Emerging evidence for compromised axonal bioenergetics and axoglial metabolic coupling as drivers of neurodegeneration.

Impaired bioenergetic capacity of the nervous system is thought to contribute to the pathogenesis of many neurodegenerative diseases (NDD). Since neuronal synapses are believed to be the major energy consumers in the nervous system, synaptic derangements resulting from energy deficits have been suggested to play a central role for the development of many of these disorders. However, long axons constitute the largest compartment of the neuronal network, require large amounts of energy, are metabolically and structurally highly vulnerable, and undergo early injurious stresses in many NDD. These stresses likely impose additional energy demands for continuous adaptations and repair processes, and may eventually overwhelm axonal maintenance mechanisms. Indeed, pathological axon degeneration (pAxD) is now recognized as an etiological focus in a wide array of NDD associated with bioenergetic abnormalities. In this paper I first discuss the recognition that a simple experimental model for pAxD is regulated by an auto-destruction program that exhausts distressed axons energetically. Provision of the energy substrate pyruvate robustly counteracts this axonal breakdown. Importantly, energy decline in axons is not only a consequence but also an initiator of this program. This opens the intriguing possibility that axon dysfunction and pAxD can be suppressed by preemptively energizing distressed axons. Second, I focus on the emerging concept that axons communicate energetically with their flanking glia. This axoglial metabolic coupling can help offset the axonal energy decline that activates the pAxD program but also jeopardize axon integrity as a result of perturbed glial metabolism. Third, I present compelling evidence that abnormal axonal energetics and compromised axoglial metabolic coupling accompany the activation of the pAxD auto-destruction pathway in models of glaucoma, a widespread neurodegenerative condition with pathogenic overlap to other common NDD. In conclusion, I propose a novel conceptual framework suggesting that therapeutic interventions focused on bioenergetic support of the nervous system should also address axons and their metabolic interactions with glia.

The LKB1-AMPK and mTORC1 Metabolic Signaling Networks in Schwann Cells Control Axon Integrity and Myelination: Assembling and upholding nerves by metabolic signaling in Schwann cells.

The Liver kinase B1 with its downstream target AMP activated protein kinase (LKB1-AMPK), and the key nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) form two signaling systems that coordinate metabolic and cellular activity with changes in the environment in order to preserve homeostasis. For example, nutritional fluctuations rapidly feed back on these signaling systems and thereby affect cell-specific functions. Recent studies have started to reveal important roles of these strategic metabolic regulators in Schwann cells for the trophic support and myelination of axons. Because aberrant intermediate metabolism along with mitochondrial dysfunction in Schwann cells is mechanistically linked to nerve abnormalities found in acquired and inherited peripheral neuropathies, manipulation of the LKB1-AMPK, and mTORC1 signaling hubs may be a worthwhile therapeutic target to mitigate nerve damage in disease. Here, recent advances in our understanding of LKB1-AMPK and mTORC1 functions in Schwann cells are covered, and future research areas for this key metabolic signaling network are proposed.

mTORC1 promotes proliferation of immature Schwann cells and myelin growth of differentiated Schwann cells.

The myelination of axons in peripheral nerves requires precisely coordinated proliferation and differentiation of Schwann cells (SCs). We found that the activity of the mechanistic target of rapamycin complex 1 (mTORC1), a key signaling hub for the regulation of cellular growth and proliferation, is progressively extinguished as SCs differentiate during nerve development. To study the effects of different levels of sustained mTORC1 hyperactivity in the SC lineage, we disrupted negative regulators of mTORC1, including TSC2 or TSC1, in developing SCs of mutant mice. Surprisingly, the phenotypes ranged from arrested myelination in nerve development to focal hypermyelination in adulthood, depending on the level and timing of mTORC1 hyperactivity. For example, mice lacking TSC2 in developing SCs displayed hyperproliferation of undifferentiated SCs incompatible with normal myelination. However, these defects and myelination could be rescued by pharmacological mTORC1 inhibition. The subsequent reconstitution of SC mTORC1 hyperactivity in adult animals resulted in focal hypermyelination. Together our data suggest a model in which high mTORC1 activity promotes proliferation of immature SCs and antagonizes SC differentiation during nerve development. Down-regulation of mTORC1 activity is required for terminal SC differentiation and subsequent initiation of myelination. In distinction to this developmental role, excessive SC mTORC1 activity stimulates myelin growth, even overgrowth, in adulthood. Thus, our work delineates two distinct functions of mTORC1 in the SC lineage essential for proper nerve development and myelination. Moreover, our studies show that SCs retain their plasticity to myelinate and remodel myelin via mTORC1 throughout life.

Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling.

The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD(+)-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.

New perspectives in diabetic neuropathy.

Diabetes prevalence continues to climb with the aging population. Type 2 diabetes (T2D), which constitutes most cases, is metabolically acquired. Diabetic peripheral neuropathy (DPN), the most common microvascular complication, is length-dependent damage to peripheral nerves. DPN pathogenesis is complex, but, at its core, it can be viewed as a state of impaired metabolism and bioenergetics failure operating against the backdrop of long peripheral nerve axons supported by glia. This unique peripheral nerve anatomy and the injury consequent to T2D underpins the distal-to-proximal symptomatology of DPN. Earlier work focused on the impact of hyperglycemia on nerve damage and bioenergetics failure, but recent evidence additionally implicates contributions from obesity and dyslipidemia. This review will cover peripheral nerve anatomy, bioenergetics, and glia-axon interactions, building the framework for understanding how hyperglycemia and dyslipidemia induce bioenergetics failure in DPN. DPN and painful DPN still lack disease-modifying therapies, and research on novel mechanism-based approaches is also covered.

Of axons that struggle to make ends meet: Linking axonal bioenergetic failure to programmed axon degeneration.

Axons are the long, fragile, and energy-hungry projections of neurons that are challenging to sustain. Together with their associated glia, they form the bulk of the neuronal network. Pathological axon degeneration (pAxD) is a driver of irreversible neurological disability in a host of neurodegenerative conditions. Halting pAxD is therefore an attractive therapeutic strategy. Here we review recent work demonstrating that pAxD is regulated by an auto-destruction program that revolves around axonal bioenergetics. We then focus on the emerging concept that axonal and glial energy metabolism are intertwined. We anticipate that these discoveries will encourage the pursuit of new treatment strategies for neurodegeneration.

Targeting NMNAT1 to axons and synapses transforms its neuroprotective potency in vivo.

Axon and synapse degeneration are common components of many neurodegenerative diseases, and their rescue is essential for effective neuroprotection. The chimeric Wallerian degeneration slow protein (Wld(S)) protects axons dose dependently, but its mechanism is still elusive. We recently showed that Wld(S) acts at a non-nuclear location and is present in axons. This and other recent reports support a model in which Wld(S) protects by extranuclear redistribution of its nuclear NMNAT1 portion. However, it remains unclear whether cytoplasmic NMNAT1 acts locally in axons and synapses or at a non-nuclear site within cell bodies. The potency of axon protection by non-nuclear NMNAT1 relative to Wld(S) also needs to be established in vivo. Because the N-terminal portion of Wld(S) (N70) localized to axons, we hypothesized that it mediates the trafficking of the NMNAT1 portion. To test this, we substituted N70 with an axonal targeting peptide derived from amyloid precursor protein, and fused this to NMNAT1 with disrupted nuclear targeting. In transgenic mice, this transformed NMNAT1 from a molecule unable to inhibit Wallerian degeneration, even at high expression levels, into a protein more potent than Wld(S), able to preserve injured axons for several weeks at undetectable expression levels. Preventing NMNAT1 axonal delivery abolished its protective effect. Axonally targeted NMNAT1 localized to vesicular structures, colocalizing with extranuclear Wld(S), and was cotransported at least partially with mitochondria. We conclude that axonal targeting of NMNAT activity is both necessary and sufficient to delay Wallerian degeneration, and that promoting axonal and synaptic delivery greatly enhances the effectiveness.

Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo.

Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld(S)) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld(S) acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld(S) significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld(S) mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld(S) for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld(S) redistribution. Cytoplasmic Wld(S) fractionated predominantly with mitochondria and microsomes. We conclude that Wld(S) can act in one or more non-nuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.

Measuring Bioenergetic Signatures of Peripheral Nerve Segments by Extracellular Flux Analysis.

Changes of energy metabolism in axons and their adjacent glia as well as alterations in metabolic axon-glia cross talk are emerging as central mechanistic components underlying axon degeneration. The analysis of extracellular flux with commercial metabolic analyzers greatly facilitates the measurement of key parameters of glycolytic and mitochondrial energy metabolism in cells and tissues. In this chapter, I describe a straightforward method to capture bioenergetic profiles of acutely isolated peripheral nerve segments using the Agilent Seahorse XFe24 platform.

A glycolytic shift in Schwann cells supports injured axons.

Axon degeneration is a hallmark of many neurodegenerative disorders. The current assumption is that the decision of injured axons to degenerate is cell-autonomously regulated. Here we show that Schwann cells (SCs), the glia of the peripheral nervous system, protect injured axons by virtue of a dramatic glycolytic upregulation that arises in SCs as an inherent adaptation to axon injury. This glycolytic response, paired with enhanced axon-glia metabolic coupling, supports the survival of axons. The glycolytic shift in SCs is largely driven by the metabolic signaling hub, mammalian target of rapamycin complex 1, and the downstream transcription factors hypoxia-inducible factor 1-alpha and c-Myc, which together promote glycolytic gene expression. The manipulation of glial glycolytic activity through this pathway enabled us to accelerate or delay the degeneration of perturbed axons in acute and subacute rodent axon degeneration models. Thus, we demonstrate a non-cell-autonomous metabolic mechanism that controls the fate of injured axons.

VCP binding influences intracellular distribution of the slow Wallerian degeneration protein, Wld(S).

Wallerian degeneration slow (Wld(S)) mice express a chimeric protein that delays axonal degeneration. The N-terminal domain (N70), which is essential for axonal protection in vivo, binds valosin-containing protein (VCP) and targets both Wld(S) and VCP to discrete nuclear foci. We characterized the formation, composition and localization of these potentially important foci. Missense mutations show that the N-terminal sixteen residues (N16) of Wld(S) are essential for both VCP binding and targeting Wld(S) to nuclear foci. Removing N16 abolishes foci, and VCP binding sequences from ataxin-3 or HrdI restore them. In vitro, these puncta co-localize with proteasome subunits. In vivo, Wld(S) assumes a range of nuclear distribution patterns, including puncta, and its neuronal expression and intranuclear distribution is region-specific and varies between spontaneous and transgenic Wld(S) models. We conclude that VCP influences Wld(S) intracellular distribution, and thus potentially its function, by binding within the N70 domain required for axon protection.

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model.

