Multi-Modal Characterization and In Vitro Modulation of HERV-K Neuroinflammation in Schizophrenia

Transcript:

One third of people with schizophrenia are treatment resistant, meaning they’ve been tried on two or more antipsychotics with little to no symptom relief, because current medications are largely non-specific, which also leads to severe side effects. In my previous project, neuroinflammation was identified to contribute to schizophrenia pathology, and this is well supported in the literature, but its mechanisms are unclear, making its characterization critical. To characterize it, I performed fixel analysis on diffusion MRI data from 147 individuals and investigated molecular drivers through RNA sequencing analysis on 9 different neuronal cell and tissue types, finding HERV-K-correlated viral activity upregulated and downregulated neuronal function. To translate these findings, I evaluated two targeted therapeutic strategies in vitro. First, I assessed an analog to a small molecule I refined to address the downstream effects on neuronal function, which showed dose-dependent, disruptions to neuronal morphology and increases in viability. Second, I developed a novel antisense oligonucleotide targeting HERV-K gag, which achieved a 69% reduction in expression. Overall, this supports precision treatments for schizophrenia which has the potential to improve quality of life. Thank you so much!

About Schizophrenia:

Schizophrenia is a chronic neurodevelopmental disorder that affects approximately 24 million people worldwide[1]. It is marked by psychotic symptoms—such as hallucinations, delusions, and disorganization—as well as negative symptoms like reduced emotional expression, motivation, and social engagement[1]. As a result, schizophrenia is among the leading causes of disability globally[1,2]. Moreover, many individuals with the disorder struggle to maintain employment and relationships, and the condition is linked to a significantly higher risk of suicide.[1,2].

Current Theories and Treatment:

Current treatment options are designed to relieve symptoms rather than provide long-term restoration of brain function and individual functioning, leaving one-third of individuals treatment-resistant [3,4]. This is because despite its severity, the biological mechanisms behind schizophrenia are not fully understood, and as a result, current treatments often fail to address the disorder’s root causes[1,4]. Moreover, current options cause severe side effects, including neuromuscular dysfunction, cognitive dysregulation, physical dependence and sexual dysfunction, making staying on treatment exceptionally difficult even if effective [1,4].

While previous theories centered disruptions in dopamine and serotonin pathways, emerging theories center neuroinflammation[1,5]. However, drivers and effects remain incompletely characterized, resulting in limited translation into targeted therapeutic strategies[5].

Objectives:

To address this gap, by:

Characterizing schizophrenia's neuroinflammatory profile to identify biomarkers of its down and upstream mechanisms.

Developing targeted therapeutic strategies to modulate identified mechanisms

And evaluating the safety and therapeutic potential of these candidates in vitro,

To ultimately work towards the development of precise schizophrenia therapeutics.

Diffusion Weighted Magnetic Resonance Imaging (DW-MRI) Analysis:

72 control and 75 schizophrenia DW-MRI scans obtained from COBRE, co-occurring disorders excluded.

Converted DICOM files to NIFTI format, b-values/b-vectors were added, and visually inspected in MRtrix.

Denoised, corrected artifiacts (Gibbs ringing), and combined with reverse phase-encoded images.

Distortion correction applied for brain mask generation.

Design/contrast matrices created; fibre density (FD), fibre cross section (FC) and fibre density cross section (FDC) analyses performed

FWE-corrected statistical maps thresholded at p < 0.05 to identify significant fixels

Single-Cell & Tissue RNA-Seq Analysis:

Differential expression and pathway analysis performed (Fig.1) on selection of control-matched genomic data from cell lines and post mortem tissue (glial, astrocytes, neural progenitors, human induced pluripotent stem cells, cortical neurons, and Putamen, Accumbens, Anterior Cingulate Cortex, & Dorsolateral Prefrontal Cortex tissue)

Therapeutic Design:

Small Molecule:

Refined structure of lead compound from previous 2025 project (Chemaxon Marvin) to improve safety and viability [C3:F substitution, C4’ (B ring): N(CH₃)₂ substitution]

Evaluated and filtered candidates based on ADME (SWISSADME) and toxicity (Tox-Prediction)

Ran binding affinity tests for candidates (AutoDOCK Vina).

