When Protein Folding Goes Wrong: Why Does It Matter So Much to the Nervous System?

Alex Davison

When Protein Folding Goes Wrong: Why does it Matter so Much to the Nervous System?

A neurodegenerative disease is one in which normal proteostasis is lost, characterised by the accumulation of toxic protein aggregates and aggregate intermediates, which lead to currently unavoidable neuronal loss1. This is the cause of the devastating proteopathies in our significantly ageing society. Research into this area has taken off since the early 1980s, when Prusiner first identified abnormal prions - small infectious particles composed of misfolded cellular prion protein2 - as the cause of a number of neuronal disorders. This discovery triggered the idea that other misfolded proteins may be the culprit behind similar diseases. As we live in an age of continual medical advance where the ability to prolong life is coveted, age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s are becoming ever more prevalent and demanding of immediate attention and research.

How protein folding normally occurs:

Protein folding is the process by which an unfolded polypeptide is modified into a highly specific three-dimensional structure, allowing it to perform its biological function. This occurs either immediately after biosynthesis of the polypeptide, or co-translationally (as translation is happening). The native conformation that the protein assumes (this is the most stable, energetically favourable state) and the exact pathway by which this is achieved is almost solely determined by the primary structure of the polypeptide3.

The process starts by the protein establishing regular secondary structures stabilised by numerous hydrogen bonds. The two typical secondary structures adopted are α-helices and β-sheets. A single polypeptide chain is able to consist of multiple regions of different secondary structures, and when these are organised into distinct functional domains the protein is said to have a super-secondary structure3.Following this, the tertiary structure is adopted; the protein’s precise 3-D shape, controlled by interactions between different secondary structure domains and individual R-groups4.

There are several ways these regions can associate, including hydrogen bonds, hydrophobic interactions, electrostatic forces, Van der Waals forces and disulphide bridges4. Interactions between hydrophobic R-groups and the aqueous environment of the cell cytoplasm is the most dominating factor in determining the tertiary structure as the hydrophobicity of these regions causes them to cluster inthe inner sheltered regions of the protein. Electrostatic forces can form between oppositely charged ionic R-groups which are closely situated, holding them together. Also, the carboxyl group of any amino acid residue is able to donate an H+ ion to the amino group, forming zwitterions which again have a strong, electrostatic attraction5. Van der Waals forces, although very weak electrostatic attractions, contribute significant stability to the tertiary structure due to their significant abundance.

How does protein folding go wrong?

Given the hugely complex nature of protein folding and its reliance on numerous chemical and physical factors, it is not surprising that the process can go wrong.There are various factors that contribute to this breakdown in normal cellular function. Firstly, the cellular environment is hugely crowded as proteins, organelles and other molecules are present in concentrations of up to 400g/dm3 in the cytoplasm6. Subsequently, unfolded or partially folded polypeptides are often closely packed and may even be in contact with each other. As nascent polypeptides have exposed hydrophobic amino groups, they are ‘sticky’, and so have a strong tendency to clump together into intractable protein aggregates. Aggregation is the process by which protein intermediates bind to each other, in search of a more thermodynamically favourable state7. Conditions in which proteins are more susceptible to aggregation include heat-shock and extremes of pH, in which proteins are more likely to become denatured, resulting in exposed hydrophobic residues. Furthermore, elevated glucose levels and the presence of oxidative agents8 increase the risk of aggregation.

The process of aggregation takes on two parts, and is initiated by a protein that is rich in hydrophobic amino acid residues and has low overall charge9. These proteins have the strongest tendency to attract and reversibly attach to similar molecules to form the expanding precursor core in the first stage of the process called nucleation. The second stage begins once this core has surpassed a critical mass threshold; further proteins are irreversibly attached to the growing aggregate6. The products of aggregation range from small unordered oligomers (protein fragments) to large ordered fibrils called amyloid. A common phenomenon in aggregation is the conformational change of a protein segment from an α-helical structure to a β-sheet rich structure, which is known as fibrillation10. This is seen in the conversion of the normal prion protein (PrPc) which is largely composed of α-helices, to the infectious and toxic prion protein (PrPsc), a predominantly β-sheet structure1.

Proteostatic mechanisms that reduce protein misfolding:

'Molecular chaperones': protein molecules that aid correct and efficient folding, mainly by recognising, binding to, and thereby shielding the exposed hydrophobic amino acid residues on nascent polypeptides11are a large contributor to maintaining proteostasis. The first chaperones to interact with a newly-synthesised protein are ribosome-associated (RA) chaperones, which bind directly to the large ribosomal subunit. The concentration of RA chaperones in eukaryotic cytosol 'exceeds ribosome concentrations by a factor of roughly 2.5x'12. This suggests that on large or particularly hydrophobic proteins, several RA molecules may bind to different hydrophobic regions at one time.Another class of molecular chaperones called 'chaperonins' form large cylindrical complexes which provide an isolated environment called the 'Anfinsen cage' in which a single protein can undergo correct folding, unimpeded by aggregation13.

