Molecules

Molecular mechanisms underlying neurophysiology

When we look at a neuron through a microscope or analyze its electrical or biochemical activity, we are, indeed, observing proteins. Proteins consist of a complexly folded string of amino-acids. They are encoded by the genes of the neuron and are the elemental building blocks of neurons and neuronal network. Proteins carry out most biological functions, for instance, proteins embedded in the plasma membrane form channels, pumps and transporters that control the ion trafficking across biological membranes, and therefore, control the electrical signalling in the nervous system. In order to elucidate the molecular mechanisms underlying various aspects of neurophysiology and neuropathology, it is mandatory to attain a deep understanding of proteins: which proteins are present in a certain type of neurons; where are they located sub-cellularly; how do they interact with each other, and what are their functional roles in physiological activities. The knowledge of how neurons function at molecular level will allow us to discover specific biomarkers and develop new medicines for brain disorders.

Proteomics

The availability of genome sequences (‘genomics’) for many organisms has generated databases of predicted protein sequences. Proteomics, complementary to genomics, is the study of the complete set of proteins of a given cell, tissue or organism at a given time. However, proteomics is not only about the assessment of ‘which’ protein is present. Proteins are complex molecules that can exist in similar layouts (isoforms), they are changed after they are transcribed and translated from genes (glycosylations and phosphorylations), they interact with other molecules in complex ways (e.g. protein–protein interactions), and they can take different forms by folding themselves differently in the three dimensional space.

Plasma membrane proteins attract particular interest in studies of neuron proteomics because they have important physiological roles. For instance, membrane channels, the proteins that are the basis for the electrical activity of neurons are all member of this class of proteins. It is also of great importance that more than two-thirds of the known targets for drugs that are used as medicaments are membrane proteins.

However, the separation and identification of plasma membrane proteins remains a challenge for proteomic technology. The reason is that they normally reside inside the membrane which is – chemically speaking - an awkward place to be. It is everyday knowledge that oil and water does not mix well. The challenge biological membranes meet is that they separate two watery compartments with an oily wall. To do this they are organized like a molecular sandwich, which at the outside is hydrophilic to connect to the watery compartments while inside it is lipophilic to realize the separation.

Proteins that reside in these membranes often have an extremely lipophilic part to adjust well to the lipophilic inside and a hydrophobic part that extrudes to the outside of the membrane and communicates with molecules that are dissolved in the watery solution of the intra- or extracellular fluid. It thus becomes clear that membrane proteins, once separated from their membrane, are difficult to handle and often change their functional properties. They need special treatment during the procedures to separate and identify proteins to keep their integrity.

Determining which proteins are present

Mass spectrometry-based proteomics has been proved to be a powerful approach for identifying and quantifying the expression, interaction and function of proteins in the nervous system. In combination with bioinformatics, the organization of dynamic, functional protein networks and macromolecular structures underlying physiological, anatomical and behavioural processes can be investigated. Neuro-proteomic studies were able to recover protein complexes that constitute transmitter receptors in synapses the elements through which neurons exchange electro-chemical messages. Furthermore, neuro-proteomics helps to identify proteins involved in brain disorders.

A mass spectrometry-based proteomics experiment generally consists of four steps:

  • In the first step, a neural tissue (e.g. different brain regions, cultured cells or subcellular fractionation) is processed to obtain a mixture of proteins. The possible contamination of the mixture must then be tested. For example, a plasma membrane fraction can be tested for the presence of factors typical for plasma membranes and factors that are typically present in the cell’s nucleus. Western plot analysis uses specific antibodies against these factors. In the case of a plasma membrane fractionation it is required that factors of plasma membranes are identified by Western blots analysis while factors of the nucleus are absent.

  • After sample preparation, the resulting protein mixture is further fractionated to reduce its complexity and facilitate the identification of low-abundance proteins, so that mass spectrometry can detect as many proteins as possible. Many problems in biochemistry are focused on separating components of a mixture from each other. Unmixing of protein solutions can employ electrical (electrophoresis) or chemical properties (liquid chromatography) of the proteins. These methods are employed in combination with enzymatic digestion of some fractions. The result is a set of protein fragments (peptides) that are purified well.

  • Once the components of the protein mixture have been separated, the resulting peptides will be ionized (i.e. electrically charged) inside the mass spectrometer and subsequently subjected to electrical and/or magnetic fields where they start to move. The path a molecule takes within such fields depends on its electrical charge as well as on its mass. This way, molecules with a different ratio of mass and charge can be effectively separated and collected. Typically a first mass spectrometry run can separate different fractions of peptides. A second run can then concentrate on different peptide fractions based on their abundance within the sample

  • The fourth and last step uses computational methods to assess the mass data to identify the peptides and in turn the parent protein.

Determining where proteins are located

After a new protein has been identified by proteomic approach, its expression in various neurons is tested. For a first orientation the messenger RNA (mRNA), a copy of the gene that is to be translated into a specific protein, can be measured. Methods to measure specific mRNA comprise Reverse Transcription Polymerase Chain Reaction (RT-PCR) and northern blots. For these methods the brain tissue has to be homogenized and information about protein content in specific brain regions, neurons, and sub-cellular compartments is lost. Much better spatial information is obtained when labelling mRNA in slices of tissue (‘in situ hybridisation’, ISH) or by labelling the proteins themselves using antibodies against them (‘immunocytochemistry’, ICC).

Double- or triple-label ICC with cell-specific markers is nowadays the standard technique to identify the neurons that express a protein of interest. As cell-specific markers, neurotransmitters and their synthetic enzymes, peptides, cytoskeletal, and calcium- binding proteins can be employed. The combination of both labelling techniques, ICC and ISH, on the same material is able to bring invaluable data about the cellular expression of a given protein, inter- or intracellular relationship between different molecules, and also functional activity of chemically identified neurons.

A second powerful strategy to localize proteins is the use of transgenic mouse lines. Transgenic organisms have additional genes incorporated in their genome. Nowadays, it is a standard procedure to generate mice that express some sort of reporter protein (e.g. a fluorescing protein that can be visualized under the microscope) whenever the protein of interest is being expressed. The trick is to assemble the reporter gene together with a so-called promotor gene. Promoter genes regulate in which cell and when a given protein is expressed. If the reporter protein is under control of the same promoter as the protein of interest, it will report when and where the protein of interest is expressed. Fluorescent reporter proteins can be monitored in real-time in vivo and in situ, and can be readily quantified. The prototype fluorescent protein reporter is green fluorescent protein (GFP) which is derived from the bioluminescent jellyfish Aequorea Victoria. But one has to keep in mind that it is quite time consuming to generate a transgenic mouse.

How do proteins interact and what is their physiological role?

The function of proteins is often defined by its interaction with other proteins. Labelling the protein of interest with an antibody can reveal its sub-cellular distribution when visualized using a high resolution optical imaging technique (e.g. confocal microscope) and electron microscopy.

Co-immunoprecipitation is a technique with which an antibody detects one known protein component of a whole protein complex. Using this trick the other components can be isolated and identified as well. An efficient method is called heterologous expression system. The trick here is to introduce the gene for the protein of interest fused with a fluorescent reporter into a standard cell culture. The protein will then be expressed and some functional properties can be studied. For instance, the cellular/sub-cellular localization and physiological function of the protein can be assessed. Moreover, pharmacological experiments or perturbing the expression of the protein can be used to determine how these perturbations affect its function.

Another approach to further elucidate its functional role in physiological activities is to express the protein of interest in neurons from specific mutant backgrounds and check whether the distinct features of the mutant phenotype can be rescued by wild type expression.