the second frequent mutant gene is parkin. It is a new gene in which different deletions and point

mutations have been revealed in patients with autosomal recessive PD [67]. The gene encoding the parkin protein has a characteristic structure of an ubiquitin-like domain in the amino-terminus and a ring-finger motif in the C-terminal. It encodes a protein which is recruited from the cytoplasm to the damaged mitochondria leading to mitochondrial breakdown by autophagy [68]. Parkin was first found to be an ubiquitin-protein ligase (E3) that is a constituent of the ubiquitin system, which is essential ATP-dependent protein degradation mechanism [72].

11.

Treatment possibilities

Probable therapeutic approaches associated to a-syn toxicity might involve decreasing the levels or accumulation of intracellular and extracellular a-syn. One way to decrease accumulation of intracellular a-syn may be to decrease the synthesis of this protein by silencing SNCA gene with microRNA or by repressing the SNCA promoter [73]. An alternative approach could be to engage the clearing a-syn by several means: activating autophagy or the proteosome, increasing proteolytic breakdown of a-syn with cathepsin D, neurosin, or promoting the binding of a-syn to chaperonelike molecules such as b-syn and HSPs. Another approach could be to reduce the post-translational modifications, such as like oxidation, nitration, phosphorylation and C-terminal cleavage [74].

12.

Recent findings

Proteins function properly only if they are folded correctly. Misfolded proteins may have deleterious effects on the cells and ultimately leads to insoluble aggregates. Gene mutation is also one of the reason for the misfolding of resulting polypeptide. The protein Ataxin-1 is highly prone to misfolding because the gene encoding it (ATXN1) has a defect [75]. Further, ATXN1 inhibition increases the levels of Ab-40 and Ab-42 by enhancing the b-secretase processing of APP [76]. In vitro metal ligands such as clioquinol increase the intracellular copper level, which diminish the level of extracellular Ab, including Ab1–40 and 1–42 [77]. There is a reduced risk of Alzheimer’s disease and an improved cognitive functioning in postmenopausal women who use hormone-replacement therapy with 17b-estradiol. There is also an improvement in Parkinson’s symptoms, and other varying hazards of neurodegenerative disease [51,75,78]. Zhao H, et al. [79] noted from the cell cultures infected with mycoplasma that the bacteria secretes a number of proteases which prevents Ab accumulation. Particularly M. hyorhinis has been shown to be effective in Ab degradation [79]. Recently, researchers focus on the development of therapy which prevents fibril formation. Nevena Todovora, et al. [80] recently shown that the cyclic derivative apo-II (60–70) called Janus cyclic peptide is an effective inhibitor of fibril formation. This cyclic molecule blocks the formation of amyloid fibrils and can be useful in the development of specific therapeutic agents to prevent amyloidosis [80]. Also, gene therapy proved to be particularly useful in protecting neural tissue from damage by amyloid

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toxicity in invitro experiments targeting the mitochondrial enzyme ABAD (amyloid-beta binding alcohol dehydrogenase) [81,82]. Other research directions include development of enzyme replacement methods and in situ synthesis of L-dopa or neurotropic factors [83].

[4]

[5]

13.

Conclusion

A large number of neurodegenerative diseases in humans result from protein misfolding and aggregation. This is believed to be the primary cause of Alzheimer’s disease and Parkinson’s disease. Generally, cells depend on molecular chaperones to seize and refold misfolded proteins. If the native state is unattainable, misfolded proteins are targeted for degradation via the ubiquitin–proteasome system. From the discussion on the mechanisms of different protein misfolding disorders, it is clear that a nascent polypeptide chain can become misfolded due to a specific gene mutation, which takes place in almost all familial neurodegenerative diseases, or a matured native protein can also achieve a misfolded conformation inside the cell. The fates of these misfolded proteins in various disorders are different; in this class of diseases misfolded proteins interact further with each other through intermolecular interaction and form structured aggregates thus gaining toxicity. Currently, there is no therapy for these diseases. However, further research in the areas of protein folding, combined with system biology analysis will hopefully provide the basis for the development of new therapeutic strategies. Gene therapy is a promising therapy candidate for both AD and PD. But, it may be possible that in the near future molecular, chemical and pharmacological chaperones might change the mode of treatment and open a new door in clinical research into the neurodegenerative diseases.

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Disclosure of interest [17]

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgement We would like to thank Vinayak Jamdade and Nitin Mundhe for their helpful discussions. We would also like to thank Kasala Eshvender Reddy and Karisetty Basappa for their valuable technical support.

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