PROTEIN AGGREGATION AND ASSOCIATED NEURO-DEGENERATIVE DISEASESSumanta Kar

Department of Biotechnology and Medical Engineering, NIT Rourkela Rourkela , Odisha; India.

Abstract— Neurodegenerative diseases are specified by selective neuronal susceptibility and neurodegeneration in specific brain regions. The pathogenesis of these disorders mainly involves abnormal accumulation and aggregation of specific proteins (such as polyglutamine, α-synuclein, amyloid β and tau), which are deposited in intracellular inclusions or extracellular aggregates that are characteristic for each disease. Protein aggregation is due to specific interactions between partially folded intermediates. Once formed, aggregates tend to be resistant to degradation. Cells have adapted a mechanism to avoid the accumulation of incorrectly folded proteins. In neurodegenerative disorders, this cellular disposal mechanism becomes ineffective and as a result, misfolded proteins accumulate and become toxic for distinctive sets of neurons. This results in clinical manifestation of disease.

Keywords— aggregation , neurodegeneration ,misfolding , amyloid β , tau , polyglutamine , α-synuclein

I. INTRODUCTION

Proteins are essential complex macromolecules of organisms and participate in actually every process within cells. Three dimensional structures of proteins play an indispensable role for biological functions. Therefore, they must undergo proper folding in order to perform these functions. However, newly synthesized proteins may not fold correctly. Consequently, they will have a strong tendency to aggregate. Heat shock proteins (chaperones) play an instrumental role in correcting protein folding and prevention of protein aggregation. Protein aggregates has toxic effects when accumulated over a certain amount in the cell. The accumulation of abnormal proteins leads to progressive loss of structure and/or function of neurons, including the death of neurons. Many diseases associated with protein aggregation such as prion, Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s diseases (HD). Many of the neurodegenerative disorders are likely to occur due to environmental and genetic factors. In addition, likelihood of being affected with /occurring AD and PD rises with increasing age. At the cellular level,many neurodegenerative disorders show similarity in the development of pathogenesis and neuronal death. Although there is a considerable debate regarding the toxicity versus protective effect of aggregates, protein misfolding appears to be a common feature of progressive neuronal dysfunction and degeneration. Moreover, mechanisms of protein aggregations and generation of neurodegenerative diseases with misfolded proteins have not been elucidated properly yet. Therefore, the aim of this review is to bring a perspective to the role of

misfolded proteins in neurodegenerative diseases in terms of molecular basis.

II. AN OVERVIEW ON PROTEIN AGGREGATION

d Protein aggregation is a phenomenon where protein clump together and accumulate to form clumps or aggregates, which are mostly toxic; in general the misfolded proteins are the one’s associated with the toxicity. Protein aggregation can be of two types: naturally occurring, productive aggregation, where in aggregation of correctly folded monomers aggregate to form polymers of the same proteins. (Eg. Monomeric G-actin, aggregates to form Filamentous F-actin – involved in maintaining the shape of cells) and unwanted aggregation, where in misfolded proteins aggregate and are toxic to the system. (proteins such as α-synuclein, β-amyloid, polyglutamine which aggregate and are involved in neuro-degenerative diseases)Proteins greater than 100 amino-acids undergo assisted protein folding with the help of chaperones and chaperonins. There is sequential folding of elementary folding units called foldons, thus the protein folds to its correct native conformational 3D shape in the cell. However there are many events that could cause the misfolding of the protein and discrepancies in the folding mechanism such as:Mutations in the coding sequence of protein ,Misprocessing of the protein polypeptide,Changes in the folding environment, Oxidative stress on protein, Association of ions with proteins hindering their folding. Mostly the misfolded proteins associated with neuro degenerative disease are related with reduced expression of chaperone or redundancy in chaperone activity which aids in the protein misfolding mechanism.

