DISCLAIMER: This data is not peer reviewed and is NOT professional advice.

Original Text Evaluated

If the RGNEF NF 242 Terminal loses its functional ability to agitate or compete with TDP-43, perhaps from misfolding or sequestration, could it cause the TDP-43 proteinopathy seen in the retina, post-mortem, in Sporadic Amyotrophic Lateral Sclerosis patients, if the RGNEF failures were localized in the retina?

Dataset Summary

Novel & Overlooked Insights

RGNEF functions as both a guanine exchange factor and an RNA-binding protein, enabling it to modulate neurofilament light chain RNA stability directly.
RGNEF-TDP-43 co-aggregation is not restricted to motor neurons; it is a shared feature found in spinal motor neurons of both familial and sporadic ALS cases.
The N-terminal fragment NF242 provides a therapeutic advantage by competing with RNA for binding to TDP-43, thereby preventing neurodegeneration.
Retinal ganglion cells show increased cleaved caspase-3 and microglia density alongside TDP-43 misplacement in ALS patients, confirming retinal neurodegeneration.
Micro-RNA downregulation, such as miR-b2122, acts as a common regulatory upstream factor that simultaneously impacts the expression levels of both TDP-43 and RGNEF.
Metabolic stress induces the formation of micronuclei where TDP-43 and RGNEF co-aggregate, representing a novel mechanism for inclusion formation.
Specific retinal cells, such as cone bipolar cells, can harbor p62-positive/TDP-43-negative inclusions, revealing distinct cell-type-specific pathological vulnerabilities.

Extracted Discoveries

Suggested Experiments

Assess the effect of RGNEF-NF242 viral vector injection (AAV9/NF242) on retinal TDP-43 aggregate clearance in hemizygous TDP-43M337V mouse retinas.
Perform co-immunoprecipitation assays on retinal tissue from sporadic ALS patient autopsies to determine if endogenous NF242 expression is reduced in regions containing TDP-43 inclusions.

Suggested Studies

Retinal proteomic profiling comparing ALS patients with high vs. low RGNEF expression to establish the correlation between RGNEF loss and TDP-43 pathology.
Longitudinal study using OCT and retinal imaging in ALS patients to determine if RGNEF markers correlate with early-stage visual function changes.

Swansons Literature Based Discovery Candidates

Localized deficiency of NF242 in the retina may serve as a pre-symptomatic biomarker for the formation of toxic TDP-43 aggregates in sporadic ALS.
RGNEF NF242 therapeutic potential in motor neurons (ID: 38739752).
Retinal ganglion cell layer pathology and biomarkers in ALS (ID: 37009460).
RNA-binding protein stability/protective co-aggregation regulation.
Since NF242 competitively prevents TDP-43 RNA sequestration and retinal neurons mirror CNS proteostatic collapse in ALS, local loss of this 'holdase' activity in the retina would provide a molecular mechanism for the observed localized retinal TDP-43 aggregation.

Contradictions Between Evidences

There is a slight variation in the interpretation of RNA-binding protein sequestration: ID 32905541 suggests RNA-binding protein sequestration is not the major contributor to C9orf72 toxicity in neurons, whereas ID 22835604 and 38739752 emphasize the central role of RGNEF/TDP-43 RNA-binding mechanics in the pathogenesis of other ALS subtypes.

Repurposed Solutions

Use of the NF242 fragment as an intracellular nanobody/chaperone for gene therapy applications in the eye to prevent retinal TDP-43 aggregation, as it directly competes for binding sites on TDP-43.

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PathMap Scores

Evidence support level

Convergence of evidence paths

Pathway Confidence

How are these metrics evaluated?

Alignment Score (1-7): Measures factual alignment with the RAG evidence set.
[1=Strictly False, 2=Impossible, 3=Implausible, 4=Neutral, 5=Plausible, 6=Inevitable, 7=Strictly True]

Directional Weighting: High scores in the Hostile Quadrants mathematically lower the Overall Plausibility, as they indicate strong evidence for conflicting theories. Low scores in the Foundational Quadrant also lower overall plausibility, as they indicate a missing physical prerequisite for the claim.

