The unsolved mysteries of protein misfolding in common neurodegenerative diseases

Throughout the 1970s, biochemist Stanley Prusiner was obsessed with trying to find the causative agent for a mysterious group of diseases. The diseases, which included kuru and Creutzfeldt–Jakob disease in humans and scrapie in sheep, were characterized by slowly-developing symptoms and neurodegeneration so severe it eventually caused the brain to take on the appearance of a sponge (due to myriad little holes that developed where grey matter was lost). By the time Prusiner began studying these diseases, they had all been experimentally transmitted to chimpanzees or other animals by injecting the animals with biological material from an infected host. So, the diseases seemed to be spread in an infectious manner, but scientists were still trying to identify the agents (e.g. viruses, bacteria, etc.) responsible for their transmission.

Prusiner focused on trying to identify the agent behind the transmission of scrapie. He expected the agent to be a virus, but was confused when his tests repeatedly suggested it was composed of protein rather than the nucleic acids that would make up viral DNA. For, although there were others before Prusiner who had proposed ways proteins might be involved in the transmission of infectious diseases, these ideas had not been widely embraced by the scientific community. If Prusiner were to accept what his tests were indicating it would mean challenging the conventional understanding of the nature of infectious agents--which were considered to at least possess DNA or RNA--as well as the fundamental characteristics of proteins, which were not previously known to have infectious capabilities.

In 1982, Prusiner published a manuscript outlining a hypothesis that scrapie was caused by a protein that had infectious qualities. He termed this protein a prion, for "proteinaceous infectious particle." Prusiner's ideas were considered somewhat heretical to virologists and others studying these diseases, and Prusiner was faced with criticism from scientists and non-scientists alike. But eventually Prusiner isolated the prion responsible for the transmission of scrapie, confirming his suspicions and silencing the naysayers. In 1997, he was awarded the Nobel Prize in Medicine for his discovery.

Now it is well established that prions are agents of disease, in that they can spread from host to host just like typical pathogens. Once inside their host, however, they also have a unique capability of "infecting" other proteins. This results in the spreading of pathology through different regions of the brain, eventually leading to widespread neurodegeneration. But the story of infectious proteins does not end with the rare group of diseases studied by Prusiner. What we are coming to learn now is that this prion-like transmission of pathology between proteins may be a characteristic of other--much more common--diseases that involve neurodegeneration. While these diseases are very different from prion diseases in that they are not spread between individuals, the evidence suggests that the pathophysiology of neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD) may involve proteins that behave like prions in their ability to infect other proteins within the brain of an affected individual.

Alzheimer's, Parkinson's, and protein misfolding

There are a number of diseases that are characterized by neurodegeneration, but AD and PD are the two neurodegenerative diseases that have the greatest impact on our society today in terms of mortality, disability, and economic burden. While both diseases involve neurodegeneration, however, they differ in the areas of the brain most affected (which plays a large role in determining their different symptomatic profiles). In AD, neurodegeneration becomes extensive in several areas of the brain, including areas of the cortex and hippocampus. These are areas that are especially important to cognition and memory, which helps to explain some of the cognitive deficits seen in the disease. In PD, neurons in structures that are part of the basal ganglia--a group of nuclei that play an important role in facilitating movement--are severely affected. The substantia nigra is particularly impacted, with somewhere between 50 and 70% of the neurons in this area being lost by the time of death. While the mechanisms underlying the large-scale neurodegeneration that occurs in AD and PD are not yet fully understood, in both diseases the neurodegeneration is associated with the accumulation of misfolded proteins in or around neurons.

Proteins are essential to an extensive list of biological functions, including many vital processes like cellular metabolism, DNA replication, and cell signaling. When a protein is formed from a chain of amino acids, one of the final steps in its synthesis is to undergo a process of folding. The chain of amino acids (known as a polypeptide) that came together to create the protein is folded into a three-dimensional structure, and it is this three-dimensional structure--known as the tertiary structure--that decides the protein's eventual function. In other words, the folding--not simply the amino acid chain that makes up the protein--is what determines the role the protein will play in the body.

Thus, protein folding is essential to synthesizing functional proteins. In AD and PD, however, proteins seem to fold incorrectly. This creates proteins with abnormal conformations composed of filaments that are pathologically twisted together instead of folded into a typical three-dimensional structure. These proteins are said to be in an amyloid state, because when this abnormal configuration was first noticed by renowned scientist Rudolf Virchow, he mistakenly identified them as starch (amyloid means starch-like). Once they have formed, these amyloid proteins have a tendency to aggregate into insoluble clumps.

