SYNJ2BP Human

What is the basic structure and localization of SYNJ2BP in human cells?

SYNJ2BP is a 145 amino acid protein localized to the outer mitochondrial membrane with a cytosolic PDZ domain that functions as a cellular signaling hub. The protein contains specific domains that facilitate protein-protein interactions, particularly with ER-associated proteins. Methodologically, subcellular fractionation coupled with western blotting can confirm its mitochondrial localization, while immunofluorescence microscopy using antibodies against both SYNJ2BP and mitochondrial markers (such as TOM20) provides visual confirmation of its outer mitochondrial membrane positioning . Proximity ligation assays (PLA) can further validate its interactions with specific binding partners in situ.

What are the primary binding partners of SYNJ2BP and how can these interactions be studied?

Based on STRING interaction network analysis, SYNJ2BP's primary binding partners include RRBP1 (Ribosome binding protein 1, score: 0.989), SYNJ2 (Synaptojanin-2, score: 0.921), ABITRAM (Protein Abitram, score: 0.826), ACVR2A (Activin receptor type-2A, score: 0.821), and RALA (Ras-related protein Ral-A, score: 0.782) . The interaction with RRBP1 is particularly important as it mediates mitochondria-ER tethering. These interactions can be studied through co-immunoprecipitation followed by mass spectrometry, which has successfully identified SYNJ2BP interactors in mitochondrial fractions . For in situ validation, proximity ligation assays can detect protein interactions occurring at distances less than 40 nm, making it ideal for confirming the spatial relationships between SYNJ2BP and its partners in intact cells .

How does SYNJ2BP expression change under cellular stress conditions?

SYNJ2BP expression increases in response to acute cellular stress. Studies in induced pluripotent stem cell (iPSC)-derived motor neurons have shown that sublethal doses of hydrogen peroxide (H₂O₂) treatment lead to elevated SYNJ2BP protein levels within two hours of exposure . This rapid upregulation suggests SYNJ2BP is part of the acute stress response pathway. Researchers can study these dynamic expression changes through time-course experiments with various stressors (oxidative stress, ER stress, hypoxia), quantifying expression changes via RT-PCR for transcript levels and western blotting for protein abundance. Transcriptional regulation can be explored through chromatin immunoprecipitation (ChIP) assays to identify transcription factors that bind to the SYNJ2BP promoter under stress conditions.

What is the mechanism by which SYNJ2BP facilitates mitochondria-ER membrane contact formation?

SYNJ2BP facilitates mitochondria-ER membrane contact sites (MERC) by functioning as a tethering protein between the outer mitochondrial membrane and the ER membrane. Specifically, SYNJ2BP on the mitochondrial outer membrane interacts with RRBP1 on the ER membrane to form MAM (mitochondrion-associated ER membrane) structures . This tethering mechanism has been confirmed through multiple methodologies: transmission electron microscopy (TEM) has visualized the detailed structure of MAMs in cells with altered SYNJ2BP expression, showing increased contact points between mitochondria and ER in SYNJ2BP-overexpressing cells . Additionally, proximity ligation assays (PLA) using mitochondrial (TOM20) and ER (KDEL) markers have quantitatively demonstrated that SYNJ2BP knockdown decreases mitochondria-ER proximity, while overexpression increases these contacts .

How do changes in SYNJ2BP expression affect mitochondrial distribution in neurons?

Alterations in SYNJ2BP expression significantly impact mitochondrial distribution in neurons, particularly in motor neurons. Overexpression of SYNJ2BP results in perinuclear clustering of mitochondria in the soma and reduces mitochondrial presence in axons and neurites, as demonstrated through live imaging . This redistribution effect can be quantified using the Mahalanobis distance of mitochondria in each cell, providing an unbiased measurement that does not assume a circular cell shape . SYNJ2BP knockout models show the opposite effect, with increased mitochondrial distribution throughout the cell. To study this phenomenon, researchers can employ time-lapse microscopy of fluorescently labeled mitochondria (using MitoTracker or mitochondria-targeted fluorescent proteins) combined with automated image analysis for tracking mitochondrial movement and distribution patterns.

What quantitative methods can be used to assess mitochondria-ER contacts in relation to SYNJ2BP function?

