The ISR downstream target ATF4 represses long-term memory in a cell type-specific manner.

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.

Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.

The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named myelin gene regulatory factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, premyelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities and die because of seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.

Therapeutic opportunities and pitfalls in the treatment of axon degeneration.

PURPOSE OF REVIEW: The current review analyzes recent findings that suggest that axon degeneration is a druggable process in the treatment of neurodegenerative disorders and a subset of traumas. RECENT FINDINGS: Emerging evidence reveals that axon degeneration is an active and regulated process in the early progression of some neurodegenerative diseases and acute traumas, which is orchestrated through a combination of axon-intrinsic and somatically derived signaling events. The identification of these pathways has presented appealing drug targets whose specificity for the nervous system and phenotypes in mouse models offers significant clinical opportunity. SUMMARY: As the biology of axon degeneration becomes clear, so too has the realization that the pathways driving axon degeneration overlap in part with those that drive neuronal apoptosis and, importantly, axon regeneration. Axon-specific disorders like those seen in CIPN, where injury signaling to the nucleus is not a prominent feature, have been shown to benefit from disruption of Sarm1. In injury and disease contexts, where involvement of somatic events is prominent, inhibition of the MAP Kinase DLK exhibits promise for neuroprotection. Here, however, interfering with somatic signaling may preclude the ability of an axon or a circuit to regenerate or functionally adapt following acute injuries.

ZFP191 is required by oligodendrocytes for CNS myelination.

The controlling factors that prompt mature oligodendrocytes to myelinate axons are largely undetermined. In this study, we used a forward genetics approach to identify a mutant mouse strain characterized by the absence of CNS myelin despite the presence of abundant numbers of late-stage, process-extending oligodendrocytes. Through linkage mapping and complementation testing, we identified the mutation as a single nucleotide insertion in the gene encoding zinc finger protein 191 (Zfp191), which is a widely expressed, nuclear-localized protein that belongs to a family whose members contain both DNA-binding zinc finger domains and protein-protein-interacting SCAN domains. Zfp191 mutants express an array of myelin-related genes at significantly reduced levels, and our in vitro and in vivo data indicate that mutant ZFP191 acts in a cell-autonomous fashion to disrupt oligodendrocyte function. Therefore, this study demonstrates that ZFP191 is required for the myelinating function of differentiated oligodendrocytes.

DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.

The cell intrinsic factors that determine whether a neuron regenerates or undergoes apoptosis in response to axonal injury are not well defined. Here we show that the mixed-lineage dual leucine zipper kinase (DLK) is an essential upstream mediator of both of these divergent outcomes in the same cell type. Optic nerve crush injury leads to rapid elevation of DLK protein, first in the axons of retinal ganglion cells (RGCs) and then in their cell bodies. DLK is required for the majority of gene expression changes in RGCs initiated by injury, including induction of both proapoptotic and regeneration-associated genes. Deletion of DLK in retina results in robust and sustained protection of RGCs from degeneration after optic nerve injury. Despite this improved survival, the number of axons that regrow beyond the injury site is substantially reduced, even when the tumor suppressor phosphatase and tensin homolog (PTEN) is deleted to enhance intrinsic growth potential. These findings demonstrate that these seemingly contradictory responses to injury are mechanistically coupled through a DLK-based damage detection mechanism.

Modeling Alzheimer's disease in primary neurons reveals DNA damage response coupled with MAPK-DLK signaling in wild-type tau-induced neurodegeneration.

Background: Alzheimer's disease (AD) is the most prevalent form of neurodegeneration. Despite the well-established link between tau aggregation and clinical progression, the major pathways driven by this protein to intrinsically damage neurons are incompletely understood. Methods: To model AD-relevant neurodegeneration driven by tau, we overexpressed wild-type human tau in primary mouse neurons and characterized the subsequent cellular and molecular changes. RNAseq profiling and functional investigation were performed as well. A direct comparison with a mutant human tau was conducted in detail. Results: We observed substantial axonal degeneration and cell death associated with wild-type tau, a process accompanied by activated caspase 3. Mechanistically, we detected deformation of the nuclear envelope and increased DNA damage response in tau-expressing neurons. Gene profiling analysis further revealed significant alterations in the mitogen-activated protein kinase (MAPK) pathway; moreover, inhibitors of dual leucine zipper kinase (DLK) and c-Jun N-terminal kinase (JNK) were effective in alleviating wild-type human tau-induced neurodegeneration. In contrast, mutant P301L human tau was less toxic to neurons, despite causing comparable DNA damage. Axonal DLK activation induced by wild-type tau potentiated the impact of DNA damage response, resulting in overt neurotoxicity. Conclusions: We have established a cellular tauopathy model highly relevant to AD and identified a functional synergy between DNA damage response and the MAPK-DLK axis in the neuronal degenerative process.

DLK-MAPK Signaling Coupled with DNA Damage Promotes Intrinsic Neurotoxicity Associated with Non-Mutated Tau.

