Where to start? Activity-dependent alternative translation initiation generates multifunctional proteoforms in the brain.

In this issue of Molecular Cell, Lee et al.1 report that alternative translation initiation can generate new proteoforms with distinct localization patterns in a neuronal activity-dependent manner.

Isoform balance of the long noncoding RNA NEAT1 is regulated by the RNA-binding protein QKI, governs the glioma transcriptome, and impacts cell migration.

The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in a variety of human cancers. Two overlapping NEAT1 isoforms, NEAT1_1 and NEAT1_2, are produced through mutually exclusive alternative 3' end formation. Previous studies extensively investigated NEAT1 dysregulation in tumors, but often failed to achieve distinct quantification of the two NEAT1 isoforms. Moreover, molecular mechanisms governing the biogenesis of NEAT1 isoforms and the functional impacts of their dysregulation in tumorigenesis remain poorly understood. In this study, we employed an isoform-specific quantification assay and found differential dysregulation of NEAT1 isoforms in patient-derived glioblastoma multiforme cells. We further showed usage of the NEAT1 proximal polyadenylation site (PAS) is a critical mechanism that controls glioma NEAT1 isoform production. CRISPR-Cas9-mediated PAS deletion reduced NEAT1_1 and reciprocally increased NEAT1_2, which enhanced nuclear paraspeckle formation in human glioma cells. Moreover, the utilization of the NEAT1 PAS is facilitated by the RNA-binding protein quaking (QKI), which binds to the proximal QKI recognition elements. Functionally, we identified transcriptomic changes and altered biological pathways caused by NEAT1 isoform imbalance in glioma cells, including the pathway for the regulation of cell migration. Finally, we demonstrated the forced increase of NEAT1_2 upon NEAT1 PAS deletion is responsible for driving glioma cell migration and promoting the expression of genes implicated in the regulation of cell migration. Together, our studies uncovered a novel mechanism that regulates NEAT1 isoforms and their functional impacts on the glioma transcriptome, which affects pathological pathways of glioma, represented by migration.

Cryptic exon inclusion is a molecular signature of LATE-NC in aging brains.

The aggregation, mislocalization, and phosphorylation of TDP-43 are pathologic hallmarks of several neurodegenerative diseases and provide a defining criterion for the neuropathologic diagnosis of Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). LATE neuropathologic changes (LATE-NC) are often comorbid with other neurodegenerative pathologies including Alzheimer's disease neuropathologic changes (ADNC). We examined whether TDP-43 regulated cryptic exons accumulate in the hippocampus of neuropathologically confirmed LATE-NC cases. We found that several cryptic RNAs are robustly expressed in LATE-NC cases with or without comorbid ADNC and correlate with pTDP-43 abundance; however, the accumulation of cryptic RNAs is more robust in LATE-NC with comorbid ADNC. Additionally, cryptic RNAs can robustly distinguish LATE-NC from healthy controls and AD cases. These findings expand our current understanding and provide novel potential biomarkers for LATE pathogenesis.

ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function.

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination.

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

Regulation of RNA granules by FMRP and implications for neurological diseases.

RNA granule formation, which can be regulated by RNA-binding proteins (RBPs) such as fragile X mental retardation protein (FMRP), acts as a mechanism to control both the repression and subcellular localization of translation. Dysregulated assembly of RNA granules has been implicated in multiple neurological disorders, such as amyotrophic lateral sclerosis. Thus, it is crucial to understand the cellular pathways impinging upon granule assembly or disassembly. The goal of this review is to summarize recent advances in our understanding of the role of the RBP, FMRP, in translational repression underlying RNA granule dynamics, mRNA transport and localized. We summarize the known mechanisms of translational regulation by FMRP, the role of FMRP in RNA transport granules, fragile X granules and stress granules. Focusing on the emerging link between FMRP and stress granules, we propose a model for how hyperassembly and hypoassembly of RNA granules may contribute to neurological diseases.

TBK1 interacts with tau and enhances neurodegeneration in tauopathy.