The protein aggregates can be classified into 2 types: amorphous aggregates – which are granular, and have disordered polypeptide chains, with enriched β-sheets which glue the macromolecular structure. (Eg. Α-crystallin found in cataract diseased condition) and amyloid fibrils – which are highly ordered and repetitive where all polypeptides adopt a common-fold (i.e. the β-sheets are oriented perpendicular to the fibril axis.)To outline the process of protein misfolding , a misfolded protein monomer is the starting component which becomes active i.e. due to its exposed hydrophobic residues attracts similar misfolded monomers to aggregate. This step is the nucleation step. The aggregation continues until it forms either an amorphous or amyloid aggregate. Usually in the system when there is a case of protein misfolding, the system tries to refold it back to its native conformation by the help of chaperone molecules such as Hsp-60, Hsp-100 (unfolders – which help unfold the misfolded protein), Hsp60 and Hsp-10 (chaperones which help in correct folding of the unfolded protein), hence the energy requirement by the misfolded protein is high compared to the normal protein which fold properly. However in case of misfolded proteins associated with neuro-degenerative diseases they are mutated and the chaperone system is also down-regulated and so the system cannot combat the protein aggregation of such misfolded proteins, which eventually forms amorphous or amyloid aggregates due to intermolecular interactions. The aggregates formed are resistant to ubitiquitin mediated proteasome destruction, and also have no affinity for chaperone and

leading to accumulation of the aggregates which is toxic.The amorphous aggregates have much smaller significance in the neuro-degenerative conditions, where in it is the amyloid fibril formation which paves way for the diseased condition.The process of amyloid fibril formation following “nucleation and elongation” model, can be divided into three phases :

The thermodynamically disfavoured lag phase where the soluble species (usually monomers) associate to form nuclei. The system tries to avert the amyloid formation by various mechanism mediated by chaperones or proteasome mediated and hence this phase is highly disfavoured in the system. It is a poorly characterized state which formation influences the overall kinetics of the amyloid reaction.

The exponential phase where the nuclei formation leads to accumulation of more monomers which aggregate in an ordered manner to form proto-fibrils. The population of these transient assemblies triggers the polymerization.

Finally, the exhaustion of monomers leads to the saturation phase where no more soluble species can associate to the ends of preformed fibrils and fibril maturation occurs, usually by lateral association of proto-fibrils.

III. HUNTINGTON’S DISEASE

Huntington disease (HD) is an autosomal dominant poly-glutamine disorder. The affected individuals exhibit involuntary jerky movements and alterations in memory and mood and there is a selective loss of striatal neurons. The gene ‘huntington’ present in the shorter arm of the 4th chromosome, gives rise to the protein ‘Huntingtin’ which is 350kDa in weight having 3144 amino acids. The Huntington protein is mostly prevalent in the neurons in the neo-cortex, cerebellar cortex, striatum and hippocampus. Huntingtin protein plays a crucial role in the nerve cell function such as intracellular transport, regulation of transcription, inhibition of programmed cell death and also responsible for development of central nervous system in the early embryo. The Huntington disease is caused when there is a mutation in this ‘huntington’s” gene leading to a mutant protein which cannot fold properly. The 5’ end of the gene has CAG repeats; CAG (Cytosine-Adenine-Guanine) triplet codon is responsible for the coding the amino acid – glutamine-(Q). The CAG codon gets expanded leading to excess number of glutamine in the defective protein. Normal huntingtin protein has 10-35

glutamine repeats, whereas mutant protein has more than 40 repeats. This polyQ tract near the N-terminus of the protein is responsible for the misfolding of the protein and formation of SDS-insoluble amyloid fibrils. The polyQ sequences convert from a largely disordered structure into a b-sheet-rich conformation upon aggregation. These fibrillar structures resemble those formed by proteins implicated in other amyloid-related neurodegenerative diseases [1]. The amino acid, glutamine is polar in nature; and the overabundance of glutamine causes links to form within and between proteins. The protein (misfolded) molecules “stick” to one another, forming strands that are held together by hydrogen bonds, rather than folding into functional proteins, they develop into tangled, rigid groupings known as protein aggregates. The protein aggregates with exposed hydrophobic patches gets aligned in an ordered manner to form amyloid fibrils, with their β-sheets perpendicular to the fibril axis.In a process similar to the formation of aggregates, the excess glutamines in the misfolded protein can also lead to a type of protein bundling known as neuronal inclusions (NI), or inclusion bodies. NIs initially forms at the axons and dendrites of nerve cells in specific areas of the human brain, producing the damaged neurons characteristic of HD.Subsequently, due to misprocessing of the mutated polypeptide or due to the alternate splicing of the mutated mRNA, smaller fragments of the defective protein are produced. These smaller misfolded defective polypeptides enter the nerve cell nuclei, forming more clumps at the centrosomes forming inclusions and leading to neuro-degenerative conditions.

