Aggregates/Nuclear inclusions: Villain or tragic hero?

Introduction
The major defining feature of neurodegenerative diseases is the progressive accumulation of nuclear inclusions comprising of irregularly folded protein aggregates. Previously it was thought that protein aggregation is the cause of neurodegeneration as it had been established that neurodegenerative diseases such Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease (AD), prion disease, and amyotrophic lateral sclerosis (ALS) all shared a common feature which was these aggregated proteins and the formation of inclusion bodies (Ross and Poirier, 2004). However, recent studies have suggested that protein aggregation may not be the cause of toxicity to cells but that it may in fact be a protective mechanism. The aggregates formed in the above-mentioned diseases can be a consequence of mutations in the sequence of the protein that is related to the disease, increased amounts of a normal protein due to a genetic variation, or even the absence of genetic variations. These may be initiated by environmental stress or aging (Ross and Margolis, 2005). The aggregated proteins can build up and form inclusion bodies, which can be either intracellular or extracellular.

There is an ongoing debate about the role of aggregation in the disease process even though one of the most common pathological features of neurodegenerative disorders is inclusion bodies. There is much evidence indicating that aggregation is associated with toxicity in these diseases. On the other hand, there are those studies that have suggested that the formation of inclusion bodies is not related to toxicity and that in actual fact are a protective response by the cell (Ross and Poirier, 2005). Therefore, the question still remains, are aggregates and nuclear inclusions the villains in neurodegenerative disease, or are they the tragic hero? The evidence collected by studies so far, indicates that the process of aggregation is associated with toxicity but the nuclear inclusions themselves are protective. The events that occur before the formation of inclusion bodies are the cause of toxicity to the cell and that the inclusion bodies themselves are just an end product of those events. These events are the actual route the disease proteins take towards aggregation forming oligomers or protofibrils, terms used to describe abnormal monomers or little groups of aggregated proteins gathered together, along the way (Ross and Poirier, 2005).

Protein misfolding
Protein molecules fold into their own distinctive structures in order to carry out their physiological functions. The folding of the protein is built upon the information within their amino acid sequence (Anfinsen, 1973). It has been shown that a large number (30%) of newly synthesized proteins in the cell are imperfect and thus they are degraded by proteasomes (Schubert et al., 2000). This displays that all normal cells have to degrade misfolded proteins regularly. There are mechanisms that cells have established in order to protect themselves from these misfolded and aggregated proteins. They have proteins called chaperones which assists with the normal folding of proteins and can also refold irregular conformations back to their natural state making them non-toxic (McClellan and Frydman, 2001). Studies have shown that these chaperones can overturn neuronal toxicity caused by mutant polyglutamine by increasing the solubility of the mutant proteins (Muchowski and Wacker, 2005).

If this mechanism does not work, the cell can target those irregular misfolded proteins and degrade them by covalently binding polyubiquitin that allows proteasomes to target them, this is known as the Ubiquitin-Proteasome Pathway (UPP) (Ciechanover and Brundin, 2003). Proteosomes are molecular components that have a space inside them in which they can unfold proteins and cut them up into short fragments. The UPP plays a pivotal role in whether a misfolded protein is degraded or its packed into an inclusion body (Chung, Dawson and Dawson, 2001). The build up of inclusion bodies was thought to consequently provoke neuronal dysfunction and/or cell death subsequently leading to neurodegeneration (Alves-Rodrigues, Gregori and Figueiredo-Pereira, 1998).

Figure 1: This figure illustrates the defensive mechanisms in the cell. Chaperons enable the folding of new proteins, they unfold/refold misfolded proteins, prevent misfolded proteins from aggregating, and guide fatally misfolded proteins for degradation by the U Ubiquitin-Proteasome System (UPS). The UPS degrades misfolded and damaged proteins along with normal unneeded proteins in the cell. The autophagy-lysosomal pathway removes protein aggregates that have formed and were not captured by chaperones or the UPS (Su and Wang, 2009).