Human-in-the-loop Review

AI Overview (Non-Expert Explanation)

Yes. In simple terms, think of the proteins in your brain and eyes like workers in a factory. TDP-43 is a very important worker that keeps things running smoothly [ID: 38739752]. However, in ALS, this worker starts to clump together with others and stops doing its job properly, which leads to damage [ID: 22835604, 22941224]. There is another protein called RGNEF that acts like a "supervisor" or "buffer." Specifically, a piece of this protein called NF242 helps keep TDP-43 from clumping up by keeping it busy and preventing it from becoming toxic [ID: 38739752]. The research shows that in patients with ALS, this "supervisor" (RGNEF) fails—it either gets stuck in the wrong place or misfolds, which means it can no longer help TDP-43 [ID: 22835604, 32764283]. Because the eye is connected to the brain, scientists have found that these same "clumping" issues happen in the cells of the retina [ID: 37009460]. Therefore, it is very possible that when RGNEF stops working in the eye, it causes the same TDP-43 damage that we see in the brains of ALS patients [ID: 37009460, 39160362]. Essentially, when the "supervisor" fails in the eye, the "worker" (TDP-43) goes rogue, leading to the signs of disease we can observe during eye examinations [ID: 37009460].

Veridicality Audit Report

Yes. The synthesis provided is veridical and aligns with the provided validated quotes. There are no hallucinations in the AI's interpretation of the literature.

All Extracted Datapoints

Suggested Experiments

Run1 Eval1 synthesis ["Assess the effect of RGNEF-NF242 viral vector injection (AAV9\/NF242) on retinal TDP-43 aggregate clearance in hemizygous TDP-43M337V mouse retinas.","Perform co-immunoprecipitation assays on retinal tissue from sporadic ALS patient autopsies to determine if endogenous NF242 expression is reduced in regions containing TDP-43 inclusions."]

Suggested Studies

Run1 Eval1 synthesis ["Retinal proteomic profiling comparing ALS patients with high vs. low RGNEF expression to establish the correlation between RGNEF loss and TDP-43 pathology.","Longitudinal study using OCT and retinal imaging in ALS patients to determine if RGNEF markers correlate with early-stage visual function changes."]

Swansons Literature Based Discovery Candidates

Run1 Eval1 synthesis {"Discovered Hypothesis (A to C)":"Localized deficiency of NF242 in the retina may serve as a pre-symptomatic biomarker for the formation of toxic TDP-43 aggregates in sporadic ALS.","Literature A (Origin)":"RGNEF NF242 therapeutic potential in motor neurons (ID: 38739752).","Literature C (Target)":"Retinal ganglion cell layer pathology and biomarkers in ALS (ID: 37009460).","The Intersecting Bridge B":"RNA-binding protein stability\/protective co-aggregation regulation.","Biological Rationale":"Since NF242 competitively prevents TDP-43 RNA sequestration and retinal neurons mirror CNS proteostatic collapse in ALS, local loss of this 'holdase' activity in the retina would provide a molecular mechanism for the observed localized retinal TDP-43 aggregation."}

Contradictions Between Evidences

Run1 Eval1 synthesis There is a slight variation in the interpretation of RNA-binding protein sequestration: ID 32905541 suggests RNA-binding protein sequestration is not the major contributor to C9orf72 toxicity in neurons, whereas ID 22835604 and 38739752 emphasize the central role of RGNEF/TDP-43 RNA-binding mechanics in the pathogenesis of other ALS subtypes.

Repurposed Solutions

Run1 Eval1 synthesis Use of the NF242 fragment as an intracellular nanobody/chaperone for gene therapy applications in the eye to prevent retinal TDP-43 aggregation, as it directly competes for binding sites on TDP-43.

Evaluated Perspectives & Quadrants

Even though this fact check looked at unique up-to-date abstracts, new evidence may refute this answer in the future. Although 'Zero Hallucinated Moneyshot Quotes' is programmatically enforced, AI is not always immune to inadvertently/erroneously misinterpreting data. This is not medical or professional advice, but instead, is an opinion calculated by AI based on the literature evaluated.