Multiple complementary techniques can quantitatively assess mitochondria-ER contacts when studying SYNJ2BP function:

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions that occur within 40 nm, making it ideal for quantifying MERC. Using antibodies against mitochondrial markers (TOM20) and ER markers (KDEL), PLA generates fluorescent dots at sites where these organelles are in close proximity .

• Confocal Microscopy with Colocalization Analysis: By labeling mitochondria and ER with different fluorescent markers, researchers can quantify overlap (yellow dots) between these organelles. The Pearson's correlation coefficient or Mander's overlap coefficient can provide numerical measures of colocalization .

• Transmission Electron Microscopy (TEM): This provides the highest resolution assessment of physical contacts between mitochondria and ER, allowing measurement of the contact length and the distance between membranes .

• FRET-based Biosensors: Genetically encoded fluorescent sensors placed at the mitochondria-ER interface can detect proximity changes through fluorescence resonance energy transfer.

These methods should be used in combination to provide comprehensive assessment of MERC formation in response to SYNJ2BP manipulation.

How is SYNJ2BP implicated in neurodegenerative diseases, particularly motor neuron disorders?

SYNJ2BP has been found to be significantly elevated in motor neurons from patients with hereditary motor neuron diseases, specifically spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis type 4 (ALS4) . In SBMA, this elevation appears to be mediated by mutant androgen receptor (AR) toxic gain of function, as knocking out the endogenous AR reduces SYNJ2BP expression. The mechanism differs in ALS4, where SYNJ2BP protein elevation may result from altered protein turnover rather than increased gene expression. Immunohistochemical staining of patient spinal cord tissue has confirmed SYNJ2BP elevation in spinal motor neurons . This abnormal elevation contributes to disease pathology by altering mitochondrial distribution and increasing mitochondria-ER contacts, which can disrupt calcium homeostasis and energy metabolism. Research approaches should include comparative studies of SYNJ2BP expression in patient-derived samples versus controls, coupled with functional assays of mitochondrial function.

What is the relationship between SYNJ2BP expression and mitochondrial dysfunction in neurological disorders?

The relationship between SYNJ2BP expression and mitochondrial dysfunction appears bidirectional. Elevated SYNJ2BP expression correlates with mitochondrial stress in both SBMA and ALS4 motor neurons, characterized by impaired oxidative function . Conversely, acute mitochondrial stress (e.g., hydrogen peroxide treatment) induces SYNJ2BP expression, suggesting it may be part of a stress response pathway . The increased SYNJ2BP leads to altered mitochondrial distribution and enhanced mitochondria-ER contacts, which can disrupt calcium homeostasis and further exacerbate mitochondrial dysfunction. Significantly, reducing SYNJ2BP levels in patient-derived cells improves mitochondrial oxidative function, indicating a potential therapeutic avenue . To investigate this relationship, researchers should employ mitochondrial function assays (oxygen consumption rate, membrane potential, ATP production) in conjunction with SYNJ2BP manipulation experiments, and utilize transgenic models with controlled SYNJ2BP expression to evaluate causality.

Could SYNJ2BP be a therapeutic target for motor neuron diseases, and how would researchers evaluate this potential?

SYNJ2BP shows promise as a therapeutic target for motor neuron diseases based on evidence that reducing SYNJ2BP levels in patient cells improves mitochondrial oxidative function . To evaluate this potential, researchers should follow a systematic approach:

• Validation Studies: Confirm the effects of SYNJ2BP knockdown on mitochondrial function across multiple patient-derived cell lines and in vivo models of motor neuron disease.

• Mechanism Elucidation: Determine whether benefits arise from normalized mitochondria-ER contacts, improved mitochondrial distribution, or other mechanisms.

• Target Engagement Assays: Develop assays to quantify SYNJ2BP activity or its critical interactions (e.g., with RRBP1) that could serve as biomarkers.

• Small Molecule Screening: Using high-throughput screening, identify compounds that modulate SYNJ2BP expression or function.

• Therapeutic Window Assessment: Determine the optimal degree of SYNJ2BP reduction that improves pathology without disrupting normal cellular functions.

• Delivery Method Development: For targeted therapies (such as antisense oligonucleotides or siRNAs), develop effective delivery methods to motor neurons.

• Long-term Safety Evaluation: Assess potential compensatory mechanisms and long-term effects of SYNJ2BP modulation.

