• Discuss how the authors used a number of technologies to conduct this study.
 
This study used snRNAseq to look at differential transcriptional activity between thousands of frontal cortex cell nuclei collected post-mortem from 48 participants in some AD studies. My questions with this technology are how well it reflects cytoplasmic RNA populations and ultimately protein populations, and whether it can capture which splice variants of genes are being produced. The selection process also seems possibly to underrepresent or exclude more transcriptionally quiet cell types.
 
Other technologies have been used here for verification of RNAseq results; FACS and RT-PCR were used to verify transcriptional differences in neuronal cells. Immunohistochemistry and fluorescence microscopy were used to visualise expression differences, and verify protein-level changes, in potential marker genes of AD pathology. They used data from previous GWAS associations with AD to verify that sets of DEGs observed are known to be associated with AD.
 
 • What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?
 
The authors did include age and gender on top of clustering of cell types. Age was controlled for as much as possible in subject selection, but was analysed in order to control for age differences when looking for AD pathology differences. It was found that age is correlated with steeper AD pathology decline, and gender was used to find an amazing dimorphism in how AD progresses in males and females.
 
48 is probably too low a number of subjects to look at racial or complex lifestyle differences in the data with very much power. Ethnicity differences in subjects aren't discussed, and that samples are nearly age matched and from the Religious Orders Study will control somewhat for lifestyle factors.

Ultimately taking all of these factors into full consideration would allow researchers to separate their effects from AD pathology, increasing the resolution of the study, and also allowing them to look at them as correlates of AD pathology.
 
 • Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA...
 
This week's paper is advocating looking at differential gene expression of all expressed genes in single cells in a research setting, whereas last week's paper was looking at the importance of tissue and transcript context in interpreting a variant. Whilst quite different in their messages, they have in common the message that extremely specific context for genetic variation is important in the function of that variation. This must have been somewhat evident even from early genetics, simply from observing the wide variety of morphology and function of human cells that develop ultimately using the same genetic code.
 
 • Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.
 
This technique is a fantastic step forward in pinpointing where the early stages of disease occur, and hence directing more specific research using appropriate models and gene targets in the investigation of the molecular mechanism of the disease. This technique potentially allows resolution of changes specific to minority sub-populations of cells that would otherwise be drowned out by bulk tissue measurements. Properly understanding a disease mechanism is a very good route to finding effective treatments and cures.

The study demonstrated the use of Single-nucleas RNA sequencing ( droplet based) in the analysis of AD. snRNA seq was used to look for different transcriptional activity  from the prefrontal cortex of 48 individuals with varying degrees of Alzheimer’s disease pathology. Across six major brain cell types, snRNA sequencing identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival.
Other technologies have been also used in this study such as FACS and RT-PCR. These technologies where able to verify transcriptional activities within neuronal cells. Transcriptional activities where differentiated between cells through immunohistochemistry and fluorescence microscopy  which enabled the study to visualise these different expressions and protein-level changes between neuronal cells. GWAS was also used to verify DEG's that are known to be linked with AD.

What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?

The study had demonstrated that age, gender, genetic ethnicity and lifestyle consideration actually cause specific cells to be differentially expressed. Differential expression of genes in different cells give us wider view about the specific type of disease we are looking at as these expressions tend to be different in different cells and this expression difference between cells will tell us more about the disease we are investigating.  Referring to the journal paper In males, there was increased pathology correlated with a global transcriptional activation in oligodendrocytes. Yet, global shifts in transcription in oligodendrocytes were not observed in females. In females, the increased pathology was correlated with a global downregulation of gene activity in both excitatory neurons and inhibitory neurons. This actually shows us that  depending on age and gender for example is vital in order to know how the disease actually function in different sexes  as different cells tend to express gene in different times and in different way in different sexes. Moreover, the study demonstrated that with age transcriptional expression differs. It was found that major transcriptional changes appeared early in pathological progression. Therefore, having such in depth knowledge on age,gender etc.. while diagnosing a disease is crucial for effective diagnosis and innovating specific effective treatments to patients with different age and gender as a disease like AD can express different genes differently in different ages and sexes and one way of knowing this is by snRNA sequencing technologies which was demonstrated in the Journal Club paper. 

Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA...

There is certainly a long way for early interpretation.  Our cells do actually contain same DNA but different cells expresses different genes differently, although they have same DNA but different cells have different functions and that function tends to be affected when these different cells express genes differently. The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, giving it its unique properties.

Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.

I think that i had also answered this question while discussing question 2.

BW 

Omar Nimri 

Here are my answers for this week’s journal club.

This study used droplet-based single-nucleus RNA sequencing (snRNA-seq) to profile over 80k cortical transcriptomes across 48 individuals (24 individuals had high levels of beta-amyloid + other pathological markers of Azhleimer’s disease (AD) and 24 had no or low beta-amyloid burden). Prior to this, immunohistochemistry was used to confirm the pathological status of the samples and bright-field and high-res confocal microscopy was used to confirm no apparent damage to the nuclei. 

Quantitative PCR with reverse transcription (RT-qPCR) and high-quality bulk RNA sequencing were used to validate the differential expressed genes (DEGs) that were found using snRNA-seq. Genome-wide association studies (GWAS) were used to link genetic risk with coordinated gene activity by quantifying the overrepresentation of genes. 

They also looked at the white matter hyperintensities from MRI data to examine the possible gender-bias in relation to white-matter pathology. 

I think the biggest finding was the difference between transcriptome expression in males and females providing support and the evidence for sexual dimorphism in AD. I found their interpretation interesting: either females have greater disease burden OR greater resilience to the disease meaning that despite strong alterations within the transcriptome, the cognitive decline remains the same between the sexes. 

With collection of data such as age, gender, genetic ethnicity etc, the authors were able to look at different groups and compare the transcriptome activity between them. This also means that they were able to account for certain modifiable risk factors (such as lifestyle etc).

I must admit, I found the last week’s journal paper quite convoluted and difficult to read, however, I think the main message from it was that the context of the tissue and the transcript was highly important in variant interpretation. This week’s paper looked at differential gene expression in single cells in a disease context. As others have commented, these approaches are a long way away from the early interpretations of the fact that all our cells contain the same DNA. We do have the same DNA, but it isn’t expressed in the same way throughout the cells due to a myriad of reasons: epigenetic marks, certain noncoding DNA functions such as enhancers being tissue specific etc. 

I read this paper with delight and I thought that the study itself was very innovative. Looking at the transcriptome allowed the authors to look at the cell-type-specific gene expression which enabled them to look closer to the disease phenotype and therefore the pathophysiology. As others already mentioned, this technique also enabled the authors to look at the early stages of disease and the changes that happen there. This is paramount as it might possibly mean that targeted therapies and preventative measures will be developed in response to better understanding of the early pathophysiology of the AD.

—

Thank you for an interesting journal club yesterday. Personally, I find the history of Alzheimer’s disease fascinating. The original case report (on one patient) presented by Alois Alzheimer at a regional meeting was promptly included by his colleague, Kraepelin, in a new psychiatry textbook with the term “Alzheimer’s disease”. A few more cases were confirmed by Alzheimer in the later years, but the term AD (back in 1910s) was largely for early-onset dementia. There was a definitive separation between senile dementia (once seen as part of normal ageing) and AD. It wasn’t until mid 1950s that the separation blurred and now I think it’s completely gone, which I think is problematic. The diagnosis of AD has to be confirmed histologically and with brain tissue that means post-mortem. Yet, clinically, this diagnosis has been given very frequently. It seems that patients with similar phenotypes (or in some cases quite differing phenotypes) have been lumped together. This is unhelpful as it is hindering research and treatment development. There was an interesting news article on the discovery of a new type of dementia called LATE and how many AD patients have been misdiagnosed (https://www.bbc.co.uk/news/health-48092570) here is the original report: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536849/. I think the more studies into the transcriptome and proteome (perhaps looking into less invasive biomarkers for early onset AD) are being done, the better our understanding will be, so I am very much excited to see what the advancing technology such as RNA-seq will offer researchers and patients. 

