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Tag: Intracellular

  • Study reveals novel approach to promote efficient uptake of ASOs into cancer cells

    Study reveals novel approach to promote efficient uptake of ASOs into cancer cells

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    Antisense oligonucleotides (ASOs) are next-generation drugs that can treat disease by blocking the transfer of harmful messages from our genes. In people with cancer, ASOs have the potential to block messages that encourage the growth and spread of the tumor. However, ASOs aren’t used for treating cancer yet. They must first get delivered inside cancer cells, but the cancer cells won’t let them in.

    Finding an effective ASO delivery system is a major challenge. Cancer cells have gatekeeper molecules that stop unwanted substances from entering. Although investigators have tried many ways of getting ASOs past the gatekeepers, success has been limited.

    Now, in a study recently published in the journal Nucleic Acids Research, researchers from Osaka University have discovered a way to deliver ASOs to their targets inside cancer cells. The team synthesized a new compound, named L687, which opens specific calcium permeable channels on the surface of cancer cells. When the calcium flows into cells through the open channels it tells the cells to let in the ASOs.

    We discovered that we could selectively activate the TRPC3/C6 calcium permeable channels with the activator L687. We then found that combination treatment with L687 and ASO promoted efficient uptake of ASO into cancer cells during laboratory tests and tumor cells inside the mouse. As a result, target gene activity was suppressed and ASO efficacy was enhanced.”


    Hiroto Kohashi, Lead Author

    Until now, ASOs have mainly been used to treat incurable diseases and had to be delivered into the liver or spinal fluid. According to the Osaka team’s research, L687 is an effective drug delivery system that may extend the benefits of ASO treatment to other parts of the body.

    “We hope that the results of our research will lead to significant progress in the development and delivery of ASOs and similar gene-targeting drugs for treating cancer,” says senior author Masahito Shimojo.

    The team believes that L687 could be a particularly effective way of delivering ASO therapy to lung or prostate cancers. These cancers have many TRPC3/C6 calcium permeable channels that can be opened by L687, potentially revealing new targets for these next-generation therapies.

    Source:

    Journal reference:

    Kohashi, H., et al. (2024) A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides. Nucleic Acids Research. doi.org/10.1093/nar/gkae245.

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  • Research identifies how leukemia develops resistance to first line treatments

    Research identifies how leukemia develops resistance to first line treatments

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    Relapses in a common form of leukemia may be preventable following new research which has identified how the cancer develops resistance to first line treatments.

    New research published in iScience by researchers from the University of Birmingham, the Institute of Cancer Research (ICR), Newcastle University, the Princess Maxima Centre of Pediatric oncology and the University of Virginia identified changes in a mutated form of acute myeloid leukemia (AML) samples from patients who relapsed after receiving FLT3 inhibitor treatment.

    The team found that the resistant cancer had up-regulated multiple other signalling pathways to overcome the drug’s action, and that the genetic change was able to be replicated in lab tests.

    These experiments revealed that by targeting RAS family proteins, using a small molecule inhibitor developed from a chemical library screen using the paratope of an inhibitory intracellular antibody by Terry Rabbitts’ team at the Weatherall Institute of Molecular Medicine University of Oxford and the ICR, increased signalling no longer rescued the cells from cell death.

    The team identified that the transcription factors AP-1 and RUNX1 were at the heart of mediating drug resistance. The two factors cooperate and bind to their target genes together, but only in the presence of growth factor signalling. The drugs targeting FLT3 rewire the cell, resulting in the upregulation of other signalling pathway associated genes, which then restored AP-1 and RUNX1 binding. Drugging RAS, which is a key component in multiple signalling pathways, prevented this restoration of RUNX1 binding, and therefore signalling from growth factors no longer rescued the cancer cells from death.

    Professor Constanze Bonifer from the Institute of Cancer and Genomic Sciences at the University of Birmingham, who has just taken up a position at the University of Melbourne, and is one of the senior authors of the paper said:

    “The pharmaceutical industry had high hopes that drugs targeting aberrant growth factor receptors such as the FLT3-ITD would prevent people from relapse. However, cancer cells are smart, and rewire their growth control machinery to use other growth factors present in the body. Targeting RAS family members prevents the cancer from rewiring and using different signalling pathways to escape cell death.”