AD is characterized by aggregations of two types of proteins, called amyloid beta and tau, respectively. Amyloid beta is always present in the brain in the extracellular space around neurons, although its function has yet to be elucidated. In AD, however, it accumulates into clusters called amyloid plaques (aka senile plaques) that form outside and around neurons. These amyloid plaques often begin to form in the cortex in earlier stages of the disease, but propagate and eventually spread to the brainstem in the later stages. Tau is found within neurons, and is normally involved in maintaining the structure of the neuron through the stabilization of microtubules. When tau becomes misfolded, it can accumulate inside neurons into clusters called neurofibrillary tangles. In AD, these tangles generally begin to form in the brainstem, then spread to the temporal lobes and cortex.

PD is characterized by the aggregation of a protein called alpha-synuclein. Alpha-synuclein is found within neurons, and may play various roles in regulating neurotransmitter release. In PD, it clumps into clusters called Lewy bodies (named for Frederic Lewy, who discovered them in 1912), which form inside the neuron. There is no consistent site of origin for Lewy bodies in the brain, but often they are first seen in the brainstem or olfactory bulb.

Just like prions, misfolded proteins in neurodegenerative diseases like AD and PD seem to be able to influence previously healthy proteins to also undergo a process of misfolding, causing them to be transformed into an amyloid state. In one of the earliest studies to provide experimental evidence of this, amyloid plaque formation was induced in the brains of monkeys after brain tissue from human AD patients was injected into the monkeys' brains. This process of the spread of pathological misfolding from one protein to the next is referred to as seeded aggregation.

Misfolding can be spread not only from protein to protein, but also from neuron to neuron. The way this happens is also still not very clear, but it is suspected that misfolded proteins may be taken up from the extracellular space into neurons or glial cells, where they begin to infect susceptible proteins and further spread pathological misfolding.

What damage does protein misfolding cause?

Although misfolded proteins are characteristic of neurodegenerative diseases, how exactly they contribute to the pathology of these diseases is still unknown. In other words, while AD and PD patients have clumps of these proteins throughout their brains, we don't actually know what role the aggregates play in causing neuronal death--or if they cause cell death at all.

According to one perspective, amyloid proteins and the aggregates they form are themselves neurotoxic and can lead to the death of neurons. Additionally, it has been hypothesized that aggregates can disrupt neuronal function by affecting the integrity of neuronal structure and impairing communication within neurons in the process (also leading to neurodegeneration). However, neuronal death is often not well correlated with the degree of aggregate formation, and often the most extensive areas of cell death occur at some distance from sites of aggregation. These findings have led to the hypothesis that it is not the amyloid proteins found in aggregates that are most harmful, but rather smaller soluble forms of amyloid (called amyloid oligomers and protofibrils) that are more detrimental to neurons. These smaller amyloid species are not easily detectable when testing the brain for the presence of amyloid, and can be found in areas where aggregation isn't apparent, potentially explaining the poor correlation between amyloid aggregates and neurodegeneration.

Another hypothesis, however, suggests that amyloid aggregates are formed by the brain as a means of controlling the spread of potentially damaging misfolded proteins. According to this perspective, molecular chaperones--proteins that facilitate the folding process of other proteins--recognize when a protein has become folded incorrectly. The chaperones can then sequester these misfolded proteins into benign aggregates, which keeps them from having toxic effects on neurons.

Questions still to be answered

We are still in the dark about many of the details of protein misfolding diseases. We know the misfolding occurs as part of the pathophysiology of these diseases, and that it is correlated with neurodegeneration in some way. But we don't know what prompts misfolding, how it seems to spread from protein to protein and neuron to neuron, or in what way it is related to the death of neurons. When the answers to some of these questions become clearer, it may open the way for more effective treatments for these diseases. If, for example, we can identify how neurodegenerative disease proteins are spread between neurons, we can try to develop ways to block such a mechanism (e.g. blocking a receptor they are utilizing to gain entry into the neuron). Until we learn more, however, we will continue to be somewhat at the mercy of these diseases that imbue proteins with a type of "infectiousness" that was unheard of less than 40 years ago.

Brettschneider, J., Tredici, K., Lee, V., & Trojanowski, J. (2015). Spreading of pathology in neurodegenerative diseases: a focus on human studies Nature Reviews Neuroscience, 16 (2), 109-120 DOI: 10.1038/nrn3887

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Know your brain: Reward system

Where is the reward system?