Figure 1: This figure illustrates the defensive mechanisms in the cell. Chaperons enable the folding of new proteins, they unfold/refold misfolded proteins, prevent misfolded proteins from aggregating, and guide fatally misfolded proteins for degradation by the U Ubiquitin-Proteasome System (UPS). The UPS degrades misfolded and damaged proteins along with normal unneeded proteins in the cell. The autophagy-lysosomal pathway removes protein aggregates that have formed and were not captured by chaperones or the UPS (Su and Wang, 2009).

The cell also has a third mechanism, called autophagy, which uses lysosomal pathways to degrade unwanted cellular components. Proteosomes and autophagy are involved in the normal turnover of proteins (Ross and Poirier, 2005). If the cells cannot deal with abnormal proteins using the above mention mechanisms, then they can isolate them via microtubule mediated transport at a cytoplasmic site generating large inclusion bodies called aggresomes (Jhonston, Ward and Kopito, 1998). Neurodegeneration may be caused the intracellular aggregates overwhelming the capabilities of the chaperones and/or the UPPS in degrading key cellular regulatory factors, this could lead to a positive feedback mechanism in which increased aggregation could in turn lead to a greater decrease in the UPP and interference of essential cellular events, in the end causing neuronal cell death (Chung, Dawson and Dawson, 2001).

Pathological Features Linked with Neurodegenerative Diseases
The residues found in neurodegenerative diseases are all formed by proteins that are distinct from each other and they gather together in discrete parts of the brain. In each disease, different parts of the brain are affected by neurodegeneration with distinctive neurons being susceptible depending on the disease. (Treusch, Cyr and Lindquist, 2009).

Alzheimer’s disease (AD) is defined by the formation of extracellular plaques containing the protein Amyloid-β and the accumulation of hyperphosphorylated tau as neurofibrillary tangles (Selkoe, 2004). The main cause of disease in AD has been postulated to be the buildup of Aβ, however there is a very weak link between the concentration of Aβ in plaques and the severity of dementia that is caused by AD (Ellison et al., 2004). The tangles formed by tau associate better with the severity of dementia in AD than Aβ plaques, however it is not clear as yet whether their aggregation is toxic or if they are the outcome of a protective mechanism (Hernandez and Avila, 2008).
Parkinson’s disease (PD) is characterized by the accumulation of the synaptic protein α-synuclein in neuronal cell bodies (Lewy bodies) and neuronal axons (Lewy neuritis) (Goedert, 2001). These Lewy bodies are most concentrated in the substantia nigra and also occur in cerebral cortical, monoaminergic and other neurons. The existence of these Lewy bodies has a weak link with the death of neurons in the substantia nigra of the brain (Margaret and William, 1997).
Another disease that involves the build-up of misfolded proteins is Creutzfeldt-Jakob disease (CJD) with the causative agent being prion proteins (Prusiner, 2001). There are various other forms of prion disease and they all involve the buildup of a proteinase-K form the prion protein PrPres. In CJD, like the other diseases discussed above, the loss of neurons is not related to the area where PrPres is deposited (Budka et al., 2003).
In Huntington’s disease (HD), the huntingtin genes contain repeat CAG expansions leading to the buildup of polyglutamine expanded huntingtin protein within inclusion bodies (Ellison et al., 2004). The length of the CAG repeat in the huntingtin gene leads to a greater concentration of inclusions. However, the concentration of the huntingtin protein or the location of huntingtin inclusions does not in fact relate with the susceptibility of the neurons being damaged (Gutekunst et al., 1999). The lack of a relationship between nuclear inclusions and cell death is in fact most evident in in HD and other polyglutamine diseases. In HD the inclusion bodies occur in high concentrations in the cerebral cortex that is only affected by minimal degeneration, in contrast, they are present in low concentrations in the substanita nigra that is affected by greater degeneration (Gutekunst et al., 1999). Another interesting aspect to note that applies to all of the above-mentioned proteins and the aggregates/nuclear inclusions they form in disease is that they have all been found to be ubiqutinated. As was discussed above, ubiquitin is added to misfolded proteins to mark them for degradation by the proteasome. Therefore, the presence of ubiquitin in every single one of the inclusions found in the neurodegenerative disease discussed shows that the neuronal cells have been unable to degrade those particular proteins and that the failure of the ubiquitin/proteasome might cause them to accumulate, disrupting fundamental cellular events and leading to neuronal cell death (Alves-Rodriques, Gregori and Fiqueiredo-Pereira, 1998). When the system fails or is overloaded, the cell sweeps all of the misfolded and mutated proteins and sequesters them in aggregates and inclusion bodies, which is why they are seen in neurodegenerative diseases (Chung, Dawson and Dawson, 2001).