One of the defining pathological features of Alzheimer's disease (AD) is the deposition of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau in the brain. Aberrant activation of kinases in AD has been suggested to enhance phosphorylation and toxicity of tau, making the responsible tau kinases attractive therapeutic targets. The full complement of tau-interacting kinases in AD brain and their activity in disease remains incompletely defined. Here, immunoaffinity enrichment coupled with mass spectrometry (MS) identified TANK-binding kinase 1 (TBK1) as a tau-interacting partner in human AD cortical brain tissues. We validated this interaction in human AD, familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) caused by mutations in MAPT (R406W & P301L) and corticobasal degeneration (CBD) postmortem brain tissues as well as human cell lines. Further, we document increased TBK1 activation in both AD and FTDP-17 and map TBK1 phosphorylation sites on tau based on in vitro kinase assays coupled to MS. Lastly, in a Drosophila tauopathy model, activating expression of a conserved TBK1 ortholog triggers tau hyperphosphorylation and enhanced neurodegeneration, whereas knockdown had the reciprocal effect, suppressing tau toxicity. Collectively, our findings suggest that increased TBK1 activation may promote tau hyperphosphorylation and neuronal loss in AD and related tauopathies.

Biased modulators of NMDA receptors control channel opening and ion selectivity.

Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.

Behavioral changes and growth deficits in a CRISPR engineered mouse model of the schizophrenia-associated 3q29 deletion.

The 3q29 deletion confers increased risk for neuropsychiatric phenotypes including intellectual disability, autism spectrum disorder, generalized anxiety disorder, and a >40-fold increased risk for schizophrenia. To investigate consequences of the 3q29 deletion in an experimental system, we used CRISPR/Cas9 technology to introduce a heterozygous deletion into the syntenic interval on C57BL/6 mouse chromosome 16. mRNA abundance for 20 of the 21 genes in the interval was reduced by ~50%, while protein levels were reduced for only a subset of these, suggesting a compensatory mechanism. Mice harboring the deletion manifested behavioral impairments in multiple domains including social interaction, cognitive function, acoustic startle, and amphetamine sensitivity, with some sex-dependent manifestations. In addition, 3q29 deletion mice showed reduced body weight throughout development consistent with the phenotype of 3q29 deletion syndrome patients. Of the genes within the interval, DLG1 has been hypothesized as a contributor to the neuropsychiatric phenotypes. However, we show that Dlg1+/- mice did not exhibit the behavioral deficits seen in mice harboring the full 3q29 deletion. These data demonstrate the following: the 3q29 deletion mice are a valuable experimental system that can be used to interrogate the biology of 3q29 deletion syndrome; behavioral manifestations of the 3q29 deletion may have sex-dependent effects; and mouse-specific behavior phenotypes associated with the 3q29 deletion are not solely due to haploinsufficiency of Dlg1.

Mechanisms Driving the Emergence of Neuronal Hyperexcitability in Fragile X Syndrome.

Hyperexcitability is a shared neurophysiological phenotype across various genetic neurodevelopmental disorders, including Fragile X syndrome (FXS). Several patient symptoms are associated with hyperexcitability, but a puzzling feature is that their onset is often delayed until their second and third year of life. It remains unclear how and why hyperexcitability emerges in neurodevelopmental disorders. FXS is caused by the loss of FMRP, an RNA-binding protein which has many critical roles including protein synthesis-dependent and independent regulation of ion channels and receptors, as well as global regulation of protein synthesis. Here, we discussed recent literature uncovering novel mechanisms that may drive the progressive onset of hyperexcitability in the FXS brain. We discussed in detail how recent publications have highlighted defects in homeostatic plasticity, providing new insight on the FXS brain and suggest pharmacotherapeutic strategies in FXS and other neurodevelopmental disorders.

The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease.

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

Reward-Related Expectations Trigger Dendritic Spine Plasticity in the Mouse Ventrolateral Orbitofrontal Cortex.

