Recombinant Human t-SNARE domain-containing protein 1 (TSNARE1)

What is TSNARE1 and what is its genetic association with psychiatric disorders?

TSNARE1 (t-SNARE domain-containing protein 1) is a high-confidence gene candidate for schizophrenia risk that was identified through genome-wide association studies (GWAS). A GWAS meta-analysis of mixed-ancestry schizophrenia, schizoaffective, and bipolar cohorts (13,394 cases and 34,676 controls) identified TSNARE1 as a significant locus (P = 1.28 × 10^-9) at chromosome 8q24.3 . Transcriptome-wide association studies (TWAS) have suggested that overexpression of TSNARE1 is associated with schizophrenia (Bonferroni P = 3.65 × 10^-7), and Hi-C results from adult prefrontal cortex revealed physical interaction between schizophrenia-associated SNPs and the promoter region of TSNARE1 . This provides a potential mechanism by which schizophrenia-associated genetic risk factors are linked to TSNARE1 expression.

What are the major isoforms of TSNARE1 expressed in the human brain?

Research has validated three to four primary isoforms of TSNARE1 expressed in human brain. All of these isoforms encode a syntaxin-like Qa SNARE domain . The gene TSNARE1 contains 15 exons, with exons 1 and 15 encoding the 5' and 3' UTR, respectively . The isoforms contain a common N-terminal sequence encoded by exon 2 and one of two C-terminal exons:

• Exon 14 that encodes a transmembrane domain

• Exon 13 that encodes a non-transmembrane domain

RNA-sequencing data from adult and fetal human brain indicates that the majority of tSNARE1 (approximately 97% in adult and similar percentage in fetal brain) lacks a transmembrane domain, which is typically thought necessary for membrane fusion . The most abundant brain isoform appears to be tSNARE1c .

What is the evolutionary origin of TSNARE1?

TSNARE1 arose from molecular domestication of a Harbinger transposon early in vertebrate evolution . Harbinger transposons were an ancient transposon superfamily that encode a transposon protein and a SANT/Myb/trihelix DNA-binding protein. While active copies have not been identified in humans, some genes in the human genome, including TSNARE1 and NAIF (nuclear apoptosis inducing factor), contain domains descended from the DNA-binding protein encoding gene from Harbinger transposons . TSNARE1 specifically encodes a fusion protein containing a Myb-like tri-helix domain and a SNARE (soluble N-ethylmaleimide-sensitive attachment receptor) domain .

What is the tissue and cell-type specific expression pattern of TSNARE1?

RNA-sequencing data from the Genotype-Tissue Expression (GTEx) database indicate that TSNARE1 expression is highest in the cortex and cerebellum . Microarray data from the Allen Brain Atlas using a probe (CUST_1054_P1416573500) that maps to a region within exon 5 (included in all predicted TSNARE1 isoforms) revealed enrichment of TSNARE1 within regions of the cortex in both fetal and adult human brain .

Single-cell RNA-sequencing results from human cortex demonstrate that TSNARE1 is highly expressed in neurons and endothelial cells compared to glial cell types. Specifically, TSNARE1 is more enriched in excitatory neurons than in inhibitory neurons . This expression pattern aligns with the cortical abnormalities observed in schizophrenia patients and the implication of excitatory pyramidal cortical neurons in schizophrenia pathophysiology.

How does tSNARE1 affect endosomal trafficking in neurons?

tSNARE1 appears to function as a negative regulator of endolysosomal trafficking. Expression studies show that either tSNARE1b or tSNARE1c (which differ only in their inclusion or exclusion of a Myb-like domain) delays the trafficking of dendritic endosomal cargo Nsg1 into late endosomal and lysosomal compartments . This suggests that tSNARE1 regulates endosomal trafficking in cortical neurons by negatively regulating early endosomal to late endosomal trafficking or maturation .

The endolysosomal system is responsible for sorting, recycling, and degradation of internalized cargo. Normally, internalized cargo arrives at Rab5+ early endosomes and can be recycled back to the plasma membrane via two separate routes, Rab11+ recycling endosomes or Rab4+ rapid recycling compartments. Alternatively, cargo is sent from Rab5+ early endosomes to Rab7+ late endosomes and LAMP1+ lysosomes for degradation . tSNARE1 appears to specifically interfere with the progression from early to late endosomes.

What is the biochemical mechanism by which tSNARE1 affects SNARE complex formation?