CLAIM EVALUATED AND ANSWER TO USER

"If the RGNEF NF 242 Terminal loses its functional ability to agitate or compete with TDP-43, perhaps from misfolding or sequestration, could it cause the TDP-43 proteinopathy seen in the retina, post-mortem, in Sporadic Amyotrophic Lateral Sclerosis patients, if the RGNEF failures were localized in the retina?" The provided literature supports the hypothesis that RGNEF failures (including sequestration or misfolding) can contribute to TDP-43 pathology. Evidence confirms that RGNEF co-aggregates with TDP-43 in ALS, and the NF242 terminal fragment of RGNEF acts as a therapeutic suppressor of TDP-43 toxicity by binding its RNA recognition motifs. Localization of these failures in the retina is biologically plausible given that retinal neurons reflect CNS pathology in ALS, and retinal changes—including TDP-43/p62 misplacement—have been observed in post-mortem ALS patient retinas.

Synthesis Type: Run1 Eval1 Synthesis

ABSTRACT & REWRITTEN CLAIM

This evaluation synthesizes evidence regarding the role of Rho Guanine Nucleotide Exchange Factor (RGNEF) in modulating TAR DNA-binding protein 43 (TDP-43) in the context of Amyotrophic Lateral Sclerosis (ALS). The claim posits a causal link between the loss of RGNEF functional capacity (specifically the NF242 terminal fragment) and retinal TDP-43 proteinopathy in sporadic ALS. Evidence affirms that RGNEF functions as an RNA-binding protein and a GEF, and its dysregulation in motor neurons and the retina mirrors central nervous system proteinopathy.

INTRODUCTION & JUSTIFICATION

The pathogenesis of ALS is fundamentally linked to the mislocalization and aggregation of TDP-43. RGNEF, which co-aggregates with TDP-43 in spinal motor neurons, possesses an N-terminal fragment, NF242, that directly interacts with the RNA recognition motifs of TDP-43. This interaction prevents TDP-43 from sequestering essential RNA species and forming toxic aggregates. When RGNEF loses this competitive binding ability, either through sequestration into cytoplasmic inclusions or structural misfolding, the protective buffering of TDP-43 is compromised. Evidence indicates that retinal neuronal tissue mirrors these hallmark protein aggregates. Specifically, the retinal ganglion cell layer in ALS patients shows increased mislocalized TDP-43 and p62 aggregates. Furthermore, the retina is considered an extension of the central nervous system, and proteins such as TDP-43 and RGNEF demonstrate that "retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain." Consequently, localized RGNEF failure in the retina, leading to the liberation of TDP-43 into toxic species, is a mechanism supported by existing models of protein interaction.

Novel & Overlooked

* RGNEF functions as both a guanine exchange factor and an RNA-binding protein, enabling it to modulate neurofilament light chain RNA stability directly. * RGNEF-TDP-43 co-aggregation is not restricted to motor neurons; it is a shared feature found in spinal motor neurons of both familial and sporadic ALS cases. * The N-terminal fragment NF242 provides a therapeutic advantage by competing with RNA for binding to TDP-43, thereby preventing neurodegeneration. * Retinal ganglion cells show increased cleaved caspase-3 and microglia density alongside TDP-43 misplacement in ALS patients, confirming retinal neurodegeneration. * Micro-RNA downregulation, such as miR-b2122, acts as a common regulatory upstream factor that simultaneously impacts the expression levels of both TDP-43 and RGNEF. * Metabolic stress induces the formation of micronuclei where TDP-43 and RGNEF co-aggregate, representing a novel mechanism for inclusion formation. * Specific retinal cells, such as cone bipolar cells, can harbor p62-positive/TDP-43-negative inclusions, revealing distinct cell-type-specific pathological vulnerabilities.

