Cases of neurodegeneration and dementia are predicted to rise with the aging population, yet conditions such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) have no cure. Developing effective therapeutic strategies requires a deeper understanding of the different cell types that are involved in the disease etiology of these neurodegenerative disorders, including how different cell types interact with one another. Just like anything, context matters!
One way of obtaining these insights is through the use of multiplexed spatial profiling techniques. Such methods have proven invaluable for oncology research, where they enable the mapping of proteins and transcripts in the tumor microenvironment (TME) to help determine how cellular dynamics influence clinical outcomes. However, while multiplexed spatial profiling of the brain holds similar promise for neurodegenerative research—such as through characterizing cells surrounding AD pathological hallmarks like amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs)—it has yet to reach its full potential.
Multiplexed spatial profiling of an adult human Alzheimer’s brain demonstrating a significant increase in phosphorylated tau-containing neurofibrillary tangles and Aβ plaques. Image from the poster Spatial Proteomic Analysis of Alzheimer’s Disease Human Brain using Multiplexed Imaging.
One hurdle uniquely faced by neurodegeneration researchers stems from the nature of the sample material, which is especially difficult to obtain for neurodegenerative research and presents several challenges for multiplexed spatial profiling. First, although human brain tissue permits direct interrogation of the disease state, unlike a mouse or cell-based model, most human brain samples are formalin-fixed and paraffin-embedded (FFPE), while murine samples are usually frozen. This means that the same antibodies cannot always be used for staining both sample types, which makes performing comparisons difficult. In addition, the use of FFPE tissue has historically limited researchers to performing a series of singleplex chromogenic measurements, which is both time-consuming and labor-intensive. Autofluorescence due to lipofuscin accumulation, and a lack of validated antibodies that are specific and sensitive enough to ensure accurate staining, introduce further issues.
Here, we focus on some of the immunohistochemical (IHC) staining methods, with particular attention to FFPE-processed tissue, that are available for multiplexed detection of proteins in brain tissue. We also look at how the Cell DIVE Multiplexed Imaging Solution from Leica Microsystems, in combination with our IF/IHC-validated antibodies, allows for spatially and molecularly characterizing AD brain samples.
Why is spatial profiling important for understanding the human brain?
Although single-cell analysis techniques such as flow cytometry and single-cell RNA sequencing (scRNA-seq) are widely used for characterizing biological samples, they cannot reveal the location of different cell types within the native tissue environment. This can lead to valuable information being missed, since cellular interactions often underlie disease etiology.
For example, in the brain, microglia and astrocytes are essential for functions including maintaining cellular homeostasis, the immune response, removal of cellular debris, isolating or repairing damage caused by toxic insults, and maintenance of the blood-brain barrier. However, their roles are incredibly complex, and these cell types can exhibit dual natures sometimes described as akin to Jekyll and Hyde-like personalities. Under normal conditions, microglia and astrocytes protect and support neural health, but in response to disease or injury, they can cause harm through chronic inflammation, neurodegeneration, or inhibition of recovery. Moreover, glia (particularly microglia), are genetically linked to neurodegenerative diseases, suggesting that these cells may play both direct and indirect roles in disease etiology. Interrogating the spatially and temporarily distinct activation patterns exhibited by these glial cells, and identifying the molecular signatures that reflect distinct functional populations, are likely to uncover new approaches for treating neurological disorders such as AD and PD.1
Spatial profiling techniques are therefore vital for investigating cellular dynamics. They allow researchers to move beyond asking basic questions like “How many of each cell type is present?” to more complex analyses such as “How might cells adapt under disease conditions to directly slow or accelerate disease progression?" and “How do pathological hallmarks, like Aβ plaques and NFTs, drive cellular responses?"
Ultimately, methods that provide answers to increasingly complex questions can help guide the development of effective therapeutic strategies.
What are the advantages of spatial proteomics?
A major advantage of spatial proteomics over other forms of spatial profiling, such as spatial transcriptomics and genomics, is that it offers insights into cellular function. This is because, in contrast to mRNA and DNA, proteins serve to directly control cellular behaviors. Although mRNA signatures have been identified for various disease states, they don't necessarily correlate with protein expression, meaning that careful validation is required to correctly interpret transcriptomics data.
