Axon Regeneration in the Central Nervous System: Facing the Challenges from the Inside.

After an injury in the adult mammalian central nervous system (CNS), lesioned axons fail to regenerate. This failure to regenerate contrasts with axons' remarkable potential to grow during embryonic development and after an injury in the peripheral nervous system (PNS). Several intracellular mechanisms-including cytoskeletal dynamics, axonal transport and trafficking, signaling and transcription of regenerative programs, and epigenetic modifications-control axon regeneration. In this review, we describe how manipulation of intrinsic mechanisms elicits a regenerative response in different organisms and how strategies are implemented to form the basis of a future regenerative treatment after CNS injury.

Neuronal maturation and axon regeneration: unfixing circuitry to enable repair.

Mammalian neurons lose the ability to regenerate their central nervous system axons as they mature during embryonic or early postnatal development. Neuronal maturation requires a transformation from a situation in which neuronal components grow and assemble to one in which these components are fixed and involved in the machinery for effective information transmission and computation. To regenerate after injury, neurons need to overcome this fixed state to reactivate their growth programme. A variety of intracellular processes involved in initiating or sustaining neuronal maturation, including the regulation of gene expression, cytoskeletal restructuring and shifts in intracellular trafficking, have been shown to prevent axon regeneration. Understanding these processes will contribute to the identification of targets to promote repair after injury or disease.

A new "spin" on recovery after spinal cord injury.

Functional recovery can occur after incomplete spinal cord injury. Takeoka et al. now report that such recovery relies on muscle spindle feedback that is necessary for neuronal circuit remodeling, suggesting novel targets to restore motor functions following spinal cord injuries.

Microtubule self-organization via protein-RNA network crosstalk.

Microtubule plus-end tracking proteins are crucial for the regulation of microtubule dynamics. Preitner et al. report that one such protein, adenomatous polyposis coli (APC), also binds RNA and identify mRNAs encoding tubulin subunits within the brain APC-RNA interactome, suggesting a new mode of microtubule self-regulation.

Key attributes of successful research institutes.

Science does not take place in a vacuum: The physical and social workplace has a profound influence on scientific discoveries. Everyone at a research institute can contribute to its scientific output and productivity, from faculty research groups to facilities and platforms staff to administration and corporate services. Although the researchers addressing exciting scientific questions are key, their efforts can be fostered and directed by the overarching strategy of the institute, interconnection with facilities and platforms, and strong and directed support of the administration and corporate services. Everybody counts and everybody should be empowered to contribute. But what are the characteristics that make scientific organizations and their people flourish? This Essay looks at the structure and culture of successful research institutes, laying out different operational strategies and highlighting points that need be taken into consideration during their implementation.

Moving through the crowd. Where are we at understanding physiological axon growth?

Axon growth enables the rapid wiring of the central nervous system. Understanding this process is a prerequisite to retriggering it under pathological conditions, such as a spinal cord injury, to elicit axon regeneration. The last decades saw progress in understanding the mechanisms underlying axon growth. Most of these studies employed cultured neurons grown on flat surfaces. Only recently studies on axon growth were performed in 3D. In these studies, physiological environments exposed more complex and dynamic aspects of axon development. Here, we describe current views on axon growth and highlight gaps in our knowledge. We discuss how axons interact with the extracellular matrix during development and the role of the growth cone and its cytoskeleton within. Finally, we propose that the time is ripe to study axon growth in a more physiological setting. This will help us uncover the physiologically relevant mechanisms underlying axon growth, and how they can be reactivated to induce axon regeneration.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Mechanisms of Axon Growth and Regeneration: Moving between Development and Disease.

Axons differ in their growth potential: whereas during development, axons rapidly grow to their targets, in the adult mammalian, CNS axons have lost their ability to grow and therefore fail to regenerate. Recent progress has enabled a better understanding of how developmental mechanisms direct axon regeneration. Focusing on neuronal polarization, where one neurite is singled out to become the axon, has uncovered the mechanisms initiating axon growth and growth restraint. This has helped to define the processes that need to be reactivated to induce axon regeneration: microtubule stabilization and actin dynamics. The molecular machinery underlying axon growth and axon regeneration is remarkably similar and includes the Rho-GTPases Cdc42, Rac-1, and RhoA, as well as the actin regulators cofilin and Myosin II. Importantly, neuron-intrinsic growth inhibitors in the adult nervous system, including the voltage-gated calcium channel subunit α2δ2 and the presynaptic active zone protein Munc13, restrain dynamics while the components driving axon growth remain largely present. The identified molecules suggest that synaptic transmission and axon growth may be processes that exclude each other. As a result, axon regeneration may be hampered by synaptic transmission and, thus, by the maturation of the CNS. This research has led to several translational avenues to induce axon regeneration and functional recovery after spinal cord injury and stroke; these include the drugs epothilones, gabapentinoids, and baclofen. Thus, the investigation of axon growth and regeneration side by side has been instrumental to coax the regenerative potential of the CNS.

New roles for the de-ubiquitylating enzyme OTUD4 in an RNA-protein network and RNA granules.

