Alzheimer's Disease Pathology: Insig-1 Loss Connects Amyloid Beta to Abnormal Lipid Synthesis

Background:

Annually there's 10 million new diagnoses of dementia, 60-80% are Alzheimer’s disease (AD), causing severe neurodegenerative effects to the brain.

Amyloid beta accumulation is a hallmark of AD, disrupting neuronal communication and causing neuronal death.

The ubiquitin-proteasome system (UPS) clears proteins with ubiquitin tagging and proteasome degradation. Many cleared proteins influence cell functions. Amyloid beta can dysregulate the UPS, which disrupts cell function.

Insulin-induced gene-1 (Insig-1) is a protein cleared by the UPS. Insig-1 prevents the activation of Sterol Regulatory Element-Binding protein (SREBP1), which activates lipid synthesis genes for cholesterol. Normally, Insig-1 is cleared by UPS allowing SREBP1 to activate and start lipid synthesis.

Knowledge Gap:

Currently, AD has no cure, partly due to incomplete understanding of how cellular mechanisms interact in AD. One of these interactions is between amyloid beta-driven UPS dysregulation on lipid synthesis.

Amyloid beta dysregulates the UPS, but consequences on lipid synthesis are unknown.

Furthermore, Insig-1's role in AD pathology and amyloid beta-driven UPS dysregulation remains poorly characterized.

Abnormal lipid and cholesterol levels are associated with AD and can increase amyloid beta production. This suggests an underdeveloped understanding of a feedback loop. Cholesterol synthesis is represented by Insig-1 and SREBP1 interaction.

Goal:

Determine if amyloid beta-driven UPS dysregulation influences lipid synthesis through Insig-1/SREBP1 pathway

Achieved through objectives:

Determine that amyloid beta dysregulates UPS

Determine if amyloid beta-driven UPS dysregulation alters Insig-1 levels

If Insig-1 levels are altered does it affect SREBP1-mediated lipid synthesis

Compare amyloid beta-driven UPS dysregulation to complete UPS inhibition

Cell Treatment:

Neuro-2a mouse cells treated with 0, 0.2, 0.5, 1, and 5µM amyloid beta for 48 hours

LDH cytotoxicity assay confirms cell viability

Protein quantification through BCA assay

Anti-Ubiquitin Western Blot:

Determine that amyloid beta dysregulates UPS

Anti-ubiquitin antibody detects polyubiquitinated proteins, proteins yet to be cleared by the proteasome. Measuring UPS dysregulation from amyloid beta

Lysates separated using SDS-PAGE

Transferred to PVDF membrane

Membrane blocked in 5% milk in TBST for 60 minutes

Probed with anti-ubiquitin primary antibody overnight at 4°C

Washed 3x with TBST

Incubated with HRP-conjugated secondary antibody for 60 minutes

Insig-1 Immunoprecipitation:

Determine if amyloid beta-driven UPS dysregulation alters Insig-1 levels

Immunoprecipitation isolates Insig-1 using antibody and magnetic beads. Insig-1 ubiquitination levels specifically can be observed, not all protein levels.

2µg of Insig-1 mouse monoclonal antibody conjugated to Dynabeads by Protein G-Sepharose at room temperature for 1.5 hours

Magnetic force isolates Insig-1-bead complex

250 µg of cell lysate to antibody-coated beads and rotated overnight at 4°C

Beads were washed 2x in PBS

Eluted using Laemmli buffer at 100°C removing Insig-1 solution from beads

Transferred to PVDF membrane overnight at 4°C

Probed with anti-Insig-1 antibody

TidyBlot Reagent-HRP eliminates interference

Re-probed with anti-ubiquitin antibody, detecting ubiquitin chains on Insig-1

SREBP1 Western Blot:

If Insig-1 levels are altered does it affect SREBP1-mediated lipid synthesis

SREBP1 activation levels using antibody across amyloid beta, which provides final output if amyloid beta-driven UPS dysregulation effects lipid synthesis.

