The Cullin-3 Connection: A Molecular Link Between Ubiquitin Dysregulation and Neuropsychiatric Disorders

Imagine someone who’s responsible for taking out the trash every Monday in a neighborhood, but they keep forgetting. At first, the buildup is just a small mess. But as the garbage continues to pile up, the smell gets worse, pests start showing up, and eventually it becomes difficult to even move around. What started as a simple oversight turns into a major disruption to daily life.

Recent research shows that buildup of unwanted waste in the cells – similar to that in the  neighbourhood –of developing embryos is strongly correlated with neurodevelopmental disorders such as Autism Spectrum Disorder (ASD), Intellectual Disabilities (ID), and Attention Deficit Hyperactivity Disorder (ADHD)[1]. The ‘clean up’ process of human cells is known as ubiquitination, and is led by a three protein cascade involving E1, E2 and E3 ligase.  It is a critical process that degrades and/or recycles proteins, helps maintain homeostasis, and regulates cell cycles. The backbone of this process involves an essential protein known as Cullin 3 [2].This protein is found in Cullin-RING E3 ubiquitin ligase (CRL3), the most commonly found E3 ligase, in the cytoplasm of neurons in the brain [3]. The expression of Cullin 3 is determined by a couple of  genes found on chromosome 16 and chromosome 2, KCTD13 and CUL 3 gene. KCTD13 encodes the protein KCTD13, and acts as a substrate recognizing adaptors for CUL3 based E3 ubiquitin ligase. Whereas, the CUL 3 gene is responsible for making the Cullin 3 protein that interacts with the RING-finger protein and a substrate adaptor protein to make the Cullin-RING E3 ubiquitin ligase [4].Therefore, the absence of, or even a duplicate of chromosome 16, can lead to dire consequences, as this directly impacts Cullin 3 production. 

Underexpression of the Cullin 3 protein in the central nervous system is directly linked to ASD and Schizophrenia. Cullin 3 activity is at its highest in the developing embryo and between childhood and adolescence. Therefore, it is crucial for the rapid and precise regulation of cell cycle progression, cytoskeletal organization, and signaling of key pathways.Cullin 3 primarily works by binding to other receptors like RBX1 and BTB substrate receptors. Therefore, when scientists genetically engineered mouse models in order to study Cullin 3’s function they found that a lack of it causes a build up of cell regulation protein like Cyclin E, which helps in the entry of the synthesis phase during DNA replication[5]. This is because the substrate receptor part of the complex, which does not function without Cullin 3,  is supposed to attach to Cyclin E and destroy it when it is not needed. Therefore, a lack of Cullin 3 results in unneeded proliferation of cells that should be stopped much earlier. This can cause DNA damage and errors in chromosome separation. In fact, Chromodomain Helicase DNA-binding protein 8 ( CHD8), one of the strongest risk genes associated with ASD, is directly related to the high activity of cyclin E2 in mammalian cells[6]. 

In addition to impaired cell regulation, protein build up in cells can also disrupt the delicate balance between excitation and inhibition. Research published in the Journal of Neuroscience found that Cullin 3 is critical for the function of dopaminergic (DA) neurons in major brain regions like the ventral tegmental area. Heightened excitability in these areas of the brain contribute to the social deficits, anxiety, and other behavioral changes that are observed in schizophrenia. Ultimately, a lack of Cullin 3 in human cells could be linked to the possible development of schizophrenia during the transition from childhood to adolescence [7]. 

Despite such strong connections between Cullin 3 and biomarkers that point towards these neurodevelopmental disorders, the exact role of Cullin 3 is still unclear. To date, research linking Cullin 3 to conditions such as autism and schizophrenia has primarily relied on the identification of de novo mutations in affected individuals and the development of mouse models with Cullin 3 deficiencies. The difficulty in creating these models without causing embryonic lethality has forced scientists to use conditional knockout. This process started being used in laboratories around the 1990s, and it inactivates a gene in a specific tissue at a specific time to analyze the gene’s role. However, its use to find Cullin 3’s role in the central nervous system did not begin abundantly until the early 2010s. This is because, even though we talk about neurodevelopmental disorders specifically, Cullin 3 is found throughout the body. So extinguishing  all of it kills the embryo itself. In addition, selectively inactivating this protein within specific tissues or regions is a complex task. So, as of now,to study Cullin 3 and its role in autism, researchers have used a haploinsufficient mouse model with a heterozygous deletion of the Cul3 gene (Cul3+/-) [8]. This model mimics human Cullin 3 and exhibits social, cognitive, and behavioral deficits, as well as neurodevelopmental abnormalities. As a result, scientists can track specific bio markers and symptoms associated with a lack of Cullin 3, but not its exact role. Therefore functions associated with Cullin 3 remain an ongoing research with new possible therapies and medications for individuals lacking this protein. 

