Structural Biochemistry/Neurodegeneration

Introduction

Amyloidogenic cascade

Neurodegenerative diseases are disorders closely linked to old age and affect more than 120 million people world wide per year. Some neurodegenerative diseases were often identified by the misfolding and clumping of their proteins - which led to neurotoxicity. But, this idea has been controversial and in light of recent research, there is a better understanding of the nature of proteins and their effect on neurodegeneracy. A prominent example of this controversial topic is Alzheimer's Disease and the aggregation of peptide β-amyloid (Aβ) in the brain. β-Amyloid peptide can exist as a non-toxic substance in the brain, however, large deposits of this peptide are found in the brains of patients with Alzheimer's Disease. The connection between these degenerative diseases and protein aggregation have been experimentally studied: genetic mutations, increased gene dosage, and post-translational gene modifications. These experiments are associated to Parkinson's Disease, Alzheimer's Disease, and Huntington's Disease, respectively. Despite these experiments, these relationships are still poorly understood. It is commonly thought that neurodegenerative diseases are caused by aging, proteasomal and mitochondrial disfunction, oxidative stress, and abnormal protein-protein interactions via cytotoxicity, a more radical proposition suggests that macroscopic proteinaceous inclusions are also an important factor in the progression of these diseases. These macroscopic inclusions were included in many models for the diseases, but were only recently observed as oligomeric and prefibrillar species in living cells.

Amyloid Hypothesis - Aberrant protein interactions, which accumulate and result in neural defects and culminate into neurodegeneration, are believed to be causally related to the insertion of aggregated proteins into ordered fibrillar structures (such as amyloid membranes). This is a result of malformed proteins - their designated chaperones cannot recognize them anymore, nor can they be destroyed by ubiquitin-proteasome system. As a result, these proteins are able to remain in the cell and have hazardous effects on the cell. These cells can then form stable and insoluble amyloid assemblies consisting of largely β-sheets. Another assembly, oligomeric species, is smaller and is predicted to be either a precursor for the amyloid fibrils or abnormal intermediates in the amyloidogenic cascade.

Neurodegenerative Diseases

Alzheimer's Disease

This is one of the most common age-associated diseases and is often associated to dementia. As the age of the population increases, so does the frequency of Alzheimer's Disease. On a molecular level, one of the most characteristic features of Alzheimer's Disease are the extracellular amyloid plaques composed of Aβ and neurofibrillary tangles. Recognizing these hallmark characteristics is not enough, because despite being able to recognize these proteins, the mechanisms of their formations are still unknown. This is partially because most Alzheimer cases occur randomly without a clear signal when the disease takes root. In order to circumvent this, conformation-dependent antibodies have been developed to help examine protein aggregates and identify prefibrillar oligomers and fibrils.

Techniques Used to Study Neurodegenerative Diseases

Optical microscopy and live-cell imaging are types of techniques often used to study the development of neurodegenerative diseases.

Bimolecular Fluorescence Complementation (BiFC) - The technique is useful because it can be used in physiological environments (rather than extraction from the body). Furthermore, it helps elucidates protein-protein interaction with spatial and temporal resolution. It also elucidates the function of protein-protein interactions within a living cell. This method involves two non-fluorescent molecules that are fragments of a reporter protein and the two proteins of interest. If the two proteins of interest interact with each other, te the two fragments of the reporter protein come together and form a structure that mimics the protein's native form. This technique has been helpful in recent years because it has helped explain the connection between oligomerization and neurodegenerative diseases. It is also predicted that BiFC will be able to help determine how the inclusion bodies and oligomeric species are formed in the brain. As a result, BiFC can aid therapeutic research by predicting appropriate targets for drugs and by suggesting new methods of treating neurodegeneration.

This technique was used starting in 2002, and has many applications beyond neurodegenerative diseases. For example, it has been used to study the interaction between basic leucine zipper and Rel family transcription factors in their physiological state. This method has also been used on several model organisms such as mammalian cell lines, plants, nematodes, yeast and bacteria. Originally, it was used only to identify singular protein-protein interactions. But in more recent experiments, it has been used to study multiple protein-protein interactions by using multiple fluorescent proteins all with different emission spectra. This advancement has allowed for the study of subcellular localization, assessment of complex formation, analysis of the control over protein-protein interactions, and the ability to simultaneously observe changes in separate protein complexes.

