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Understanding the neurobiology of the ageing brain
- 1. Understanding the neurobiology of the ageing brain Developing a synaptoneursome isolation protocol Dr. Declan King, University of Edinburgh, 2020, CC BY
- 2. Understanding healthy ageing • We live in an ageing society • By 2040, ~1 in 7 people in UK will be >75 • Cognitive decline most feared aspect of old age • Great clinical need to understand and address this • Q1: What do we currently know about the ageing brain?
- 3. What about dementia? • In dementia it is well established that synaptic degeneration is the strongest pathological correlate of cognitive decline • However in normal ageing – synapse loss increases with age; is region specific and also correlates with cognitive change • Very few studies have assessed synaptic changes in “normal ageing”
- 4. • Study brains of healthy, aged individuals • Q2: What technologies could we use to study brain tissue changes? • Use specific synaptic based technologies to highlight changes in brain that may play a role in cognitive decline Approach
- 5. • Analyse regional brain transcriptional and translational changes in synapses? • This multi-system approach will fully capture all aspects of cellular activity • Identifying factors that predispose individuals to a faster rate of cognitive decline is important for developing intervention /treatment strategies aimed at maintaining cognition and brain health into older age Method
- 6. • Focus on isolating synaptic enriched syanptoneurosome fractions from PM brain • Q3. What is a synaptoneursome? • Develop a method to isolate both protein and RNA-fractions from each brain sample Method development
- 7. • We need both protein and RNA fractions from the same tissue sample? • Solution: The method described in next slide allows for the isolation of both protein and RNA synaptic preparations that can be used for both downstream proteomic and RNA-seq analysis • The following animation shows how preparations can be isolated from two brain regions of interest Method problem
- 8. 8 Syn-p Syn-r TH TH Syn-p Syn-r TH-p TH-pTH-r TH-r Syn Syn Animation of protocol
- 9. • The proposed method described here allows for efficient isolation of both protein and RNA fractions from the same sample • Using data from both complementary systems (proteomic and RNA-seq) will provide datasets capable of discovering novel biological processes involved in healthy ageing Results/ Conclusions
- 10. Clues to Questions Q1: What do we currently know about the ageing brain? LBC Cohort and Cognitive ageing Q2: What technologies could we use to study brain tissue? Think about scanning technologies such as MRI / video, DTI, PET, ultrastructure EM Q3. What is a synaptoneursome? Synaptoneursome information
Editor's Notes
- Slide 1: In this CC BY licensed PowerPoint presentation, Dr. Declan King, who is a post-doc in the Centre for Discovery Brain Sciences at the Edinburgh Medical School: Biomedical Sciences’ at the University of Edinburgh gives a brief explanation of the ageing brain and briefly describes a useful method for isolating both protein and RNA preparations from enriched synaptic fractions from post-mortem brain tissue. The PowerPoint style lecture was made into an OER by Dr. King on April 2020 as part of the Digital Education module for the Postgraduate Certificate (PgCert) in Academic Practice at The University of Edinburgh.
- Slide 2: We live in an ageing society and by the year 2035, older adults will outnumber children under the age of 18 for the first time in history (U.S. News 2019). In the UK, nearly one in seven people will be > 75yrs old (Government Office of Science, 2016). This is obviously a good thing and is due to significant advances in modern medicine, however diseases and problems of older age will unavoidably result from our ageing population. Cognitive decline is the most feared aspect of old age, and therefore there’s already a great clinical need to understand and be able to address this. Q1: What do we currently know about the ageing brain? Links to clues at end of presentation.
- Slide 3: In dementia, it is well established that synaptic degeneration is the strongest pathological correlate of cognitive decline. However, early post-mortem studies have also revealed that low levels of Alzheimer’s-related pathology is often found in ”healthy” aged brains, and these present as Tau+ve tangles prominent in the hippocampus and amyloid+ve plaques common in the cortex. Indeed synaptic loss also increases with age, is region specific and correlates with cognitive change. Unfortunately, very few studies have assessed these synaptic changes in “normal ageing”. We therefore need a much greater understanding of the normal ageing process before we can identify pathological changes in diseases such as Alzheimer’s.
- Slide 4: To improve our understandings of normal ageing we need to study the brains of healthy aged individuals. Obviously we can’t do this in detail at a synaptic level whilst the person is living. Our approach will therefore focus on working with kindly donated post-mortem brains with specific emphasis on studying these brains at the synaptic level. Q2: What technologies could we use to study brain tissue changes? Links to clues at end of presentation.
- Slide 5: To address and monitor synaptic changes in the brain we will focus on regional brain transcriptional and translational changes at the synaptic level. This method encompasses two approaches – one focusing on transcriptional changes (which allows us to see which genes are up-or-down regulated relative to control state but this does not provide information about post-translational modifications that are critical for signaling) and the other on translational changes (which addresses limitation of transcriptomics alone). This is advantageous as no single data set fully captures all aspects of cellular activity in a giving experimental setting. Using the proposed multi-system approach based on complementary datasets jointly will allow us to discover novel biological processes associated with specific perturbations associated with ageing. Identifying novel factors that predispose individuals to a faster rate of cognitive decline is important for developing intervention /treatment strategies aimed at maintaining cognition and brain health into older age.
- Slide 6: So far we have introduced the concepts that understanding ageing at the synaptic level is crucial to broaden our understandings of healthy ageing. To expand our knowledge in this area we can focus our efforts on isolating synaptic enriched fractions also known as synaptoneurosomes from post-mortem brain samples. These samples will then be used to obtain both RNA for transcriptomic studies and protein for proteomics. Q3: What is a synaptoneurosome? Links to clues at end of presentation. In order to isolate both protein and RNA-fractions we need to develop a method that will allow both isolates to be obtained from a single tissue.
- Slide 7: Isolating either RNA or protein from a tissue sample is quite a standard procedure in the laboratory. Incorporating this into a synaptoneurosome islolation protocol is a little more difficult. And isolating two fractions (both RNA and protein) adds another layer of complexity to the method.
- Slide 8: This animation was designed to briefly describe how we addressed these issues and successfully developed a protocol to isolate both protein and RNA fractions from a brain sample. The animation starts displaying a cartoon of a human brain which is divided into two regions of interest, highlighted in red. As we mentioned earlier synaptic loss can be region specific so looking at two regions or more is advantageous. From this sample we can make a total homogenate (TH) which is then passed through a filter (standard size) that is then split into three fractions. One to create TH-p for protein, one to create TH-r for RNA and the remainder will be further filtered (through a smaller specialized filter) to produce synaptoneurosomes fractions for both protein (Syn-p) and RNA (Syn-r) downstream isolations. This protocol therefore allows us to successfully obtain multiple synaptoneurosome fractions for further downstream investigations to be carried out.
- Slide 9: The animation demonstates a method that allows for efficient isolation of both protein and RNA fractions from the same brain tissue sample. Using data from both complementary systems (proteomic and RNA-seq) will provide datasets capable of discovering novel biological processes involved in healthy ageing and cognition and therefore will broaden our understandings of this understudied area.
- Slide 10: Links provided here to scientific publications, images and videos.