Therapies at the moment focus on managing symptoms and providing care for the patient as needed. This may include speech therapy, feeding therapy, and any medication that helps manage symptoms that impede the quality of day to day life [9]. However, ongoing research has shown potential direction for clinical treatments that can be used in the future. Using animal models, like mice, researchers have mainly targeted signaling pathways disrupted by the lack of Cullin 3. For example, one potential treatment targets the processes disrupted by an accumulation of RhOA in the vascular smooth muscle cells, the main type of cell in the walls of arteries responsible for regulating blood pressure [10] This treatment administers Rhosin, a pharmacological inhibitor of RhOA. This, as a result, restores neuronal activity and dendritic length that have been impaired by the accumulation of RhOA. Other treatments focus on CRISPR gene editing to correct genetic defects. Essentially, CRISPR gene editing uses a guide RNA and the Cas 9 enzyme, to cut the faulty part of the DNA out and replace it with the right pair respectively [11]. This helps restore the functional gene that makes Cullin 3 as needed, and therefore all of its functionality. This is still a highly experimental and complex process, at the moment and is used mainly for research. However, possible treatments like these offer promising insights into how more specific, mechanism-based treatments might eventually be developed.

References

1. Lin, Ping, et al. “Current Trends of High-Risk Gene CUL3 in Neurodevelopmental Disorders.” Frontiers, Frontiers, 28 July. 2023.

2. Morandell, Jasmin, Lena A. Schwarz, Bernadette Basilico, Saren Tasciyan, Georgi A. Dimchev, Armel Nicolas, Christoph M. Sommer, et al. “Cul3 Regulates Cytoskeleton Protein Homeostasis and Cell Migration during a Critical Window of Brain Development.” Nature Communications, vol. 12, no. 1, 2021, article 3058.

3. Dubiel, Wolfgang, et al. “Cullin 3‑Based Ubiquitin Ligases as Master Regulators of Mammalian Deubiquitinating Enzymes.” Genome Biology, vol. 12, no. 4, 2011, article 220.

4. Akopian, D., et al. “Co‑adaptor Driven Assembly of a CUL3 E3 Ligase Complex.” Molecular Cell, vol. 82, no. 5, 2022, pp. 847–862.e8, PMC.

5. Zhou, Weihua, et al. “Genetically Engineered Mouse Models for Functional Studies of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases.” Nature News, Nature Publishing Group, 26 Mar. 2013,

6. Weissberg, Orly, and Evan Elliott. “The mechanisms of CHD8 in neurodevelopment and autism spectrum disorders.” Genes, vol. 12, no. 8, 2021, article 1133.

7. Gao, N., et al. “Deficiency of Cullin 3, a Protein Encoded by a Schizophrenia and Autism Risk Gene, Impairs Behaviors by Enhancing the Excitability of Ventral …” The Journal of Neuroscience, vol. 43, no. 36, 2023, pp. 6249.

8. Amar, Megha, et al. “Autism‑linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling.” Molecular Psychiatry, vol. 26, no. 7, 2021, pp. 3586–3613.

9. Montenegro, E. M. da Silva, et al. “Meta-Analyses Support Previous and Novel Autism Candidate Genes: Outcomes of an Unexplored Brazilian Cohort.” Autism Research, vol. 13, no., 2019.

10. Zhu, Yi, et al. “Calcium in Vascular Smooth Muscle Cell Elasticity and Adhesion: Novel Insights into the Mechanism of Action.” Frontiers, Frontiers, 6 Aug. 2019, www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2019.00852/full. 

11. Persons, Timothy M., et al. “CRISPR Gene Editing.” U.S. Government Accountability Office, 2020, report no. GAO‑20‑478SP.

12. “Conditional Gene Knockout.” ScienceDirect Topics, Elsevier.

13. Harper, J. W., and M. J. Tan. “Understanding Cullin-RING E3 Biology through Proteomics-Based Substrate Identification.” Molecular & Cellular Proteomics, vol. 11, no. 12, Dec. 2012, pp. 1541–1550. PMC, PMC3518111.

14. Sarikas, Antonio, Thomas Hartmann, and Zhen‑Qiang Pan. “The cullin protein family.” Genome Biology, vol. 12, no. 4, 2011, article 220.

15. “What Is CUL3‑Related Neurodevelopmental Disorder?” MedlinePlus Genetics, U.S. National Library of Medicine.

16. Zeliadt, Nicholette. “Loss of Autism Gene May Lead to Pile-up of Proteins in Neurons.” The Transmitter, 19 Dec. 2019.