If interested (and have time), here is an article on the historical context of AD: https://academic.oup.com/brain/article/138/12/3816/415650. 

Viktorija

In this study, using different methods like Immunohistochemistry and droplet-based single-nucleus RNA-seq, they identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. Further techniques include RNA in situ hybridization and Quantitative PCR with reverse transcription, all these techniques helped reaching the results which indicate that all major cell types are affected at the transcriptional level by AD pathology,

Disease-associated changes appeared early in pathological progression and were highly cell-type specific, whereas genes upregulated at late stages were common across cell types and primarily involved in the global stress response.

scientist observed that cells isolated from female individuals were overrepresented in many of the AD-pathology-associated cell subpopulations and that responses between sexes were contrasting and qualitatively distinct, however, the degree of pathophysiological and cognitive decline remains similar in females and males.

comparing the levels of gene expression in cells isolated from AD-pathology versus no-pathology individuals by cell type (Methods), and identifying 1,031 unique differentially expressed genes.

Alzheimer’s disease (AD) is a slowly progressing neurodegenerative disorder that starts with mild memory loss and culminates in severe, AD involves interactions between neuron and glia; in support of this, transcriptomic and epigenomic analyses were performed and it reveals downregulation of neuronal functions and upregulation of innate immune responses in AD brains, Cell-type-specific regulatory complexity may therefore need to be taken into account in the planning of therapeutic interventions in AD.

 
Thank you,
Aljowhara alageel

 

Droplet-based single-nucleus RNA sequencing was used to assess changes in the transcriptome, and thus changes in gene expression, in 48 individuals with Alzheimer’s disease of varying degrees of severity. This technique and others, such as immunohistochemistry and RT-PCR, were used to investigate the transcriptome of affected individuals to further understand Alzheimer’s disease and characterise its pathology.

Further knowledge into the significance of the factors mentioned in Alzheimer’s disease pathology could be gained, helping to better understand the disease and determine risk factors for individuals. Lifestyle considerations could be particularly valuable for individuals who may found to be at a higher risk of the disease due to other factors, as changing their lifestyle could potentially delay onset or reduce severity. 

I imagine a lot has changed since the early interpretations. The last two journal clubs have further highlighted the extreme complexity of gene expression, both spatially and temporally. The fact all cells in an individual contain the same DNA does not mean the cells contain the same type or amount of protein, even within a tissue. This needs to be considered when analysing variation involved in disease.

This type of investigation can be used to interrogate the pathology of Alzheimer’s disease, on both molecular and cellular levels. It can be used to determine differences in gene expression in affected individuals. It enables key features which might be of significance to be highlighted, such as their conclusion that myelination may have a key role. The more that is understood about the disease, the more likely it is that an idea for an effective intervention or treatment will be identified. 

Including background information about the individuals lifestyle could give a better indication of risk factors associated with the development and progression of AD. This may help identify the impact of specific lifestyle factors such as diet on a AD diagnosis and provide patients with the opportunity to change elements of their lifestyle to have a more positive impact on the disease outcome.  

These two studies demonstrate how far our understanding has come, the recognition of the complexity of our genome and we are only just beginning to understand how much we have to learn about genome regulation by the non-coding regions. We understand now that the difference between cell types is due to expression of different genes at different times.

·       What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?

The authors found significant differences between gene expression and cell type dependent upon 🤬, additional information would have helped to highlight any additional correlation between molecular changes and the above mentioned traits. This could potentially lead to differential treatment for AD in the era of “personalised medicine”.
 
·       Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA...

 ·        Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.

The authors profiled selected tissue for pathological status using Immunohistochemistry.

Single-cell RNA sequencing was used to enable the study of individual cells involved in Alzheimer’s disease. Through snRNA- sequencing, they aimed to understand the cellular-level view of Alzheimer’s disease; shared and specific gene expression between cell types, 🤬-based, age, transcripts expressions. 

Reverse Transcriptase Quantitative PCR for validation in cells nuclei positive for the neuronal marker for some genes tested.

