PTBP1 regulates injury responses and sensory pathways in adult peripheral neurons.

Polypyrimidine tract binding protein 1 (PTBP1) is thought to be expressed only at embryonic stages in central neurons. Its down-regulation triggers neuronal differentiation in precursor and non-neuronal cells, an approach recently tested for generation of neurons de novo for amelioration of neurodegenerative disorders. Moreover, PTBP1 is replaced by its paralog PTBP2 in mature central neurons. Unexpectedly, we found that both proteins are coexpressed in adult sensory and motor neurons, with PTBP2 restricted mainly to the nucleus, while PTBP1 also shows axonal localization. Levels of axonal PTBP1 increased markedly after peripheral nerve injury, and it associates in axons with mRNAs involved in injury responses and nerve regeneration, including importin ß1 (KPNB1) and RHOA. Perturbation of PTBP1 affects local translation in axons, nociceptor neuron regeneration and both thermal and mechanical sensation. Thus, PTBP1 has functional roles in adult axons. Hence, caution is required before considering targeting of PTBP1 for therapeutic purposes.

A conditional null allele of Dync1h1 enables targeted analyses of dynein roles in neuronal length sensing.

Size homeostasis is a fundamental process in biology and is particularly important for large cells such as neurons. We previously proposed a motor-dependent length-sensing mechanism wherein reductions in microtubule motor levels would be expected to accelerate neuronal growth, and validated this prediction in dynein heavy chain 1 Loa mutant (Dync1h1Loa) sensory neurons. Here, we describe a new mouse model with a conditional deletion allele of exons 24 and 25 in Dync1h1. Homozygous Islet1-Cre-mediated deletion of Dync1h1 (Isl1-Dync1h1-/-), which deletes protein from the motor and sensory neurons, is embryonic lethal, but heterozygous animals (Isl1-Dync1h1+/-) survive to adulthood with ∼50% dynein expression in targeted cells. Isl1-Dync1h1+/- sensory neurons reveal accelerated growth, as previously reported in Dync1h1Loa neurons. Moreover, Isl1-Dync1h1+/- mice show mild impairments in gait, proprioception and tactile sensation, similar to what is seen in Dync1h1Loa mice, confirming that specific aspects of the Loa phenotype are due to reduced dynein levels. Isl1-Dync1h1+/- mice also show delayed recovery from peripheral nerve injury, likely due to reduced injury signal delivery from axonal lesion sites. Thus, conditional deletion of Dync1h1 exons 24 and 25 enables targeted studies of the role of dynein in neuronal growth.

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin ß1 Interactomes.

Importin ß1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin ß1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin ß1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Importin α3 regulates chronic pain pathways in peripheral sensory neurons.

How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.

DYNLRB1 is essential for dynein mediated transport and neuronal survival.

The cytoplasmic dynein motor complex transports essential signals and organelles from the cell periphery to the perinuclear region, hence is critical for the survival and function of highly polarized cells such as neurons. Dynein Light Chain Roadblock-Type 1 (DYNLRB1) is thought to be an accessory subunit required for specific cargos, but here we show that it is essential for general dynein-mediated transport and sensory neuron survival. Homozygous Dynlrb1 null mice are not viable and die during early embryonic development. Furthermore, heterozygous or adult knockdown animals display reduced neuronal growth, and selective depletion of Dynlrb1 in proprioceptive neurons compromises their survival. Conditional depletion of Dynlrb1 in sensory neurons causes deficits in several signaling pathways, including ß-catenin subcellular localization, and a severe impairment in the axonal transport of both lysosomes and retrograde signaling endosomes. Hence, DYNLRB1 is an essential component of the dynein complex, and given dynein's critical functions in neuronal physiology, DYNLRB1 could have a prominent role in the etiology of human neurodegenerative diseases.

Cell size sensing-a one-dimensional solution for a three-dimensional problem?

Individual cell types have characteristic sizes, suggesting that size sensing mechanisms may coordinate transcription, translation, and metabolism with cell growth rates. Two types of size-sensing mechanisms have been proposed: spatial sensing of the location or dimensions of a signal, subcellular structure or organelle; or titration-based sensing of the intracellular concentrations of key regulators. Here we propose that size sensing in animal cells combines both titration and spatial sensing elements in a dynamic mechanism whereby microtubule motor-dependent localization of RNA encoding importin ß1 and mTOR, coupled with regulated local protein synthesis, enable cytoskeleton length sensing for cell growth regulation.

Translatome Regulation in Neuronal Injury and Axon Regrowth.

Transcriptional events leading to outgrowth of neuronal axons have been intensively studied, but the role of translational regulation in this process is not well understood. Here, we use translatome analyses by ribosome pull-down and protein synthesis characterization by metabolic isotopic labeling to study nerve injury and axon outgrowth proteomes in rodent dorsal root ganglia (DRGs) and sensory neurons. We identify over 1600 gene products that are primarily translationally regulated in DRG neurons after nerve injury, many of which contain a 5'UTR cytosine-enriched regulator of translation (CERT) motif, implicating the translation initiation factor Eif4e in the injury response. We further identified approximately 200 proteins that undergo robust de novo synthesis in the initial stages of axon growth. ApoE is one of the highly synthesized proteins in neurons, and its receptor binding inhibition or knockout affects axon outgrowth. These findings provide a resource for future analyses of the role of translational regulation in neuronal injury responses and axon extension.

Locally translated mTOR controls axonal local translation in nerve injury.

