Doublecortin and the Death of a Dogma

By Isabella Savin

Doublecortin (DCX) is a microtubule-associated protein (MAP) that stabilises microtubules, dynamic protein polymers within cells that are critical to cell motility and migration. DCX contains two separate domains, namely CDC and NDC, which cryo-electron microscopy studies have shown to contribute to the nucleation of the tubulin subunits and its long-term stability, respectively (see Fig. 1).1 DCX’s association with microtubules also prevents them from depolymerising, allowing neuroblasts (undifferentiated neuron precursor cells) to migrate to the correct brain regions.

DCX also interacts with other MAPs, in particular, neurofascin, which controls axon guidance and outgrowth of neuronal projections.2 Furthermore, knocking out the DCX gene is associated with expression of intercellular adhesion molecules and cadherins,3 key for helping neurons adhere to surrounding cells. Without DCX, therefore, brains do not properly develop. In humans, mutations in the DCX gene results in a “double cortex” (hence the protein’s name) in hippocampal regions in females, and lissencephaly (a “smooth brain”) in males.4

The involvement of DCX in neural development and migration led to its growing popularity as a biomarker in neurogenesis, the process by which new neurons form from neural stem cells. Neurogenesis was first documented in the 1960s and has enormous therapeutic potential in treating strokes and neurodegenerative diseases. Promoting endogenous proliferation of neural stem cells, while incredibly challenging, could outweigh the loss of neural growth factors and reduce the cognitive decline associated with neurodegeneration.5 Thus, documenting and quantifying neurogenesis in the human brain is crucial.

DCX’s ability to pass through the blood-brain barrier and its non-toxicity, unlike other markers such as bromodeoxyuridine (a thymidine-like molecule which integrates into the DNA of newly dividing cells3), have rendered it the gold standard for neurogenesis research. DCX also exhibits time-specific expression patterns, with highest levels of expression in neuroblasts and immature neurons, useful for marking the trajectory of neuronal maturation. DCX is usually fluorescently labelled and measured in combination with neural nuclear antigen (NeuN) protein, a marker of mature neurons; DCX downregulation occurs on the 10th day of newly formed neurons of the adult human olfactory bulb, and NeuN is upregulated concurrently.6

However, DCX’s efficacy as a biomarker has recently been disputed. Its specificity to neuronal cells was initially attractive, however immunostaining now suggests DCX is also expressed in other cells of the central nervous system. Verwer et al. found DCX-positive (DCX+) astrocytes in post-mortem human hippocampal slices and found few DCX+ cells with the classical bipolar morphology of migrating neuroblasts.7 Similarly, DCX mRNA has been found in cells expressing glial cell markers.8 These data call into question the accuracy of DCX and suggest a need to systematically evaluate current neuron-specific biomarkers.

DCX+ cells are frequently found within canonical neurogenesis regions, such as the hippocampus and dentate gyrus (DG), regions involved in memory and learning, and the walls of the lateral ventricles. For instance, Klempin et al. used DCX conjugated with green fluorescent protein (DCX-GFP) to localise DCX expression in transgenic mice and observed fluorescence in the DG as hypothesised.10 However, searching for DCX+ cells in these regions alone creates selection bias, inflating the frequency of positive results. Thus, the entire brain’s neurogenic capacity needs to be fully characterised by region; indeed, DCX+ cells have been observed in regions not considered neurogenic, such as the rodent piriform10 and cerebral11 cortices. However, extrapolating such results to humans should be done cautiously. Even though these regions show high DCX expression, research is required to confirm whether immature DCX+ cells are properly integrated into existing neural circuits, a matter of debate since the initial discovery of neurogenesis.

Numerous techniques have also demonstrated fluctuations in DCX expression with age, generating controversy over whether neurogenesis is maintained throughout life. Western blotting to quantify DCX protein expression within the human occipital cortex found DCX is highly expressed during embryonic development, which decreases to an almost indistinguishable level in adult cells.9 More recently, Sorrells et al. used single-molecule in situ hybridisation, a technique enabling localisation of a nucleic acid sequence in cells, combined with fluorescence microscopy to localise DCX mRNA transcripts in post-mortem cells near the human DG at various stages of life. They observed a sharp decline in DCX transcripts from birth, and no fluorescence was seen from 19 years onwards.8

Therefore, ironically, the use of DCX as a biomarker has provided evidence against neurogenesis. However, the inconsistencies of DCX measurements have ushered in the use of novel techniques which provide a more holistic, representative, and quantitative approach to quantifying neurogenesis than DCX immunostaining, which only investigates small groups of cells and is specific to only one marker. Only last month, Franjic et al. employed single-nucleus RNA sequencing (snRNA-seq) to analyse the total RNA expression in nuclei of cells from the adult human DG. Remarkably, out of the 139,187 nuclei sequenced, only 2 showed appropriate transcriptomes for neural precursor cells,12 significantly lower than estimates with DCX staining and suggesting adult human neurogenesis is rare, if it occurs at all. Functional magnetic resonance imaging (fMRI) has also been employed to assess neurogenesis in vivo, however not all neurogenesis is detectable with fMRI, and it is often unclear whether regional changes are attributable to increased plasticity of existing neurons or the growth of new ones.13 High-throughput proteomic analyses in various brain cell types, to highlight differentially expressed proteins and their functions and signalling pathways, currently show promise for identifying more specific markers,14 as do single-cell sequencing analyses like those performed by Franjic and colleagues.  