    Targeting RAS blocks rewiring

    The small molecule inhibitors used to target RAS in this study were developed using intracellular antibody technology. This technology involves screening a large number of antibody fragments to identify those which bind to the target protein in cells and prevent their protein-protein interactions. Small molecule inhibitors are can be screened from chemical libraries that interact with the parts of the target protein where these antibody fragments bind (the paratope). Due to the unparalleled natural specificity of these antibody fragments, this technology (called Antibody derived or Abd technology) can be used to target difficult to drug proteins and identify new parts of the protein which can be targeted to prevent protein-protein interactions.

    Professor Terry Rabbitts from the Institute of Cancer Research who developed these drugs said:

    “The strength of the Antibody-derived technology approach is that intracellular antibodies can selected to essentially any protein. In turn, their specific binding sites can be employed to select chemical compounds for drug discovery against hard to drug proteins. Mutant RAS was considered undruggable, but the Abd technology facilitated the development of the RAS-binding compounds used in the current study of cancer cell re-wiring. Abd technology will allow development of a new generation of drugs to hard-to-drug and intrinsically disordered proteins.“

    AML with a FLT3-ITD mutation occurs in nearly 30% of all patients and is a highly aggressive disease with a poor prognosis. This genetic change causes the expression of a mutant growth factor receptor which is always active and therefore cancer cells expressing it grow uncontrollably. While inhibitors which specifically target the FLT3 protein are now in use in the clinic, patients treated with these inhibitors frequently relapse.

    This work was funded by Leukaemia Research UK, the Medical Research Council, Blood Cancer Research UK, the Royal Society, the Wellcome and Cancer Research UK. The first author, Daniel Coleman is a John Goldman Fellow of Leukaemia UK.

    Source:

    Journal reference:

    Daniel J.L., et al. (2024). Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1. iScience. doi.org/10.1016/j.isci.2024.109576.

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  • Rare variants in the YKT6 gene cause new neurological disorder, study finds

    Rare variants in the YKT6 gene cause new neurological disorder, study finds

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    A recent collaborative study has discovered rare variants in the YKT6 gene as the cause of a new neurological disorder characterized by developmental delays along with severe progressive liver disease and a potential risk for liver cancer. The study, published in Genetics in Medicine, was led by Dr. Hugo Bellen, Distinguished Service Professor at Baylor College of Medicine and Principal Investigator at the Jan and Dan Duncan Neurological Research Institute (Duncan NRI) at Texas Children’s Hospital, and Dr. Wendy Chung, the Chief of the Department of Pediatrics at Boston Children’s Hospital. 

    “It is known that the YKT6 gene plays important roles in many intracellular vesicular trafficking events in the cells but this is the first time it has been linked to a genetically inherited disorder,” Dr. Bellen said. “This study, using patient samples and fruit flies, provides a solid experimental foundation for future studies to better understand this new disease and to develop therapies.”

    YKT6 gene variants disrupt brain development and sometimes, liver function

    In collaboration with Dr. Mythily Ganapathi at Columbia University Irving Medical Center, Drs. Paula Hertel and Davut Pehlivan at Texas Children’s Hospital and Dr. James Lupski at Baylor College of Medicine, and by using the GeneMatcher tool and Baylor Genetics clinical diagnostics laboratory, this team of researchers and clinicians found three unrelated individuals with missense (analogous to misspellings in a word) variants in both copies of the YKT6 gene. 

    All three individuals had early onset of disease (four to six months of age) with failure to thrive. Two of them had an identical missense variant because of which the tyrosine amino acid at position 185 was changed to cysteine (Tyr185Cys). On the other hand, the third child carried a variant that caused the same amino acid change but in a different location (Tyr64Cys) of the YKT6 protein. Interestingly, in addition to developmental delays and neurological defects which were observed in all three children, only the two individuals with the Tyr185Cys variant had liver dysfunction and a potential risk for developing liver cancer.

