CTSS Mouse

What is CTSS and why is it studied in mouse models?

CTSS (Cathepsin S) is a eukaryotic cysteine protease predominantly expressed in professional antigen presenting cells (APCs). It plays crucial roles in various physiological and pathological processes, including antigen processing and presentation via MHC class II, inflammation regulation, and immune response modulation . Mouse models are invaluable for studying CTSS because they allow researchers to investigate its specific functions in different disease contexts through genetic manipulation (knockout, knockdown, or overexpression). Recent studies have revealed CTSS's involvement in autoimmune diseases, cardiovascular disorders, and neurodegenerative conditions, making it an important therapeutic target . Unlike previous assumptions that CTSS is exclusively expressed in microglia, newer research has demonstrated that neurons also express CTSS, with expression levels positively correlating with age .

What types of CTSS mouse models are currently available for research?

Several CTSS mouse models have been developed for different research applications:

• CTSS-deficient mice (CTSS-/-): Genetically engineered mice with complete deletion of the CTSS gene, allowing researchers to study the consequences of CTSS absence in various physiological and pathological processes .

• Wild-type mice (CTSS+/+): Used as controls in comparative studies with CTSS-deficient mice .

• AAV-mediated CTSS overexpression models: Adeno-associated virus vectors can be used to overexpress CTSS in specific brain regions like the hippocampus in young mice, enabling the study of CTSS's effects on spatial learning and memory .

• AAV-mediated CTSS knockdown models: Similarly, AAV can be used to knockdown CTSS expression in specific brain regions of aging mice .

• CD25KO mice: An autoimmune mouse model of Sjögren's disease driven by autoreactive T cells, used to study the effects of CTSS inhibition on disease progression .

• APP/PS1 mice: Alzheimer's disease model mice used to study the role of CTSS in neurodegenerative processes .
  These diverse models allow researchers to investigate CTSS functions in different physiological contexts and disease states.

How does CTSS expression change with aging in mouse models?

RNA sequencing and protein analyses have revealed that CTSS expression is significantly upregulated in the hippocampus of aging mice compared to young mice . This upregulation occurs in both neurons and microglia, contradicting earlier assumptions that CTSS is expressed exclusively in microglia . Principal Component Analysis (PCA) of transcriptional patterns between young and aging mice shows substantial differences, with CTSS emerging as one of the significantly upregulated genes in aging mice .
The increased CTSS expression in aging mice correlates with cognitive deficits, particularly impaired recognition abilities. Similar age-related increases in CTSS concentration have been observed in human serum samples, with elderly individuals showing significantly higher CTSS levels than younger individuals . Additionally, aging mice exhibit increased beta-amyloid 1-42 (Aβ1-42) in the hippocampus, which partially colocalizes with CTSS, suggesting potential involvement of CTSS in Aβ1-42 transition or clearance mechanisms .

What are reliable methods for detecting CTSS activity in mouse samples?

A significant challenge in CTSS research has been the development of reliable detection methods for mouse samples. A novel approach using the fluorogen substrate Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2 has been adapted for mouse samples . This method offers several advantages:

• Specificity: The substrate specifically detects CTSS activities in mouse antigen presenting cells.

• Accessibility: The protocol requires standard laboratory equipment without specialized instrumentation.

• Quantitative results: Unlike some previous methods, this approach delivers reliable quantitative measurements of CTSS activity.

• Reproducibility: The modified protocol is designed to be easy, quick, and reproducible across different laboratory settings .
  This method is particularly valuable since most basic CTSS research is performed in mice, filling a methodological gap in the field and enabling quantitative CTSS activity detection in almost any laboratory setting .

How can CTSS be effectively inhibited in mouse models for experimental studies?

CTSS inhibition can be achieved through both pharmacological and genetic approaches:

• Pharmacological inhibition:

  • Selective CTSS inhibitors like LY3000328 have demonstrated efficacy in rescuing AD-related pathological features in APP/PS1 mice .

  • CTSS inhibitor supplementation in the diet has been shown to improve autoimmune signs in CD25KO mice, leading to better cornea sensitivity, improved lacrimal gland inflammatory scores, and increased lifespan (approximately 30% longer) .

• Genetic inhibition:

  • CTSS-deficient mice (CTSS-/-) provide a complete genetic knockout model .