Queried optimized structure in ZINC database

Identified closest purchasable analog

Antisense Oligonucleotide (ASO):

Obtained HERV-K coding sequence

Identified accessible regions for targeting.

Generated candidate ASO sequences in Pfred based on accessibility and efficacy.

Optimized (ASOptimizer) and evaluated stability (OligoAnalyzer)

Generated scrambled control (GenScript) and ordered as duplexed RNA oligos

In Vitro Validation:

Small Molecule (See Fig. 2-3):

Rat cortical neurons (DIV 12) treated (20–80 µM)

Viability (live/dead) and synaptic structure (DAPI, Synaptophysin, PSD-95, Neurofilament)

Quantified using FIJI and SynBot

ASO (See Fig. 4):

HEK293T cells treated with 50uM ASO solution and scramble control after reaching 70-90% confluency (see Fig. 3)

RT-qPCR was performed with HERV-K primers (env, pol, gag)

Relative expression quantified using 2⁻ΔΔCt normalization to control and housekeeping gene.

DW-MRI Analysis Results:

See Fig. 5 for analysis of altered tracts.

Fixel-based analysis identified 18 statistically significant clusters with fibre density and cross section alterations (FWE-corrected, p < 0.05). These clusters mapped onto 9 major white matter tracts, with several clusters representing distinct segments of the same pathway. Only clusters with anatomically plausible mappings and literature-supported relevance were retained for interpretation, leaving 14 clusters total but retaining the 9 tracts. Of the 14 significant clusters, 9 anatomically corresponded to regions identified in the fMRI functional connectivity analysis, indicating partial structural-functional convergence. Moreover, 12/14 clusters were primarily driven by fibre density reductions, indicating axonal integrity loss instead of large-scale atrophy. Regions overlapped in parietal, supramarginal, thalamic, and hippocampal areas, which indicates convergence in networks related to attention, sensory integration, and memory.

These findings suggest that neuroinflammation-driven axonal disruption may underlie network dysfunction, meaning targeted interventions aimed at reducing inflammation can also work to preserve neuronal, functional, and structural integrity.

Single-Cell & Tissue RNA-Seq Analysis:

Upregulated genes revealed strong enrichment in viral RNA sensing, interferon signaling, and endogenous retroviral repression pathways, which support an activation of innate immune responses and antiviral defense mechanisms, consistent with a neuroinflammatory state that may contribute to downstream neuronal dysfunction (see Fig. 6).

Downregulated genes revealed alterations across immune/viral response pathways as well, but most notably in intracellular signaling networks, and regulators. Together, these patterns suggest that inflammatory and viral-like processes may disrupt neuronal viability particularly through kinase-mediated signaling and neurotrophic dysregulation (see Fig. 7).

Human Endogenous Retrovirus-K (HERV-K) Activation Hypothesis:

Differential expression showed a strong viral-like / innate antiviral response despite no evidence of active exogenous infection, which points toward endogenous viral elements (EVEs) rather than an external pathogen[6]. EVEs are viral sequences integrated into the host genome and inherited across generations[6,7]. HERVs are also part of the larger family of transposable elements which propagate by exploiting RNA to DNA reverse transcription and are supposed to be kept epigenetically repressed[7].

HERV-K specifically is one of the best-preserved HERV families (ancient retroviruses integrated in the human genome), with retained retroviral features and is among the HERV groups still capable of coding viral products[6]. This suggests that silencing it may reduce this observed neuroinflammatory response, potentially restoring the downstream neuronal dysregulation observed and serving as a therapeutic target[6,7].

Design of Therapeutic Candidates:

See Fig. 8.

In Vitro Results:

ASO-mediated silencing resulted in ~69% reduction in HERV-K gag expression. This demonstrates that endogenous retroviral activity can be selectively modulated, providing a promising avenue for precision-based intervention.