Biochemical data demonstrate that tSNARE1 can compete with Syntaxin 12 (Stx12) for incorporation into an endosomal SNARE complex, supporting its possible role as an inhibitory SNARE . A typical SNARE complex contains one R-SNARE and three Q-SNAREs (further classified as Qa, Qb, and Qc based on sequence homology) .

Recombinant protein studies with human Syntaxin6, Syntaxin12, Vti1a, and tSNARE1 show that tSNARE1 can interact with SNARE components and potentially interfere with normal complex formation . The majority of tSNARE1 isoforms lack a transmembrane domain or other predicted membrane attachment site, which is typically required for the free energy of SNARE complex formation to be translated into membrane fusion . This structural feature is consistent with tSNARE1 acting as a negative regulator rather than facilitating membrane fusion.

How do schizophrenia-associated variants affect TSNARE1 expression and function?

Studies have found that schizophrenia-associated genetic variants near the TSNARE1 gene appear to affect its expression rather than altering specific isoforms. Isoform QTLs (isoQTL) for TSNARE1 were not associated with schizophrenia, suggesting that it is overexpression of all TSNARE1 transcripts that is associated with schizophrenia, rather than overexpression of a specific isoform .

A recent study investigated a functional schizophrenia-associated genetic variant (rs4129585) near the TSNARE1 and ADGRB1 genes. The researchers introduced each allele into human induced pluripotent cells and differentiated isogenic clones homozygous for the risk allele and non-risk allele into neural progenitor cells. RNA sequencing revealed that the two alleles yield significant transcriptional differences in the expression of 109 genes , suggesting that this variant influences gene regulatory networks involving TSNARE1.

What are the recommended approaches for expressing and purifying recombinant tSNARE1 protein?

Based on published protocols, recombinant human tSNARE1 can be purified as follows:

• Express tSNARE1 as a GST-fusion protein (e.g., using pGEX4P1 expression vector)

• Purify using glutathione Sepharose beads

• Cleave the GST-fusion protein with HRC 3C protease overnight at 4°C in buffer containing:

  • 50 mM Tris, pH 7.5

  • 150 mM NaCl

  • 1 mM EDTA

  • 0.5% Triton X-100

  • 0.5 mM DTT

For interaction studies with other SNARE proteins, the following conditions have been used successfully:

• Mix soluble recombinant SNARE proteins with GST-tagged binding partners immobilized on glutathione Sepharose beads

• Use binding buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 3 mM MgCl₂

• Incubate overnight at 4°C or for 2 hours at 4°C for inhibition assays

• Wash beads 4 times in binding buffer

• Elute by boiling in SDS sample buffer

• Analyze by Coomassie staining and/or immunoblotting

What experimental systems are appropriate for studying tSNARE1 localization and function?

Several experimental systems have been successfully used to study tSNARE1:

• Cell Culture Systems:

  • Human neural progenitor cells

  • Human neuroblastoma cells

  • Mouse cortical neurons (primary culture from E15.5 embryos)

  • Rat neurons

• Expression Constructs:

  • GFP-tSNARE1 isoforms (tSNARE1a, tSNARE1b, tSNARE1c) in pEGFP-C2 vectors

  • Co-expression with spectrally distinct red-tagged markers of the endosomal system:

    • tagRFP-Rab4, tagRFP-Rab5, tagRFP-Rab11, tagRFP-Rab7

    • LAMP1-mCherry

    • mCherry-Stx12

• Imaging Approaches:

  • Live-cell imaging of fluorescently tagged proteins

  • Colocalization analysis with endosomal markers

  • Cargo trafficking assays using HaloTag-labeled cargo proteins (e.g., Nsg1-HaloTag)

How can researchers measure the effects of tSNARE1 on endosomal trafficking?