Western blot steps in red are repeated

However, probed with anti-SREBP1 primary antibody

Proteasome Inhibition Transcriptomic Analysis:

Compare amyloid beta-driven UPS dysregulation to complete UPS inhibition

Dataset GSE22465 observes lactystin-treated (proteasome inhibited) and control mouse cortical neurons. Analyzed using iDEP's differential gene expression analysis and InnateDB's pathway over-representation analysis to observe differences in pathways.

Determine that amyloid beta dysregulates UPS:

Anti-ubiquitin western blot revealed high-molecular weight bands (~200kDa) representing polyubiquitinated proteins, this can be interpreted as proteins waiting to be degraded. Signal increased with amyloid beta dose, indicating overwhelm in the UPS and a growing backlog of proteins.

Densitometry quantification of proteins confirmed this trend, using integrated density, this is the sum of pixel intensity values. One-way ANOVA of the fold change of integrated density showed amyloid beta concentrations greater than 0.2µM had significantly greater ubiquitin levels than the control (p=0.03). Dose-response trend showed ubiquitin signals change significantly between amyloid beta concentrations (p=0.023).

Results demonstrates amyloid beta does dysregulate UPS indicated by a progressive increase in ubiquitinated proteins with amyloid beta concentration.

Determine if amyloid beta-driven UPS dysregulation alters Insig-1 levels:

To examine Insig-1 specifically, immunoprecipitation was performed followed by re-probing with anti-ubiquitin antibody.

Active Insig-1 bands (~35kDa, in sky blue box), decreased with increasing amyloid beta concentration. Bands show that functional Insig-1, that actively prevents SREBP1 activation, declines with more intense amyloid beta-driven UPS dysregulation.

Polyubiquitinated Insig-1 bands (in teal box), showed an even more pronounced decrease, suggesting rapid UPS-mediated degradation of Insig-1. This is consistent with the anti-ubiquitin results, which suggested a backlogged UPS accelerating protein degradation.

One-way ANOVA of the fold change of integrated density showed that all concentrations of amyloid beta had significantly lower levels of Insig-1 than compared to control (p=0.006). Insig-1 levels were shown to significantly decrease progressively with amyloid beta concentration using dose-response trend (p=0.0092).

Overall, Insig-1 immunoprecipitation results affirmed that amyloid beta-driven UPS dysregulation alters Insig-1 levels negatively by increasing degradation of Insig-1.

If Insig-1 levels are altered does it affect SREBP1-mediated lipid synthesis:

SREBP1 western blot demonstrated an increase in signal with amyloid beta concentration (~55kDA), indicating higher rates of lipid synthesis initiation.

SREBP1 activation in all amyloid beta concentrations are significantly greater than control levels using one-way ANOVA (p=0.012). Furthermore, dose-response trend demonstrated that SREBP1 progressively increased significantly with amyloid beta concentration (p=0.017).

Elevated SREBP1 activation signals the initiation of cholesterol lipid synthesis through downstream gene activation, providing evidence that amyloid beta-driven UPS dysregulation abnormally influences lipid synthesis via the Insig-1/SREBP1 pathway.

Compare amyloid beta-driven UPS dysregulation to complete UPS inhibition:

A transcriptomic dataset from proteasome-inhibited mouse cortical neurons was analyzed. Since the wet lab demonstrated an overwhelmed UPS, opposite results are expected with complete UPS inhibition.

Of 128 significantly differentially expressed genes, a cluster of 16 showed significant downregulation in pathways including SREBP-mediated gene activation, cholesterol biosynthesis, and cholesterol metabolism in the UPS inhibition group.

Contrast reinforces that the UPS is a major regulator of SREBP1-driven lipid synthesis, and that amyloid beta-driven UPS dysregulation abnormally elevates this process.

Conclusion

All objectives were met and proved through evidence from the assays with a logical pathway by observing relationships between amyloid beta, UPS, Insig-1, and SREBP1. Collectively, the evidence demonstrates that amyloid beta-driven UPS dysregulation does abnormally influence lipid synthesis.