    Interestingly, both individuals with the Tyr185Cys variant belong to the Syrian/Saint Thomas Christians of Kerala, India, a group currently estimated to be comprised of about 5 million individuals worldwide. Our genetic lineage analysis suggests this variant likely originated from a common ancestor before the community split.”


    Dr. Mythily Ganapathi at Columbia University Irving Medical Center 

    YKT6 gene variants impair autophagy

    To assess how YKT6 variants result in the observed disease pathologies, the Bellen team studied the fruit fly version of this gene which is quite similar to its human counterpart.

    “We found that the fly version of this protein is expressed in the fat body and brain which are analogous to the human liver and central nervous system respectively,” Dr. Mengqi Ma, one of the first authors and a postdoctoral fellow in the Bellen lab, said. “Moreover, fly strains with loss of function mutations in this gene were lethal.”

    Further, they observed that Ykt6 mutant flies expressing the normal fly version of the Ykt6 gene flies had an average lifespan. However, transgenic flies expressing the fly versions of the disease variants were less effective in restoring lifespan and other symptoms. While Ykt6 mutant flies expressing Tyr65Cys (equal to human Tyr64Cys) had normal lifespan and locomotion, those expressing Tyr186Cys (equal to human Tyr185Cys) had severely reduced lifespan and locomotor defects. “Our results showed that the fly Ykt6 Tyr186Cys cause more severe defects than Tyr65Cys,” Dr. Ma added, “suggesting that the corresponding human YKT6 Tyr185Cys is a more severe variant than Tyr64Cys.”

    To understand why these variants behaved differently, they delved deeper into their biology.

    YKT6 belongs to the SNARE family of proteins that regulate the flow of protein traffic to various compartments within the cell. In mammalian cells, YKT6 mediates the fusion of two cellular organelles – the autophagosomes and lysosomes to form autolysosomes – within which ‘used’ cellular proteins, lipids, and other molecules are degraded and recycled back for future use. This process called autophagy is critical for the proper function and health of the cells.

    The team found that the loss of fly Ykt6 led to an abnormal accumulation of proteins involved in autophagosome formation and autophagic cargo receptor, indicating a block in the autophagy pathway. Further studies revealed that just like lethality and other defects, fly Tyr186Cys (equal to human Tyr185Cys) was less efficient in reverting the symptoms compared to a normal copy of the Ykt6 gene. Furthermore, they observed that while autophagy initiation was normal, the steps involved in the breakdown of cellular waste were impaired in the absence of Ykt6.

    “Based on our findings, we recommend the YKT6 gene as a candidate for carrier screening in the Syrian/Saint Thomas Christian community of Kerala,” Dr. Mythily Ganapathi said.

    “Our work suggests children diagnosed with YKT6 liver disease will also need to be screened for hepatocellular carcinoma,” Dr. Paula Hertel said.

    “In summary, we have discovered YKT6 variants as the cause of a novel developmental disorder affecting brain function and in certain cases, also liver function, providing us valuable insights into a new genetic disease. However, additional studies with more patients will be needed to precisely understand the pathogenesis and to identify potential therapeutic targets for this condition,” Dr. Bellen added.

    Source:

    Texas Children’s Hospital

    Journal reference:

    Ma, M., et al. (2024). Homozygous missense variants in YKT6 result in loss of function and are associated with developmental delay, with or without severe infantile liver disease and risk for hepatocellular carcinoma. Genetics in Medicine. doi.org/10.1016/j.gim.2024.101125.

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  • Newly discovered adhesion GPCR mayo controls midgut development in Drosophila

    Newly discovered adhesion GPCR mayo controls midgut development in Drosophila

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    Adhesion GPCRs belong to the large family of G protein-coupled receptors (GPCRs). There are about 700 variants in humans, which are responsible for sensory impressions, hormonal cycles, controlling the cardiovascular system and more. GPCRs translate stimuli that hit a cell from outside into an intracellular biochemical signal.