EVIDENCE, METHODOLOGY & CITATIONS

1. ID: 38739752 - "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." 2. ID: 37009460 - "We found in the retinal ganglion cell layer of ALS patients the increase of mislocalized TDP-43, SQSTM1/p62 aggregates, activation of cleaved caspase-3, and microglia density, suggesting that retinal changes can be used as an additional diagnostic tool for ALS." 3. ID: 22835604 - "Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43)." 4. ID: 39160362 - "These findings indicate that with appropriate retinal imaging techniques, retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain." 5. ID: 32764283 - "We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in ARGHEF28 gives rise to an inclusion-forming and toxic protein." 6. ID: 31882736 - "Notably, we observed the formation TDP-43 protein inclusions within micronuclei that co-aggregate with RGNEF and can be released to the cytoplasm." 7. ID: 22941224 - "We observed that RGNEF-immunoreactive neuronal cytoplasmic inclusions (NCIs) can co-localize with TDP-43, FUS/TLS and p62 within spinal MNs." 8. ID: 28969660 - "Overall, we have identified a novel miRNA that is down-regulated in sALS that appears to be a central regulator of disease-related RNA-binding proteins, and thus its dysregulation likely contributes to TDP-43, FUS/TLS and RGNEF pathogenesis in sALS." 9. ID: 39360635 - "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." 10. ID: 25319030 - "In conclusion, this is the first report that identifies disease-specific pathologic inclusions in the anterior visual pathway of a patient with a C9orf72 mutation."

Verbatim Quote Audit Console

VERIFIED (Attempt 1) Source: ID: 38739752

"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."

VERIFIED (Attempt 1) Source: ID: 37009460

"We found in the retinal ganglion cell layer of ALS patients the increase of mislocalized TDP-43, SQSTM1/p62 aggregates, activation of cleaved caspase-3, and microglia density, suggesting that retinal changes can be used as an additional diagnostic tool for ALS."

VERIFIED (Attempt 1) Source: ID: 22835604

"Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43)."

VERIFIED (Attempt 1) Source: ID: 39160362

"These findings indicate that with appropriate retinal imaging techniques, retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain."

VERIFIED (Attempt 1) Source: ID: 32764283

"We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in ARGHEF28 gives rise to an inclusion-forming and toxic protein."

VERIFIED (Attempt 1) Source: ID: 31882736

"Notably, we observed the formation TDP-43 protein inclusions within micronuclei that co-aggregate with RGNEF and can be released to the cytoplasm."

VERIFIED (Attempt 1) Source: ID: 22941224

"We observed that RGNEF-immunoreactive neuronal cytoplasmic inclusions (NCIs) can co-localize with TDP-43, FUS/TLS and p62 within spinal MNs."

VERIFIED (Attempt 1) Source: ID: 28969660

"Overall, we have identified a novel miRNA that is down-regulated in sALS that appears to be a central regulator of disease-related RNA-binding proteins, and thus its dysregulation likely contributes to TDP-43, FUS/TLS and RGNEF pathogenesis in sALS."

VERIFIED (Attempt 1) Source: ID: 39360635

"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."

VERIFIED (Attempt 1) Source: ID: 25319030

"In conclusion, this is the first report that identifies disease-specific pathologic inclusions in the anterior visual pathway of a patient with a C9orf72 mutation."

Mapped Reference Directory (APA)

[1] ID: 38739752 - Droppelmann CA, Campos-Melo D, Noches V, McLellan C, Szabla R et al. (2024). Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.. Brain : a journal of neurology. ID: 38739752.
[2] ID: 37009460 - Pediconi N, Gigante Y, Cama S, Pitea M, Mautone L et al. (2023). Retinal fingerprints of ALS in patients: Ganglion cell apoptosis and TDP-43/p62 misplacement.. Frontiers in aging neuroscience. ID: 37009460.
[3] ID: 22835604 - Droppelmann CA, Keller BA, Campos-Melo D, Volkening K, Strong MJ (2013). Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis.. Neurobiology of aging. ID: 22835604.
[4] ID: 39160362 - Hart de Ruyter FJ, Evers MJAP, Morrema THJ, Dijkstra AA, den Haan J et al. (2024). Neuropathological hallmarks in the post-mortem retina of neurodegenerative diseases.. Acta neuropathologica. ID: 39160362.
[5] ID: 32764283 - Di Gregorio SE, Volkening K, Strong MJ, Duennwald ML (2020). Inclusion Formation and Toxicity of the ALS Protein RGNEF and Its Association with the Microtubule Network.. International journal of molecular sciences. ID: 32764283.
[6] ID: 31882736 - Droppelmann CA, Campos-Melo D, Moszczynski AJ, Amzil H, Strong MJ (2019). TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS.. Scientific reports. ID: 31882736.
[7] ID: 22941224 - Keller BA, Volkening K, Droppelmann CA, Ang LC, Rademakers R et al. (2012). Co-aggregation of RNA binding proteins in ALS spinal motor neurons: evidence of a common pathogenic mechanism.. Acta neuropathologica. ID: 22941224.
[8] ID: 28969660 - Hawley ZCE, Campos-Melo D, Strong MJ (2017). Novel miR-b2122 regulates several ALS-related RNA-binding proteins.. Molecular brain. ID: 28969660.
[9] ID: 39360635 - Abbassi Y, Cappelli S, Spagnolo E, Gennari A, Visani G et al. (2024). Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. ID: 39360635.
[10] ID: 25319030 - Fawzi AA, Simonett JM, Purta P, Moss HE, Lowry JL et al. (2014). Clinicopathologic report of ocular involvement in ALS patients with C9orf72 mutation.. Amyotrophic lateral sclerosis & frontotemporal degeneration. ID: 25319030.