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin ß1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.

Nucleolin-Mediated RNA Localization Regulates Neuron Growth and Cycling Cell Size.

How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin ß1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin ß1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin ß1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.

Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3ß (GSK3ß) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gßγ subunits. Li(+) did not impair direct activation of GIRK channels by Gßγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gßγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

Axon-soma communication in neuronal injury.

The extensive lengths of neuronal processes necessitate efficient mechanisms for communication with the cell body. Neuronal regeneration after nerve injury requires new transcription; thus, long-distance retrograde signalling from axonal lesion sites to the soma and nucleus is required. In recent years, considerable progress has been made in elucidating the mechanistic basis of this system. This has included the discovery of a priming role for early calcium waves; confirmation of central roles for mitogen-activated protein kinase signalling effectors, the importin family of nucleocytoplasmic transport factors and molecular motors such as dynein; and demonstration of the importance of local translation as a key regulatory mechanism. These recent findings provide a coherent mechanistic framework for axon-soma communication in the injured nerve and shed light on the integration of cytoplasmic and nuclear transport in all eukaryotic cells.

Cell length sensing for neuronal growth control.

Neurons exhibit great size differences, and must coordinate biosynthesis rates in cell bodies with the growth needs of different lengths of axons. Classically, axon growth has been viewed mainly as a consequence of extrinsic influences. However, recent publications have proposed at least two different intrinsic axon growth-control mechanisms. We suggest that these mechanisms form part of a continuum of axon growth-control mechanisms, wherein initial growth rates are pre-programmed by transcription factor levels, and subsequent elongating growth is dependent on feedback from intrinsic length-sensing enabled by bidirectional motor-dependent oscillating signals. This model might explain intrinsic limits on elongating neuronal growth and provides a mechanistic framework for determining the connections between genome expression and cellular growth rates in neurons.

WIS-NeuroMath enables versatile high throughput analyses of neuronal processes.

Automated analyses of neuronal morphology are important for quantifying connectivity and circuitry in vivo, as well as in high content imaging of primary neuron cultures. The currently available tools for quantification of neuronal morphology either are highly expensive commercial packages or cannot provide automated image quantifications at single cell resolution. Here, we describe a new software package called WIS-NeuroMath, which fills this gap and provides solutions for automated measurement of neuronal processes in both in vivo and in vitro preparations. Diverse image types can be analyzed without any preprocessing, enabling automated and accurate detection of neurites followed by their quantification in a number of application modules. A cell morphology module detects cell bodies and attached neurites, providing information on neurite length, number of branches, cell body area, and other parameters for each cell. A neurite length module provides a solution for images lacking cell bodies, such as tissue sections. Finally, a ganglion explant module quantifies outgrowth by identifying neurites at different distances from the ganglion. Quantification of a diverse series of preparations with WIS-NeuroMath provided data that were well matched with parallel analyses of the same preparations in established software packages such as MetaXpress or NeuronJ. The capabilities of WIS-NeuroMath are demonstrated in a range of applications, including in dissociated and explant cultures and histological analyses on thin and whole-mount sections. WIS-NeuroMath is freely available to academic users, providing a versatile and cost-effective range of solutions for quantifying neurite growth, branching, regeneration, or degeneration under different experimental paradigms.

Subcellular knockout of importin ß1 perturbs axonal retrograde signaling.

Subcellular localization of mRNA enables compartmentalized regulation within large cells. Neurons are the longest known cells; however, so far, evidence is lacking for an essential role of endogenous mRNA localization in axons. Localized upregulation of Importin ß1 in lesioned axons coordinates a retrograde injury-signaling complex transported to the neuronal cell body. Here we show that a long 3' untranslated region (3' UTR) directs axonal localization of Importin ß1. Conditional targeting of this 3' UTR region in mice causes subcellular loss of Importin ß1 mRNA and protein in axons, without affecting cell body levels or nuclear functions in sensory neurons. Strikingly, axonal knockout of Importin ß1 attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. Thus, localized translation of Importin ß1 mRNA enables separation of cytoplasmic and nuclear transport functions of importins and is required for efficient retrograde signaling in injured axons.

A motor-driven mechanism for cell-length sensing.

Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing.Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development.Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1.Thus, molecular motors critically influence cell length sensing and growth control.

Axonal transcription factors signal retrogradely in lesioned peripheral nerve.

Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

Axoplasm isolation from rat sciatic nerve.

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

Signaling to transcription networks in the neuronal retrograde injury response.

Retrograde signaling from axon to soma activates intrinsic regeneration mechanisms in lesioned peripheral sensory neurons; however, the links between axonal injury signaling and the cell body response are not well understood. Here, we used phosphoproteomics and microarrays to implicate approximately 900 phosphoproteins in retrograde injury signaling in rat sciatic nerve axons in vivo and approximately 4500 transcripts in the in vivo response to injury in the dorsal root ganglia. Computational analyses of these data sets identified approximately 400 redundant axonal signaling networks connected to 39 transcription factors implicated in the sensory neuron response to axonal injury. Experimental perturbation of individual overrepresented signaling hub proteins, including Abl, AKT, p38, and protein kinase C, affected neurite outgrowth in sensory neurons. Paradoxically, however, combined perturbation of Abl together with other hub proteins had a reduced effect relative to perturbation of individual proteins. Our data indicate that nerve injury responses are controlled by multiple regulatory components, and suggest that network redundancies provide robustness to the injury response.