    The use of the fruit fly as a model animal allows researchers in this field to gain a deep understanding of human diseases, because the animals are genetically very similar to humans. Scientists estimate that around 75 per cent of the genes involved in human diseases are also found in fruit flies.

    The research team at the Rudolf Schönheimer Institute of Biochemistry at the Faculty of Medicine has discovered three new adhesion GPCR genes in the genome of the fruit fly, or Drosophila. One of them is very old in evolutionary terms, and has been called mayo. In the current publication, the Leipzig scientists demonstrate the functions of this adhesion GPCR using the fruit fly as a living model. “We found that mayo affects the development of the midgut in Drosophila by promoting the growth of enterocytes, the epithelial cells of the intestinal mucosa,” says Dr Beatriz Blanco-Redondo, corresponding author of the study.

    In their publication, the Leipzig scientists also show that the loss of mayo in the intestine accelerates the heart rate of the animals and that they develop dangerous palpitations. The results indicate that the functions of the intestine and heart are linked through the role of mayo in the proliferation of enterocytes. These regulate and secondarily govern ion uptake, systemic potassium levels and heart rate.

    The researchers at Leipzig University studied animals in which the mayo gene had been switched off. They found that these “knockout animals” displayed elongated guts. A similar genetic picture was observed after overexpression of another adhesion GPCR in mouse intestinal cells, resulting in a mega-intestine. The study shows that adhesion GPCRs are also involved in the development of the gastrointestinal tract in other species and that these phenomena may be relevant in humans.

    We are only at the beginning of this research project. The main goal is to identify the signaling pathway in which the adhesion GPCR mayo is involved in order to find out how it controls intestinal development.” 


    Tobias Langenhan, Professor of General Biochemistry at the Rudolf Schönheimer Institute and corresponding author of the study

    Source:

    Journal reference:

    Contreras, F. V., et al. (2024). The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila. Cell Reports. doi.org/10.1016/j.celrep.2023.113640.

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  • DNA origami vaccine DoriVac paves way for personalized cancer immunotherapy

    DNA origami vaccine DoriVac paves way for personalized cancer immunotherapy

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    Therapeutic cancer vaccines are a form of immunotherapy in the making that could not only destroy cancer cells in patients, but keep a cancer from coming back and spreading. Multiple therapeutic cancer vaccines are being studied in clinical trials, but despite their promise, they are not routinely used yet by clinical oncologists to treat their patients. 

    The central ingredient of therapeutic cancer vaccines is antigens, which are preferentially produced or newly produced (neoantigens) by tumor cells and enable a patient’s immune system to search and destroy the cancerous cells. In most cases, those antigens cannot act alone and need the help of adjuvant molecules that trigger a general alarm signal in immune cells known as antigen-presenting cells (APCs). APCs internalize both antigen and adjuvant molecules and present the antigens to different types of T cells. Those T cells then launch an immediate attack against the tumor, or preserve a longer-lasting memory of the tumor for future defense.

    A cancer vaccine’s effectiveness depends on the level and duration of the “alarm” its adjuvants can ring in APCs. Previously, researchers found that delivering adjuvant and antigen molecules to APCs simultaneously using nanostructures like DNA origami can increase APC activation. However, none of these approaches systematically investigated how the number and nanoscale arrangement of adjuvant molecules affect downstream tumor-directed immunity. 

    Now, a research team at the Wyss Institute at Harvard University, Dana-Farber Cancer Institute (DFCI), Harvard Medical School (HMS), and Korea Institute of Science and Technology (KIST) has created a DNA origami platform called DoriVac, whose core component is a self-assembling square block-shaped nanostructure. To one face of the square block, defined numbers of adjuvant molecules can be attached in highly tunable, nanoprecise patterns, while the opposite face can bind tumor antigens. The study found that molecules of an adjuvant known as CpG spaced exactly 3.5 nanometers apart from each other resulted in the most beneficial stimulation of APCs that induced a highly-desirable profile of T cells, including those that kill cancer cells (cytotoxic T cells), those that cause beneficial inflammation (Th-1 polarized T cells), and those that provide a long-term immune memory of the tumor (memory T cells). DoriVac vaccines enabled tumor-bearing mice to better control the growth of tumors and to survive significantly longer than control mice. Importantly, the effects of DoriVac also synergized with those of immune checkpoint inhibitors, which are a highly successful immunotherapy that is already widely used in the clinic. The findings are published in Nature Nanotechnology.