RNA-in situ hybridization to verify the data gotten from snRNA-seq.

Differences in gene expression between the cell types involved in Alzheimer's and regulatory complexity. Variations in expression among the sexes with over-representation among the females in many of the pathology associated cells.

It shows that regardless of the high similarity at the DNA level, the variation as a result of transcriptional and translational processes control how genes are expressed.

This type of studies provides better understanding at the cellular level, factors influencing a disease and findings can be applied for developing treatment means. 

 

Single cell RNA sequencing was the primary method used to determine cell type-specific pathology in AD. Further techniques used included immunohistochemistry and confocal imaging to detect AD-related cellular pathology. RT-qPCR was used to confirm up or down-regulation of the six genes tested. 

 

 

 

Most cases of AD do not show  a clear familial inheritance pattern, suggesting an interaction of genetic predisposition and potential environmental/ external triggers. Interestingly, the authors found evidence or sexual dimorphism, with more pronouced early pathology in female cells. Further information on patient background and lifestyle factors may improve understanding of mechanisms driving AD. 

 

 

 

This study once again confirms how important cell-specific differences can be in disease. Especially in disease-modeling, it is important to investigate a wide range of cells and tissues, in order to get a more complete understanding of pathology. New research appears to be moving towards a more complex view of disease mechanisms even within similar tissues. 

 

 

This study was particularly interesting, as it confirmed the importance of investigating disease mechanisms at a single-cell level. Crucially, results suggest a highly variable early disease state, and role of myelination in AD pathology. This may provide clues for novel therapeutic targets. 

 

Best wishes,

Johanna

The analyses conducted were highly complex due to changes in cell composition during neurodegeneration and the opposing protective and damaging molecular processes.

Methods used: FACS, qRTPCR, RNA Seq, snRNA Seq, in situ hybrididation, 

 

·  What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?

The finding of sexual dimorphism in AD was interesting and suggests that including and controlling for a wider range of variables, as suggested above, could identify further significant changes in expression.

 

·  Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA...

Knowledge of regulatory mechanisms such as epigenetics and post transcriptional regulation has grown significantly. We are beginning to understand the role of these mechanisms in disease, making it important to understand this variation to improve our ability to manage and treat disease. Just considering tissue level resolution masks differences across and within cell types – especially in less abundant cell types. This study demonstrates that expression changes and directionality of these changes can depend on cell type proving the importance of single cell over tissue wide measurements.

 

·   Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.

The study found that transcriptional changes happen before development of severe pathology and therefore there is potential for development of screening and early interventions in AD.

They also showed how complex the transcriptional response is and further work can help to separate neuroprotective mechanisms from pathogenic mechanisms which will also further improve our ability to develop AD treatments.

snRNA-seq profiling of tissue from the pre-frontal cortex in two groups of individuals to see the genes expressed. RT-qPCR was done to see the extent of expression of the genes identified and studied. Immunohistochemistry for β-amyloid confirmed the pathological status of the samples. GWAS was done to see the risk the genes had on the disease. 

It was interesting to see that the authors found that the cells obtained from female individuals were found to be associated with the AD-associated cells, despite the phenotype of the disease being similar in both sexes. The authors, however, did acknowledge this bias in the molecular processes and suggested that a larger, follow-up study should be conducted to ascertain the relationship between transcriptional and pathological differences between sexes. 

We all know how extra-genetic factors can influence the phenotypes of disease. Therefore, in order to capture a truly realistic picture of the disease, other factors such as age, ethnicity, etc., should be considered. Saying that it is understandable that to combine all of these factors concurrently into one study would be asking for too much. 

It is understandable why the authors decided to focus on a single cell type from a specific region; the subject of interest here is AD, and the PFC is a region of the brain which is affected in AD. 

All of our cells may contain the same genome, but as we have learnt in the past, through a wide range of mechanism, specific cells express specific proteins. Otherwise, we would all be just one big goop of the same cells with the same proteins expressed in the same amount. 

