Research Projects



Nucleocytoplasmic Transport

Both G4C2 repeat RNAs and DPRs disrupt nucleocytoplasmic transport (Fox and Tibbetts, Nature, 2015).

Previously, we have identified defects in nucleocytoplasmic transport as a critical pathogenic mechanism in c9ALS/FTD (Zhang et al., Nature, 2015). We have found that the G4C2 RNA binds and sequesters RanGAP, a key regulator of nucleocytoplasmic transport. As a result, importin-mediated transport is disrupted and some nuclear proteins are mislocalized to the cytoplasm. Importantly, a nuclear export inhibitor, KPT-276, suppress neurodegeneration in a c9ALS/FTD Drosophila model, suggesting that abnormal nucleocytoplasmic transport can be a therapeutic target of c9ALS/FTD. In addition, other groups have showed that DPRs can also disrupt nucleocytoplasmic transport. Together, these studies have highlighted the importance of nucleocytoplasmic transport in c9ALS/FTD pathogenesis.

After our discoveries, nucleocytoplasmic transport defects have been identified in other forms of ALS, including sporadic ALS, as well as several other neurodegenerative diseases, including Alzheimer’s and Huntington’s diseases. 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 can 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?

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;525(7567):56-61.

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;94(1):93-107.


Stress Granules

Stress granule assembly disrupts nucleocytoplasmic transport and contributes to neurodegeneration (Zhang et al., Cell, 2018).

As cytoplasmic protein aggregates disrupt nucleocytoplasmic transport, it is possible that disrupted nucleocytoplasmic transport can be a general cellular response to protein misfolding stress. Upon 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 upon 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 (Zhang et al., Cell, 2018). 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/organelles other than nucleocytoplasmic transport (e.g. DNA damage, cytoskeleton dynamics, etc.), and if yes, how?

  • Can stress granules be a therapeutic target for neurodegenerative diseases with protein aggregations?

Zhang K, Daigle JG, Cunningham KM, Coyne AC, Ruan K, Grima JC, Bowen KE, Wadhwa H, Yang P, Rigo F, Taylor JP, Gitler AD, Rothstein JD, Lloyd TE. Stress granule assembly disrupts nucleocytoplasmic transport, Cell. 2018;173(4):958-971.e17.


Liquid-liquid Phase Separation (LLPS) and Membrane-less Organelles in Cells

Liquid-liquid phase separation (from left to right): Solutes (red dots) evenly dissolved in a solution (blue mesh) undergoes LLPS to form a denser phase (red circles) that separates from the less dense phase.

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 membrane-less organelles, including nucleoli, speckles, Cajal bodies, heterochromatin protein complexes, etc., also 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 more than one separated liquid phases with different solute concentration are formed. In cells, LLPS is essential for the assembly and function of cellular membrane-less organelles, whereas abnormal LLPS can impair the formation and/or function of these organelles and contribute to neurodegeneration. Indeed, many proteins that aggregate in neurodegenerative diseases, such as DPRs, undergo LLPS. Furthermore, DPRs disrupt the LLPS of proteins in membrane-less organelles, such as stress granules, and disrupt the organelle function in c9ALS/FTD.


Using fluorescent recovery after photobleaching (FRAP) to distinguish a liquid condensate (the 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 a condensate with lasers, and monitor the fluorescence recovery. The fluorescence in dynamic condensates, e.g. stress granules, usually recover in seconds or minutes, whereas the fluorescence in solid aggregates do 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 technique, the figure below shows 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 GFP intensity is quantified on the right, with the pre-bleaching intensity set as 1 and the intensity immediately after bleaching set as 0.

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 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 the research mentioned above, the Zhang lab also studies the role of other cellular organelles and/or 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.