    “DoriVac’s DNA origami vaccine technology merges different nanotechnological capabilities that we have developed over the years with an ever-deepening knowledge about cancer-suppressing immune processes,” said Wyss Core Faculty member William Shih, Ph.D., who led the Wyss Institute team together with first-author Yang (Claire) Zeng, M.D., Ph.D. “We envision that in the future, antigens identified in patients with different types of tumors could be quickly loaded onto prefabricated, adjuvant-containing DNA origami to enable highly effective personalized cancer vaccines that can be paired with FDA-approved checkpoint inhibitors in combination therapies.”

    Shih is also a Professor at HMS and DFCI’s Department of Cancer Biology and, as some of the other authors, a member of the NIH-funded cross-institutional “Immuno-engineering to Improve Immunotherapy” (i3) Center based at the Wyss. 

    DNA origami rationale

    The CpG adjuvant is a synthetic strand of DNA made up of repeated CpG nucleotide motifs that mimic the genetic material from immune cell-invading bacterial and viral pathogens. Like its natural counterparts, CpG adjuvants bind to a “danger receptor” called TLR9 in immune cells, which in turn induces an inflammatory (innate) immune response that works in concert with the antigen-induced (adaptive) immune response. 

    “We knew from previous work that to trigger strong inflammatory responses, TLR9 receptors need to dimerize and aggregate into multimeric complexes binding to multiple CpG molecules. The nanoscale distances between the CpG-binding domains in effective TLR9 assemblies revealed by structural analysis fell right into the range of what we hypothesized we could mirror with DNA origami structures presenting precisely spaced CpG molecules,” explained Zeng, who was an Instructor in Medicine at the time of the study and now is a senior scientist at DFCI and Harvard Medical School (HMS). In addition to Shih, Zeng was also mentored on the project by senior authors Ju Hee Ryu, Ph.D., a Principal Researcher at KIST, and Wyss Founding Core Faculty member David Mooney, Ph.D., who also is Professor at Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), and one of the i3 Center’s Principal Investigators. 

    Zeng and the team fabricated DoriVac vaccines in which different numbers of CpG strands were spaced at 2.5, 3.5, 5, or 7 nanometers apart from each other on one face of the square block, and a model antigen was attached to the opposite face. They protected their structures from being degraded in the body using a chemical modification method that Shih’s group had developed earlier. When internalized by different types of APCs, including dendritic cells (DCs), which orchestrate tumor-directed T cell responses, the DoriVac vaccines improved the uptake of antigens compared to controls consisting of free antigen molecules. A CpG spacing of 3.5 nanometers produced the strongest and most beneficial responses in APCs, and significantly outperformed a control vaccine containing only free CpG molecules. “We were excited to find that the DoriVac vaccine preferentially induced an immune activation state that supports anti-tumor immunity, which is what researchers generally want to see in a good vaccine,” said Zeng. 

    Besides spacing, the numbers of CpG molecules in DoriVac vaccines also mattered. The team tested vaccines containing between 12 to 63 optimally spaced CpG molecules and found that 18 CpG molecules provided the best APC activation. This meant that their approach can also help limit the dosage of CpG molecules and thus minimize commonly observed toxic side effects observed with adjuvants.

    Gained in (tumor) translation

    Importantly, these in vitro trends translated to in vivo mouse tumor models. When prophylactically injected under the skin of mice, DoriVac vaccines accumulated in the closest lymph nodes where they stimulated DCs. A vaccine loaded with a melanoma antigen prevented the growth of subsequently injected aggressive melanoma cells. While all control animals had succumbed to the cancer by day 42 of the experiment, DoriVac-protected animals all were alive. DoriVac vaccines also inhibited tumor growth in mice in which the formation of melanoma tumors was already underway, with a 3.5 nanometer spacing of 18 CpG molecules again providing maximum effects on DC and T cells, and the strongest reduction in tumor growth.

