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Abstract Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G > A, p.(Trp402*). The variant was associated with a ~ 31% reduction in full-length protein levels in the patient’s brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression.

A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation.

Abstract Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits.

Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G > A, p.(Trp402*). The variant was associated with a ~ 31% reduction in full-length protein levels in the patient's brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits. [ABSTRACT FROM AUTHOR]

A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ishiguro and Ishihama.)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid-liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the content of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)