Neurons contribute to pathology in a mouse model of Krabbe disease in a cell-autonomous manner.

In this issue of PLOS Biology, Kreher and colleagues show in a mouse model that in vivo, neurons and not only myelinating glia are primary effectors of disease progression in Krabbe disease. The neuron-specific model generated allows the unprecedented capacity to investigate the neuronal autonomous component of this disorder.

Sensory neurons have an axon initial segment that initiates spontaneous activity in neuropathic pain.

The axon initial segment is a specialized compartment of the proximal axon of CNS neurons where action potentials are initiated. However, it remains unknown whether this domain is assembled in sensory dorsal root ganglion neurons, in which spikes are initiated in the peripheral terminals. Here we investigate whether sensory neurons have an axon initial segment and if it contributes to spontaneous activity in neuropathic pain. Our results demonstrate that myelinated dorsal root ganglion neurons assemble an axon initial segment in the proximal region of their stem axon, enriched in the voltage-gated sodium channels Nav1.1 and Nav1.7. Using correlative immunofluorescence and calcium imaging, we demonstrate that the Nav1.7 channels at the axon initial segment are associated with spontaneous activity. Computer simulations further indicate that the axon initial segment plays a key role in the initiation of spontaneous discharges by lowering their voltage threshold. Finally, using a Cre-based mouse model for time-controlled axon initial segment disassembly, we demonstrate that this compartment is a major source of spontaneous discharges causing mechanical allodynia in neuropathic pain. Thus, an axon initial segment domain is present in sensory neurons and facilitates their spontaneous activity. This study provides a new insight in the cellular mechanisms that cause pathological pain and identifies a new potential target for chronic pain management.

Variants in ADD1 cause intellectual disability, corpus callosum dysgenesis, and ventriculomegaly in humans.

PURPOSE: Adducins interconnect spectrin and actin filaments to form polygonal scaffolds beneath the cell membranes and form ring-like structures in neuronal axons. Adducins regulate mouse neural development, but their function in the human brain is unknown. METHODS: We used exome sequencing to uncover ADD1 variants associated with intellectual disability (ID) and brain malformations. We studied ADD1 splice isoforms in mouse and human neocortex development with RNA sequencing, super resolution imaging, and immunoblotting. We investigated 4 variant ADD1 proteins and heterozygous ADD1 cells for protein expression and ADD1-ADD2 dimerization. We studied Add1 functions in vivo using Add1 knockout mice. RESULTS: We uncovered loss-of-function ADD1 variants in 4 unrelated individuals affected by ID and/or structural brain defects. Three additional de novo copy number variations covering the ADD1 locus were associated with ID and brain malformations. ADD1 is highly expressed in the neocortex and the corpus callosum, whereas ADD1 splice isoforms are dynamically expressed between cortical progenitors and postmitotic neurons. Human variants impair ADD1 protein expression and/or dimerization with ADD2. Add1 knockout mice recapitulate corpus callosum dysgenesis and ventriculomegaly phenotypes. CONCLUSION: Our human and mouse genetics results indicate that pathogenic ADD1 variants cause corpus callosum dysgenesis, ventriculomegaly, and/or ID.

Effects of early intravesical administration of resiniferatoxin to spinal cord-injured rats in neurogenic detrusor overactivity.

OBJECTIVES: To investigate if intravesical administration during spinal shock of resiniferatoxin (RTX), an ultrapotent desensitizing agonist of transient receptor potential vanilloid-1 (TRPV1), would silence TRPV1-expressing bladder afferents at an early stage of disease progression and modulate neurogenic detrusor overactivity (NDO) emergence. MATERIALS AND METHODS: Rats submitted to largely incomplete spinal cord transection at T8/9 spinal segment were treated with intravesical RTX (50 nM) or its vehicle during spinal shock. Four weeks after spinal lesion, bladder-reflex activity was evaluated by cystometry under urethane anesthesia, after which the bladder, spinal cord, and dorsal root ganglia were collected and processed. RESULTS: We found improvements on bladder function several weeks after early intravesical RTX administration, including a marked decrease of intravesical pressures and amplitude of bladder contractions. Such strong long-lasting urodynamic effects resulted from the very potent desensitizing activity of RTX on peripheral terminals of sensory afferents, an effect restricted to the bladder. CONCLUSION: Our results support that an early intervention with RTX could potentially attenuate NDO development and ensuing urinary incontinence, with a dramatic impact on the quality of life of spinal cord injury patients.