The most promising approach may involve partial rather than complete inhibition of SYNJ2BP function, aiming to normalize rather than eliminate mitochondria-ER contacts.

What are the most effective approaches for studying SYNJ2BP in human cellular models?

The most effective research approaches for studying SYNJ2BP in human cellular models involve a combination of genetic manipulation, imaging techniques, and functional assays:

• Genetic Manipulation:

  • CRISPR/Cas9 for knockout studies as demonstrated in HEK293T cells

  • siRNA for transient knockdown, particularly useful in primary cells

  • Overexpression systems using appropriate vectors with epitope tags for detection

  • Inducible expression systems to study temporal effects of SYNJ2BP manipulation

• Imaging Techniques:

  • Confocal microscopy with co-localization analysis of mitochondria and ER

  • Transmission electron microscopy (TEM) for high-resolution visualization of MAM structures

  • Proximity ligation assay (PLA) for quantifying protein-protein interactions in situ

  • Live-cell imaging to track mitochondrial dynamics and distribution

• Functional Assays:

  • Mitochondrial function assessments (oxygen consumption, membrane potential, ATP production)

  • Calcium imaging to assess ER-mitochondria calcium transfer

  • Stress response assays to evaluate how SYNJ2BP responds to various cellular stressors

Particularly powerful are patient-derived models such as induced pluripotent stem cell (iPSC)-derived motor neurons, which maintain patient-specific genetic backgrounds and have successfully been used to study SYNJ2BP in disease contexts .

What proteomics approaches are most informative for identifying SYNJ2BP interactors in different cellular compartments?

For identifying SYNJ2BP interactors across cellular compartments, several complementary proteomics approaches have proven informative:

• Co-Immunoprecipitation Mass Spectrometry:

  • Pull-down of SYNJ2BP from mitochondrial fractions followed by mass spectrometry has successfully identified mitochondrial SYNJ2BP interactors

  • This should be performed with appropriate controls (IgG, SYNJ2BP-null cells) and normalization methods

• Proximity-Dependent Biotinylation (BioID or APEX):

  • Fusion of SYNJ2BP to promiscuous biotin ligases allows labeling of proximal proteins

  • Different compartment-specific variants can identify interactors in distinct subcellular locations

• Cross-linking Mass Spectrometry (XL-MS):

  • Chemical cross-linking of proteins in close proximity followed by MS analysis

  • Particularly useful for capturing transient interactions at membrane contact sites

• Fractionation-based Approaches:

  • Isolation of MAM fractions compared to pure mitochondrial or ER fractions

  • Quantitative proteomics comparing SYNJ2BP-enriched versus depleted fractions

  • LOPIT (localization of organelle proteins by isotope tagging) for spatial proteomics

• Targeted Approaches:

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can validate and quantify specific interactions identified in discovery experiments

These methods should be combined with functional validation through imaging and biochemical assays to confirm the biological relevance of identified interactions .

How can researchers effectively manipulate SYNJ2BP expression in primary neuronal cultures?

Effectively manipulating SYNJ2BP expression in primary neuronal cultures requires techniques that achieve efficient gene modulation while maintaining neuronal viability:

• Viral Vector Delivery:

  • Lentiviral or adeno-associated viral (AAV) vectors provide efficient gene delivery to primary neurons

  • For overexpression, vectors containing SYNJ2BP under neuron-specific promoters (e.g., synapsin) ensure targeted expression

  • For knockdown, vectors expressing shRNA or sgRNA (for CRISPR-Cas9) can reduce SYNJ2BP levels

• Nucleofection:

  • Direct delivery of expression plasmids, siRNAs, or CRISPR-Cas9 components via electroporation

  • Should be performed prior to plating or at early stages of culture

• Antisense Oligonucleotides (ASOs):

  • Can achieve temporary knockdown with reduced toxicity compared to viral approaches

  • Beneficial for temporal studies of SYNJ2BP function

• Inducible Expression Systems:

  • Tet-On/Tet-Off systems allow temporal control of SYNJ2BP expression

  • Particularly useful for studying dynamic processes like stress responses

• Verification Approaches:

  • Confirm manipulation efficiency using qPCR for transcript levels

  • Western blot analysis for protein levels

  • Immunofluorescence to assess subcellular distribution changes

When working with primary neurons, timing is critical - manipulation before key developmental stages (e.g., synaptogenesis) may yield different results than manipulation in mature neurons. Based on studies in iPSC-derived motor neurons, researchers should monitor for changes in mitochondrial distribution and function following SYNJ2BP manipulation .