    Next, the team asked whether DoriVac vaccines could also boost immune responses produced by small “neoantigens” emerging in melanoma tumors. Neoantigens are ideal targets because they are exclusively made by tumor cells. However, they often are not very immunogenic themselves, which make highly effective adjuvants an important component in neoantigen vaccines. A DoriVac vaccine customized with four neoantigens enabled the researchers to significantly suppress growth of the tumor in mice that produced the neoantigens.

    Finally, the researchers asked whether DoriVac could synergize with immune checkpoint therapy, which reactivates T cells that have been silenced in tumors. In mice, the two therapies combined resulted in the total regression of melanoma tumors, and prevented them from growing back when the animals were exposed to the same tumor cells again four months later. The animals had built up an immune memory of the tumor. The team obtained a similar vaccination efficiency in a mouse lymphoma model.

    We think that DoriVac’s value for determining a sweet spot in adjuvant delivery and enhancing the delivery and effects of coupled antigens can pave the way to more effective clinical cancer vaccines for use in patients with a variety of cancers.”


    Yang (Claire) Zeng, M.D., Ph.D., First Author

    The team is currently translating the DoriVac platform toward its clinical application, which is supported by the study’s assessment of vaccine distribution and vaccine-directed antibodies in mice, as well as cytokines produced by immune cells in response to the vaccines in vivo. 

    “The DoriVac platform is our first example of how our pursuit of what we call Molecular Robotics – synthetic bioinspired molecules that have programmable shape and function – can lead to entirely new and powerful therapeutics. This technology opens an entirely new path for development of designer vaccines with properties tailored to meet specific clinical challenges. We hope to see its rapid translation into the clinic,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at SEAS.

    Other authors on the study are Olivia Young, Christopher Wintersinger, Frances Anastassacos, James MacDonald, Giorgia Isinelli, Maxence Dellacherie, Miguel Sobral, Haiqing Bai, Amanda Graveline, Andyna Vernet, Melinda Sanchez, Kathleen Mulligan, Youngjin Choi, Thomas Ferrante, Derin Keskin, Geoffrey Fell, Donna Neuberg, Cathrine Wu, and Ick Chan Kwon. The study was funded by the Wyss Institute’s Validation Project and Institute Project programs, Claudia Adams Barr Program at DFCI, Korean Fund for Regenerative Medicine (award #21A0504L1), Intramural Research Program of KIST (award #2E30840), and National Institutes of Health (under the i3 Center supporting U54 grant (award #CA244726-01).

    Source:

    Wyss Institute at Harvard University

    Journal reference:

    Zeng, Y. C., et al. (2024). Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination. Nature Nanotechnology. doi.org/10.1038/s41565-024-01615-3.

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  • How cell types and location influence Parkinson’s disease

    How cell types and location influence Parkinson’s disease

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    In a recent study published in Cell Reports, researchers conducted a single-cell spatial transcriptome analysis on murine brain expression in age and disease using a Parkinson’s disease (PD) transgenic model, focusing on dopaminergic neurons (DA) spanning 29 cell types.

    Study: Single-cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, aging, and disease. Image Credit: solarseven/Shutterstock.comStudy: Single-cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, aging, and disease. Image Credit: solarseven/Shutterstock.com

    Background

    The spatially structured brain contains various cells, each with a distinct purpose. PD is a neurodegenerative condition characterized by DA loss and alpha-synuclein buildup due to overexpression via locus multiplication.

    Single-cell ribonucleic acid sequencing (RNS-seq) has improved the knowledge of cell-based expression in organs such as the brain, but current methods fail to attain high-throughput resolution. Fluorescent in situ hybridization (FISH) technologies provide higher sensitivity at an individual level.

    About the study

    In the present study, researchers performed single-cell spatial transcriptomic and translatomic profiling of DA to find markers for healthy and aged cells.

    The researchers crossed Rosa26fsTRAP::DATIREScre (DAT-TRAP) mice with SNCA-OVX mice to enable DA messenger RNA (mRNA) capture in a PD model.