  • AAV-mediated CTSS knockdown in specific brain regions (e.g., hippocampus) has been successful in aging mice, with significant reduction in CTSS fluorescence intensity .
    When designing CTSS inhibition experiments, researchers should consider the timing, tissue specificity, and potential compensatory mechanisms that might affect experimental outcomes. For example, inhibiting CTSS in CD25KO mice resulted in a significant decrease in the frequency of CD4+ immune cells and a significant increase in the frequency of CD8+ immune cells in the draining lymph nodes .

What protocols are recommended for CTSS overexpression in mouse models?

For CTSS overexpression in specific brain regions, the following protocol has proven effective:

• AAV vector preparation: Use adeno-associated virus carrying the CTSS gene under a neuron-specific promoter.

• Stereotactic injection: Perform targeted injection into the desired brain region (e.g., hippocampus) in young mice (approximately 2 months old).

• Recovery period: Allow approximately 15 days for recovery and robust expression.

• Verification of overexpression: Confirm successful overexpression through:

  • mRNA expression analysis (qPCR)

  • Fluorescence intensity measurement

  • Verification of expression in specific subregions (CA1, CA3, dentate gyrus) .
    After CTSS overexpression in the hippocampus of young mice, behavioral tests like the Morris Water Maze can be used to assess effects on spatial learning and memory. Successful overexpression typically results in longer escape latencies during training, less time spent in the target quadrant, increased probe time, and decreased crossing number, indicating impaired spatial learning and memory abilities .

How do CTSS mouse models enhance our understanding of autoimmune diseases?

CTSS mouse models have provided critical insights into the pathogenesis and potential treatment of several autoimmune diseases:

• Sjögren's Syndrome:

  • CD25KO mice serve as a model of Sjögren's disease driven by autoreactive T cells.

  • CTSS inhibition in these mice improves cornea sensitivity and lacrimal gland inflammatory scores.

  • CTSS inhibitor treatment significantly decreases the frequency of CD4+ immune cells and Th1/Th17 cells in lacrimal glands and draining lymph nodes.

  • Gene expression analysis shows decreased levels of Ifng, Ciita, and Casp8 mRNA in CTSS inhibitor-treated mice .

• Multiple Sclerosis:

  • CTSS activity regulation has been implicated in the pathogenesis of multiple sclerosis.

  • Mouse models help elucidate the role of CTSS in antigen processing and presentation, a critical process in MS pathology .

• Inflammatory Bowel Disease (IBD):

  • CTSS activity detection in mouse models provides insights into IBD mechanisms .
    These models demonstrate that CTSS inhibition represents a promising therapeutic approach for autoimmune diseases affecting the eye and lacrimal gland, as evidenced by improved clinical parameters and extended lifespan in treated mice .

What role does CTSS play in stress-related thrombosis as revealed by mouse models?

Research using wild-type (CTSS+/+) and CTSS-deficient (CTSS-/-) mice exposed to immobilization stress has revealed critical roles of CTSS in stress-related thrombosis:

• Stress-induced thrombosis enhancement:

  • In wild-type mice, stress significantly increases the lengths and weights of thrombi formed after iron chloride (FeCl3)-induced carotid thrombosis surgery.

  • Stress also increases arterial tissue CTSS expression .

• Prothrombotic marker modulation:

  • Stress alters levels of key factors including PAI-1 (plasminogen activation inhibitor-1), ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 13 motifs), and vWF (von Willebrand factor) .

• Protection in CTSS-deficient mice:

  • CTSS-deficient mice show reduced arterial thrombus area and endothelial loss compared to stressed wild-type mice.

  • They exhibit decreased levels of pro-inflammatory and pro-thrombotic markers (PAI-1, vWF, TNF-α, IL-1β, TLR-4) and increased levels of protective factors (ADAMTS13, SOD-1/2, eNOS) .

• Pharmacological relevance:

  • Pharmacological inhibition of CTSS demonstrates vasculoprotective effects, suggesting potential therapeutic applications .
    These findings establish CTSS as a key modulator of stress-related thrombosis, operating through mechanisms involving inflammation, oxidative stress, and vascular endothelial function.

How are CTSS mouse models advancing neuroinflammation and Alzheimer's disease research?

CTSS mouse models have provided valuable insights into neuroinflammation and Alzheimer's disease (AD):

• Age-related neuroinflammation:

  • RNA sequencing of young and aging mouse hippocampus revealed CTSS upregulation in aging, correlating with increased inflammation.