Another important element of proteostasis is that of protein degradation performed through two main pathways in eukaryotic cells; the Ubiquitin-Proteasome Pathway (UPP) and Lysosomal Proteolysis (LP). UPP uses ubiquitin (a 76-amino acid polypeptide) to mark defective proteins for proteolysis by a proteasome: a multi-subunit protease complex14. The ubiquitin chain is attached to the amino group of a lysine residue, and then further ubiquitin molecules are added, forming a polyubiquinated protein which can be recognised by the proteasome. LP involves the use of lysosomes to uptake proteins by endocytosis, and digest them with proteolytic enzymes14.

Proteostasis is controlled by cell responses; the unfolded protein response (UPR) is a negative feedback mechanism activated when there is a rise in the level of misfolded proteins in the cytosol, or under conditions of endoplasmic reticulum (ER) stress, such as heat shock, when proteins are more prone to aggregation. The UPR has three main actions: firstly, it increases the production of molecular chaperones. Secondly, it upregulates ER associated degradation (ERAD), the process by which misfolded proteins are transported to the cytosol, ubiquitinated and destroyed by a proteasome. Finally, the UPR reduces the rate of global protein synthesis through the PERK pathway. PERK is a stress-signal transducer which, when initiated during UPR, phosphorylates the α-subunit of translation initiation factor 2, thereby inactivating it, which reduces global protein synthesis15. The UPR is largely controlled by the binding immunoglobulin protein (BiP). Under normal conditions BiP is bonded to the three main stress-signal transducers: PERK, IRE1 and ATF6, inactivating them. When the level of misfolded proteins in the cell rises, BiP dissociates from these transducers, allowing them to initiate UPR. BiP is then free to act as a molecular chaperone, and assist correct protein folding15.

How misfolded proteins damage the nervous system:

In the nervous system, misfolded proteins present two critical problems: firstly, they do not perform their intended function, and secondly they disrupt normal neuronal function. Amyloidoses, a group of diseases that demonstrate the impact of both these problems, are caused by the accumulation of amyloid (an insoluble fibrous protein aggregate) in organs and tissues. David Eisenberg et al used x-ray microcrystallography to investigate the structure of amyloid; they found that each fibril consists of a pair of β-sheets, and each sheet is made up of many β-strands18, suggesting that fibrillation plays a large role in the conversion of healthy proteins to amyloid.Many neurodegenerative diseases are types of amyloidosis; Alzheimer’s disease is caused by the build-up of small amyloid – β peptides, taken from the amyloid precursor protein (APP). These peptides fibrillate, aggregate and accumulate to form amyloid plaques. The brain is most affected by these plaques because neurons are one of the largest expressers of APP, as it acts as a growth factor for neuronal stem cells19. Another protein that damages neuronal function is misfolded α-synuclein, in the form of soluble oligomeric protofibrils, which disrupt synaptic function, eventually leading to neuronal death20. These are small β-sheet rich clusters of amino acid residues that are in the unstable state before developing into a fibrous protein. This is the characteristic feature of Parkinson’s disease and Lewy-body dementia, which is caused by the accumulation of Lewy-bodies: large fibrillar aggregates mainly composed of α-synuclein.

Prion diseases are also the result of a deposition of amyloid. According to the ‘protein only’ hypothesis developed by Pruisner21, this amyloid is created by the conformational change of the cellular prion protein (PrPc), predominantly found on the surface of neurons, to PrPsc, an insoluble isoform which is the cause of the transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt–Jakob–Disease. Forloniet al22 have shown that a specific domain of PrPsc spanning amino acid residues 106-126 can form protease-resistant fibrills and other β-sheet rich structures which have a neurotoxic effect on rat hippocampal neurons in vitro. A unique feature of the misfolded PrPsc protein is that it is transmissible; according to the ‘refolding model’23, when a PrPsc molecule encounters its correctly – folded counterpart, it initiates a kinetically–controlled conformational change into its own misfolded structure. There is a high activation enthalpy barrier between the two states that prevents spontaneous conversion of PrPc without a PrPsc molecule present24.

