Frontotemporal dementia-like disease progression elicited by seeded aggregation and spread of FUS.

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fused in sarcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic Frontotemporal lobar degeneration (FTLD). Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.

Frontotemporal dementia-like disease progression elicited by seeded aggregation and spread of FUS.

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fu sed in s arcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic FTLD. Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.

14-3-3θ, a novel player in TDP-43 pathophysiology: Implications for ALS/FTD.

In this issue of Neuron, Ke et al.1 report a novel non-canonical interaction between 14-3-3θ and TDP-43 that impacts loss-of-function and gain-of-toxic pathology in TDP-43 proteinopathies. The authors further provide proof of principle for a 14-3-3θ-targeted gene therapy to reduce TDP-43-induced deficits in transgenic TDP-43 mutant mice.

Current Methods In ALS Research.

ARTICLES DISCUSSED: Asakawa, K., Handa, H., Kawakami, K. Optogenetic phase transition of TDP-43 in spinal motor neurons of zebrafish larvae. Journal of Visualized Experiments. (180), e62932 (2022). Coyne, A. N., Rothstein, J. D. Nuclei isolation and super-resolution structured illumination microscopy for examining nucleoporin alterations in human neurodegeneration. (175), e62789 (2021). Currey, H. N., Liachko, N. F. Evaluation of motor impairment in C. elegans models of amyotrophic lateral sclerosis. (175), e62699 (2021). Hayes, L. R., Duan, L., Vidensky, S., Kalab, P. Nuclear transport assays in permeabilized mouse cortical neurons. (173), e62710 (2021). Krishnamurthy, K., Trotti, D., Pasinelli, P., Jensen, B. Real-time fluorescent measurements of synaptic functions in models of amyotrophic lateral sclerosis. (173), e62813 (2021). Loganathan, S., Ball H. E., Manzo, E., Zarnescu, D. C. Measuring glucose uptake in Drosophila models of TDP-43 proteinopathy. (174), e62936 (2021). Stilwell, G., Agudelo, A. Dissection and immunohistochemistry of the Drosophila adult leg to detect changes at the neuromuscular junction for an identified motor neuron. (180), e62844 (2022) Taga, A. et al. Establishment of an electrophysiological platform for modeling ALS with regionally-specific human pluripotent stem cell-derived astrocytes and neurons. (174), e62726 (2021). Stoklund Dittlau, K. et al., Generation of human motor units with functional neuromuscular junctions in microfluidic devices. (175), e62959 (2021).

Lost in local translation: TDP-43 and FUS in axonal/neuromuscular junction maintenance and dysregulation in amyotrophic lateral sclerosis.

Key early features of amyotrophic lateral sclerosis (ALS) are denervation of neuromuscular junctions and axonal degeneration. Motor neuron homeostasis relies on local translation through controlled regulation of axonal mRNA localization, transport, and stability. Yet the composition of the local transcriptome, translatome (mRNAs locally translated), and proteome during health and disease remains largely unexplored. This review covers recent discoveries on axonal translation as a critical mechanism for neuronal maintenance/survival. We focus on two RNA binding proteins, transactive response DNA binding protein-43 (TDP-43) and fused in sarcoma (FUS), whose mutations cause ALS and frontotemporal dementia (FTD). Emerging evidence points to their essential role in the maintenance of axons and synapses, including mRNA localization, transport, and local translation, and whose dysfunction may contribute to ALS. Finally, we describe recent advances in omics-based approaches mapping compartment-specific local RNA and protein compositions, which will be invaluable to elucidate fundamental local processes and identify key targets for therapy development.

Wild-type FUS corrects ALS-like disease induced by cytoplasmic mutant FUS through autoregulation.

Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (Fus∆NLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two Fus∆NLS alleles, and improved the age-dependent motor deficits and reduced lifespan caused by heterozygous expression of mutant FUS∆NLS. Mechanistically, wild-type FUS decreased the load of cytoplasmic FUS, increased retention of introns 6 and 7 in the endogenous mouse Fus mRNA, and decreased expression of the mutant mRNA. Thus, the wild-type FUS allele activates the homeostatic autoregulatory loop, maintaining constant FUS levels and decreasing the mutant protein in the cytoplasm. These results provide proof of concept that an autoregulatory competent wild-type FUS expression could protect against this devastating, currently intractable, neurodegenerative disease.

Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB.

Methods to enhance adult neurogenesis by reprogramming glial cells into neurons enable production of new neurons in the adult nervous system. Development of therapeutically viable approaches to induce new neurons is now required to bring this concept to clinical application. Here, we successfully generate new neurons in the cortex and dentate gyrus of the aged adult mouse brain by transiently suppressing polypyrimidine tract binding protein 1 using an antisense oligonucleotide delivered by a single injection into cerebral spinal fluid. Radial glial-like cells and other GFAP-expressing cells convert into new neurons that, over a 2-month period, acquire mature neuronal character in a process mimicking normal neuronal maturation. The new neurons functionally integrate into endogenous circuits and modify mouse behavior. Thus, generation of new neurons in the dentate gyrus of the aging brain can be achieved with a therapeutically feasible approach, thereby opening prospects for production of neurons to replace those lost to neurodegenerative disease.

HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells.

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding-deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named "anisosomes", have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)-dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43-containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.

Reduced C9ORF72 function exacerbates gain of toxicity from ALS/FTD-causing repeat expansion in C9orf72.

Hexanucleotide expansions in C9orf72, which encodes a predicted guanine exchange factor, are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although repeat expansion has been established to generate toxic products, mRNAs encoding the C9ORF72 protein are also reduced in affected individuals. In this study, we tested how C9ORF72 protein levels affected repeat-mediated toxicity. In somatic transgenic mice expressing 66 GGGGCC repeats, inactivation of one or both endogenous C9orf72 alleles provoked or accelerated, respectively, early death. In mice expressing a C9orf72 transgene with 450 repeats that did not encode the C9ORF72 protein, inactivation of one or both endogenous C9orf72 alleles exacerbated cognitive deficits, hippocampal neuron loss, glial activation and accumulation of dipeptide-repeat proteins from translation of repeat-containing RNAs. Reduced C9ORF72 was shown to suppress repeat-mediated elevation in autophagy. These efforts support a disease mechanism in ALS/FTD resulting from reduced C9ORF72, which can lead to autophagy deficits, synergizing with repeat-dependent gain of toxicity.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Phenotypic Suppression of ALS/FTD-Associated Neurodegeneration Highlights Mechanisms of Dysfunction.

A fundamental question regarding the etiology of amyotrophic lateral sclerosis (ALS) is whether the various gene mutations associated with the disease converge on a single molecular pathway or act through multiple pathways to trigger neurodegeneration. Notably, several of the genes and cellular processes implicated in ALS have also been linked to frontotemporal dementia (FTD), suggesting these two diseases share common origins with varied clinical presentations. Scientists are rapidly identifying ALS/FTD suppressors that act on conserved pathways from invertebrates to vertebrates to alleviate degeneration. The elucidation of such genetic modifiers provides insight into the molecular pathways underlying this rapidly progressing neurodegenerative disease, while also revealing new targets for therapeutic development.

An endogenous peptide marker differentiates SOD1 stability and facilitates pharmacodynamic monitoring in SOD1 amyotrophic lateral sclerosis.

The discovery of novel biomarkers has emerged as a critical need for therapeutic development in amyotrophic lateral sclerosis (ALS). For some subsets of ALS, such as the genetic superoxide dismutase 1 (SOD1) form, exciting new treatment strategies, such as antisense oligonucleotide-mediated (ASO-mediated) SOD1 silencing, are being tested in clinical trials, so the identification of pharmacodynamic biomarkers for therapeutic monitoring is essential. We identify increased levels of a 7-amino acid endogenous peptide of SOD1 in cerebrospinal fluid (CSF) of human SOD1 mutation carriers but not in other neurological cases or nondiseased controls. Levels of peptide elevation vary based on the specific SOD1 mutation (ranging from 1.1-fold greater than control in D90A to nearly 30-fold greater in V148G) and correlate with previously published measurements of SOD1 stability. Using a mass spectrometry-based method (liquid chromatography-mass spectrometry), we quantified peptides in both extracellular samples (CSF) and intracellular samples (spinal cord from rat) to demonstrate that the peptide distinguishes mutation-specific differences in intracellular SOD1 degradation. Furthermore, 80% and 63% reductions of the peptide were measured in SOD1G93A and SOD1H46R rat CSF samples, respectively, following treatment with ASO, with an improved correlation to mRNA levels in spinal cords compared with the ELISA measuring intact SOD1 protein. These data demonstrate the potential of this peptide as a pharmacodynamic biomarker.