  • Transcriptome and FACS analysis showed that neuronal CTSS overexpression aggravates brain inflammatory milieu, activating microglia toward an M1 pro-inflammatory phenotype .

• Neuron-microglia communication:

  • Neuronal CTSS activates the CX3CL1-CX3CR1 axis and JAK2-STAT3 pathway.

  • This represents a novel mechanism of neuron-microglia "crosstalk" mediated by CTSS .

• Alzheimer's disease connections:

  • Elevated CTSS expression has been observed in multiple brain regions of AD patients, including the hippocampus.

  • In aging mice, CTSS partially colocalizes with Aβ1-42, suggesting involvement in amyloid processing or clearance.

  • CTSS selective inhibitor (LY3000328) rescues AD-related pathological features in APP/PS1 mice .

• Cognitive function:

  • CTSS overexpression in hippocampal neurons impairs spatial learning and memory in young mice.

  • Conversely, CTSS knockdown in hippocampal neurons rescues spatial learning and memory deficits in aging mice .
    These findings establish neuronal CTSS as a potential biomarker for aging and a therapeutic target for AD, highlighting the value of mouse models in elucidating the relationship between CTSS, neuroinflammation, and cognitive function.

How does CTSS deletion affect inflammatory and cellular protection pathways?

CTSS deletion produces complex effects on multiple inflammatory and cellular protection pathways:

• Inflammatory marker modulation:

  • CTSS-deficient mice show significantly decreased levels of pro-inflammatory markers including:

    • TNF-α (tumor necrosis factor-alpha)

    • Interleukin-1β

    • Toll-like receptor-4

    • ICAM-1 (intercellular adhesion molecule-1)

    • MCP-1 (monocyte chemoattractant protein-1)

    • MyD88 (myeloid differentiation primary response 88)

    • MMP-2/-9 (matrix metalloproteinase-2/-9)

• Apoptotic pathway modulation:

  • CTSS deletion reduces levels of apoptosis markers including:

    • Cleaved-caspase 3

    • Cytochrome c

    • p16 INK4A

• Oxidative stress reduction:

  • Decreased levels of oxidative stress markers:

    • gp91 phox

    • p22 phox

• Enhanced protective signaling:

  • Increased levels of protective factors:

    • SOD (superoxide dismutase)-1/-2

    • eNOS (endothelial NO synthase)

    • p-Akt (phospho-protein kinase B)

    • Bcl-2 (B-cell lymphoma-2)

    • p-GSK3α/β (phospho-glycogen synthase kinases alpha and beta)

    • p-Erk1/2 (phospho-extracellular signal-regulated kinase 1 and 2)
      These findings demonstrate that CTSS deletion has broad protective effects against inflammation, oxidative stress, and apoptosis, potentially through modulation of multiple signaling pathways that influence cell survival and inflammatory responses.

What are the molecular mechanisms of CTSS-mediated neuron-microglia communication?

Recent research has revealed novel insights into how neuronal CTSS mediates communication with microglia:

How do age-related changes in CTSS expression impact molecular pathways and cognition?

Age-related increases in CTSS expression trigger a cascade of molecular events that ultimately affect cognitive function:

• Transcriptional changes with aging:

  • RNA sequencing of young vs. aging mouse hippocampus reveals 204 differentially expressed genes (113 upregulated, 91 downregulated).

  • CTSS is among the significantly upregulated genes in aging mice .

• Gene Ontology enrichment patterns:

  • Upregulated processes in aging include:

    • Antigen processing and presentation via MHC class II

    • Innate immune response regulation

    • Learning and memory processes

    • Recognition mechanisms

  • Downregulated processes include:

    • Extracellular matrix organization

    • Cell-substrate adhesion

    • Nervous system regulation

• Cognitive impact:

  • CTSS overexpression in hippocampal neurons of young mice impairs spatial learning and memory, as measured by:

    • Longer escape latencies during training in the Morris Water Maze

    • Less time spent in the target quadrant

    • Increased probe time

    • Decreased crossing number

• Reversal of age-related deficits:

  • CTSS knockdown in hippocampal neurons of aging mice rescues spatial learning and memory deficits .