A validated methodology to measure tSNARE1's effects on endosomal trafficking involves:

• Cargo Trafficking Assay:

  • Transfect cells with a cargo protein tagged with HaloTag (e.g., Nsg1-HaloTag)

  • Co-transfect with endosomal markers (tagRFP-Rab4, tagRFP-Rab5, tagRFP-Rab7, tagRFP-Rab11, or LAMP1-mCherry)

  • Include or exclude GFP-tSNARE1 to test its effect

  • Label HaloTag with a fluorescent ligand (e.g., AlexaFluor-660 HaloTag ligand) prior to imaging

• Quantification:

  • Track the colocalization of cargo with different endosomal compartments over time

  • Compare trafficking kinetics in the presence versus absence of tSNARE1

  • Analyze differences in early-to-late endosomal progression

• Recombinant Protein Interaction Assays:

  • Use GST pulldown assays to test competition between tSNARE1 and Stx12

  • Vary tSNARE1 concentrations (0-6 μM) while keeping other SNARE proteins constant (0.4 μM)

  • Analyze complex formation by SDS-PAGE and western blotting

What are the challenges in studying TSNARE1 in the context of schizophrenia research?

Several methodological challenges exist when studying TSNARE1 in schizophrenia research:

• Expression Level Control:

  • Since schizophrenia is associated with overexpression of TSNARE1 rather than specific isoform changes , designing experiments that accurately model pathological expression levels is critical.

• Cell Type Specificity:

  • TSNARE1 is enriched in excitatory neurons , necessitating cell-type specific approaches rather than whole-brain or mixed culture systems.

• Functional Readouts:

  • Establishing the connection between altered endosomal trafficking and schizophrenia-relevant phenotypes requires development of appropriate functional assays.

• Genetic Background:

  • When testing the effects of schizophrenia-associated variants, controlling for genetic background using isogenic cell lines is important, as demonstrated in recent studies .

• Temporal Dynamics:

  • Understanding developmental aspects of TSNARE1 function requires comparing expression and function between fetal and adult contexts.

What are the unexplored aspects of TSNARE1 function that warrant investigation?

Several aspects of TSNARE1 remain unexplored and represent valuable research directions:

• Synaptic Function:

  • While tSNARE1 localizes to dendritic shafts and spines, suggesting a role at the postsynapse , the specific impact on synaptic function, plasticity, and neurotransmission requires further investigation.

• Isoform-Specific Functions:

  • The functional differences between tSNARE1 isoforms beyond their differential effects on endosomal trafficking need to be characterized.

• Interaction Partners:

  • A comprehensive interactome analysis would help identify all binding partners beyond the known SNARE proteins.

• Regulatory Mechanisms:

  • The mechanisms regulating TSNARE1 expression, including transcription factors and epigenetic modifications, especially in the context of schizophrenia-associated variants.

• Animal Models:

  • Development of transgenic models with altered TSNARE1 expression to study behavioral, cognitive, and neurophysiological phenotypes relevant to schizophrenia.

How might understanding TSNARE1 function contribute to therapeutic strategies for schizophrenia?

Understanding TSNARE1 function could inform therapeutic approaches through:

• Target Validation:

  • Confirming TSNARE1's role in schizophrenia pathophysiology would validate it as a potential therapeutic target.

• Endosomal Pathway Modulation:

  • Identifying compounds that can normalize endosomal trafficking disrupted by TSNARE1 overexpression.

• Expression Regulation:

  • Developing strategies to normalize TSNARE1 expression levels in patients with schizophrenia-associated variants.

• Biomarker Development:

  • Using TSNARE1 expression or associated endosomal trafficking defects as biomarkers for disease subtypes or treatment response.

• Precision Medicine Approach:

  • Stratifying patients based on TSNARE1-related genetic variants to guide personalized treatment strategies.

How does tSNARE1 differ from other SNARE proteins in structure and function?

A comparative analysis of tSNARE1 and other SNARE proteins reveals several key differences:

tSNARE1 appears to act more as a regulatory SNARE rather than directly facilitating membrane fusion, likely by competing with conventional SNAREs like Stx12 for incorporation into SNARE complexes .

How do research findings on TSNARE1 connect with other schizophrenia risk genes?

TSNARE1 findings connect with other schizophrenia risk genes in several ways:

• Endosomal Trafficking Pathway:

  • Multiple schizophrenia risk genes, including TSNARE1, are involved in endosomal trafficking and synaptic function .

• Neurodevelopmental Processes:

  • TSNARE1 expression in both fetal and adult brain suggests involvement in developmental processes, similar to other schizophrenia risk genes .

• Excitatory Neuron Function:

  • The enrichment of TSNARE1 in excitatory neurons aligns with the excitatory/inhibitory imbalance hypothesis of schizophrenia .

• Polygenic Risk:

  • As one of many high-confidence schizophrenia risk genes, TSNARE1 contributes to the polygenic architecture of the disorder .