The proteins that misfold in the above mentioned diseases are different, but in each disease, the protein deposits are a weak marker of the loss of neurons. This leads to the assumption that these aggregates and inclusion bodies may not actually be the villains they were once thought to be, but that they aid the cell in dealing with misfolded proteins and when neurons cannot make these aggregates and inclusion bodies, that is when they become susceptible to the disease. So, if the aggregates and nuclear inclsions are not the villains, then what causes degeneration of neurons and the brain in these diseases?

Aggregate Intermediates
The process of aggregation creates many intermediate species such as oligomers and protofibrils and these species may be the cause of toxicity. Oligomers are proteins gathered together in a small group and are an aggregation intermediate. Protofibrils are small fibril like structures but they are soluble and are also an aggregation intermediate. It has been shown that fibrils are formed by the addition of oligomers and protofibrils; they are added to the growing fibril (Collins et al., 2004). Studies looking at the formation of Aβ in AD found that the levels of soluble Aβ related directly to the degeneration of those areas of the brain (Lue et al., 1999). These oligomers are probably formed before the formation of fibrils as was shown in the above study, their concentration correlates with neurodegeneration. Another study was carried out where they injected Aβ into the brain of a rat and observed that it had toxic effects. They then treated the Aβ before injection so that all monomers would be degraded and they still observed a toxic effect. This led them to rule out monomers as the toxic species and indicated that soluble oligomers are in fact the causative agents (Walsh et al., 2002).
Figure 2: Possible pathways for aggregate and inclusion body formation.
Figure 2: Possible pathways for aggregate and inclusion body formation.
The aggregates formed by α-synuclein have been shown to take several different forms, including globular oligomers, protofibrils, and annular intermediates. These annular intermediates are doughnut shaped oligomers that have pore-like properties and damage the membrane (Volles et al., 2001). In one study, it was shown that a particular mutated form of α-synuclein (A30P) found in oligomeric form in the brain of patients had a property that caused α-synuclein oligomers to form much more rapidly and not go on to form fibrils, these patients had a faster progression of the disease (Miller et al., 2004). This shows that oligomeric intermediates of α-synuclein are the cytotoxic species related to the protein not the mature fibrils (Uversky, 2007).
There have also been studies looking at the pathway of aggregation for poluglutamine and they have found several distinct kinds of aggregates of huntingtin that form before the actual aggregate and that they are fundamental to toxicity (Poirier et al., 2002). They also found that Congo red blocks the aggregation pathway at an early stage. Another group used this on mice and found that their behaviour improved and they also survived longer (Sanchez, Mahlke and Yuan , 2003). These studies showed that huntingin intermediates are the ones that have a role in neuronal death.

Conclusion
Taking all of the evidence that has been presented above, it can be concluded that the original understanding of aggregates and nuclear inclusions as the villains in neurodegenerative disease was in fact incorrect. Accumulating evidence has shown soluble intermediates of the abnormal disease causing proteins exist and that these have a toxic effect on the cell leading to neuronal cell death. The presence of ubiquitin in aggregates shows that the cell tries to degrade the misfolded protein and when it cannot, it sequesters them away in an aggregate. It may not be the best solution but it better than leaving the soluble and more toxic form free in the cell. Therefore, in can be concluded that aggregates and inclusion bodies are the tragic hero’s in neurodegenerative diseases.

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