The Dyslexia-susceptibility Protein KIAA0319 Inhibits Axon Growth Through Smad2 Signaling.

KIAA0319 is a transmembrane protein associated with dyslexia with a presumed role in neuronal migration. Here we show that KIAA0319 expression is not restricted to the brain but also occurs in sensory and spinal cord neurons, increasing from early postnatal stages to adulthood and being downregulated by injury. This suggested that KIAA0319 participates in functions unrelated to neuronal migration. Supporting this hypothesis, overexpression of KIAA0319 repressed axon growth in hippocampal and dorsal root ganglia neurons; the intracellular domain of KIAA0319 was sufficient to elicit this effect. A similar inhibitory effect was observed in vivo as axon regeneration was impaired after transduction of sensory neurons with KIAA0319. Conversely, the deletion of Kiaa0319 in neurons increased neurite outgrowth in vitro and improved axon regeneration in vivo. At the mechanistic level, KIAA0319 engaged the JAK2-SH2B1 pathway to activate Smad2, which played a central role in KIAA0319-mediated repression of axon growth. In summary, we establish KIAA0319 as a novel player in axon growth and regeneration with the ability to repress the intrinsic growth potential of axons. This study describes a novel regulatory mechanism operating during peripheral nervous system and central nervous system axon growth, and offers novel targets for the development of effective therapies to promote axon regeneration.

Cell intrinsic control of axon regeneration.

Although neurons execute a cell intrinsic program of axonal growth during development, following the establishment of connections, the developmental growth capacity declines. Besides environmental challenges, this switch largely accounts for the failure of adult central nervous system (CNS) axons to regenerate. Here, we discuss the cell intrinsic control of axon regeneration, including not only the regulation of transcriptional and epigenetic mechanisms, but also the modulation of local protein translation, retrograde and anterograde axonal transport, and microtubule dynamics. We further explore the causes underlying the failure of CNS neurons to mount a vigorous regenerative response, and the paradigms demonstrating the activation of cell intrinsic axon growth programs. Finally, we present potential mechanisms to support axon regeneration, as these may represent future therapeutic approaches to promote recovery following CNS injury and disease.

CNS axons globally increase axonal transport after peripheral conditioning.

Despite the inability of CNS axons to regenerate, an increased regenerative capacity can be elicited following conditioning lesion to the peripheral branch of dorsal root ganglia neurons (DRGs). By in vivo radiolabeling of rat DRGs, coupled to mass spectrometry and kinesin immunoprecipitation of spinal cord extracts, we determined that the anterograde transport of cytoskeleton components, metabolic enzymes and axonal regeneration enhancers, was increased in the central branch of DRGs following a peripheral conditioning lesion. Axonal transport of mitochondria was also increased in the central branch of Thy1-MitoCFP mice following a peripheral injury. This effect was generalized and included augmented transport of lysosomes and synaptophysin- and APP-carrying vesicles. Changes in axonal transport were only elicited by a peripheral lesion and not by spinal cord injury. In mice, elevated levels of motors and of polyglutamylated and tyrosinated tubulin were present following a peripheral lesion and can explain the increase in axonal transport induced by conditioning. In summary, our work shows that a peripheral injury induces a global increase in axonal transport that is not restricted to the peripheral branch, and that, by extending to the central branch, allows a rapid and sustained support of regenerating central axons.

Neuronal deletion of GSK3ß increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.

BACKGROUND: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. RESULTS: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3ß (GSK3ß) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3ß regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3ß activity in neurons. Following spinal cord injury, reduced GSK3ß levels or complete neuronal deletion of GSK3ß led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3ß substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3ß-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3ß-CRMP-2 relevance during axon regeneration. CONCLUSIONS: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3ß-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.

Transthyretin is a metallopeptidase with an inducible active site.