Another variation of BiFC uses the reporter protein in conjunction with bioluminescence resonance energy transfer spectroscopy as an alternative to multicolor BiFC.

The Advantages of BiFC

There are two main advantages of BiFC over many of the older techniques.

1. This technique uses molecules that cause fluorescence after the protein-protein interaction occurs, because this interaction is so specific, it unlikely that any other interaction with be able to cause a strong enough fluorescence in the reporter protein.

2. The use of fluorescence complexes allows the protein-protein interactions to be studied within the organism, rather than removing the proteins of interest from its natural environment to stain and track its actions.

The Disadvantages of BiFC

1. This technique is an indirect method of identifying the protein-protein interaction and requires the use of a very specific, fluorescing reporter protein. As a result, it is not possible to use this method to study the interactions between the proteins if one of them is unknown. This issue as been overcome by producing libraries in which different cDNAs are attached to fluorescent protein fragments.

2. One of the limitations in using this technique to study neurodegeneracy is that when the fluorophore recombines when the proteins interact, than the complexed fluorophore will stabilize the interacting proteins which will prevent the desired interactions from being observed. Despite this disadvantage, it could also be beneficial because it allows for a more selective study of oligomeric and dimeric species. For neurodegenerative studies, this effect might be particularly useful because by stabilizing the protein complex, the complex will be able to exist longer in its interacting state. This will make it easier to study the more ephemeral protein-protein interactions. This technique now makes it possible to study the protein aggregation process because parts of the formation involve these short lived protein-protein interactions. 3. This method does not distinguish between dimers, oligomeric and higher-ordered species. Other techniques have to be used to accomplish this, such as flow cytometry or SDS-PAGE.

Electron cryomicroscopy - In conjunction with nuclear magnetic resonance (NMR) spectroscopy, this technique has be useful in determining the structure of β-Amyloid peptide-derived assemblies. This is important because a better understanding of the assembly of the Aβ peptide will help explain the effect of Aβ aggregation in the brain.

Key Characteristics of Neurodegenerative Diseases

Aberrant Protein-Protein Interactions (PPi)

Although protein-protein interactions are not yet clearly understood with current research techniques, it is known that they are important in understanding the resultant proteins and how they function in physiological conditions. A common result of aberrant protein interaction is the presence of cytoplasmic, nuclear, or extracellular inclusions. These inclusions are generally a result of a build up of misfolded proteins into insoluble nuclear or cytoplasmic amyloid deposits. The changes in protein-protein interactions are believed to lead to the formation of these inclusions. Although the formation of the inclusion mechanism is not completely understood, studies suggest that the formations of inclusions follow a generally uniform pattern, even when the misfolded proteins are different. As a result, it is necessary for more conclusive data about inclusion bodies to be determined in order to develop therapeutic techniques for neurodegeneration treatment.

The study of Protein-Protein Interactions In the past, protein-protein interactions were studied via co-immunoprecipitation and co-purification. In these procedures, the protein-protein interaction could only be observed by removing the proteins from their physiological conditions. The more recent protein microarrays also work in a similar fashion. Removing the protein from its environment introduces a source of error because protein-protein interaction is also affected by its environment and its removal from physiological conditions means that the proteins won't be acting as they usually do in the cell. In comparison, Protein-Fragment Complementation Assays (PCAs), functional analysis of compensatory mutations, and imaging-based techniques are able to more accurately identify protein-protein interactions by avoiding the removal of proteins from the cell.

Older techniques used to make protein-protein interaction images often times used fluorescence or bioluminescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, and image correlation spectroscopy. Fluorescence resonance energy transfer microscopy is useful because it labels the two interacting proteins with two different fluorophores in vivo. When the donor fluorophore is excited, it will transfer energy to to the second fluorophore, the acceptor fluorophore, which will label the second protein. The distance between the two proteins is determined by the difference between the lifetimes of the fluorophores. This technique is limited though, because the distance between the two proteins must be less than ten nanometers, otherwise the emission spectrum of the two fluorophores will not overlap. This technique has been used to study the effect of mutations in the gene for amyloid precursor protein and its interaction with presenilin-1. This technique can also be used to characterize intramolecular and intermolecular interactions.

Fluorescene Correlation Spectroscopy is a bioimaging technique that uses theoretical analysis to interpret fluctuations and diffusion rates of fluorescently labeled molecules. Specific fluctuations are associated to specific interactions and aggregation patterns of interacting proteins. This method has been used to study how peptide β-amyloid aggregates form.