In contrast, treatment with the flavone-based compound Eutropoflavin did not produce significant changes in synaptic colocalizations or neurite length across tested doses (see Fig. 9). However, qualitative imaging revealed dose-dependent morphological deterioration, which included neurite fragmentation and compromised cell integrity. As visualized in Fig. 9, an increase in metabolic viability was observed at higher doses, which suggests that cells remained viable despite structural degeneration. This ultimately suggests an overactivation of trophic signaling, an important consideration for TRKB agonists in therapeutic development.

Discussion:

Using multimodal analysis, I investigated the neuroinflammatory profile of schizophrenia and identified HERV-K–associated endogenous retroviral pathways as a key contributor. Finally, I evaluated targeted therapeutic strategies in vitro to modulate this mechanism, both through its potential upstream driver and downstream consequences.

The identified neuroinflammatory mechanism was gleaned from the analysis of diffusion-weighted magnetic resonance imaging analysis that suggested convergent structural-functional disruption and cellular vulnerability (FD reductions) rather than gross anatomical loss. Hypothesis-driven RNA sequencing analysis revealed a dysregulation of antiviral immune response genes, which correlated with an activation of evolutionarily young endogenous retroviral elements, implicating HERV-K as an upstream driver of neuroinflammation.

When tested in vitro, the analog small molecule aimed at restoring the convergent structural-functional dysregulation showed dose dependent, sublethal effects on neuronal morphology, and the novel antisense oligonucleotide (ASO) targeting HERV-K gag achieved a 69% reduction in expression. These results demonstrate the limitations of downstream targeting as they are susceptible to unintended consequences and the effectiveness of targeting upstream pathogenic drivers.

Conclusion:

A precision approach moves away from symptomatic therapeutics to targeting key drivers

Schizophrenia involves multi-modal disruption, with neuroinflammation driven by upstream mechanisms. Specifically, convergent imaging and gene expression implicated HERV-K as a potential driver. Through in vitro testing, it is evident that downstream approaches may appear effective, but can induce unintended signaling effects not captured by standard assays. In contrast, upstream targeting using ASOs can allow for precise modulation. While this work remains preliminary, it ultimately supports the development of more precise schizophrenia therapeutics.

Future Directions:

1. Validate HERV-K mechanism directly

Directly quantify HERV-K transcript/protein activity in schizophrenia datasets.

Distinguish which HERV-K region is most pathogenic.

Test the effect of HERV-K suppression on downstream inflammatory responses.

2. Validate therapeutic candidates in disorder models

Test therapeutic strategies in models representative of schizophrenia neurobiology.

Assess whether ASO treatment can lead to restored neuronal function to validate upstream hypothesis.

3. Refining dosing, delivery, and safety

Dose-response optimization and assessment of larger range of doses.

Assess repeated versus single-dose treatment and long-term toxicity.

Assess off-target effects.

Consider in vivo delivery mechanisms for the ASO.

Thank you to the Naweed Syed Lab including Dr. Syed, Zainab Khan, Fahad Iqbal, Badra Abbas, and all other members for training me and supporting me during the in vitro validation aspect of this project.

Thank you to Dr. MacEachern and all members at the Precision Neurodevelopment Lab for the opportunity to work at the Precision Neurodevelopment Lab. My experience motivated this project since its inception and allowed me to explore this field meaningfully.

Thank you to Youth Science Canada, for the support and advice during the ISEF selection development program.

Thank you to school science fair coordinator, Ms. O'Keefe.

Thank you to the CYSF for the opportunity to attend the CWSF and my delegates for volunteering their time, support and feedback.

And finally, thank you to my teachers, friends, family, and all other supporters for keeping me going!

[1] Patel, K. R., Cherian, J., Gohil, K., & Atkinson, D. (2014, September). Schizophrenia: Overview and treatment options. P & T: a peer-reviewed journal for formulary management. https://pmc.ncbi.nlm.nih.gov/articles/PMC4159061/

[2] Sher, L., & Kahn, R. S. (2019). Suicide in Schizophrenia: An Educational Overview. Medicina (Kaunas, Lithuania), 55(7), 361. https://doi.org/10.3390/medicina55070361

[3] Chokhawala, K. (2023, February 26). Antipsychotic medications. StatPearls. https://www.ncbi.nlm.nih.gov/books/NBK519503/