• Amyloid interaction:

  • CTSS partially colocalizes with Aβ1-42 in aging mouse hippocampus, suggesting involvement in amyloid processing or clearance .
    These findings establish a direct link between age-related increases in CTSS expression and cognitive decline, mediated through neuroinflammatory processes and altered neuron-microglia communication, with important implications for understanding and potentially treating age-related cognitive disorders.

What are common technical challenges in CTSS activity detection and how can they be addressed?

Researchers face several technical challenges when measuring CTSS activity in mouse samples:

• Specificity issues:

  • Challenge: Many traditional substrates for cathepsin activity lack specificity for CTSS.

  • Solution: Use the fluorogen substrate Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, which shows high specificity for CTSS in mouse samples .

• Quantification difficulties:

  • Challenge: Many methods fail to deliver reliable quantitative results.

  • Solution: The modified protocol using fluorogen substrate provides quantitative measurements using standard laboratory equipment .

• Technical requirements:

  • Challenge: Some detection methods require specialized equipment not available in standard laboratories.

  • Solution: The recommended protocol requires only equipment commonly available in standard laboratories .

• Sample preparation:

  • Challenge: Proper sample preparation is critical for reliable activity measurements.

  • Solution: Follow standardized protocols for tissue homogenization and protein extraction to maintain enzyme activity while preventing degradation .

• pH sensitivity:

  • Challenge: CTSS activity is pH-dependent, with optimal activity at acidic pH.

  • Solution: Ensure proper buffer conditions during activity assays to maintain the appropriate pH for CTSS activity .
    By addressing these technical challenges, researchers can obtain reliable and quantitative measurements of CTSS activity in mouse samples, facilitating more robust and reproducible studies.

How can researchers distinguish between neuronal and microglial sources of CTSS?

Distinguishing between neuronal and microglial sources of CTSS requires specific methodological approaches:

• Co-immunostaining techniques:

  • Perform double immunofluorescence staining using:

    • CTSS antibody combined with neuronal markers (e.g., NeuN, MAP2)

    • CTSS antibody combined with microglial markers (e.g., Iba1, CD11b)

  • This allows visualization and quantification of CTSS expression in different cell types .

• Cell-specific genetic manipulation:

  • Use neuron-specific or microglia-specific promoters to drive CTSS overexpression or knockdown.

  • For example, AAV vectors with neuron-specific promoters can be used to manipulate CTSS expression exclusively in neurons .

• Cell isolation and sorting:

  • Fluorescence-activated cell sorting (FACS) can be used to separate neuronal and microglial populations.

  • RNA or protein can then be extracted from these purified populations for CTSS expression analysis .

• In vitro validation:

  • Primary neuronal and microglial cultures can be used to validate CTSS expression and function in different cell types.

  • In vitro studies have shown that CTSS silencing and overexpression affect apoptosis of human umbilical vein endothelial cells under stress conditions .
    These techniques enable researchers to determine the relative contribution of neuronal versus microglial CTSS to observed phenotypes, providing a more nuanced understanding of CTSS functions in different cell types and disease contexts.

What factors should be considered when interpreting behavioral results in CTSS mouse models?

When interpreting behavioral results in CTSS mouse models, researchers should consider several potential confounding factors:

• Motor function assessment:

  • Challenge: Impaired motor function could be misinterpreted as cognitive deficits.

  • Solution: Include control tests like the open field test to assess exploratory activity and rule out motor deficits as confounding factors. For example, CTSS overexpression in the hippocampus did not affect exploratory activity in the open field test .

• Anxiety levels:

  • Challenge: Anxiety-like behaviors can affect performance in cognitive tests.

  • Solution: Evaluate anxiety behaviors using measures such as time spent in the center of the open field. Studies have shown that CTSS overexpression does not induce anxiety-like behaviors .

• Age considerations:

  • Challenge: Age strongly influences both CTSS expression and cognitive performance.

  • Solution: Carefully match experimental and control groups for age, and consider age as a variable when interpreting results .

• Regional specificity:

  • Challenge: CTSS effects may vary across brain regions.

  • Solution: When manipulating CTSS expression, verify that the manipulation is limited to the intended brain region (e.g., hippocampus) and does not spread to other regions .

• Sex differences:

  • Challenge: CTSS expression and its effects may differ between male and female mice.

  • Solution: Consider sex as a biological variable and analyze data separately for male and female mice when possible. By addressing these potential confounding factors, researchers can more confidently attribute behavioral changes to CTSS manipulation rather than to other variables, strengthening the validity of their findings.