TTR (transthyretin) was found recently to possess proteolytic competency besides its well-known transport capabilities. It was described as a cryptic serine peptidase cleaving multiple natural substrates (including ß-amyloid and apolipoprotein A-I) involved in diseases such as Alzheimer's disease and atherosclerosis. In the present study, we aimed to elucidate the catalytic machinery of TTR. All attempts to identify a catalytic serine residue were unsuccessful. However, metal chelators abolished TTR activity. Proteolytic inhibition by EDTA or 1,10-phenanthroline could be reversed with Zn2+ and Mn2+. These observations, supported by analysis of three-dimensional structures of TTR complexed with Zn2+, led to the hypothesis that TTR is a metallopeptidase. Site-directed mutagenesis of selected amino acids unambiguously confirmed this hypothesis. The TTR active site is inducible and constituted via a protein rearrangement resulting in ~7% of proteolytically active TTR at pH 7.4. The side chain of His88 is shifted near His90 and Glu92 establishing a Zn2+-chelating pattern HXHXE not found previously in any metallopeptidase and only conserved in TTR of humans and some other primates. Point mutations of these three residues yielded proteins devoid of proteolytic activity. Glu72 was identified as the general base involved in activation of the catalytic water. Our results unveil TTR as a metallopeptidase and define its catalytic machinery.

HDAC-6 inhibition ameliorates the early neuropathology in a mouse model of Krabbe disease.

Introduction: In Krabbe disease (KD), mutations in ß-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport. Methods: We tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice. Results: Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria. Discussion: Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction.

Coronal brain atlas in stereotaxic coordinates of the African spiny mouse, Acomys cahirinus.

The African spiny mouse (Acomys cahirinus) is an emerging model of mammalian epimorphic regeneration that has aroused the interest of the scientific community in the last decade. To date, studies on brain repair have been hindered by the lack of knowledge on the neuroanatomy of this species. Here, we present a coronal brain atlas in stereotaxic coordinates, which allows for three-dimensional identification and localization of the brain structures of this species. The brain of 12-week-old spiny mice was mapped in stereotaxic coordinates using cresyl violet-stained brain sections obtained from coronal cryosectioning of the brain after transcardial perfusion with fixative. The atlas is presented in 42 plates representing sections spaced 240 µm apart. Stereotaxic coordinates were validated using both a model of Parkinsonian lesion of the striatum with 6-hydroxydopamine and labeling of the corticospinal tract in the spiny mouse spinal cord using AAV1/2-GFP intracortical injections. This work presents a new tool in A. cahirinus neurobiology and opens new avenues of research for the investigation of the regenerative ability of A. cahirinus in models of brain disorders.

Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transection.

Regeneration of adult mammalian central nervous system (CNS) axons is abortive, resulting in inability to recover function after CNS lesion, including spinal cord injury (SCI). Here, we show that the spiny mouse (Acomys) is an exception to other mammals, being capable of spontaneous and fast restoration of function after severe SCI, re-establishing hind limb coordination. Remarkably, Acomys assembles a scarless pro-regenerative tissue at the injury site, providing a unique structural continuity of the initial spinal cord geometry. The Acomys SCI site shows robust axon regeneration of multiple tracts, synapse formation, and electrophysiological signal propagation. Transcriptomic analysis of the spinal cord following transcriptome reconstruction revealed that Acomys rewires glycosylation biosynthetic pathways, culminating in a specific pro-regenerative proteoglycan signature at SCI site. Our work uncovers that a glycosylation switch is critical for axon regeneration after SCI and identifies ß3gnt7, a crucial enzyme of keratan sulfate biosynthesis, as an enhancer of axon growth.

The cytoskeleton as a modulator of tension driven axon elongation.

Throughout development, neurons are capable of integrating external and internal signals leading to the morphological changes required for neuronal polarization and axon growth. The first phase of axon elongation occurs during neuronal polarization. At this stage, membrane remodeling and cytoskeleton dynamics are crucial for the growth cone to advance and guide axon elongation. When a target is recognized, the growth cone collapses to form the presynaptic terminal. Once a synapse is established, the growth of the organism results in an increased distance between the neuronal cell bodies and their targets. In this second phase of axon elongation, growth cone-independent molecular mechanisms and cytoskeleton changes must occur to enable axon growth to accompany the increase in body size. While the field has mainly focused on growth-cone mediated axon elongation during development, tension driven axon growth remains largely unexplored. In this review, we will discuss in a critical perspective the current knowledge on the mechanisms guiding axon growth following synaptogenesis, with a particular focus on the putative role played by the axonal cytoskeleton.