How does SYNJ2BP regulate mitochondrial calcium homeostasis, and what experimental approaches best capture this function?

SYNJ2BP regulates mitochondrial calcium homeostasis primarily through its role in maintaining and regulating mitochondria-ER membrane contact sites (MERC), which are critical calcium exchange points between these organelles. When SYNJ2BP is overexpressed, the increased formation of MERC facilitates calcium transfer from ER to mitochondria . This process affects mitochondrial metabolism, as calcium is a key regulator of mitochondrial enzymes involved in ATP production.

To experimentally measure this function, researchers can employ:

• Genetically Encoded Calcium Indicators:

  • Targeted calcium sensors like mito-GCaMP (mitochondrial) and ER-GCaMP (ER-specific) can monitor calcium dynamics in real-time

  • Dual-color imaging allows simultaneous monitoring of both compartments

• Calcium Flux Measurements:

  • Measuring calcium flux across mitochondrial membranes using calcium-sensitive dyes

  • Patch-clamp electrophysiology of mitochondrial calcium uniporter activity

• ER-Mitochondria Calcium Transfer Assays:

  • Release of calcium from ER stores (using thapsigargin or IP3-generating agonists) followed by measurement of mitochondrial calcium uptake

  • Comparing transfer efficiency between control and SYNJ2BP-manipulated cells

• Functional Consequences Assessment:

  • Measure mitochondrial enzyme activities affected by calcium (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase)

  • Monitor mitochondrial ATP production in response to calcium signals

• In situ Calcium Microdomains:

  • Super-resolution microscopy to visualize calcium microdomains at MERC

  • Correlation with SYNJ2BP localization

These approaches should be combined with genetic manipulation of SYNJ2BP to establish causality between SYNJ2BP levels, MERC formation, calcium transfer, and downstream functional outcomes.

What is the interplay between SYNJ2BP and the mitochondrial protein import machinery?

The interplay between SYNJ2BP and mitochondrial protein import machinery represents an emerging area of research with important implications. As an outer mitochondrial membrane protein, SYNJ2BP persists on the OMM surface after disruption of protein synthesis and binds several mRNAs of oxidative phosphorylation (OXPHOS) proteins . This suggests SYNJ2BP may function as an RNA-binding protein (RBP) that participates in the localized translation and import of specific mitochondrial proteins.

The potential mechanisms and experimental approaches include:

• mRNA Binding Analysis:

  • RNA immunoprecipitation (RIP) or CLIP-seq (cross-linking immunoprecipitation and sequencing) to identify SYNJ2BP-bound mRNAs

  • Focus on mRNAs encoding mitochondrial proteins to determine specificity

• Localized Translation Studies:

  • Proximity-specific ribosome profiling to identify mRNAs being translated near SYNJ2BP

  • Visualization of translation events using puromycylation assays combined with proximity ligation

• Import Assay Systems:

  • In vitro import assays using isolated mitochondria from cells with manipulated SYNJ2BP levels

  • Real-time tracking of fluorescently labeled mitochondrial precursor proteins

• Interaction with Import Machinery:

  • Co-immunoprecipitation and mass spectrometry to identify interactions between SYNJ2BP and components of the mitochondrial import machinery (TOM/TIM complexes)

  • Structural studies to determine interaction domains

• Functional Impact Assessment:

  • Measurement of import efficiency for specific mitochondrial proteins in cells with altered SYNJ2BP expression

  • Analysis of mitochondrial proteome composition using quantitative proteomics

While current published experiments remain limited and contain contradictions , this represents an important frontier in understanding SYNJ2BP's multifaceted roles in mitochondrial biology and potentially in age-related mitochondrial dysfunction.

How do SYNJ2BP-mediated mitochondria-ER contacts influence cellular responses to stress conditions?