The term reward system refers to a group of structures that are activated by rewarding or reinforcing stimuli (e.g. addictive drugs). When exposed to a rewarding stimulus, the brain responds by increasing release of the neurotransmitter dopamine and thus the structures associated with the reward system are found along the major dopamine pathways in the brain. The mesolimbic dopamine pathway is thought to play a primary role in the reward system. It connects the ventral tegmental area (VTA), one of the principal dopamine-producing areas in the brain, with the nucleus accumbens, an area found in the ventral striatum that is strongly associated with motivation and reward. Another major dopamine pathway, the mesocortical pathway, travels from the VTA to the cerebral cortex and is also considered part of the reward system. So, the reward system is generally considered to be made up of the main dopamine pathways of the brain (especially the mesolimbic pathway) and structures like the VTA and nucleus accumbens, which are connected by these dopamine pathways.

What is the reward system and what does it do?

In the 1950s, James Olds and Peter Milner implanted electrodes in the brains of rats and allowed the animals to press a lever to receive a mild burst of electrical stimulation to their brains. Olds and Milner discovered that there were certain areas of the brain that rats would repeatedly press the lever to receive stimulation to. They found a region known as the septal area, which lies just below the front end of the corpus callosum, to be the most sensitive. One of the rats in their experiment pressed a lever 7500 times in 12 hours to receive electrical stimulation here.

Olds and Milner's experiments were significant because they appeared to verify the existence of brain structures that are devoted to mediating rewarding experiences. For, if the rats were lever-pressing repeatedly to receive stimulation to these areas, it suggested they were enjoying the experience. Subsequent studies attempted to more thoroughly map out these "reward areas," and it was discovered that some of the most sensitive areas are situated along the medial forebrain bundle. The medial forebrain bundle is a large collection of nerve fibers that travels between the VTA and the lateral hypothalamus, making many other connections along the way. Some areas of the medial forebrain bundle were found to be so sensitive that rats would choose receiving stimulation to them over food or sex.

Eventually it was recognized that dopamine neurons are activated during this type of rewarding brain stimulation, and researchers found that they could cause rats to stop lever pressing by administering a dopamine antagonist (a drug that blocks the effects of dopamine). In other words, without the activity of dopamine the rats were less likely to find brain stimulation reinforcing, and so they stopped pressing the lever altogether. Other evidence, such as the discovery that dopamine antagonists seemed to reduce the rewarding qualities of drugs like amphetamines, further supported the importance of dopamine's role in reward.

Based on brain stimulation experiments and the increasingly recognized importance of dopamine in reward, attention began to turn toward major dopamine pathways as playing an important part in mediating rewarding experiences. The medial forebrain bundle connects the dopamine-rich VTA with the nucleus accumbens and is considered part of the mesolimbic dopamine pathway. It eventually became recognized that, when we use an addictive drug or experience something otherwise rewarding, dopamine neurons in the VTA are activated. These neurons project to the nucleus accumbens via the mesolimbic dopamine pathway, and their activation causes dopamine levels in the nucleus accumbens to rise. Furthermore, disrupting this pathway in rodents that had become addicted to pressing a lever for brain stimulation or a drug reward caused them to stop lever-pressing, suggesting these areas are crucially important to the occurrence of addictive behavior.

As the mesolimbic dopamine pathway is activated whenever we use an addictive drug, it has come to be considered the primary pathway of the reward system. However, dopaminergic projections from the VTA travel to the frontal cortex as well; they comprise the mesocortical dopamine pathway. These fibers are also thought to be involved in reward and motivation, although their contribution to rewarding experiences is less clear than that of the mesolimbic pathway.

It's important to note that since the earliest research on the reward system our perspective on dopamine's role in reward has changed slightly. At one time dopamine was considered to be the neurotransmitter responsible for causing the experience of pleasure, but it is now thought to be involved with aspects of reward other than the direct experience of enjoyment. While the details are still being worked out, some have suggested dopamine is involved in encoding memories about a reward (e.g. how to get it, where it was obtained) and attributing importance to environmental stimuli that are associated with the reward.

While the reward system is implicated in pleasurable and potentially addictive behaviors, the substrates of pleasure are not confined to the structures mentioned above and dopamine is not the only neurotransmitter involved. The reward system refers to a group of structures that seem to be frequently involved in mediating rewarding experiences, but the actual network dedicated to creating the feelings we associate with these experiences is likely more complex.

Wise RA (1998). Drug-activation of brain reward pathways. Drug and alcohol dependence, 51 (1-2), 13-22 PMID: 9716927