An essential aspect of goal-directed decision-making is selecting actions based on anticipated consequences, a process that involves the orbitofrontal cortex (OFC) and potentially, the plasticity of dendritic spines in this region. To investigate this possibility, we trained male and female mice to nose poke for food reinforcers, or we delivered the same number of food reinforcers non-contingently to separate mice. We then decreased the likelihood of reinforcement for trained mice, requiring them to modify action-outcome expectations. In a separate experiment, we blocked action-outcome updating via chemogenetic inactivation of the OFC. In both cases, successfully selecting actions based on their likely consequences was associated with fewer immature, thin-shaped dendritic spines and a greater proportion of mature, mushroom-shaped spines in the ventrolateral OFC. This pattern was distinct from spine loss associated with aging, and we identified no effects on hippocampal CA1 neurons. Given that the OFC is involved in prospective calculations of likely outcomes, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for solidifying durable expectations. To investigate causal relationships, we inhibited the RNA-binding protein fragile X mental retardation protein (encoded by Fmr1), which constrains dendritic spine turnover. Ventrolateral OFC-selective Fmr1 knockdown recapitulated the behavioral effects of inducible OFC inactivation (and lesions; also shown here), impairing action-outcome conditioning, and caused dendritic spine excess. Our findings suggest that a proper balance of dendritic spine plasticity within the OFC is necessary for one's ability to select actions based on anticipated consequences.SIGNIFICANCE STATEMENT Navigating a changing environment requires associating actions with their likely outcomes and updating these associations when they change. Dendritic spine plasticity is likely involved, yet relationships are unconfirmed. Using behavioral, chemogenetic, and viral-mediated gene silencing strategies and high-resolution microscopy, we find that modifying action-outcome expectations is associated with fewer immature spines and a greater proportion of mature spines in the ventrolateral orbitofrontal cortex (OFC). Given that the OFC is involved in prospectively calculating the likely outcomes of one's behavior, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for maintaining durable expectations.

RNA-mediated toxicity in C9orf72 ALS and FTD.

A GGGGCC hexanucleotide repeat expansion in the first intron of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Compelling evidence suggests that gain of toxicity from the bidirectionally transcribed repeat expanded RNAs plays a central role in disease pathogenesis. Two potential mechanisms have been proposed including RNA-mediated toxicity and/or the production of toxic dipeptide repeat proteins. In this review, we focus on the role of RNA mediated toxicity in ALS/FTD caused by the C9orf72 mutation and discuss arguments for and against this mechanism. In addition, we summarize how G4C2 repeat RNAs can elicit toxicity and potential therapeutic strategies to mitigate RNA-mediated toxicity.

Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage.

Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.

Dysregulation and restoration of translational homeostasis in fragile X syndrome.

Fragile X syndrome (FXS), the most-frequently inherited form of intellectual disability and the most-prevalent single-gene cause of autism, results from a lack of fragile X mental retardation protein (FMRP), an RNA-binding protein that acts, in most cases, to repress translation. Multiple pharmacological and genetic manipulations that target receptors, scaffolding proteins, kinases and translational control proteins can rescue neuronal morphology, synaptic function and behavioural phenotypes in FXS model mice, presumably by reducing excessive neuronal translation to normal levels. Such rescue strategies might also be explored in the future to identify the mRNAs that are critical for FXS pathophysiology.

Bidirectional control of mRNA translation and synaptic plasticity by the cytoplasmic polyadenylation complex.

Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation, and mGluR signaling.

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

Sonic Hedgehog Guides Axons via Zipcode Binding Protein 1-Mediated Local Translation.

Sonic hedgehog (Shh) attracts spinal cord commissural axons toward the floorplate. How Shh elicits changes in the growth cone cytoskeleton that drive growth cone turning is unknown. We find that the turning of rat commissural axons up a Shh gradient requires protein synthesis. In particular, Shh stimulation increases ß-actin protein at the growth cone even when the cell bodies have been removed. Therefore, Shh induces the local translation of ß-actin at the growth cone. We hypothesized that this requires zipcode binding protein 1 (ZBP1), an mRNA-binding protein that transports ß-actin mRNA and releases it for local translation upon phosphorylation. We found that Shh stimulation increases phospho-ZBP1 levels in the growth cone. Disruption of ZBP1 phosphorylation in vitro abolished the turning of commissural axons toward a Shh gradient. Disruption of ZBP1 function in vivo in mouse and chick resulted in commissural axon guidance errors. Therefore, ZBP1 is required for Shh to guide commissural axons. This identifies ZBP1 as a new mediator of noncanonical Shh signaling in axon guidance.SIGNIFICANCE STATEMENT Sonic hedgehog (Shh) guides axons via a noncanonical signaling pathway that is distinct from the canonical Hedgehog signaling pathway that specifies cell fate and morphogenesis. Axon guidance is driven by changes in the growth cone in response to gradients of guidance molecules. Little is known about the molecular mechanism of how Shh orchestrates changes in the growth cone cytoskeleton that are required for growth cone turning. Here, we show that the guidance of axons by Shh requires protein synthesis. Zipcode binding protein 1 (ZBP1) is an mRNA-binding protein that regulates the local translation of proteins, including actin, in the growth cone. We demonstrate that ZBP1 is required for Shh-mediated axon guidance, identifying a new member of the noncanonical Shh signaling pathway.

Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.