Spatial proteomics also allows for studying post-translational modifications (PTMs), which are often dysregulated in neurodegenerative disease. For example, the microtubule-associated protein tau is known to be hyperphosphorylated in AD brains, leading to the formation of cytotoxic tau aggregates in NFTs. In contrast, a lesser known PTM of tau, termed O-GlcNAcylation (O-GlcNAc), is thought to attenuate tau hyperphosphorylation and could thus provide therapeutic benefit for AD patients.2
Antibody Selection for Studying Brain Tissue Samples
When generating spatial proteomics data with precious brain tissue samples, it is critically important to select antibodies that have been validated for the application in which they will be used. A common mistake is to assume that an antibody that is validated for IHC staining of FFPE tissue will also work with frozen tissue samples, which often may not be the case. When working with FFPE tissues, antibodies must be rigorously validated for use with this sample type because harsh processing steps, such as formaldehyde fixation, deparaffinization, and antigen retrieval, can lead to epitope damage.
At CST, we follow stringent validation principles when developing and testing our antibodies. This means generating validation data on an application- and protocol-specific basis, since we learned early on in our 25 years of developing high-quality antibodies that performance in one application does not guarantee performance in another. We offer over 800 antibodies that are validated for use with FFPE tissue, in addition to a large portfolio of antibodies that have been validated on fresh frozen tissues.
The below table includes some of the key antibodies we offer for AD biomarkers and other neurological disorders, and includes information about the tissue type they are validated for:
You can also explore our full catalog of antibodies validated for IHC with either FFPE tissue or frozen tissue.
Spatial Proteomics Methods
Once you've selected the FFPE-compatible antibodies, you can use them in a growing list of methods that enable spatial proteomics. The following methods using FFPE brain tissue differ in terms of complexity and the number of markers they are able to measure. Methods that are supported by CST include the following:
- Leica Cell DIVE: Using CST antibodies validated for the Cell DIVE, FFPE tissue is stained with sequential rounds of up to four fluorescently labeled antibodies, and a gentle chemical dye inactivation process is used between cycles.
- Traditional IHC with direct detection: Fluorophore-labeled primary antibodies are used for direct detection, which involves fewer protocol steps than indirect detection and avoids the risk of secondary antibody cross-reactivity.
- Multiplexing with multiple species: Primary antibodies from different host species (e.g., mouse, rabbit, goat) are used in combination with species-specific secondary antibodies, each conjugated to a unique fluorophore, allowing simultaneous detection of three to five targets in a single staining round.
- SignalStar™ Multiplex IHC: SignalStar technology amplifies multiple biomarkers simultaneously in FFPE tissue with high sensitivity and specificity. Oligonucleotide-conjugated antibodies simultaneously bind up to eight analytes, then complementary oligonucleotides with fluorescent dyes amplify the signal to enable target detection.
The research poster below details how the Leica Cell DIVE can be used to spatially define populations of cells, such as astrocytes, microglia, and neurons, in the context of Aβ and tau expression in the AD brain, as well as to computationally examine synaptic processes. Learn more and download the poster:
You can also find more information on multiplexing methods in the following blogs:
- Pros and Cons of Different Multiplexing Techniques for IHC and IF
- CST Antibodies Validated on the Leica Microsystems Cell DIVE Multiplexed Imaging Solution
Future Challenges for Neurodegeneration Research
As researchers’ understanding of neurodegeneration continues to grow, methods that enable spatial profiling of the brain must evolve to keep pace. While antibodies are already demonstrating broad utility for describing the cellular dynamics of AD and other neurodegenerative disorders, future challenges will include increasing the number of proteins that can simultaneously be detected to tens or even hundreds of targets, generating antibody products for newly discovered biomarkers, and developing ever more sophisticated tools and technologies for performing multi-omics analyses of cells in the context of disease.
Select References
- Wu Y, Eisel ULM. Microglia-Astrocyte Communication in Alzheimer's Disease. J Alzheimers Dis. 2023;95(3):785-803. doi:10.3233/JAD-230199
- Zhu Y, Shan X, Yuzwa SA, Vocadlo DJ. The emerging link between O-GlcNAc and Alzheimer disease. J Biol Chem. 2014;289(50):34472-34481. doi:10.1074/jbc.R114.601351
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