Mechanisms that regulate the formation of membrane-less cellular organelles, such as neuronal RNA granules and stress granules, have gained increasing attention over the past years. These granules consist of RNA and a plethora of RNA-binding proteins. Mutations in RNA-binding proteins have been found in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). By performing pulldown experiments and subsequent mass spectrometry on mouse brain lysates, we discovered that the de-ubiquitylating enzyme OTU domain-containing protein 4 (OTUD4) unexpectedly is part of a complex network of multiple RNA-binding proteins, including core stress granule factors, such as FMRP (also known as FMR1), SMN1, G3BP1 and TIA1. We show that OTUD4 binds RNA, and that several of its interactions with RNA-binding proteins are RNA dependent. OTUD4 is part of neuronal RNA transport granules in rat hippocampal neurons under physiological conditions, whereas upon cellular stress, OTUD4 is recruited to cytoplasmic stress granules. Knockdown of OTUD4 in HeLa cells resulted in defects in stress granule formation and led to apoptotic cell death. Together, we characterize OTUD4 as a new RNA-binding protein with a suggested function in regulation of translation.

Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations.

The nonlysosomal glucosylceramidase ß2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren-like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein-protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.

Can injured adult CNS axons regenerate by recapitulating development?

In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS.

Neuronal polarization: From spatiotemporal signaling to cytoskeletal dynamics.

Neuronal polarization establishes distinct molecular structures to generate a single axon and multiple dendrites. Studies over the past years indicate that this efficient separation is brought about by a network of feedback loops. Axonal growth seems to play a major role in fueling those feedback loops and thereby stabilizing neuronal polarity. Indeed, various effectors involved in feedback loops are pivotal for axonal growth by ultimately acting on the actin and microtubule cytoskeleton. These effectors have key roles in interconnecting actin and microtubule dynamics - a mechanism crucial to commanding the growth of axons. We propose a model connecting signaling with cytoskeletal dynamics and neurite growth to better describe the underlying processes involved in neuronal polarization. We will discuss the current views on feedback loops and highlight the current limits of our understanding.

Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

Assembly of a new growth cone after axotomy: the precursor to axon regeneration.

The assembly of a new growth cone is a prerequisite for axon regeneration after injury. Creation of a new growth cone involves multiple processes, including calcium signalling, restructuring of the cytoskeleton, transport of materials, local translation of messenger RNAs and the insertion of new membrane and cell surface molecules. In axons that have an intrinsic ability to regenerate, these processes are executed in a timely fashion. However, in axons that lack regenerative capacity, such as those of the mammalian CNS, several of the steps that are required for regeneration fail, and these axons do not begin the growth process. Identification of the points of failure can suggest targets for promoting regeneration.

A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.

AAV-mediated expression of BAG1 and ROCK2-shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury.

A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

Mouse model of CADASIL reveals novel insights into Notch3 function in adult hippocampal neurogenesis.

Could impaired adult hippocampal neurogenesis be a relevant mechanism underlying CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)? Memory symptoms in CADASIL, the most common hereditary form of vascular dementia, are usually thought to be primarily due to vascular degeneration and white matter lacunes. Since adult hippocampal neurogenesis, a process essential for the integration of new spatial memory occurs in a highly vascularized niche, we considered dysregulation of adult neurogenesis as a potential mechanism for the manifestation of dementia in CADASIL. Analysis in aged mice overexpressing Notch3 with a CADASIL mutation, revealed vascular deficits in arteries of the hippocampal fissure but not in the niche of the dentate gyrus. At 12 months of age, cell proliferation and survival of newborn neurons were reduced not only in CADASIL mice but also in transgenic controls overexpressing wild type Notch3. At 6 months, hippocampal neurogenesis was altered in CADASIL mice independent of overt vascular abnormalities in the fissure. Further, we identified Notch3 expression in hippocampal precursor cells and maturing neurons in vivo as well as in cultured hippocampal precursor cells. Overexpression and knockdown experiments showed that Notch3 signaling negatively regulated precursor cell proliferation. Notch3 overexpression also led to deficits in KCl-induced precursor cell activation. This suggests a cell-autonomous effect of Notch3 signaling in the regulation of precursor proliferation and activation and a loss-of-function effect in CADASIL. Consequently, besides vascular damage, aberrant precursor cell proliferation and differentiation due to Notch3 dysfunction might be an additional independent mechanism for the development of hippocampal dysfunction in CADASIL.

The DLK signalling pathway--a double-edged sword in neural development and regeneration.

Dual leucine zipper kinase (DLK), a mitogen-activated protein kinase kinase kinase, controls axon growth, apoptosis and neuron degeneration during neural development, as well as neurodegeneration after various insults to the adult nervous system. Interestingly, recent studies have also highlighted a role of DLK in promoting axon regeneration in diverse model systems. Invertebrates and vertebrates, cold- and warm-blooded animals, as well as central and peripheral mammalian nervous systems all differ in their ability to regenerate injured axons. Here, we discuss how DLK-dependent signalling regulates apparently contradictory functions during neural development and regeneration in different species. In addition, we outline strategies to fine-tune DLK function, either alone or together with other approaches, to promote axon regeneration in the adult mammalian central nervous system.

Serum Response Factor (SRF)-cofilin-actin signaling axis modulates mitochondrial dynamics.

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.