[4] Pandey, A., & Kalita, K. N. (2022a, August 30). Treatment-resistant schizophrenia: How far have we traveled?. Frontiers in psychiatry. https://pmc.ncbi.nlm.nih.gov/articles/PMC9468267/

[5] Wang, Q., et al. (2024). Neuroinflammatory mechanisms in psychiatric disorders. Translational Psychiatry. https://doi.org/10.1038/s41398-024-02775-5

Elsevier. (n.d.). Interferon. In ScienceDirect Topics. https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/interferon

[6] Grandi, N., & Tramontano, E. (2019). Human endogenous retroviruses are ancient acquired elements still shaping innate immune responses. Virology, 538, 230–241. https://doi.org/10.1016/j.virol.2019.08.020

[7] Turelli, P., Trono, D. (2015). Editing out endogenous retroviruses: Mechanisms and consequences. Nature Reviews Genetics. https://pmc.ncbi.nlm.nih.gov/articles/PMC5127361/

Additional References

Borrego-Ruiz, A.,& Borrego, J. J. (2025, March 10). Involvement of virus infections and antiviral agents in schizophrenia. Psychological medicine. https://pmc.ncbi.nlm.nih.gov/articles/PMC12055031/

​

Dhuri, K., Bechtold, C., Quijano, E., Pham, H., Gupta, A., Vikram, A., & Bahal, R. (2020, June 26). Antisense oligonucleotides: An emerging area in drug discovery and development. Journal of clinical medicine. https://pmc.ncbi.nlm.nih.gov/articles/PMC7355792/

Hubrecht, R. C., & Carter, E. (2019, September 30). The 3Rs and humane experimental technique: Implementing change. Animals : an open access journal from MDPI. https://pmc.ncbi.nlm.nih.gov/articles/PMC6826930/

​

Kim E; Carreira Figueiredo I; Simmons C; Randall K; Rojo Gonzalez L; Wood T; Ranieri B; Sureda-Gibert P;Howes O;Pariante C;Nima Consortium None;P asternak O;Dell’Acqua F;TurkheimerF;Cash D; (n.d.). Mapping acute neuroinflammation in vivo with diffusion-MRI in rats given a systemic lipopolysaccharide challenge. Brain, behavior, andimmunity. https://pubmed.ncbi.nlm.nih.gov/37482203/

​

Klein, H. C. (2017a, December 13). Silencing of viral elements: An available cure for schizophrenia?. Frontiers in psychiatry. https://pmc.ncbi.nlm.nih.gov/articles/PMC5733551/

​

Kotsiri, I., Resta, P., Spyrantis, A., Panotopoulos, C., Chaniotis, D., Beloukas, A., & Magiorkinis, E. (2023, June 9). Viral infections and schizophrenia: A comprehensive review.Viruses. https://pmc.ncbi.nlm.nih.gov/articles/PMC10302918/

​

Liu, X., Chan, C.-B., Jang, S.-W., Pradoldej, S., Huang, J., He, K., Phun, L. H., France, S., Xiao, G., Jia, Y., Luo, H. R., & Ye, K. (2010, December 9). A synthetic 7,8-dihydroxyflavone derivative promotes neurogenesis and exhibits potent antidepressant effect. Journal of medicinal chemistry. https://pmc.ncbi.nlm.nih.gov/articles/PMC3150605/

Rakhra, G., & Rakhra, G. (2021). Zinc finger proteins: Insights into the transcriptional and post-transcriptional regulation of immune response. Molecular Biology Reports, 48, 5735–5743. https://doi.org/10.1007/s11033-021-06556-x

Yang, P., Wang, Y., & Macfarlan, T. S. (2017). The role of KRAB-ZFPs in transposable element repression and mammalian evolution. Trends in Genetics, 33(11), 871–881. https://doi.org/10.1016/j.tig.2017.08.006

Lander, E. S. (2011). Initial impact of the sequencing of the human genome. Nature Reviews Molecular Cell Biology, 12(10), 693–698. https://doi.org/10.1038/nrm3270

Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2021). Fastp: An ultra-fast all-in-one FASTQ preprocessor. Nucleic Acids Research, 49(10), 5943–5956.https://doi.org/10.1093/nar

Grow, E. J., Flynn, R. A., Chavez, S. L., et al. (2015). Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature, 522(7555), 221–225. https://pmc.ncbi.nlm.nih.gov/articles/PMC4509593/

Zhang, Y., Li, X., & Wang, Z. (2021). Antisense oligonucleotide therapeutics: Mechanisms and applications. Asian Journal of Pharmaceutical Sciences, 16(5), 567–578. https://doi.org/10.1016/j.ajps.2021.04.005

Grandi, N., & Tramontano, E. (2019). Human endogenous retroviruses are ancient acquired elements still shaping innate immune responses. Virology, 538, 230–241. https://doi.org/10.1016/j.virol.2019.08.020

Tycko, J., DelRosso, N., Hess, G. T., et al. (2020). High-throughput discovery and characterization of human transcriptional effectors. Science Advances, 6(49), eabc3020. https://doi.org/10.1126/sciadv.abc3020

Turelli, P., Trono, D. (2015). Editing out endogenous retroviruses: Mechanisms and consequences. Nature Reviews Genetics. https://pmc.ncbi.nlm.nih.gov/articles/PMC5127361/

Steullet, P., Cabungcal, J. H., Cuenod, M., & Do, K. Q. (2013). Oxidative stress-driven parvalbumin interneuron impairment as a common mechanism in models of schizophrenia. Frontiers in Integrative Neuroscience, 7, 9. https://doi.org/10.3389/fnint.2013.00009

Zhang, X., et al. (2024). [Preprint on gene regulation / HERV-related mechanisms]. bioRxiv. https://doi.org/10.1101/2024.07.07.602402

Khandaker, G. M., Cousins, L., Deakin, J., Lennox, B. R., Yolken, R., & Jones, P. B. (2018). Inflammation and immunity in schizophrenia: Implications for pathophysiology and treatment. Schizophrenia Bulletin, 44(Suppl. 1), S287–S294. https://doi.org/10.1093/schbul/sby014

Li, W., et al. (2025). Targeting molecular pathways in neuroinflammation. Theranostics, 15, 3234–3250. https://www.thno.org/v15p3234.pdf

Balestrieri, E., et al. (2020). Human endogenous retroviruses and neuroinflammation in neurological disease. Frontiers in Microbiology. https://pmc.ncbi.nlm.nih.gov/articles/PMC7606184/

​

Plank , JR, CA;Dell’Acqua F;Sundram F;Hoeh NR;Muthukumaraswamy S;Lin JC; (n.d.). Mapping neuroinflammation with diffusion-weighted magnetic resonance imaging: A randomized crossover study. Biological psychiatry. Cognitive neuroscience and neuroimaging. https://pubmed.ncbi.nlm.nih.gov/40379248/

Rinaldi, C., & Wood, M. J. A. (2017, December1). Antisense oligonucleotides: The next frontier for treatment of neurological disorders. Nature News. https://www.nature.com/articles/nrneurol.2017.148​

​

Leon-Carrion, J., Leon-Dominguez, U., Pollonini, L., Wu, M. H., Frye, R. E., Dominguez-Morales, M. R., & Zouridakis, G. (2012). Synchronization between the anterior and posterior cortex determines consciousness level in patients with traumatic brain injury (TBI). Brain research, 1476, 22–30. https://doi.org/10.1016/j.brainres.2012.03.055

Barreda Tomás, F. J., Turko, P., Heilmann, H., Trimbuch, T., Yanagawa, Y., Vida, I., & Münster-Wandowski, A. (2020). BDNF Expression in Cortical GABAergic Interneurons. International journal of molecular sciences, 21(5), 1567. https://doi.org/10.3390/ijms21051567

Brambilla, P., Cipriani, A., Hotopf, M., & Barbui, C. (2005). Side-effect profile of fluoxetine in comparison with other SSRIs, tricyclic and newer antidepressants: a meta-analysis of clinical trial data. Pharmacopsychiatry, 38(2), 69–77. https://doi.org/10.1055/s-2005-837806