SYNJ2BP-mediated mitochondria-ER contacts play critical roles in cellular stress responses through several interconnected mechanisms:

• Adaptive Stress Response:

  • SYNJ2BP expression increases rapidly (within two hours) in response to oxidative stress (H₂O₂ treatment)

  • This suggests SYNJ2BP upregulation represents an adaptive response to acute stress

• ER Stress Modulation:

  • Overexpression of SYNJ2BP can reduce ER stress induced by viral proteins

  • This protective effect likely stems from improved calcium and ATP transfer from mitochondria to ER, supporting ER functions during stress

• ATP Availability:

  • MAMs formed through SYNJ2BP-RRBP1 interaction provide more ATP to the ER

  • This enhanced energetic supply is critical during stress conditions when cellular energy demands increase

• Calcium Homeostasis:

  • SYNJ2BP-mediated MAMs facilitate calcium transfer between ER and mitochondria

  • This regulated calcium exchange is essential for stress signaling pathways and mitochondrial metabolic adaptation

• Zinc Homeostasis:

  • SYNJ2BP plays a critical role in maintaining mitochondrial Zn²⁺ homeostasis in nucleus pulposus cells during intervertebral disc degeneration

  • This represents another metal ion regulatory function of SYNJ2BP relevant to cellular stress

To study these relationships, researchers should employ stress induction protocols (oxidative stress, ER stress, nutrient deprivation) combined with SYNJ2BP manipulation, while monitoring:

• Unfolded protein response (UPR) markers

• ATP levels in different cellular compartments

• Calcium and zinc dynamics using compartment-specific sensors

• Mitochondrial respiratory capacity under stress

• Cell survival and recovery kinetics

The paradoxical aspect of SYNJ2BP function is that while acute upregulation appears protective, chronic elevation (as seen in neurodegenerative diseases) becomes detrimental, suggesting a time-dependent switch in its functional impact that warrants further investigation .

What are the most significant SYNJ2BP protein-protein interactions identified to date?

How does SYNJ2BP expression compare across different disease models?

What experimental designs are most appropriate for investigating SYNJ2BP function in different research contexts?

This structured approach to experimental design provides researchers with a roadmap for investigating SYNJ2BP function across multiple contexts, from basic cellular mechanisms to disease relevance and therapeutic potential.

What are the most promising unexplored aspects of SYNJ2BP biology that warrant further investigation?

Several promising unexplored aspects of SYNJ2BP biology warrant further investigation:

• Tissue-Specific Functions: While current research has focused on motor neurons and a few cell types, SYNJ2BP's function likely varies across tissues. Systematic comparison of SYNJ2BP's role across neural, muscle, liver, and other tissues would provide insights into tissue-specific vulnerabilities in diseases with SYNJ2BP dysregulation.

• Post-Translational Modifications: The regulatory mechanisms controlling SYNJ2BP activity remain largely unexplored. Phosphorylation, ubiquitination, and other modifications likely influence SYNJ2BP's interaction with binding partners and its role in MAM formation. Phosphoproteomic approaches could identify regulatory sites and responsible kinases/phosphatases.

• Temporal Dynamics: The kinetics of SYNJ2BP-mediated MAM formation and disassembly remain poorly understood. Real-time imaging of fluorescently tagged SYNJ2BP combined with optogenetic tools could reveal how these dynamics respond to cellular needs and stresses.

• Signaling Integration: SYNJ2BP contains a PDZ domain that likely serves as a hub for integrating various signaling pathways. A systematic analysis of how different cellular signals converge on SYNJ2BP to regulate mitochondria-ER communication would provide valuable insights.

• Role in Mitochondrial Quality Control: The potential involvement of SYNJ2BP in mitophagy, mitochondrial fission/fusion, or other quality control mechanisms remains unexplored despite its strategic location on the outer mitochondrial membrane.

• Contribution to Aging: Given SYNJ2BP's relationship with mitochondrial function and stress responses, its potential role in age-related mitochondrial decline represents an important area for investigation .

• Interaction with microRNAs and Long Non-coding RNAs: As SYNJ2BP appears to have RNA-binding capabilities, its potential interaction with regulatory RNAs could reveal novel mechanisms of post-transcriptional regulation.

These areas represent fertile ground for discoveries that could significantly advance our understanding of cellular homeostasis and disease pathogenesis.

How might advances in imaging and proteomics technologies enhance our understanding of SYNJ2BP function?