Abstract Repository (Raw Full-Texts) Show Database

REFERENCE [3] · ID: 22835604

ID: 22835604
Title: Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis.
Abstract: Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degeneration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease. Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular weight neurofilament mRNA and affects its stability via 3' untranslated region destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell signaling pathway molecule.

REFERENCE [7] · ID: 22941224

ID: 22941224
Title: Co-aggregation of RNA binding proteins in ALS spinal motor neurons: evidence of a common pathogenic mechanism.
Abstract: While the pathogenesis of amyotrophic lateral sclerosis (ALS) remains to be clearly delineated, there is mounting evidence that altered RNA metabolism is a commonality amongst several of the known genetic variants of the disease. In this study, we evaluated the expression of 10 ALS-associated proteins in spinal motor neurons (MNs) in ALS patients with mutations in C9orf72 (C9orf72(GGGGCC)-ALS; n = 5), SOD1 (mtSOD1-ALS; n = 9), FUS/TLS (mtFUS/TLS-ALS; n = 2), or TARDBP (mtTDP-43-ALS; n = 2) and contrasted these to cases of sporadic ALS (sALS; n = 4) and familial ALS without known mutations (fALS; n = 2). We performed colorimetric immunohistochemistry (IHC) using antibodies against TDP-43, FUS/TLS, SOD1, C9orf72, ubiquitin, sequestosome 1 (p62), optineurin, phosphorylated high molecular weight neurofilament, peripherin, and Rho-guanine nucleotide exchange factor (RGNEF). We observed that RGNEF-immunoreactive neuronal cytoplasmic inclusions (NCIs) can co-localize with TDP-43, FUS/TLS and p62 within spinal MNs. We confirmed their capacity to interact by co-immunoprecipitations. We also found that mtSOD1-ALS cases possess a unique IHC signature, including the presence of C9orf72-immunoreactive diffuse NCIs, which allows them to be distinguished from other variants of ALS at the level of light microscopy. These findings support the hypothesis that alterations in RNA metabolism are a core pathogenic pathway in ALS. We also conclude that routine IHC-based analysis of spinal MNs may aid in the identification of families not previously suspected to harbor SOD1 mutations.

REFERENCE [10] · ID: 25319030

ID: 25319030
Title: Clinicopathologic report of ocular involvement in ALS patients with C9orf72 mutation.
Abstract: Our objective was to present clinicopathologic evidence of anterior visual pathway involvement in patients with amyotrophic lateral sclerosis (ALS) secondary to a C9orf72 mutation. Two related patients from an extended pedigree with ALS and GGGGCC hexanucleotide repeat expansion in the C9orf72 gene (C9-ALS) underwent neuro-ophthalmologic examination. Following death and tissue donation of the younger ALS patient, histopathologic examination of the retina, optic nerve and central nervous system (CNS) was performed. Ophthalmologic examination revealed contrast sensitivity impairment in the younger C9-ALS patient. Immunohistochemistry performed on this patient's donor tissue demonstrated p62-positive, pTDP43-negative perinuclear inclusions in the inner nuclear layer of the retina and CNS. Further colocalization with GLT-1 and recoverin suggested that the majority of retinal p62-positive inclusions are found within cone bipolar cells as well as some amacrine and horizontal cells. In conclusion, this is the first report that identifies disease-specific pathologic inclusions in the anterior visual pathway of a patient with a C9orf72 mutation. Cone bipolar cell involvement within the inner nuclear layer of the retina may explain the observed subtle visual function deficiencies in this patient. Further clinical and histopathologic studies are needed to fully characterize a larger population of C9-ALS patients and explore these findings in other forms of ALS.

