Our research is focused on basic mechanisms of ribonucleoprotein (RNP) assembly, localization, and local translation in neuron development, plasticity, and maintenance, and how perturbation of these mechanisms contributes to the neurodegenerative diseases spinal muscular atrophy (SMA) and the amyotrophic lateral sclerosis (ALS) / frontotemporal dementia (FTD) disease spectrum.
Currently, we are using model systems such as yeast, primary cortical and motor neurons, mouse models, and patient-derived induced pluripotent stem cell models to study 1) the assembly of RNA and proteins into RNP granules that regulate mRNA processing, localization, and local translation, 2) the aggregation of RNA-binding proteins, and their effect on protein transport and degradation pathways in neurons, and how dysfunction in RNA processing and protein homeostasis can cause neurodegenerative diseases.
How do low levels of the SMN proteins cause spinal muscular atrophy (SMA)?
Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease that represents the most common genetic cause of infant death. SMA is caused by reduced levels of functional survival of motor neuron (SMN) protein, leading to cell autonomous defects at the neuromuscular junctions, axon degeneration, and loss of motor neurons in the spinal cord. The ubiquitously expressed SMN protein has a well characterized essential function in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) in all tissues, but it is still unclear to what extent pre-mRNA splicing defects contribute to SMA. It is a central question in the field why spinal motor neurons are more severely affected by low SMN protein levels than other cell types.
Our findings led to the hypothesis that SMN plays an important role in neuronal mRNP biology by regulating the dynamic assembly of mRNAs with proteins into neuronal transport granules, their axonal trafficking, and the local translation at the axon terminals, which may explain neuron specific defects in SMA. We are currently using advanced imaging methods to investigate the role of SMN in mRNP assembly.
The spectrum of RNA processing defects caused by SMN-deficiency, and their contribution to axonal pathology and motor neuron degeneration observed in SMA is not known. While specific defects in mRNA localization have been described in vitro, there is a critical need to identify the whole range of affected transcripts, to characterize the molecular pathways affected by the disruption of SMN-dependent RNP assembly and RNA processing pathways, and to investigate their contribution to the disease phenotype in SMA mouse models in vivo. We are using patient stem cell-derived neurons and mouse models to determine mRNA processing defects in SMA spinal cords and how they contribute to motor neuron degeneration.
Molecular mechanisms that regulate TDP-43 protein aggregation and toxicity
Pathologic and genetic studies have established the RNA-binding protein TDP-43 as a key player in the disease process of the vast majority of familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Therefore, understanding and manipulating the pathways that mediate TDP-43 protein aggregation may benefit the large majority of ALS patients.
In a search for modulators of pathogenic TDP-43, we have identified poly(A)-binding protein nuclear 1 (PABPN1) as a potent suppressor of TDP-43 proteinopathy. Our recently published data demonstrate that PABPN1 reduces aggregation and cytoplasmic mislocalization of pathological TDP-43 fragments and rescues nuclear localization of TDP-43 and protects from TDP-43 toxicity in cell culture and animal models.
We hypothesize that reducing the cytoplasmic accumulation of pathological TDP-43 fragments can restore normal cellular function and form the basis of novel strategies for therapeutic intervention. The aim of this project is to elucidate in detail the molecular pathways and mechanisms governing the suppression of TDP-43 toxicity by reducing the burden of pathological TDP-43 aggregates.
Intracellular transport defects as emerging disease mechanisms in ALS/FTD
Nearly all sporadic and familial ALS cases are characterized by cytoplasmic aggregations of hyper-phosphorylated, ubiquitinated, and cleaved TDP-43 fragments and the loss of nuclear TDP-43. Although the specific composition of these detergent-insoluble inclusions may hold important clues to the pathological process in TDP-43 proteinopathies, it was previously unknown.
To address this question, we have adapted a new technique that allows us to efficiently isolate biotinylated proteins present in detergent-insoluble pathological aggregates by affinity capture under denaturing conditions, and to identify them via mass spectrometry. Quantitative proteomics and gene ontology analysis show that the TDP-CTF interactome can be functionally categorized in translation, RNA processing, protein degradation, and intracellular transport. Our findings demonstrate that expression of TDP-CTF and full length TDP-43 carrying ALS-specific point mutations cause defects in the localization of proteins within the cell. This is further supported by genetic interaction data from a Drosophila model of TDP-43 proteinopathy and staining of human brain tissue. Taken together, our data show that TDP-43 toxicity causes the disruption of intracellular transport pathways may contribute to the neurodegeneration observed in ALS/FTD, and potentially other neurologic disorders.
Currently, we are employing these methods to investigate the composition of pathological aggregates containing the RNA-binding protein FUS and the microtubule-associated protein tau, and how these contribute to disease phenotypes in ALS and FTD.