Projects

Nucleocytoplasmic transport

Previously, we identified defects in nucleocytoplasmic transport as a critical pathogenic mechanism in C9orf72-mediated amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS-FTD). Since our discovery, nucleocytoplasmic transport defects have been identified in other forms of amyotrophic lateral sclerosis (ALS), including sporadic ALS, as well as in several other neurodegenerative diseases, including Alzheimer's disease and Huntington's disease. Furthermore, cytoplasmic protein aggregates, a common feature observed in many neurodegenerative diseases, have been shown to disrupt nucleocytoplasmic transport, suggesting that defects in nucleocytoplasmic transport could be a general mechanism of neurodegeneration.

Open questions

  • What is the downstream effect of disrupted nucleocytoplasmic transport in c9ALS-FTD?
  • Can disrupted nucleocytoplasmic transport be a therapeutic target for neurodegenerative diseases other than c9ALS-FTD?
  • Is disrupted nucleocytoplasmic transport a primary cause of neurodegeneration?
  • Does disrupted nucleocytoplasmic transport contribute to aging and other age-related diseases, and if yes, how?

Related publications

  • Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, Daley EL, Miller SJ, Cunningham KM, Vidensky S, Gupta S, Thomas MA, Hong I, Chiu SL, Huganir RL, Ostrow LW, Matunis MJ, Wang J, Sattler R, Lloyd TE, Rothstein JD. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015; doi:10.1038/nature14973.
  • Fox, B., Tibbetts, R. Problems at the nuclear pore. Nature. 2015; doi.org/10.1038/nature15208.
  • Grima JC, Daigle JG, Arbez N, Cunningham KC, Zhang K, Ochaba J, Geater C, Morozko E, Stocksdale J, Glatzer JC, Pham JT, Ahmed I, Peng Q, Wadhwa H, Pletnikova O, Troncoso JC, Duan W, Snyder SH, Ranum LPW, Thompson LM, Lloyd TE, Ross CA, Rothstein JD. Mutant Huntingtin disrupts the nuclear pore complex. Neuron. 2017; doi:10.1016/j.neuron.2017.03.023.

Stress granules

As cytoplasmic protein aggregates disrupt nucleocytoplasmic transport, it is possible that disrupted nucleocytoplasmic transport could be a general cellular response to protein misfolding stress. Under protein misfolding stress, cells halt their translation to cope with the chaperone burden. In this case, the large ribosomal subunit dissociates from the mRNA, which then recruits many RNA-binding proteins, leading to the formation of protein-RNA condensates called stress granules. The assembly of these condensates is mediated by the liquid-liquid phase separation (LLPS) of RNA-binding proteins, a process that can cause these proteins to aggregate. As protein aggregation is a common feature in neurodegenerative diseases, stress granules are considered a crucial pathogenic contributor to several neurodegenerative diseases.

We found that under cellular stress, key nucleocytoplasmic transport factors, including karyopherins (importins and exportins), Ran GTPase and nucleoporins translocate to stress granules, leading to defective nucleocytoplasmic transport. Importantly, inhibiting stress granule assembly suppresses these defects, as well as neurodegeneration, in c9ALS-FTD models. Together, these findings connected two pathophysiological processes — stress granule assembly and nucleocytoplasmic transport disruption — in a unified pathway that contributes to pathogenesis.

Open questions

  • What regulates stress granule assembly and dynamics?
  • What proteins are sequestered in stress granules?
  • Do stress granules functionally relate to cellular processes and organelles other than nucleocytoplasmic transport, such as DNA damage and cytoskeleton dynamics? If yes, how?
  • Can stress granules be a therapeutic target for neurodegenerative diseases with protein aggregations?

Related publications

LLPS and membraneless organelles in cells

Stress granules are RNA-protein condensates with liquid-like characteristics. Unlike solid aggregates, these condensates are dynamic, dissolvable and functional compartments devoid of surrounding membranes. In addition to stress granules, many other forms of membraneless organelles, including nucleoli, speckles, Cajal bodies, heterochromatin protein complexes and others, also are present in cells. The formation of these condensates is mediated by LLPS of their protein components.

LLPS is a process by which a solute de-mixes within the solvent so that multiple separated liquid phases with different solute concentration are formed. In cells, LLPS is essential for the assembly and function of cellular membrane-less organelles; however, abnormal LLPS can impair the formation and function of these organelles and contribute to neurodegeneration. Indeed, many proteins that aggregate in neurodegenerative diseases, such as dipeptide repeats (DPRs), undergo LLPS. Furthermore, DPRs disrupt the LLPS of proteins in membraneless organelles, such as stress granules, and disrupt organelle function in c9ALS-FTD.

Diagram showing how fluorescent recovery after photobleaching (FRAP) is used to distinguish liquid from solid

Using fluorescent recovery after photobleaching (FRAP) to distinguish a liquid condensate (green circle surrounded by red spotted lines, left) from a solid aggregate (right)

To study the dynamics of protein condensates in cells, we use the fluorescent recovery after photobleaching (FRAP) technique to measure how fast proteins exchange between the condensates and their surroundings. We fluorescently label the proteins that can phase-separate to form condensates, bleach the fluorescence of these condensates with lasers and monitor the fluorescence recovery. The fluorescence in dynamic condensates, such as stress granules, usually recovers in seconds or minutes, whereas the fluorescence in solid aggregates does not recover for hours. As functional compartments in cells are usually dynamic, the FRAP technique allows us to assess the function of these condensates.

Using this method, the figure below shows that stress granules are dynamic.

FRAP experiment results showing that stress granules are dynamic

FRAP experiment showing that stress granules are dynamic, liquid-like compartments. Red circles indicate the stress granule analyzed. Numbers indicate the recovery time after photobleaching. The recovered green fluorescent protein (GFP) intensity is quantified on the right, with the pre-bleaching intensity set as 1 and the intensity immediately after bleaching set as 0.

Open questions

  • How does abnormal LLPS cause neurodegeneration?
  • What regulates the LLPS of DPRs and how?
  • Does LLPS relate to DPR toxicity?
  • Can abnormal LLPS be a therapeutic target of neurodegenerative diseases?

Other cellular organelles and processes

In addition to undertaking the research mentioned above, the Zhang lab studies the role of other cellular organelles and processes in neurodegeneration. We perform unbiased genetic screens in Drosophila models to identify genes that play roles in neurodegeneration and then study the function of these genes. After that, we study the cellular mechanisms in human cell lines and then, verify our findings in patient iPSC-derived neuron and mouse models.