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated α-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated α-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating α-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.

Transthyretin internalization by sensory neurons is megalin mediated and necessary for its neuritogenic activity.

Mutated transthyretin (TTR) causes familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR deposition in the peripheral nervous system (PNS). The origin/reason for TTR deposition in the nerve is unknown. Here we demonstrate that both endogenous mouse TTR and TTR injected intravenously have access to the mouse sciatic nerve. We previously determined that in the absence of TTR, both neurite outgrowth in vitro and nerve regeneration in vivo were impaired. Reinforcing this finding, we now show that local TTR delivery to the crushed sciatic nerve rescues the regeneration phenotype of TTR knock-out (KO) mice. As the absence of TTR was unrelated to neuronal survival, we further evaluated the Schwann cell and inflammatory response to injury, as well as axonal retrograde transport, in the presence/absence of TTR. Only retrograde transport was impaired in TTR KO mice which, in addition to the neurite outgrowth impairment, might account for the decreased regeneration in this strain. Moreover, we show that in vitro, in dorsal root ganglia neurons, clathrin-dependent megalin-mediated TTR internalization is needed for TTR neuritogenic activity. Supporting this observation, we demonstrate that in vivo, decreased levels of megalin lead to decreased nerve regeneration and that megalin's action as a regeneration enhancer is dependent on TTR. In conclusion, our work unravels the mechanism of TTR action during nerve regeneration. Additionally, TTR presence in the nerve, as is here shown, may underlie its preferential deposition in the PNS of familial amyloid polyneuropathy patients.

Aboard transthyretin: From transport to cleavage.

Transthyretin (TTR) is a plasma and cerebrospinal fluid protein mainly recognized as the transporter of thyroxine (T(4)) and retinol. Mutated TTR leads to familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR amyloid deposition particularly in peripheral nerves. Beside its transport activities, TTR is a cryptic protease and participates in the biology of the nervous system. Several studies have been directed at finding new ligands of TTR to further explore the biology of the protein. From the identified ligands, some were in fact TTR protease substrates. In this review, we will discuss the existent information concerning TTR ligands/substrates.

Substrate specificity of transthyretin: identification of natural substrates in the nervous system.

Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTR(prot-) (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine alpha-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTR(prot-), was measured. Cells grown with TTR(prot-) displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.

Non-Muscle Myosin II in Axonal Cell Biology: From the Growth Cone to the Axon Initial Segment.

By binding to actin filaments, non-muscle myosin II (NMII) generates actomyosin networks that hold unique contractile properties. Their dynamic nature is essential for neuronal biology including the establishment of polarity, growth cone formation and motility, axon growth during development (and axon regeneration in the adult), radial and longitudinal axonal tension, and synapse formation and function. In this review, we discuss the current knowledge on the spatial distribution and function of the actomyosin cytoskeleton in different axonal compartments. We highlight some of the apparent contradictions and open questions in the field, including the role of NMII in the regulation of axon growth and regeneration, the possibility that NMII structural arrangement along the axon shaft may control both radial and longitudinal contractility, and the mechanism and functional purpose underlying NMII enrichment in the axon initial segment. With the advances in live cell imaging and super resolution microscopy, it is expected that in the near future the spatial distribution of NMII in the axon, and the mechanisms by which it participates in axonal biology will be further untangled.

Profilin as a dual regulator of actin and microtubule dynamics.

Although originally identified as G-actin sequestering proteins, profilins are emerging as critical regulators of actin dynamics, capable of interacting with multiple acting binding proteins, and being able to link membrane lipids to cytoskeleton components. Recently, in addition to its actin, poly-proline, and phosphatidylinositol binding domains, profilin has been shown to contain residues specialized in microtubule binding. Here we will discuss in a critical perspective the emerging body of data supporting that profilins are central mediators of actin microfilament and microtubule interaction. We will also address the unanswered questions in the field, including the nature of the interaction of profilin with microtubules, and its effect on microtubule dynamics. These recent discoveries deepen our understanding on how different cytoskeleton components are integrated within cells.

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.