Liquid liquid phase separation:
Liquid liquid phase separation (LLPS) brings together functionally related proteins through the intrinsic biophysics of proteins in a process that is driven by reducing free energy and maximizing entropy. The process of LLPS allows proteins to form structures, termed membraneless organelles, that help the proteins work together to carry out specific functions. RNA and DNA present long chained charged polymers that promote LLPS. Consequently, many RNA binding proteins (RBPs) and DNA binding proteins form membraneless organelles. However, the highly concentrated phase separated state creates conditions that also promote formation of irreversible protein aggregates. Mutations in RNA and DNA binding proteins that increase the stability of irreversible aggregates also increase the accumulation of irreversible aggregates from membraneless organelles, as well as directly. Many of the RBPs that exhibit disease-linked mutations carry out cytoplasmic actions through stress granules, which are a pleiotropic type of RNA granule that regulates the translational response to stress. Phosphorylation and oligomerization convert the microtubule associated protein tau to a state where it also regulates RNA translation through interactions with RBPs and ribosomal proteins; we propose that this is a major reason that tau becomes phosphorylated with stress. Persistent stress, such as occurs in disease, leads to the accumulation of irreversible aggregates composed of RBPs or tau, which then cause toxicity and form many of the hallmark pathologies of major neurodegenerative diseases. This pathophysiology ultimately leads to multiple forms of neurodegenerative diseases, the specific type of which reflects the temporal and spatial accumulation of different aggregating proteins.
Understanding the relationship between tau and RNA metabolism has taken on an increasingly prominent footprint in the Wolozin laboratory. Tau has been known interact with RNA since 1996, when it was demonstrated that RNA promotes tau fibrillization. The significance of this interaction has only become apparent recently. Biophysical studies demonstrate that tau phase separates in a manner that is increased by the presence of RNA. This sensitivity to RNA belies a more profound biological role for tau in RNA metabolism. Hyperphosphorylated tau (pTau) rapidly generates oligomeric tau (oTau), and these species selectively interact with many components of RNA metabolism. pTau (and presumably oTau) regulates the translational stress response; it both promotes and is required for a normal translational stress response, which is associated with stress granule formation. pTau also binds to the ribosomal protein RPS6. Tau also appears to play a role in ribosomal genesis, because the tau isoform, 2N4R, localizes to the nucleolus under basal conditions, but moves away from the nucleolus to other parts of the nucleus upon phosphorylation during stress. Many proteins change function upon phosphorylation or oligomerization. For example, the binding profiles of p53 differs markedly between monomeric and oligomeric forms. Oligomerization is also known to initiate signaling of many receptors, and also regulates the function of many intracellular signaling cascades.
Work from the Wolozin laboratory increasingly indicates that the biological logic of tau phosphorylation and oligomerization flows from the exact same processes, where phosphorylation and oligomerization of tau switches its binding partners and functions from that of regulating microtubules in the axon to that of regulating the translational stress response in the cytoplasm.
Research from our laboratory places RNA binding proteins such as TIA1, HNRNPA2B1 and EIFs at the center of the biology of tau in stress. We are developing many tools for investigating this biology including optogenetics, proteomics, conditional knockout mice, 3D iPSC organoids, synthetic biology and advanced biophysical techniques.
Amyotrophic Lateral Sclerosis (ALS):
The relationship between RNA binding proteins and disease first became apparent with the discovery of Tar DNA binding protein – 43 (TDP-43) as the major protein that accumulates in the spinal cords of patients with Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is a nuclear protein that normally functions as a component of the splicesome, but translocates to the cytoplasm during times of stress, associating with SGs. TDP-43 can phase separate due to interactions in its LCD. TDP-43 fibrils are also capable of inducing further TDP-43 aggregation in cells. Disease associated mutations in TDP-43 tend to occur in the LCD, and increase the propensity of the protein to aggregate, which shifts the balance between LLPS and irreversible aggregation such that aggregated TDP-43 accumulates in the neurons over time. The aggregates typically accumulate in the cytoplasm, occurring with a corresponding loss of nuclear TDP-43. In some cases of FTD (Type IV), visible accumulations of TDP-43 aggregates are present in the nucleus, but studies suggest that stress can cause TDP-43 can to form insoluble, non-functional nuclear aggregates even when no visible aggregates are present in the nucleus. The loss of nuclear TDP-43 function, perhaps combined with the loss of TDP-43 function at other sites outside of the nucleus (e.g., at synapses) is thought to precipitate the neurodegeneration that we associate with ALS.
The work on TDP-43 and ALS has been powered by a strong collaboration with the Emili laboratory using advanced proteomics. The Emili laboratory recently developed a novel approach for interrogating molecular complexes in an unbiased manner, which is termed BraInMap, and using this we identified the endogenous complexes that interact with TDP-43. We are extending this work by examining models of ALS, human tissues and adding other advanced techniques to our repertoire, such as proximity profile labeling. These tools allow us to map out pathways of biology and disease in a spatially and temporally specific manner in neurons during disease progression.
The research on Parkinson Disease focuses on genetic factors implicated in Parkinson’s disease, including LRRK2, α-synuclein, parkin and DJ-1. Research in our laboratory suggests that genetic mutations linked to Parkinson’s disease converge onto two main cellular pathways, mitochondrial function or management of misfolded proteins Using genetically modified cells (e.g., primary neuronal cultures or cell lines) and genetically modified animals (C. elegans and transgenic mice), we have demonstrated that LRRK2 enhances the sensitivity of dopaminergic neurons to toxins such as rotenone, a mitochondrial inhibitor, and we are currently investigating fascinating interactions between mitochondrial functions and autophagy. Our work also suggests particular biochemical pathways that mediate the actions of LRRK2. We have have found that LRRK2 binds to MKK6, a kinase that lies upstream of p38 and regulates the stress response. We also discovered that LRRK2 binds rac1, which is a small GTPase; binding to rac1 translocates LRRK2 from the cytoplasm to the membrane and stimulates process outgrowth. We are using systems biology approaches, including context likelihood of relatedness (CLR) and mode-of-action by network identification (MNI), to identify the LRRK2 regulatory network. Through this work we have developed a systems network regulatory map for LRRK2 action, and are currently identifying which of the pathways modulate the neurodegenerative pathways associated with Parkinson’s disease.