Glaucoma is a leading cause of blindness caused by progressive degeneration of retinal ganglion cells (RGCs) and their axons. The pathogenesis of glaucoma remains incompletely understood, but optic nerve (ON) axonal injury appears to be an important trigger of RGC axonal and cell body degeneration. Rat models are widely used in glaucoma research to explore pathogenic mechanisms and to test novel neuroprotective approaches. Here we investigated the mechanism of axon loss in glaucoma, studying axon degeneration in slow Wallerian degeneration (Wld(S)) rats after increasing intraocular pressure. Wld(S) delays degeneration of experimentally transected axons for several weeks, so it can provide genetic evidence for Wallerian-like degeneration in disease. As apoptosis is unaffected, Wld(S) also provides information on whether cell death results from axon degeneration or arises independently, an important question yet to be resolved in glaucoma. Having confirmed expression of Wld(S) protein, we found that Wld(S) delayed ON axonal degeneration in experimental rat glaucoma for at least 2 weeks, especially in proximal ON where wild-type axons are most severely affected. The duration of axonal protection is similar to that after ON transection and crush, suggesting that axonal degeneration in glaucoma follows a Wallerian-like mechanism. Axonal degeneration must be prevented for RGCs to remain functional, so pharmacologically mimicking and enhancing the protective mechanism of Wld(S) could offer an important route towards therapy. However, Wld(S) did not protect RGC bodies in glaucoma or after ON lesion, suggesting that combination treatments protecting both axons and cell bodies offer the best therapeutic prospects.

Multiple lines of inhibitory feedback on AKT kinase in Schwann cells lacking TSC1/2 hint at distinct functions of mTORC1 and AKT in nerve development.

During nerve development, Schwann cells (SCs) build multilayered myelin sheaths around axons to accelerate nerve conduction. The mechanistic target of rapamycin complex 1 (mTORC1) downstream of PI3K/AKT signaling lately emerged as a central anabolic regulator of myelination. Using mutant mice with sustained mTORC1 hyperactivity in developing SCs we recently uncovered that mTORC1 impedes developmental myelination by promoting proliferation of immature SCs while antagonizing SC differentiation. In contrast, mTORC1 stimulates myelin production, rather than SC proliferation, in already differentiated SCs. Importantly, these diametrical mTORC1 functions were unmasked under settings of greatly suppressed AKT signaling. Here we demonstrate, inter alia, additional mechanisms of feedback inhibition of AKT by mTORC1, such as strikingly elevated PTEN levels in SCs with disruption of the mTORC1 inhibitory complex, TSC1/2. These data lead us to propose a model wherein mTORC1 and AKT have distinct roles in developing SCs that have to be precisely coordinated for normal myelinogenesis.

Axon degeneration: make the Schwann cell great again.

Axonal degeneration is a pivotal feature of many neurodegenerative conditions and substantially accounts for neurological morbidity. A widely used experimental model to study the mechanisms of axonal degeneration is Wallerian degeneration (WD), which occurs after acute axonal injury. In the peripheral nervous system (PNS), WD is characterized by swift dismantling and clearance of injured axons with their myelin sheaths. This is a prerequisite for successful axonal regeneration. In the central nervous system (CNS), WD is much slower, which significantly contributes to failed axonal regeneration. Although it is well-documented that Schwann cells (SCs) have a critical role in the regenerative potential of the PNS, to date we have only scarce knowledge as to how SCs 'sense' axonal injury and immediately respond to it. In this regard, it remains unknown as to whether SCs play the role of a passive bystander or an active director during the execution of the highly orchestrated disintegration program of axons. Older reports, together with more recent studies, suggest that SCs mount dynamic injury responses minutes after axonal injury, long before axonal breakdown occurs. The swift SC response to axonal injury could play either a pro-degenerative role, or alternatively a supportive role, to the integrity of distressed axons that have not yet committed to degenerate. Indeed, supporting the latter concept, recent findings in a chronic PNS neurodegeneration model indicate that deactivation of a key molecule promoting SC injury responses exacerbates axonal loss. If this holds true in a broader spectrum of conditions, it may provide the grounds for the development of new glia-centric therapeutic approaches to counteract axonal loss.

The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice.

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

Axon degeneration: Linking axonal bioenergetics to myelin.

The mechanisms by which axonal degeneration occurs, even in the presence of apparently normal myelin sheaths, remain unknown. In this issue, Yin et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201607099) study mutant mice in which proteolipid protein is replaced by the peripheral myelin protein P0 and describe a number of early axonal abnormalities, which together suggest that aberrant mitochondrial energy metabolism precedes axonal degeneration.