While DCX has clearly outlived its utility as a neurogenesis biomarker, it has encouraged scientists to turn to unbiased analyses outside predetermined regions and markers, which will surely stimulate further debate and research on neurogenesis in the coming years.

References:

  1. Manka SW, Moores CA. Pseudo‐repeats in doublecortin make distinct mechanistic contributions to microtubule regulation. EMBO Reports. 2020;21(12). https://doi.org/10.15252/embr.202051534.
  2. Ayanlaja AA, Xiong Y, Gao Y, Ji G, Tang C, Abdikani Abdullah Z, et al. Distinct Features of Doublecortin as a Marker of Neuronal Migration and Its Implications in Cancer Cell Mobility. Frontiers in Molecular Neuroscience. 2017;28(10). https://doi.org/10.3389/fnmol.2017.00199.
  3. Shahsavani M, Pronk RJ, Falk R, Lam M, Moslem M, Linker SB, et al. An in vitro model of lissencephaly: expanding the role of DCX during neurogenesis. Molecular Psychiatry. 2017;23(7): 1674–84. https://doi.org/10.1038/mp.2017.175.
  4. GeneCards. DCX Gene – Doublecortin. https://www.genecards.org/cgi-bin/carddisp.pl?gene=DCX. [Accessed 25th February 2022].
  5. Brinton DR, Wang JM. Therapeutic Potential of Neurogenesis for Prevention and Recovery from Alzheimer’s Disease: Allopregnanolone as a Proof of Concept Neurogenic Agent. Current Alzheimer Research. 2006;3(3): 185–90. https://doi.org/10.2174/156720506777632817.
  6. Brown JP, Couillard-Després S, Cooper-Kuhn CM, Winkler J, Aigner L, Kuhn HG. Transient expression of doublecortin during adult neurogenesis. Journal of Comparative Neurology. 2003;467(1): 1–10. https://doi.org/10.1002/cne.10874.
  7. Verwer RWH, Sluiter AA, Balesar RA, Baayen JC, Noske DP, Dirven CMF, et al. Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin. Brain. 2007;130(12): 3321–35. https://doi.org/10.1093/brain/awm264.
  8. Sorrells SF, Paredes MF, Cebrian-Silla A, Sandoval K, Qi D, Kelley KW, et al. Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature. 2018;555(7696): 377–81.
  9. Gleeson JG, Lin PT, Flanagan LA, Walsh CA. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron. 1999;23(2): 257–71. https://doi.org/10.1016/s0896-6273(00)80778-3.
  10. Klempin F, Kronenberg G, Cheung G, Kettenmann H, Kempermann G. Properties of Doublecortin-(DCX)-Expressing Cells in the Piriform Cortex Compared to the Neurogenic Dentate Gyrus of Adult Mice. PLoS ONE. 2011;6(10). https://doi.org/10.1371/journal.pone.0025760.
  11. Kremer T, Jagasia R, Herrmann A, Matile H, Borroni E, Francis F, et al. Analysis of Adult Neurogenesis: Evidence for a Prominent “Non-Neurogenic” DCX-Protein Pool in Rodent Brain. PLoS ONE. 2013;8(5): e59269. https://doi.org/10.1371/journal.pone.0059269.
  12. Franjic D, Skarica M, Ma S, Arellano JI, Tebbenkamp ATN, Choi J, et al. Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells. Neuron. 2021;110(3): 452–69.e14. https://doi.org/10.1016/j.neuron.2021.10.036.
  13. Ho NF, Hooker JM, Sahay A, Holt DJ, Roffman JL. In vivo imaging of adult human hippocampal neurogenesis: progress, pitfalls and promise. Molecular Psychiatry. 2013 Feb 26;18(4):404–16. https://doi.org/10.1038/mp.2013.8.
  14. Mathew B, Mansuri MS, Williams KR, Nairn AC. Exosomes as Emerging Biomarker Tools in Neurodegenerative and Neuropsychiatric Disorders—A Proteomics Perspective. Brain Sciences. 2021 Feb 1;11(2):258. https://doi.org/10.3390/brainsci11020258.

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