Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

Epigenetics in the formation of pathological aggregates in amyotrophic lateral sclerosis.

The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target.

Phase Separation of SARS-CoV-2 Nucleocapsid Protein with TDP-43 Is Dependent on C-Terminus Domains.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

The Integral Role of RNA in Stress Granule Formation and Function.

Stress granules (SGs) are phase-separated, membraneless, cytoplasmic ribonucleoprotein (RNP) assemblies whose primary function is to promote cell survival by condensing translationally stalled mRNAs, ribosomal components, translation initiation factors, and RNA-binding proteins (RBPs). While the protein composition and the function of proteins in the compartmentalization and the dynamics of assembly and disassembly of SGs has been a matter of study for several years, the role of RNA in these structures had remained largely unknown. RNA species are, however, not passive members of RNA granules in that RNA by itself can form homo and heterotypic interactions with other RNA molecules leading to phase separation and nucleation of RNA granules. RNA can also function as molecular scaffolds recruiting multivalent RBPs and their interactors to form higher-order structures. With the development of SG purification techniques coupled to RNA-seq, the transcriptomic landscape of SGs is becoming increasingly understood, revealing the enormous potential of RNA to guide the assembly and disassembly of these transient organelles. SGs are not only formed under acute stress conditions but also in response to different diseases such as viral infections, cancer, and neurodegeneration. Importantly, these granules are increasingly being recognized as potential precursors of pathological aggregates in neurodegenerative diseases. In this review, we examine the current evidence in support of RNA playing a significant role in the formation of SGs and explore the concept of SGs as therapeutic targets.

TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease with no known etiology. The formation of pathological protein inclusions, including RNA-binding proteins such as TDP-43 and rho guanine nucleotide exchange factor (RGNEF) are a hallmark of ALS. Despite intensive research, the mechanisms behind protein aggregate formation in ALS remains unclear. We have investigated the role of metabolic stress in protein aggregate formation analyzing how it is relevant to the co-aggregation observed between RGNEF and TDP-43 in motor neurons of ALS patients. Metabolic stress was able to induce formation of micronuclei, small nuclear fragments, in cultured cells. Notably, we observed the formation TDP-43 protein inclusions within micronuclei that co-aggregate with RGNEF and can be released to the cytoplasm. We observed that the leucine-rich domain of RGNEF is critical for its interaction with TDP-43 and localization in micronuclei. Finally, we described that micronuclei-like structures can be found in brain and spinal cord of ALS patients. This work is the first description of protein inclusion formation within micronuclei which also is linked with a neurodegenerative disease. The formation of TDP-43 inclusions within micronuclei induced by metabolic stress is a novel mechanism of protein aggregate formation which may have broad relevance for ALS and other neurodegenerative diseases.

A novel overlapping NLS/NES region within the PH domain of Rho Guanine Nucleotide Exchange Factor (RGNEF) regulates its nuclear-cytoplasmic localization.

Rho Guanine Nucleotide Exchange Factor (RGNEF) is a 190 kDa protein implicated in both amyotrophic lateral sclerosis (ALS) and cancer. Under normal physiological conditions, RGNEF is predominantly cytoplasmic with moderate levels of nuclear localization. We have identified a 23-amino acid region containing a bipartite nuclear localization signal (NLS) within the Pleckstrin Homology (PH) domain of RGNEF, which when deleted or mutated abolishes the nuclear localization of this protein. Fusion proteins containing only the PH domain demonstrated that this region by itself is able to translocate a 160 kDa protein to the nucleus. Interestingly, we also detected a nuclear export signal (NES) within the linker region of this bipartite NLS which is able to export from the nucleus a fusion protein containing two NLSs. Experiments using Leptomycin-B -an inhibitor of nuclear export- confirmed that this region promotes nuclear export in an exportin-1 dependent manner. This study is the first report demonstrating either of these signals embedded within a PH domain. Notably, this is also the first description of a functional overlapped NLS/NES signal.

MotomiRs: miRNAs in Motor Neuron Function and Disease.

MiRNAs are key regulators of the mammalian transcriptome that have been increasingly linked to degenerative diseases of the motor neurons. Although many of the miRNAs currently incriminated as participants in the pathogenesis of these diseases are also important to the normal development and function of motor neurons, at present there is no knowledge of the complete miRNA profile of motor neurons. In this review, we examine the current understanding with respect to miRNAs that are specifically required for motor neuron development, function and viability, and provide evidence that these should be considered as a functional network of miRNAs which we have collectively termed MotomiRs. We will also summarize those MotomiRs currently known to be associated with both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), and discuss their potential use as biomarkers.

Rho guanine nucleotide exchange factor (RGNEF) is a prosurvival factor under stress conditions.

Rho guanine nucleotide exchange factor (RGNEF) is a 190kDa RNA binding protein (RBP) that also contains a Dbl/PH domain capable of RhoA activation. Consistent with a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS), RGNEF forms pathological neuronal cytoplasmic inclusions in degenerating spinal motor neurons. To further understand the role of RGNEF in the stress response, we first observed that the expression of RGNEF is upregulated in murine spinal motor neurons following distal sciatic nerve injury. Secondly, in response to in vitro cellular stress (500µM sodium arsenite for 1h; or 400mM sorbitol 1 hour exposure; as an oxidative or osmotic stress, respectively), we observed a significant survival benefit in RGNEF-transfected HEK293T cells. Using deletion constructs, we found that the NH2-terminus domain is essential for this protective effect. Interestingly, we observed that under stress conditions RGNEF associates with Staufen1 positive granules but not TIA-1-positive stress granules. These findings support the hypothesis that RGNEF plays a critical role both in RNA homeostasis and in the response to cell stress.