Different technologies have been used to conduct this study. First of all. snRNAseq to analyse the transcriptional activity of more than 80000 post mortem frontal cortex cell nuclei from 48 different individuals, half of whom had high levels of AD pathological markers including beta-amyloid, and half who had low levels of AD pathological markers. Then, different methods including FACs and RT-PCR had been used them to verify the expressed genes and evaluate the differences in transcriptomes level and gene expression. Furthermore, GWAS study was used to evaluate the pathology and severity associated with the different genes. Immunohistochemistry and high- resolution confocal microscopy were used to confirm the pathological state of the disease.

·       What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?

In addition, the researchers have noticed that the cell associated with AD pathology were more prevalent in female cells which may predict a higher occurrence of this disease in female patients. Moreover, the datasets collected helped identify the risk factors for AD, which can potentially help patients at risk to alter their lifestyle, such as quit smoking, to eventually delay the onset of AD. 

• Discuss how the authors used a number of technologies to conduct this study. 

There were three primary methodologies used by the authors of these studies. One was the level of gene expression was analysed using RT-qPCR RNA sequencing between those with AD and those without. Gwas was also used to link gene activity to the genetic risk of an individual having these genes. MRI data was also used to to analyse white matter to negate risk of a gender bias.

• What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found? 

They already observed a higher commonality of female cells associated with disease which possibly suggests that other associations with factors such as age and ethnicity may occur. If included they may have been able to associate the disease with other factors that are part of lifestyle.

• Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA... 

Whilst most DNA within cells is the same the activation and expression of DNA within the cells greatly differs depending on the tissue and cell type, though using a single cell may not completely detect the DNA even in most cells of the body due to somatic mosaicism.

• Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.

This paper highlighted how examining the transcriptome can be a viable way to investigate disease but in my opinion to get a better understanding of the way in which AD can be evaluated for the intervention and treatment of the disease it must be evaluated in multiple ways to give a complete understanding of how novel treatments for the disease can be made in the future. Such other progressions could be through the novel discoveries through PET scanning. 

 

·        Discuss how the authors used a number of technologies to conduct this study

They were able to use clinical data, genetic, epigenomic, transcriptomic, proteomic, and metabolomic to produce a full picture of cellular activity. As discussed in the previous weeks, the study of omics is defined by a specific tissue at a certain point in time. Thus, by collecting data from these different sources, the authors of the study were able to develop and timeline of cell activity, divide the cells by cluster of activity, and explain differences in expression at biological levels. It presents a complex picture of different omics fitted together to better understand a disease as complicated as Alzheimer’s dementia.

·        What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?

As Alzheimer’s is a progressive neurodegenerative disorder, the disease evolves over time and space. Thus, by taking into consideration these other factors, it would have been better possible to create a timeline of cell activity between populations thus tracking its evolution and changes depending on these epidemiological factors to better adapt analysis and treatment research to the patient.

·        Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA...

As we better understand epigenomics, transcriptomics, metabolomics, proteomics and the vastly expanding omics domains, we realise that there are so many different factors that go into the functioning of each cell. And these factors change wildly between cells and within cell tissues to allow for all the varied process for the functioning of the whole organism.

·         Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.

This type of investigation uncovers the reality of the complex interactions of different cells within the same tissue. Thus, by understanding this heterogeneity in the development of disease we can better understand resistance to treatments, better map out the inner workings of complex diseases such as cancer, and possibly develop better targeted therapies. Furthermore, it can allow for better experimental studies and interpretation when making cell colonies and testing out reactions and treatments.

https://cellandbioscience.biomedcentral.com/articles/10.1186/s13578-019-0314-y#:~:text=Compared%20with%20traditional%20sequencing%20technology,cells%2C%20and%20delineating%20cell%20maps. 

·       What further knowledge might the authors have gained by including age, gender, genetic ethnicity and lifestyle considerations on top of the clusters of cell-types they found?

·       Last week's journal article tackled tissue specificity and this week is advocating the use of single cells from within the same tissue. Discuss how far this is from early interpretations of the fact that all our cells contain the same DNA...

·        Reflect on the strength of this type of investigation in understanding disease and our ability to intervene for treatment or cure.