Feedback Loop and Reframing Lipids in AD:

Findings point toward a feedback loop in AD pathology that is underdeveloped. Amyloid beta overwhelms the UPS, degrading Insig-1 and freeing SREBP1 to drive excessive lipid and cholesterol synthesis, in turn producing more amyloid beta, restarting the cycle.

This positions lipid dysregulation not as a downstream consequence of AD, but a contributor to progression. Cholesterol-rich lipid rafts facilitate cleavage of amyloid precursor protein into amyloid beta. This contextualizes the high SREBP1 activation to directly impact amyloid beta production.

Results further explain the progressive nature of AD by mechanistically incorporating lipid synthesis and better characterizing a feedback loop of AD surrounding lipids.

Insig-1: Novel Target for Treatment and Diagnoses:

Three molecules appear as viable targets the UPS, SREBP1, and Insig-1. Repairing UPS function can interevent progression. SREBP1 inhibitors already used in cancer and metabolic disease could be repurposed for AD. Tracking ubiquitination and SREBP1 levels could make earlier, more accurate diagnosis possible.

However, Insig-1 stands out.

Downstream effects of the UPS and SREBP1 has begun to be investigated in AD research, but Insig-1 has not been meaningfully characterized in AD context, upstream or downstream. These findings are among the first to contextualize Insig-1 in AD, making it a genuinely novel target for both treatment and diagnosis.

Societal Impact:

Results identify Insig-1 as a mechanistic link between current knowledge and lipids, offering a novel druggable target and a new direction for treatment in a disease affecting 55 million people worldwide with no cure.

This study identified Insig-1 as a potential target for AD by observing pathology through lipids, opening potential therapeutic applications focusing on inhibiting SREBP1 to develop a cure for AD. This can be achieved by preventing the degradation of Insig-1 by the UPS. A potential candidate is Insig-1-derived inhibitory peptide (Indip), which blocks degradation of Insig-1. Developing Indip as a neuroprotective agent can lead to a therapeutic against AD using lipids.

Quantifying lipid activation would provide more confidence, rather than inferring lipid synthesis from known aspects of SREBP1. Lipid metabolism activity assays using lipophilic dyes or enzymatic assays would achieve this.

I would like to thank Dr. Taghibiglou who helped provide resources and helped mentor me throughout the project, answering many of my questions. Nishat Nawal who was generous enough to help me in the lab. I would also like to extend my thanks to all those who were in the lab with me and everyone at the neuroscience cluster at USASK.

Finally, thank you to all the people in my life who were incredibly patient, understanding, and supportive of me throughout the project.

Previous Project:

Chang, B. (n.d.). Linking proteasomal dysfunction and alzheimer’s disease through novel Biomarker Identification. Youth Science Canada. https://partner.projectboard.world/ysc/project/linking-proteasomal-dysfunction-and-alzheimers-disease-through-novel-biomarker-discovery

Images:

Estimated growth in number of people with dementia 2019-2050. (n.d.). Alzheimer’s Disease International. Retrieved from https://www.alzint.org/u/Estimated-growth-in-number-of-people-with-dementia-2019%E2%80%932050_Final-e1630609971569.jpg.

Hegde, A. N. (n.d.). Perturbations of Ubiquitin-Proteasome-Mediated Proteolysis in Aging and Alzheimer’s Disease. Frontiers in Aging Neuroscience. Retrieved from https://www.frontiersin.org/journals/aging-neuroscience/articles/10.3389/fnagi.2019.00324/full.

Immunoprecipitation worklow (. (n.d.). Rockland. Retrieved from https://www.rockland.com/resources/immunoprecipitation-technique/?srsltid=AfmBOoqo2KJ6xE3d1LdeVN8yVcwzRFPpFEEe55Opw6GF4LNrdWvIzojx.

Lipid Rafts Figure. (n.d.). ResearchGate. Retrieved from https://www.researchgate.net/figure/Schematic-representation-of-amyloid-precursor-protein-APP-palmitoylation-modulating_fig1_334965868.

Western Blot Protocol, Troubleshooting, and Applications. (n.d.). The Scientist. Retrieved from https://www.the-scientist.com/western-blot-protocol-troubleshooting-and-applications-71995.