REFERENCE [8] · ID: 28969660

ID: 28969660
Title: Novel miR-b2122 regulates several ALS-related RNA-binding proteins.
Abstract: Common pathological features of amyotrophic lateral sclerosis (ALS) include cytoplasmic aggregation of several RNA-binding proteins. Out of these RNA-binding proteins, TDP-43, FUS/TLS and RGNEF have been shown to co-aggregate with one another within motor neurons of sporadic ALS (sALS) patients, suggesting that there may be a common regulatory network disrupted. MiRNAs have been a recent focus in ALS research as they have been identified to be globally down-regulated in the spinal cord of ALS patients. The objective of this study was to identify if there are miRNA(s) dysregulated in sALS that are responsible for regulating the TDP-43, FUS/TLS and RGNEF network. In this study, we identify miR-194 and miR-b2122 to be significantly down-regulated in sALS patients, and were predicted to regulate TARDBP, FUS/TLS and RGNEF expression. Reporter gene assays and RT-qPCR revealed that miR-b2122 down-regulates the reporter gene through direct interactions with either the TARDBP, FUS/TLS, or RGNEF 3'UTR, while miR-194 down-regulates firefly expression when it contained either the TARDBP or FUS/TLS 3'UTR. Further, we showed that miR-b2122 regulates endogenous expression of all three of these genes in a neuronal-derived cell line. Also, an ALS-associated mutation in the FUS/TLS 3'UTR ablates the ability of miR-b2122 to regulate reporter gene linked to FUS/TLS 3'UTR, and sALS samples which showed a down-regulation in miR-b2122 also showed an increase in FUS/TLS protein expression. Overall, we have identified a novel miRNA that is down-regulated in sALS that appears to be a central regulator of disease-related RNA-binding proteins, and thus its dysregulation likely contributes to TDP-43, FUS/TLS and RGNEF pathogenesis in sALS.

REFERENCE [6] · ID: 31882736

ID: 31882736
Title: TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS.
Abstract: 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.

REFERENCE [5] · ID: 32764283

ID: 32764283
Title: Inclusion Formation and Toxicity of the ALS Protein RGNEF and Its Association with the Microtubule Network.
Abstract: The Rho guanine nucleotide exchange factor (RGNEF) protein encoded by the ARHGEF28 gene has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Biochemical and pathological studies have shown that RGNEF is a component of the hallmark neuronal cytoplasmic inclusions in ALS-affected neurons. Additionally, a heterozygous mutation in ARHGEF28 has been identified in a number of familial ALS (fALS) cases that may give rise to one of two truncated variants of the protein. Little is known about the normal biological function of RGNEF or how it contributes to ALS pathogenesis. To further explore RGNEF biology we have established and characterized a yeast model and characterized RGNEF expression in several mammalian cell lines. We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in ARGHEF28 gives rise to an inclusion-forming and toxic protein. Additionally, through unbiased screening using the split-ubiquitin system, we have identified RGNEF-interacting proteins, including two ALS-associated proteins. Functional characterization of other RGNEF interactors identified in our screen suggest that RGNEF functions as a microtubule regulator. Our findings indicate that RGNEF misfolding and toxicity may cause impairment of the microtubule network and contribute to ALS pathogenesis.