The mechanism of how amyloid aggregates are actually neurotoxic is not yet fully understood. The amyloid cascade hypothesis (ACH) devised by Hardy and Allsop in 1991 suggests that it is the large fibrillar aggregates that are the primary cause of neuronal damage in amyloidoses25. This hypothesis is supported by in vitro experiments showing the toxicity of fibrillar amyloid-β to cultured neuronal cells26 and in vivo observations showing neuronal damage after injecting fibrillar amyloid-β into aged rhesus monkeys27. However, there is a growing belief that the soluble oligomer intermediates of the amyolid formation process are more toxic to the nervous system than the final product28. These intermediates take the form of micelles, protofibrills or diffusible ligands (small, soluble chemicals) and are in a temporarily unstable state before the transition to their ultimate fibrillar conformation. McLean et al29 found that the concentration of soluble oligomeric aggregates, rather than the concentration of large fibrillar aggregates, is proportional to the extent of neuronal damage.

Possible reasons for the significant neurotoxicity of soluble oligomers are that they are able to breach the phospholipid bilayer, thereby disrupting ion concentrations on either side of the cell membrane, inhibiting critical intracellular proteins from functioning and disturbing proteostatic mechanisms such as chaperone molecules and proteasome complexes30. It is also possible that micelle-like oligomers distort transmembrane ion channels, increasing membrane permeability which leads to a higher concentration of calcium ions inside the neuron, thereby inducing an ER stress response culminating in apoptosis31.An indirect impact of both soluble oligomers and large fibrillar aggregates is the neuroinflammatory responses they provoke. Chronic inflammation in the nervous system can initiate a chain of cellular actions which again end in apoptosis32.

Another situation in which protein misfolding is damaging to the nervous system is the misfolding of the proteolipid protein (PLP) or myelin basic protein, which are both essential components of all myelin tissue16leading to a dysfunctional myelin sheath. The myelin sheath is composed of an elongated plasma membrane, originating from Schwann cells in the peripheral nervous system and oligodendroglial cells in the central nervous system, which is wrapped around the axon16. The periodic non-myelinated sections of the axon are called the nodes of Ranvier, where sodium ion channels are concentrated, and depolarisation happens. The myelin sheath allowssaltatory conduction to take place; the electrical impulse jumps between nodes as the cytoplasm conducts sufficient electrical charge to depolarise the consecutive node17. This massively speeds up conduction, which is critical to neuronal function.Dysfunctional myelin tissue results in electrical impulses being transmitted at a much slower rate than is necessary for normal co-ordination, therefore parts of the nervous system are unable to communicate effectively. This is a possible cause of multiple sclerosis: an autoimmune demyelinating disorder in which oligodendrocytes, the cells responsible for creating and maintaining the myelin sheath, are destroyed33. The exact causes of MS are unknown, but it is thought that misfolded proteins contribute to oligodendrocyte death34both through disrupting myelin function and activation the UPR, potentially leading to apoptosis.

Although the translation and folding of proteins is a constant occurrence in almost all cell types throughout the body, neurons are the most adversely affected by misfolded protein aggregation35. This is because neurons are long-lived cells that remain in the G0 phase of the cell cycle indefinitely, and do not usually undergo mitosis36. This makes them particularly susceptible to the accumulation of aggregates and non-functional proteins. Furthermore, neurons which undergo apoptosis in result of the UPR are not replaced, magnifying the neurodegenerative effect of misfolded proteins on the nervous system. The symptoms of the wide-scale neuronal death caused by these various proteopathies ranges from insignificant memory loss to debilitating loss of control over both physical and mental activity. The impact that this has on the sufferer and everyone connected to them is often enormous, as these diseases have the power to cruelly take away both their future and their past simultaneously.

Hope for the future:

Protein misfolding and the damage it causes, specifically to the nervous system remains a conundrum in modern science and medicine. However, recent advances in research methods and tools have increased the possibility of understanding and working towards a treatment for the various neurodegenerative disorders that result from protein misfolding. The key questions that we must answer in order to continue this are: How exactly are misfolded proteins neurotoxic? Why do normal cellular proteostatic mechanisms fail with age? And finally, how can protein aggregates be removed, and ultimately how can their formation be prevented?There are numerous ideas regarding the treatment of proteopathies currently being explored, for example the up-regulation of chaperone molecules that activate the UPR such as BiP has been shown to have a protective effect on dopaminergenicneurones in a rat model of Parkinson's disease1. Other possibilities involve drug-like molecules that either inhibit aggregation by stabilising the native conformation of proteins prone to misfolding, or stimulate cell defence pathways such as the UPR, which increase misfolded protein degradation.These fast expanding areas of research have established new opportunities for combating the growing devastation caused by neurodegenerative proteopathies in our ageing society.

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