The first protein we’ve characterized related to ALS is FUS. Discovered to be involved in ALS in 2009, mutations in FUS remain tied as the third leading cause of familial ALS. Our earliest studies found FUS to be a broad regulator of transcription in cells. We then found that RNA stimulates FUS to form fibrous assemblies and this activity is required for FUS interactions with the transcription machinery. Armed with this knowledge, we explored cells recovered from skin biopsies donated by ALS patients to research. We found that FUS assemblies are altered in ALS patients, losing their dynamics, their ability to assemble and disassemble rapidly, and they no longer function effectively as regulators of transcription. Ongoing projects address: (1) the role of FUS in transcription, RNA processing, and ALS pathology; and (2) development of approaches to purify FUS containing granules out of cells, both non-disease and cells derived from ALS patients.

The Normal Function of FUS: (A) FUS is a multi-domain RNA-binding protein with an N-terminal Low Complexity (LC) domain. The most prevalent ALS-causing mutations in FUS are found in the nuclear localization signal (NLS). (B) FUS binds RNA Pol II (RNAP2) and co-localizes with the polymerase to active gene promoters. (C) FUS is one of the most abundant proteins found in the nucleus. It targets transcription by recognizing the earliest transcripts produced by RNA Pol II, and then forms a fibrous scaffold, which recruits more RNA Pol II, other transcription machinery, and regulates phosphorylation of the CTD of RNA Pol II.

Study of FUS function in ALS patient-derived cells: (A) An invaluable resource to ALS research are fibroblast cells donated by ALS patients. (B) Despite mutations to the nuclear localization signal of FUS, in patient samples most of the FUS protein remains in the nucleus. Whereas pathology had been presumed to be associated with toxic cytoplasmic activity of the mislocalized protein, we have explored whether nuclear dysfunction may contribute significantly to pathology.

Aberrant FUS function in ALS patients: Despite the majority of FUS being in the nucleus in ALS patients bearing FUS mutations (A), nuclear FUS is trapped in aggregates that cannot migrate on an SDS-PAGE gel and not functional (B). (C) Genes actively transcribed by RNA Pol II are localized within the cell nucleus to discrete foci and this open chromatin stains weaker for DAPI. For ALS patients with FUS mutations, this organization is disrupted and the foci of RNA Pol II becomes fragmented. (D) Our model is that FUS forms droplets, which are scaffolds surrounding expressed genes. We find in ALS patient cells, FUS droplets in the nucleus become aggregate and no longer function to regulate transcription or help organize the transcription machinery.

Relevant Publications:

Schwartz JC, Cech TR, Parker RR, “Biochemical properties and biological functions of FET proteins”, Annu. Rev. Biochem. 2015; 84:355-79. (PMID: 25494299)

Schwartz JC, Podell ER, Han SSW, Berry JD, Eggan KC, Cech TR, “FUS is sequestered in nuclear aggregates in ALS patient fibroblasts” Mol Biol Cell, 2014; 25(17):2571-8. (PMID: 25009283)

Schwartz JC, Wang X, Podell ER, Cech TR, “RNA seeds higher order assembly of FUS protein” Cell Reports, 2013; 5(4):918-25. (PMID: 24268778)

Schwartz JC, Ebmeier CC, Podell ER, Heimiller J, Taatjes DJ, Cech TR, “FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2” Genes Dev, 2012; 26:2690-95. (PMID: 23249733)

Tied with FUS as the third leading cause of familial ALS is the RNA-binding protein TDP-43. We hypothesize that it is the intersection of overlapping functions for proteins involved in ALS pathology that will reveal to us the molecular basis for this disease. Moreover, TDP-43 aggregation is a nearly ubiquitous feature found in ALS patients, even those who do not possess the disease-causing mutations in this gene. Finally, TDP-43 provides the link connecting ALS pathology to other neurodegenerative diseases and notably, dementia. It is now appreciated that Frontal Temporal Dementia (FTD) is a disease that is closely related to ALS. Some mutations in the same genes, such as TDP-43, cause both diseases. Pathological aggregation of the same proteins, such as FUS, is observed in both diseases. One shared function between FUS and TDP-43 is the ability to regulate gene expression at the level of transcription. We are exploring whether TDP-43 possess other functional overlaps with FUS or pathological mechanisms, including those involving DNA damage repair. Our hypothesis is that while each ALS-associated gene has many cellular functions, it is the overlap of those where the most likely mechanism of disease will lie.

Affinity-enrichment mass spectrometry reveals interactions shared by FUS and TDP-43: Use of improved mass spectrometry approaches greatly enhances sensitivity. Our preliminary analysis of recent data revealed known interactors, such as NONO, FXR1, and hnRNPU. We also found interactors with known significance for ALS pathology, such as hnRNPA2B1, PPIA, GABBR1, and TUBBA4A.

The role of TDP43 in transcription regulation: (A) Using a next-generation sequencing technique, GRO-seq, we can visualize transcription as it is occurring across the entire genome. Then by removing TDP-43 by siRNA knockdown, we can determine the affect TDP43 has on transcription. (B) Multiple models may explain TDP43 regulation of transcription. TDP43 may be recruited to regulate transcription by binding the transcribed RNA (i) or the DNA (ii). It may bind enhancer RNAs (eRNA) upstream of the gene (iii). TDP43 is a constitutive member of paraspeckles, which raises the possibility of a connect between these bodies and transcription (iv, West et al., J Cell Biol 2016). (C) By replacing TDP43 with those deleting key domains, the mechanism of transcription regulation can be further elucidated.

A pressing question about ALS pathology is why do only neurons die and why does the disease mostly appear in old age. The hypothesis that DNA damage repair defects might contribute to the pathology neatly connects these two observations from the clinic. Recently, studies from many labs support the conclusion that DNA damage in the nervous system accumulates with age and the mechanisms for DNA damage repair seem to weaken with age. In fact, several of the genes implicated in ALS have links to DNA damage repair. The most well known connection is that FUS is required for DNA damage repair and is one of the earliest proteins recruited to a DNA damage site. A recent paper has suggested and we have multiple lines of evidence supporting a role for TDP-43 in DNA damage repair (Hill et al., PNAS 2016).

Multiple genes implicated in ALS are known to be involved in ALS pathology: FUS, VCP, and SETX have all been implicated as directly interacting with DNA repair machinery. SOD1 and p62 have roles in autophagy, which is linked to DNA damage through regulating the production of radical oxidative species (ROS). The role of TDP-43 remains to be determined.

Identification of proteins interacting with FUS and TDP-43 after DNA damage: Heat maps of z-scores indicate change in levels of proteins detected after DNA damage, with green increases and red decreases. Proteins where changes rose to the level of statistic significance indicated by blue bars. Currently, we hypothesize that FUS and TDP-43 cohabit several nuclear bodies, though not interacting directly. After DNA damage, FUS leaves many of its unique complexes and increases its association with complexes that also contain TDP-43. Few interactions for TDP-43 significantly change. Of known interactors of FUS and TDP-43, paraspeckles and the snRNP complex seem unchanged by DNA damage, RNA exosome interactions increase, and hnRNP interactions decrease.