IV. ALZHEIMER’S DISEASE

Alzheimer’s disease (AD) is the most frequent type of neurodegenerative disorder, common cause of cognitive impairment in elderly people around the world. In 1906, AD was first described by psychiatrist and pathologist Dr. Alois Alzheimer and then the disease was named with his surname. The AD affected individuals suffer from a massive loss of neurons. Although AD patients develop memory impairment, they survive 7–10 years after the onset of symptoms. The histo-pathological signs of AD involve the presence of copious amount of neuritic plaques and neurofibrillary tangles (NFTs). The senile plaques mostly consist of β-amyloid (Aβ) peptide and the NFTs are intracellular aggregates of hyperphosphorylated microtubular tau proteins. It has been thought that the formation of plaques facilitates the initiation the formation of NFT. AD is a genetically complex disorder of the elderly and family history is associated with increasing risk of developing AD. It is most likely associated with genes and environmental factors. In familial AD, a

genetic contribution is clearly evident. To date, three genes: β-amyloid precursor protein (APP) gene (located on chromosome 21), presenilin 1 (PS1) (on chromosome 14) and presenilin 2 (PS2) (chromosome 1) when mutated cause early-onset familial AD. In addition, polymorphisms in four genes: apolipoprotein E (apo E), α-2 microglobulin, very low density lipoprotein receptor and low density lipoprotein receptor-related protein (LRP) are shown to be risk factors for AD pathogenesis. In this respect, it is interesting that apo E, amyloid β protein and α-2 microglobulin are ligands to LRP and they are present in senile plaques.The presence of the ε4 allele of apo E is associated with an increased risk of AD in white and Asian population as opposed to Black and Hispanic, but the pathogenic mechanism is unknown. All three mutant genes (APP, presenilins 1 and 2) increase the production of β-amyloid peptide which is deposited in neuritic plaques. The β-amyloid gene encodes a transmembrane amyloid precursor protein. The insoluble Aβ peptide production requires proteolytic cleavage of APP. This involves two steps: the first cleavage is achieved by β-secretase at methionine 671 of APP and the second cleavage is at the C-terminal of A by γ-secretase [2]. The microtubule associated protein tau is the major constituent of NFT and is mainly found in axons. Hyperphosphorylated tau may destabilize the microtubule network because of its inability to bind microtubules and promote their assembly. It may also impair axon transport and contribute to NFT formation and ultimately neuronal death [2].

V. PARKINSON’S DISEASEParkinson’s disease is a late-onset neurodegenerative disorder, which is characterized by muscular rigidity, postural instability and resting tremor. It is a slowly progressive disorder and the pathology of PD involves the degeneration of dopaminergic neurons in the substantia nigra. The clinical hallmark of PD is the deposition of intra-cytoplasmic inclusion bodies called Lewy bodies in brain cells. The mechanism by which dopaminergic neurons are selectively lost is not understood. The role of genetics in PD has always been controversial. However, familial aggregation studies suggest that late-onset PD has a significant genetic etiology. Heritable forms of PD are caused by mutations in genes. To date, three genes (α-synuclein, parkin and ubiquitin C-terminal hydrolase L1 (UCHL1)) are shown to be associated with familial forms of PD [3,4] .Autosomal dominant PD results in mutations in the α-synuclein gene and autosomal recessive PD is due to mutations in the parkin gene. In the case of the parkin gene, it is not only the recessive loss of parkin that is a risk factor for juvenile and early-onset Parkinsonism, but heterozygous mutation could also be a risk factor. Among other factors, apolipoprotein E ε2/ε4 genotype might be a risk factor for sporadic PD. However, Several lines of evidence suggest that in all known forms of PD, protein aggregation in dopaminergic neurons of the substantia nigra is the common mechanism of neurodegeneration. For instance,

all three proteins mentioned above are present in Lewy bodies in sporadic PD and in dementia with Lewy bodies. Accumulation of α-synuclein in cultured human cells also selectively degenerates dopaminergic neurons in presence of dopamine but not non-dopaminergic neurons suggesting a selective toxicity of its accumulation. It is possible that α-synuclein may protect and help neurotransmitter vesicles to transport them from cell body to synapse. Its aggregate may exert its toxicity by creating pores in the lipid membrane. This may lead to loss of dopamine from vesicle to cytoplasm.