    The mice were aged to 18 months to assess the influence of human ⍺-synuclein overexpression and aging on dopaminergic neuron genetic expression and study the effect of healthy and Parkinsonian aging.

    The researchers created a single-cell-level spatial transcriptomic map of gene expression in the adult mouse brain and a high-fidelity translatome-level profile of DA neuron expression.

    They analyzed stereo-seq arrays with special transcript maps. They converted the transcript expression maps to segmented individual cells, identifying 29 types of cells, including astrocytes and inhibitory cortical neurons, in 18 brain slices.

    The team filtered the segmented cells by transcriptome size and complexity and analyzed spatially distinct cortex, hippocampus, and thalamus populations. They also examined the ventral midbrain and striatum to enrich transcripts confined to the cell body and putative axon.

    They used data from gene enrichment and genome-wide association studies (GWAS) to identify potential disease-causing genes.

    They evaluated each cell’s location and compared DA neuronal gene expression in various cells.

    The researchers examined enhanced green fluorescent protein (eGFP)-tagged ribosomes in DAT-expressing cells and confirmed eGFP colocalization with tyrosine hydroxylase (TH), a DA neuronal marker.

    They split cells from stereo-seq brain slices, filtering them based on detected genes, and performed Uniform Manifold Approximation and Projection (UMAP) analysis.

    They also performed short- and long-read RNA sequencing of the translating mRNA collected by TRAP and used stereo-sequencing and TRAP data to rank prospective genes for inquiry into sporadic PD.

    The researchers demonstrated specific enrichment of DA marker genes and depletion of marker genes of other neighboring cell types in DAT-TRAP mRNA.

    They confirmed specific calcium-sensing receptor (CASR) protein expression in mouse ventral midbrain neurons and investigated CASR expression in human-induced pluripotent stem cell-derived DA neurons. They also examined age-related gene variations in cells revealed by stereo-seq.

    Results

    The team studied PD in young and aged brains to identify genes having spatially varying expression in dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and particular markers such as copine-7 (Cpne7) and Solute carrier family 10 member 4 (Slc10a4) genes.

    They also detected splice variants unique to DA. They demonstrated ways of using TRAP and stereo-sequencing expression specificity measurements to identify potentially relevant genes from GWAS areas, indicating that CASR regulates intracellular DA neuronal calcium.

    The findings demonstrated substantia nigra-specific DA neuronal loss and increased microglial activation with age. They highlighted aging- and disease-associated genetic alterations in various cells, including dopaminergic neurons, across many PD-related pathways.

    Stereo-seq detected expression alterations caused by aging and illness across different cell types, loss of nigral DA neurons, and the neuroinflammatory expansion of microglia.

    Pathway enrichment research revealed that various biological processes were altered, including axon ensheathment, synaptic transmission modulation, intracellular calcium ion homeostasis, and catecholamine secretion control.

    The team extracted 355,307 transcriptomes of high quality with spatial coordinate details from 18 murine brains, identifying a total of 14,494 genes.

    They observed synaptosomal-associated protein, 25kDa (Snap25) expression, and structured localization in places like the thalamus or hippocampus differentiated neurons from glia.

    They also examined neuronal cells in the CA1 region, CA3 region, subiculum, and dentate gyrus in the hippocampus region or gamma-aminobutyric acid (GABA)-releasing nuclei in the midbrain.  

    Marker expression enabled oligodendrocyte, astrocyte, microglia, and erythrocyte identification. In DAT-TRAP samples, classical markers of dopaminergic neurons showed significant enrichment, whereas those of other cell types decreased in the ventral midbrain.

    Conclusion

    Overall, the study identified 29 unique brain cell types by examining variations in spatial gene expression linked to aging and illness. The stereo-seq data indicated differences in transcript use across more than a thousand genes.

    There were 817 occurrences of alternative splicing, suggesting that more genes were being translated than gene-level count data showed. The study also discovered an age-dependent drop in SN DA neuron cell number, which supports earlier findings.

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