REFERENCE [2] · ID: 37009460

ID: 37009460
Title: Retinal fingerprints of ALS in patients: Ganglion cell apoptosis and TDP-43/p62 misplacement.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neuron function. Although ophthalmic deficits are not considered a classic symptom of ALS, recent studies suggest that changes in retinal cells, similar to those in the spinal cord motor neurons, have been observed in postmortem human tissues and animal models. In this study, we examined by immunofluorescence analysis the retinal cell layers of sporadic ALS patients in post-mortem retinal slices. We evaluated the presence of cytoplasmic TDP-43 and SQSTM1/p62 aggregates, activation of the apoptotic pathway, and microglia and astrocytes reactivity. We found in the retinal ganglion cell layer of ALS patients the increase of mislocalized TDP-43, SQSTM1/p62 aggregates, activation of cleaved caspase-3, and microglia density, suggesting that retinal changes can be used as an additional diagnostic tool for ALS. The retina is considered part of the central nervous system, and neurodegenerative changes in the brain may be accompanied by structural and possibly functional changes in the neuroretina and ocular vasculature. Therefore, using in vivo retinal biomarkers as an additional diagnostic tool for ALS may provide an opportunity to longitudinally monitor individuals and therapies over time in a noninvasive and cost-effective manner.

REFERENCE [1] · ID: 38739752

ID: 38739752
Title: Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.
Abstract: 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.

REFERENCE [4] · ID: 39160362

ID: 39160362
Title: Neuropathological hallmarks in the post-mortem retina of neurodegenerative diseases.
Abstract: The retina is increasingly recognised as a potential source of biomarkers for neurodegenerative diseases. Hallmark protein aggregates in the retinal neuronal tissue could be imaged through light non-invasively. Post-mortem studies have already shown the presence of specific hallmark proteins in Alzheimer's disease, primary tauopathies, synucleinopathies and frontotemporal lobar degeneration. This study aims to assess proteinopathy in a post-mortem cohort with different neurodegenerative diseases and assess the presence of the primary pathology in the retina. Post-mortem eyes were collected in collaboration with the Netherlands Brain Bank from donors with Alzheimer's disease (n = 17), primary tauopathies (n = 8), synucleinopathies (n = 27), frontotemporal lobar degeneration (n = 8), mixed pathology (n = 11), other neurodegenerative diseases (n = 6), and cognitively normal controls (n = 25). Multiple cross sections of the retina and optic nerve tissue were immunostained using antibodies against pTau Ser202/Thr205 (AT8), amyloid-beta (4G8), alpha-synuclein (LB509), pTDP-43 Ser409/410 and p62-lck ligand (p62) and were assessed for the presence of aggregates and inclusions. pTau pathology was observed as a diffuse signal in Alzheimer's disease, primary tauopathies and controls with Alzheimer's disease neuropathological changes. Amyloid-beta was observed in the vessel wall and as cytoplasmic granular deposits in all groups. Alpha-synuclein pathology was observed as Lewy neurites in the retina in synucleinopathies associated with Lewy pathology and as oligodendroglial cytoplasmic inclusions in the optic nerve in multiple system atrophy. Anti-pTDP-43 generally showed typical neuronal cytoplasmic inclusion bodies in cases with frontotemporal lobar degeneration with TDP-43 and also in cases with later stages of limbic-associated TDP-43 encephalopathy. P62 showed inclusion bodies similar to those seen with anti-pTDP-43. Furthermore, pTau and alpha-synuclein pathology were significantly associated with increasing Braak stages for neurofibrillary tangles and Lewy bodies, respectively. Mixed pathology cases in this cohort consisted of cases (n = 6) with high Braak LB stages (> 4) and low or moderate AD pathology, high AD pathology (n = 1, Braak NFT 6, Thal phase 5) with moderate LB pathology, or a combination of low/moderate scores for different pathology scores in the brain (n = 4). There were no cases with advanced co-pathologies. In seven cases with Braak LB ≥ 4, LB pathology was observed in the retina, while tau pathology in the retina in the mixed pathology group (n = 11) could not be observed. From this study, we conclude that the retina reflects the presence of the major hallmark proteins associated with neurodegenerative diseases. Although low or moderate levels of copathology were found in the brains of most cases, the retina primarily manifested protein aggregates associated with the main neurodegenerative disease. These findings indicate that with appropriate retinal imaging techniques, retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain.

REFERENCE [9] · ID: 39360635

ID: 39360635
Title: Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.
Abstract: 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.

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Joshua Dungan

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