VI. PRION DISEASE Prion disease is the best example to show that protein conformational change can even lead toinfectious disease.In the year of 1982, prion term coined by Stanley Prusiner and co-workers from “proteinaceous infectious particle”. Prion protein is found in two different forms: a cellular form of prion protein (PrPc) and scrapie isoform of prion protein (PrPSc) [5]. Properly folded form is denoted as PrPc while misfolded form is denoted as PrPSc . The PrPc is an α-helix-rich glycoprotein that is approximately 250 amino acids in length. It is encoded by the prion protein gene (Prpn) which is located on chromosome 20 [7]. PrPc is commonly found on neuronal cell membrane by a glycosyl phosphatidylinositol (GPI). However, it is also expressed on other cells such as leukocytes and dendritic cells. PrPc is highly conserved protein among mammals during evolution. When we examine the primary structure of the protein, PrPc consist of a signal peptide , five octapeptide repeats , a highly conserved hydrophobic domain, and a GPI (glycolsyl phosphatidyl-inositol) anchor. Furthermore, PrPc contains two N-linked glycosylation sites. PrPSc can be defined as an infectious isoform of PrPc and causes fatal prion diseases. PrPSc is formed by misfolding of PrPc with a lost in α-helical content. PrPSc has same amino acid sequence with PrPc, but their secondary, tertiary, and quarternary structures are different [7]. Approximately, PrPc includes 3% β-structure and 47% α-helix structure, but nonetheless PrPSc is composed of 43% β-structure and 30% α-helix structure. It becomes non-soluble and resists to proteolytic degradation with conformational changes, whereas PrPc is soluble and protease sensitive. This insoluble protein accumulates in brain and causes a variety of prion diseases in human and animals [7]. Furthermore, interaction of PrPc with membrane lipid layers play a significant role in conversion of PrPc to PrPSc. PrPc is localized in cholesterol and sphingomyelin rich area on cell surface (known as lipid raft) [6]. PrPc is bound to lipid membranes through its GPI anchor. While leaving PrPc from the membranes by catalysis of phosphatidylinositol phospholipase C (PIPLC), PrPSc show resistance to PIPLC. When binding of PrPc to the lipid membranes, PrPc is degraded or converted into PrPSc form [7]. The group of prion diseases, including Creutzfeldt-Jakob (CJD), fatal familial insomnia (FFI), Kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS) are seen in humans, and in similar fashion scrapie, bovine spongiform encephalopathy (mad cow

disease), chronic wasting diseases (CWD), transmissible mink encephalopathy (TME), feline spongiform encephalopathy (FSE) diseases are observed in animals [7]. All of these diseases give similar neurological symptoms such as dysmnesia, depression, sense disturbances, and psychosis [7].

VII. CONCLUSIONSAlzheimer's disease, Huntington's disease, prion diseases and Parkinson's disease are characterized by progressive nervous system dysfunction. They involve deposition of misfolded proteins such as β-amyloid , huntingtin , prion and α-synuclein respectively. Protein aggregation is due to specific interactions between partially folded intermediates. Once formed, aggregates tend to be resistant to degradation. Cells have adapted a mechanism to avoid the accumulation of incorrectly folded proteins. In neurodegenerative disorders, this cellular disposal mechanism becomes ineffective and as a result, misfolded proteins accumulate and become toxic for distinctive sets of neurons. This results in clinical manifestation of disease. Although many of these disorders form protein aggregates we are still ignorant about their composition, causes of misfolding and how accumulation of misfolded protein becomes toxic for the cell . There is strong evidence over the past decade that suggests that adult human brain has an endogenous population of stem cells. These cells are multipotent and at least in the rodent brain they can be induced to proliferate by various growth factors. However, these cells do not appear to replace the lost cells in neurodegenerative disorders. This could be due to several factors such as failure to produce appropriate signals or production of inhibitory signals by degenerating neurons. Alternatively, it is also possible that the rate of cell loss is greater in the disease state than the rate of stem cell division.

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