Alzheimer's disease (AD) is the most prevalent form of neurodegeneration. Despite the well-established link between tau aggregation and clinical progression, the major pathways driven by this protein to intrinsically damage neurons are incompletely understood. To model AD-relevant neurodegeneration driven by tau, we overexpressed non-mutated human tau in primary mouse neurons and observed substantial axonal degeneration and cell death, a process accompanied by activated caspase 3. Mechanistically, we detected deformation of the nuclear envelope and increased DNA damage response in tau-expressing neurons. Gene profiling analysis further revealed significant alterations in the mitogen-activated protein kinase (MAPK) pathway; moreover, inhibitors of dual leucine zipper kinase (DLK) and c-Jun N-terminal kinase (JNK) were effective in alleviating wild-type human tau-induced neurodegeneration. In contrast, mutant P301L human tau was less toxic to neurons, despite causing comparable DNA damage. Axonal DLK activation induced by wild-type tau potentiated the impact of DNA damage response, resulting in overt neurotoxicity. In summary, we have established a cellular tauopathy model highly relevant to AD and identified a functional synergy between the DLK-MAPK axis and DNA damage response in the neuronal degenerative process.

DLK-dependent mitochondrial fission drives axon degeneration and neuronal cell death.

Currently there are no effective treatments for an array of neurodegenerative disorders to a large part because cell-based models fail to recapitulate disease. Here we developed a robust human iPSCbased model where laser axotomy causes retrograde axon degeneration leading to neuronal cell death. Time-lapse confocal imaging revealed that damage triggers a wave of mitochondrial fission proceeding from the site of injury to the soma. We demonstrated that mitochondrial fission and resultant cell death is entirely dependent on phosphorylation of dynamin related protein 1 (DRP1) by dual leucine zipper kinase (DLK). Importantly, we show that CRISPR mediated Drp1 depletion protected mouse retinal ganglion neurons from mitochondrial fission and degeneration after optic nerve crush. Our results provide a powerful platform for studying degeneration of human neurons, pinpoint key early events in damage related neural death and new focus for therapeutic intervention.

Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by Atf4 following optic nerve injury.

Previously we showed that neurodegeneration initiated by axonal insults depends in part on the stress-responsive kinase Perk (Larhammar et al., 2017). Here we show that Perk acts primarily through Activating Transcription Factor-4 (Atf4) to stimulate not only pro-apoptotic but also pro-regenerative responses following optic nerve injury. Using conditional knockout mice, we find an extensive Perk/Atf4-dependent transcriptional response that includes canonical Atf4 target genes and modest contributions by C/ebp homologous protein (Chop). Overlap with c-Jun-dependent transcription suggests interplay with a parallel stress pathway that couples regenerative and apoptotic responses. Accordingly, neuronal knockout of Atf4 recapitulates the neuroprotection afforded by Perk deficiency, and Perk or Atf4 knockout impairs optic axon regeneration enabled by disrupting the tumor suppressor Pten. These findings contrast with the transcriptional and functional consequences reported for CRISPR targeting of Atf4 or Chop and reveal an integral role for Perk/Atf4 in coordinating neurodegenerative and regenerative responses to CNS axon injury.

Remyelination protects neurons from DLK-mediated neurodegeneration.

Chronic demyelination is theorized to contribute to neurodegeneration and drive progressive disability in demyelinating diseases like multiple sclerosis. Here, we describe two genetic mouse models of inducible demyelination, one distinguished by effective remyelination, and the other by remyelination failure and persistent demyelination. By comparing these two models, we find that remyelination protects neurons from apoptosis, improves conduction, and promotes functional recovery. Chronic demyelination of neurons leads to activation of the mitogen-associated protein kinase (MAPK) stress pathway downstream of dual leucine zipper kinase (DLK), which ultimately induces the phosphorylation of c-Jun in the nucleus. Both pharmacological inhibition and CRISPR/Cas9-mediated disruption of DLK block c-Jun phosphorylation and the apoptosis of demyelinated neurons. These findings provide direct experimental evidence that remyelination is neuroprotective and identify DLK inhibition as a potential therapeutic strategy to protect chronically demyelinated neurons.

Differential clustering of Caspr by oligodendrocytes and Schwann cells.

Formation of the paranodal axoglial junction (PNJ) requires the presence of three cell adhesion molecules: the 155-kDa isoform of neurofascin (NF155) on the glial membrane and a complex of Caspr and contactin found on the axolemma. Here we report that the clustering of Caspr along myelinated axons during development differs fundamentally between the central (CNS) and peripheral (PNS) nervous systems. In cultures of Schwann cells (SC) and dorsal root ganglion (DRG) neurons, membrane accumulation of Caspr was detected only after myelination. In contrast, in oligodendrocytes (OL)/DRG neurons cocultures, Caspr was clustered upon initial glial cell contact already before myelination had begun. Premyelination clustering of Caspr was detected in cultures of oligodendrocytes and retinal ganglion cells, motor neurons, and DRG neurons as well as in mixed cell cultures of rat forebrain and spinal cords. Cocultures of oligodendrocyte precursor cells isolated from contactin- or neurofascin-deficient mice with wild-type DRG neurons showed that clustering of Caspr at initial contact sites between OL processes and the axon requires glial expression of NF155 but not of contactin. These results demonstrate that the expression of membrane proteins along the axolemma is determined by the type of the contacting glial cells and is not an intrinsic characteristic of the axon.

NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.

Axons dictate whether or not they will become myelinated in both the central and peripheral nervous systems by providing signals that direct the development of myelinating glia. Here we identify the neurotrophin nerve growth factor (NGF) as a potent regulator of the axonal signals that control myelination of TrkA-expressing dorsal root ganglion neurons (DRGs). Unexpectedly, these NGF-regulated axonal signals have opposite effects on peripheral and central myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes. These findings indicate a novel role for growth factors in regulating the receptivity of axons to myelination and reveal that different axonal signals control central and peripheral myelination.

Intrinsic Neuronal Stress Response Pathways in Injury and Disease.

From injury to disease to aging, neurons, like all cells, may face various insults that can impact their function and survival. Although the consequences are substantially dictated by the type, context, and severity of insult, distressed neurons are far from passive. Activation of cellular stress responses aids in the preservation or restoration of nervous system function. However, stress responses themselves can further advance neuropathology and contribute significantly to neuronal dysfunction and neurodegeneration. Here we explore the recent advances in defining the cellular stress responses within neurodegenerative diseases and neuronal injury, and we emphasize axonal injury as a well-characterized model of neuronal insult. We highlight key findings and unanswered questions about neuronal stress response pathways, from the initial detection of cellular insults through the underlying mechanisms of the responses to their ultimate impact on the fates of distressed neurons.

Nerve regeneration: regrowth stumped by shared receptor.

Three different myelin proteins, Nogo, MAG, and OMgp, inhibit regenerating axons after CNS injury. New work reveals that they all share a common receptor and that blockade of this receptor promotes CNS repair and functional recovery.

Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult.

The PKR-like endoplasmic reticulum kinase (PERK) arm of the Integrated Stress Response (ISR) is implicated in neurodegenerative disease, although the regulators and consequences of PERK activation following neuronal injury are poorly understood. Here we show that PERK signaling is a component of the mouse MAP kinase neuronal stress response controlled by the Dual Leucine Zipper Kinase (DLK) and contributes to DLK-mediated neurodegeneration. We find that DLK-activating insults ranging from nerve injury to neurotrophin deprivation result in both c-Jun N-terminal Kinase (JNK) signaling and the PERK- and ISR-dependent upregulation of the Activating Transcription Factor 4 (ATF4). Disruption of PERK signaling delays neurodegeneration without reducing JNK signaling. Furthermore, DLK is both sufficient for PERK activation and necessary for engaging the ISR subsequent to JNK-mediated retrograde injury signaling. These findings identify DLK as a central regulator of not only JNK but also PERK stress signaling in neurons, with both pathways contributing to neurodegeneration.

Myelinating cocultures of rat retinal ganglion cell reaggregates and optic nerve oligodendrocyte precursor cells.

This protocol describes the generation of a rapidly myelinating central nervous system coculture for the study of complex neuronal-glial interactions in vitro. Postnatal rat retinal ganglion cells (RGCs) purified by immunopanning are promoted to cluster into reaggregates and then allowed to extend dense beds of radial axons for 10-14 d. Subsequently, rodent oligodendrocyte precursor cells are purified by immunopanning, transfected if desired, and seeded on top of the RGC reaggregates. Under the conditions described here, compact myelin can be observed within 6 d.

Myelinating cocultures of purified oligodendrocyte lineage cells and retinal ganglion cells.

In this article, we introduce methods for generating rapidly myelinating cocultures with reaggregates of purified retinal ganglion cells and optic nerve oligodendrocyte precursor cells. This coculture system facilitates the study of complex central nervous system neuronal-glial interactions and myelination. It enables control of the extracellular environment and allows the use of transfected, virally infected, mutant, or knockout neurons and/or glial cell types. It is therefore possible to assess the role of various signaling pathways and genes in myelination and node of Ranvier formation.

JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.

Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitin-proteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults.

Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.

Mechanistic studies of CNS myelination have been hindered by the lack of a rapidly myelinating culture system. Here, we describe a versatile CNS coculture method that allows time-lapse microscopy and molecular analysis of distinct stages of myelination. Employing a culture architecture of reaggregated neurons fosters extension of dense beds of axons from purified retinal ganglion cells. Seeding of oligodendrocyte precursor cells on these axons results in differentiation and ensheathment in as few as 3 days, with generation of compact myelin within 6 days. This technique enabled (1) the demonstration that oligodendrocytes initiate new myelin segments only during a brief window early in their differentiation, (2) identification of a contribution of astrocytes to the rate of myelin wrapping, and (3) molecular dissection of the role of oligodendrocyte gamma-secretase activity in controlling the ensheathment of axons. These insights illustrate the value of this defined system for investigating multiple aspects of CNS myelination.