Tools:

Breuer, K., Foroushani, A. K., Laird, M. R., Chen, C., Sribnaia, A., Lo, R., Winsor, G. L., Hancock, R. E., Brinkman, F. S., & Lynn, D. J. (2012). InnateDB: Systems Biology of innate immunity and beyond—recent updates and continuing curation. Nucleic Acids Research, 41(D1). https://doi.org/10.1093/nar/gks1147

Ge, S. X., Son, E. W., & Yao, R. (2018). IDEP: An integrated web application for differential expression and pathway analysis of RNA-Seq Data. BMC Bioinformatics, 19(1). https://doi.org/10.1186/s12859-018-2486-6

Journals:

Alzheimer's Disease, Amyloid Beta Oligomers & Neuronal Dysfunction

Dinamarca, M. C., Raveh, A., Schneider, A., Fritzius, T., Früh, S., Rem, P. D., Bian, X., Bhatt, D. L., Bhattacharya, A., Bhattacharya, A., & Bhatt, D. (2021). Amyloid β-based therapy for Alzheimer's disease: Challenges, successes and future. Signal Transduction and Targeted Therapy, 8, 248. https://doi.org/10.1038/s41392-023-01484-7

Ferreira, S. T., Lourenco, M. V., Oliveira, M. M., & De Felice, F. G. (2015). Soluble amyloid-β oligomers as synaptotoxins leading to cognitive impairment in Alzheimer's disease. Frontiers in Cellular Neuroscience, 9, 191. https://doi.org/10.3389/fncel.2015.00191

Ubiquitin-Proteasome System (UPS) in Alzheimer's Disease

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Sulistio, Y. A., & Heese, K. (2016). The ubiquitin-proteasome system and molecular chaperone deregulation in Alzheimer's disease. Molecular Neurobiology, 53(2), 905–931. https://doi.org/10.1007/s12035-014-9063-4

Zhang, K., Chen, S., Yang, Q., Guo, S., Chen, Q., Li, Z., Liu, H., Jiang, M., Kaminski, N., Jiang, D., Zhou, Y., & Liang, J. (2022). The ubiquitin-proteasome system in Alzheimer's disease: Mechanism of action and current status of treatment. Frontiers in Aging Neuroscience, 14, 990749. https://doi.org/10.3389/fnagi.2022.990749

Insig-1, SREBP1 & Lipid Metabolism Regulation

Lee, J. N., & Ye, J. (2004). Proteolytic activation of sterol regulatory element-binding protein induced by cellular stress through depletion of Insig-1. Journal of Biological Chemistry, 279(43), 45257–45265. https://doi.org/10.1074/jbc.M408235200

Nohturfft, A., & Zhang, S. C. (2009). Coordination of lipid metabolism in membrane biogenesis. Annual Review of Cell and Developmental Biology, 25, 539–566. https://doi.org/10.1146/annurev.cellbio.24.110707.175308

Ye, J., & DeBose-Boyd, R. A. (2011). Regulation of cholesterol and fatty acid synthesis. Cold Spring Harbor Perspectives in Biology, 3(7), a004754. https://doi.org/10.1101/cshperspect.a004754

Lipid Dysregulation in Alzheimer's Disease

Di Paolo, G., & Kim, T.-W. (2011). Linking lipids to Alzheimer's disease: Cholesterol and beyond. Nature Reviews Neuroscience, 12(5), 284–296. https://doi.org/10.1038/nrn3012

Grimm, M. O. W., Grimm, H. S., & Hartmann, T. (2012). The role of APP proteolytic processing in lipid metabolism. Experimental Brain Research, 217(3–4), 365–375. https://doi.org/10.1007/s00221-011-2975-6

Picard, C., Bhatt, D. L., Bhattacharya, A., & Bhatt, D. (2021). Involvement of lipids in Alzheimer's disease pathology and potential therapies. Frontiers in Physiology, 12, 703. https://doi.org/10.3389/fphys.2020.00598