In this blog I will go into the last two disease process associated with MND from the review paper “Decoding ALS: from genes to mechanism”. This week we are going to focus on topics e and f which discuss changes in transport along the axon of the motor neuron and disturbances in RNA metabolism. Firstly, I will give a brief introduction into the process of generating RNA, how RNA then makes protein and how these processes are disrupted in MND.
Figure 1. Mechanisms of disease implicated in MND (adapted from Taylor JP et al., 2016)
The central dogma of biology
The central dogma or biology describes the molecular processes that lead to the making of proteins from genes. In short: DNA makes RNA which makes protein (Figure 2). These processes are incredibly complex and very tightly regulated and understanding these will help show why any disruption to these processes can have huge wide-spread effects. (If you would like to watch some great videos about genes, DNA and protein there are lot of great 3D-animations on the DNA learning center website).
Figure 2. The central dogma of molecular biology.
DNA exists as a double strand of genetic code which, when written in particular order, make all the genes in the body. Each cell contains 3 billion letters of code and surprisingly, only 1% of all the genetic material codes for proteins – the rest is non-coding. When making protein, DNA first undergoes a process called transcription where it is split into two single strands and makes a copy of itself. This single-stranded copy of the DNA message is called RNA. If the RNA contains the code for a protein it is called messenger RNA or mRNA. If the RNA sequence doesn’t code for a protein it is a non-coding RNA (later we will discuss one type, microRNA).
The structure of mRNA is shown below in Figure 3. Here it is shown in its pre-mRNA state which contains regions called introns and exons. Introns are non-coding and only the exons contain the mRNA codes to make proteins.
Figure 3. Structure of RNA showing the coding regions (exons 1 – 4) and the non-coding regions (introns).
Pre-mRNA will then undergo a process called splicing, where special splicing machinery will chop the sequence at the beginning and end of each of the introns and stitch the exons together (Figure 4). This is the mature mRNA sequence, which is used to build proteins in the process known as translation.
Figure 4. Pre-mRNA is “spliced” to remove the non-coding sections and generate the mature mRNA
When DNA is transcribed into RNA, it can encode more than 1 gene or protein. How does it do this? Through a process called alternative splicing. This is when the splicing machinery chops out the introns, but includes different combinations of the exons. When one or more of the exons is excluded, the code is changed and so the subsequent protein is also changed (Figure 5). Alternative splicing is a common feature of many genes and each different variation of the mRNA, known as the splice variant, can have a particular function.
Figure 5. Different proteins can be made from the same gene via alternative splicing.
Topic e. Cytoskeletal defects and altered axonal transport.
Motor neurons are uniquely structured cells, consisting of the cell body (soma) located in the brain and spinal cord and incredibly long axons that can extend all the way to the terminal end which connect with the muscles in the periphery. Motor neurons rely on fast and efficient transport to send proteins between the terminal ends and the cell body. To overcome the long distance of their axons, neurons can also make proteins at sites away from the cell body, closer to the terminal end. This is called local translation and requires all the cell machinery as well as the mRNA for making proteins to be transported along the axon to the local site. (watch mRNAs transported along an axon under the microscope ).
In MND, prior to the loss of neurons, transport of proteins along the axon is slowed in both directions making the cells less responsive to stimulation and stress. In addition, this transport process is controlled by a group of similar proteins known as RNA-binding proteins. Interestingly, a number the MND-linked genes code for RNA-binding proteins, which points to disturbances in RNA-binding protein function as a disease-causing mechanism in MND.
Topic f. Disturbances in RNA metabolism.
In 2006, the discovery of the RNA-binding protein TDP-43 in protein aggregates inside spinal cord motor neurons suggested that TDP-43 might play a role in MND. Also, mutations in the genes encoding TDP-43 and other RNA-binding proteins (FUS and hnRNP A1) have been identified In MND patients. As their name suggests, RNA-binding proteins interact/bind to RNA. They regulate production, editing and breakdown of RNA and are known to interact with thousands of different RNA sequences. This means that even small changes in these proteins can have global effects on gene expression. In addition, RNA-binding proteins also control the levels of themselves and other RNA-binding proteins, meaning that the disruption of one can affect the others.
Mislocalization of RNA-binding proteins
RNA-binding proteins shuttle between the nucleus of the cell and the cytoplasm (see Figure 6 for a refresher!) and have specific functions in each area of the cell. One of the key pathologies seen in the motor neurons of MND patients and in cell and animal models of MND is the change in the location of RNA-binding proteins from the nucleus of the cell to the cytoplasm (example shown of FUS in Figure 6). The build-up of RNA-binding proteins can trap RNA and other proteins into aggregates and are toxic to the cell. As well as this, loss of RNA-binding proteins from the nucleus can disrupt the alternative splicing of RNAs and mRNAs. In a mouse model where levels of TDP-43 have been experimentally lowered, hundreds of splicing events in the brain were altered causing the loss of several RNAs and subsequently several proteins that are known to be important for neurons.
Figure 6. RNA-binding protein FUS (in green) becomes mislocalized away from the nucleus into the cell’s cytoplasm where it gets trapped and builds up into small and eventually large aggregates which can be seen in MND patients (adapted from Dormann & Haass 2011).
RNA-binding protein complexes and non-coding microRNAs
Both TDP-43 and FUS function as part of a larger protein structure that generates microRNAs, the small non-coding RNAs mentioned above. microRNAs are particularly important for maintaining the structure and function of the nerve terminal, where the motor neuron innervates the muscle. Thus, any changes to the level of microRNAs can be critical for motor neuron health and function.
The discovery of an expansion in the gene C9orf72
In 2011, while searching MND patient’s gene sequences for mutations, scientist identified a huge expansion in the DNA code of a gene of unknown function, which has been called C9orf72. In this gene, the DNA code repeats a short section of itself, GGGGCC. In a healthy person, this section is repeated anywhere between 2-23 times, but in an MND patient it can be repeated hundreds or thousands of times. In MND patients with the expansion, C9orf72 is seen as small, RNA aggregates (called foci) within the nucleus of neurons in the brain and spinal cord and these foci are proposed to be toxic. The RNA foci also trap RNA-binding proteins, leading to wide-spread changes in splicing. Another interesting thing about this repeat expansion is that is found in the non-coding region of the gene, thus it doesn’t code for a protein. However, the repeat expansion is capable of translating itself using an alternative translation process, generating a number of small proteins called DPRs. DPRs accumulate in the cytoplasm, block transport between the cytoplasm and the nucleus and are considered to be toxic to the cell.
Disease mechanisms in MND
As you can see from the disease mechanism blogs over the last few months there are a huge range of factors that contribute to disease progression in MND. Many of the concepts described are still theories developed by researchers, hypothesizing about what is causing the pathology that is observed. It is unknown if there is a single root cause of MND, or if each of the mechanisms described here enhance or trigger each other as the disease progresses. What we do know is that researchers have made huge advances in the last 10 years and that each discovery is another piece of the MND puzzle waiting to be solved.