Membralin deficiency dysregulates astrocytic glutamate homeostasis leading to ALS-like impairment.

Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin KO motor cortex indicated significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsf1a. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results shown that activation of TNF receptor (TNFR1)-NFκB pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in SOD1G93A astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in SOD1G93A mice significantly prolonged mouse survival. Together, our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potentials in ALS therapy.

Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death.

While cytoplasmic aggregation of TDP-43 is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia, how aggregates form and what drives its nuclear clearance have not been determined. Here we show that TDP-43 at its endogenous level undergoes liquid-liquid phase separation (LLPS) within nuclei in multiple cell types. Increased concentration of TDP-43 in the cytoplasm or transient exposure to sonicated amyloid-like fibrils is shown to provoke long-lived liquid droplets of cytosolic TDP-43 whose assembly and maintenance are independent of conventional stress granules. Cytosolic liquid droplets of TDP-43 accumulate phosphorylated TDP-43 and rapidly convert into gels/solids in response to transient, arsenite-mediated stress. Cytoplasmic TDP-43 droplets slowly recruit importin-α and Nup62 and induce mislocalization of RanGap1, Ran, and Nup107, thereby provoking inhibition of nucleocytoplasmic transport, clearance of nuclear TDP-43, and cell death. These findings identify a neuronal cell death mechanism that can be initiated by transient-stress-induced cytosolic de-mixing of TDP-43.

Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis.

Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.

Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from patients with sporadic ALS and familial ALS with GGGGCC repeat expansion in the C9orf72 gene, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS-FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.

ALS/FTD-Linked Mutation in FUS Suppresses Intra-axonal Protein Synthesis and Drives Disease Without Nuclear Loss-of-Function of FUS.

Through the generation of humanized FUS mice expressing full-length human FUS, we identify that when expressed at near endogenous murine FUS levels, both wild-type and ALS-causing and frontotemporal dementia (FTD)-causing mutations complement the essential function(s) of murine FUS. Replacement of murine FUS with mutant, but not wild-type, human FUS causes stress-mediated induction of chaperones, decreased expression of ion channels and transporters essential for synaptic function, and reduced synaptic activity without loss of nuclear FUS or its cytoplasmic aggregation. Most strikingly, accumulation of mutant human FUS is shown to activate an integrated stress response and to inhibit local, intra-axonal protein synthesis in hippocampal neurons and sciatic nerves. Collectively, our evidence demonstrates that human ALS/FTD-linked mutations in FUS induce a gain of toxicity that includes stress-mediated suppression in intra-axonal translation, synaptic dysfunction, and progressive age-dependent motor and cognitive disease without cytoplasmic aggregation, altered nuclear localization, or aberrant splicing of FUS-bound pre-mRNAs. VIDEO ABSTRACT.

Misfolded SOD1 is not a primary component of sporadic ALS.

A common feature of inherited and sporadic ALS is accumulation of abnormal proteinaceous inclusions in motor neurons and glia. SOD1 is the major protein component accumulating in patients with SOD1 mutations, as well as in mutant SOD1 mouse models. ALS-linked mutations of SOD1 have been shown to increase its propensity to misfold and/or aggregate. Antibodies specific for monomeric or misfolded SOD1 have detected misfolded SOD1 accumulating predominantly in spinal cord motor neurons of ALS patients with SOD1 mutations. We now use seven different conformationally sensitive antibodies to misfolded human SOD1 (including novel high affinity antibodies currently in pre-clinical development) coupled with immunohistochemistry, immunofluorescence and immunoprecipitation to test for the presence of misfolded SOD1 in high quality human autopsy samples. Whereas misfolded SOD1 is readily detectable in samples from patients with SOD1 mutations, it is below detection limits for all of our measures in spinal cord and cortex tissues from patients with sporadic or non-SOD1 inherited ALS. The absence of evidence for accumulated misfolded SOD1 supports a conclusion that SOD1 misfolding is not a primary component of sporadic ALS.

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.