Emerging technologies in imaging and proteomics offer unprecedented opportunities to deepen our understanding of SYNJ2BP function:

• Super-Resolution Microscopy:

  • STED, STORM, or PALM microscopy can resolve MAM structures at nanometer resolution

  • Live-cell super-resolution imaging could capture dynamic SYNJ2BP redistribution during stress responses

  • Multicolor super-resolution could simultaneously track SYNJ2BP, its binding partners, and organelle markers

• Cryo-Electron Tomography:

  • Can visualize native MAM architecture at molecular resolution

  • Could reveal how SYNJ2BP organizes protein complexes at the mitochondria-ER interface

• Volumetric Electron Microscopy (FIB-SEM):

  • Provides 3D reconstruction of entire cells at nanometer resolution

  • Could map the complete network of SYNJ2BP-mediated contacts throughout the cell

• Spatial Proteomics:

  • APEX-based proximity labeling combined with mass spectrometry can map the protein landscape around SYNJ2BP with nanometer precision

  • Multiplexed ion beam imaging (MIBI) or imaging mass cytometry could visualize protein neighborhoods in situ

• Single-Cell Proteomics:

  • Would reveal cell-to-cell variability in SYNJ2BP expression and interactome

  • Could identify distinct subpopulations with different SYNJ2BP functions

• Crosslinking Mass Spectrometry:

  • Can capture direct protein-protein interaction interfaces

  • Would reveal the structural basis of SYNJ2BP's interactions with partners like RRBP1

• Proteome Dynamics:

  • Pulse-chase SILAC combined with mass spectrometry to study turnover rates

  • Could reveal how SYNJ2BP and interacting proteins are regulated during stress

• Integrative Omics:

  • Combining transcriptomics, proteomics, and metabolomics data from cells with altered SYNJ2BP expression

  • Would provide a systems-level understanding of SYNJ2BP's impact on cellular physiology

These technologies, especially when used in combination, have the potential to resolve current contradictions in the literature and provide a comprehensive understanding of SYNJ2BP's multifaceted roles in cellular homeostasis.

What contradictions exist in the current SYNJ2BP literature, and how might these be resolved through improved experimental approaches?

Several significant contradictions and knowledge gaps exist in the current SYNJ2BP literature that warrant methodological refinement:

• Contextual Function Discrepancies:

  • Contradiction: SYNJ2BP upregulation appears protective in some contexts (viral infection ) but detrimental in others (neurodegenerative diseases )

  • Resolution Approach: Systematic comparison across cell types, stress conditions, and expression levels using standardized assays for mitochondrial function and ER stress; time-course studies to distinguish acute versus chronic effects

• Molecular Mechanism Inconsistencies:

  • Contradiction: The precise molecular interactions by which SYNJ2BP tethers mitochondria to ER remain incompletely characterized, with different studies emphasizing different binding partners

  • Resolution Approach: Structural biology approaches (crystallography, cryo-EM) of the SYNJ2BP-RRBP1 complex; comprehensive mutation analysis to map interaction domains; in vitro reconstitution of tethering with purified components

• Physiological versus Pathological Roles:

  • Contradiction: The normal physiological role of SYNJ2BP versus its role in disease states is not clearly delineated

  • Resolution Approach: Tissue-specific conditional knockout models with careful phenotyping across multiple physiological parameters; developmental time-course studies

• Transcriptional Regulation Uncertainties:

  • Contradiction: Different regulatory factors (AR in SBMA , stress response in other contexts) have been implicated in controlling SYNJ2BP expression

  • Resolution Approach: Comprehensive promoter analysis; ChIP-seq for multiple transcription factors; reporter assays with systematic mutation of regulatory elements

• Mitochondrial Protein Import Role:

  • Contradiction: Limited and contradictory evidence regarding SYNJ2BP's role in mitochondrial protein import

  • Resolution Approach: Selective import assays for specific mitochondrial proteins; temporal analysis of import events; direct visualization of import using split fluorescent proteins

• Therapeutic Potential Ambiguities:

  • Contradiction: Unclear whether SYNJ2BP inhibition or activation would be beneficial in different disease contexts

  • Resolution Approach: Dose-response studies with partial inhibition/activation; temporal manipulation at different disease stages; combination approaches targeting multiple aspects of MERC formation