Dominant mutations in the gene encoding the nucleic acid binding protein FUS cause ~5% of familial amyotrophic lateral sclerosis (ALS). Our long-term objectives are to discern the mechanism(s) by which FUS mutants injure aging motor neurons and to develop novel therapeutic approaches to increase the defenses against these insults. Our laboratory has identified a novel and robust nuclear phenotype caused by ALS- linked FUS mutants: impaired stress-responsive processing of sub-nuclear assemblies known as promyelocytic leukemia (PML) nuclear bodies. PML nuclear bodies are induced by a variety of cellular stresses and regulate nuclear protein homeostasis, transcription, DNA-damage pathways, and cellular senescence, yet their potential role in ALS has not been explored. We observed that PML bodies were abnormally enlarged both in cell lines and in primary ALS human fibroblasts expressing mutant FUS. Furthermore, proteasome activities were decreased, and exposure to mild oxidative stress or proteasome inhibition in FUS mutant but not control cells stalled the turnover of expanded PML bodies. We hypothesize that the observed abnormality of PML nuclear bodies may report upon altered nuclear homeostasis resulting from FUS mutant expression.
In Aim 1 of this project, we will develop an imaging-based phenotypic screening assay to identify small molecule compounds that modulate the observed PML nuclear body enlargement in cells expressing FUS mutants. We will test compound libraries that include FDA-approved drugs and diverse CNS-active agents predicted to cross the blood-brain barrier. We will prioritize initial hits using dose-response studies and will determine whether proteins known to be targeted to the proteasome following stress are more effectively eliminated upon treatment with hit compounds. We have established transgenic mice harboring ALS-linked FUS variants and have observed a phenotype of age-dependent loss of the connection between motor nerves and muscle in mice that express the R495X mutant.
In Aim 2 of this project, we will validate hit compounds and analyze pathways related to PML nuclear body function in CNS cells and tissues from our mutant FUS transgenic mice. We will test whether a subset of the prioritized hit compounds from Aim 1 ameliorate defects of nuclear protein homeostasis or proteasome activity in transgenic primary cortical neurons, glial cells, or motor neurons. In further experiments, which do not depend upon the success of obtaining modulator compounds in Aim 1, we will define more precisely which pathways related to PML nuclear body function are most relevant to FUS- mediated ALS. We will purify proteasomes from the CNS of our FUS mice at pre-symptomatic and symptomatic ages and will quantify subunit expression, assembly, and activities in collaboration with Dr. Fred Goldberg's group. We will also use a high-resolution tissue monolayer preparation to identify nuclear insults related to altered PML body function in aging neurons from our FUS transgenic mice.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that strikes insidiously in midlife. Recent genetic discoveries implicate mutant proteins that regulate RNA processing in the pathogenesis of ALS. One of these proteins, FUS, is broadly distributed throughout the body but is particularly abundant within the cell nucleus of motor neurons that die prematurely in ALS. We engineered cell lines that express either normal or mutant FUS variants and identified a novel pathology in the nucleus specific to the ALS mutants. We observed that assemblies known as PML nuclear bodies, which normally serve as master regulators to coordinate multiple stress responses in cells, are expanded chronically in the mutant cells and also in primary human fibroblasts harboring mutant FUS. Furthermore, FUS mutants impair the normal dynamics of these structures in response to imposed stresses. In this project, we will develop a cellular imaging assay suitable for small molecule high-throughput screening to identify compounds that ameliorate this nuclear abnormality both in cell lines and in primary human fibroblasts. Hit compounds from this assay will then be validated in secondary screens using neurons and glial cells obtained from transgenic mice we have established that express normal or mutant human FUS. In further experiments, we will evaluate the effects of FUS mutants upon specific functions linked to PML bodies, including protein quality control within the nucleus and the repair response to DNA damage, which may be relevant to motor neuron survival in ALS.
