Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS.

Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair

NgR1 and NgR3 are Receptors for Chondroitin Sulfate Proteoglycans

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin–associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. Here we show that NgR1 and NgR3 bind with high–affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (NgR123−/−), but not single mutants, show enhanced axonal regeneration following retro–orbital optic nerve crush injury. The combined loss of NgR1 and NgR3 (NgR13−/−), but not NgR1 and NgR2 (NgR12−/−), is sufficient to mimic the NgR123−/− regeneration phenotype. Regeneration in NgR13−/− mice is further enhanced by simultaneous ablation of RPTPσ, a known CSPG receptor. Collectively, these results identify NgR1 and NgR3 as novel CSPG receptors, demonstrate functional redundancy among CSPG receptors, and provide unexpected evidence for shared mechanisms of MAI and CSPG inhibition

Transient Demyelination Increases the Efficiency of Retrograde AAV Transduction

Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)–induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms

Multitasking: Dual Leucine Zipper–Bearing Kinases in Neuronal Development and Stress Management

The dual leucine zipper-bearing kinase (DLK) and leucine zipper-bearing kinase (LZK) are evolutionarily conserved MAPKKKs of the mixed-lineage kinase family. Acting upstream of stress-responsive JNK and p38 MAP kinases, DLK and LZK have emerged as central players in neuronal responses to a variety of acute and traumatic injuries. Recent studies also implicate their function in astrocytes, microglia, and other nonneuronal cells, reflecting their expanding roles in the multicellular response to injury and in disease. Of particular note is the potential link of these kinases to neurodegenerative diseases and cancer. It is thus critical to understand the physiological contexts under which these kinases are activated, as well as the signal transduction mechanisms that mediate specific functional outcomes. In this review we first provide a historical overview of the biochemical and functional dissection of these kinases. We then discuss recent findings on regulating their activity to enhance cellular protection following injury and in disease, focusing on but not limited to the nervous system

Axon regeneration after spinal cord injury: insight from genetically modified mouse models

The use of genetically modified mice to study axon regeneration after spinal cord injury has served as a useful in vivo model for both loss-of-function and gain-of-function analysis of candidate proteins. This review discusses the impact of genetically modified mice on axon regeneration after spinal cord injury in the context of axon growth inhibition by myelin, the glial scar, and chemorepellent molecules. We also discuss the use of mice which transgenically express fluorescent proteins in specific axons for increasing our understanding of how spinal cord axons behave after injury

Role of myelin-associated inhibitors in axonal repair after spinal cord injury

Myelin-associated inhibitors of axon growth, including Nogo, MAG and OMgp, have been the subject of intense research. A myriad of experimental approaches have been applied to investigate the potential of targeting these molecules to promote axonal repair after spinal cord injury. However, there are still conflicting results on their role in axon regeneration and therefore a lack of a cohesive mechanism on how these molecules can be targeted to promote axon repair. One major reason may be the lack of a clear definition of axon regeneration in the first place. Nevertheless, recent data from genetic studies in mice indicate that the roles of these molecules in CNS axon repair may be more intricate than previously envisioned

Thirteen years of manipulating the mouse genome: A personal history

In 1974, Dr. Ralph Brinster published a paper describing the consequences of injecting embryonal carcinoma cells, the predecessors of embryonic stem cells, into mouse blastocysts. Despite their early promise, embryonal carcinoma cells would not efficiently populate the germ line of mice. A decade later Elizabeth Robertson and I described the efficient generation of germline chimaeras from cultured embryonic stem cells and shortly afterwards the genetic manipulation of the mouse germline using ES cells. Our demonstration of the potency of Embryonic Stem cells gave birth to a new era in manipulative mouse genetics, one in which endogenous genes can now be mutated at will using gene targeting of retroviral mutagenesis. This review focuses on the development and testing of concepts and techniques during the thirteen years after we knew germline modification of endogenous genes in the mouse would be possible. This period is one in which more and more sophisticated tools for manipulating the mouse germline were developed and implemented. In this review I have taken the rare opportunity to reveal some of my thought processes, frustrations, successes and failures as we moved through this exciting period of rapid technological change. As I look forward to the next thirteen years, I feel that this will be an equally exciting period for manipulative genetics as we struggle to formulate concepts and design experiments that enable us to understand gene function in an era when the sequence of all genes will be known.Link_to_subscribed_fulltex

Understanding the axonal response to injury by in vivo imaging in the mouse spinal cord: A tale of two branches.

Understanding the basic properties of how axons respond to injury in the mammalian central nervous system (CNS) is of fundamental value for developing strategies to promote neural repair. Axons possess complex morphologies with stereotypical branching patterns. However, current knowledge of the axonal response to injury gives little consideration to axonal branches, nor do strategies to promote axon regeneration. This article reviews evidence from in vivo spinal cord imaging that axonal branches markedly impact the degenerative and regenerative responses to injury. At a major bifurcation point, depending on whether one or both axonal branches are injured, neurons may choose either a more self-preservative response or a more dynamic response. The stabilizing effect of the spared branch may underlie a well-known divergence in neuronal responses to injury, and illustrates an example where in vivo spinal cord imaging reveals insights that are difficult to elucidate with conventional histological methods

Genetic mouse models for studying inhibitors of spinal axon regeneration

The laboratory mouse has emerged as a primary model organism for studying axon regeneration after experimental spinal cord injury, owing to its genetic amenability. Mutant mouse models are contributing significantly to our understanding of the molecular mechanisms of axon regeneration failure in the adult mammalian central nervous system (CNS), in particular regarding the role of axon-growth inhibitors. Here, we discuss recent advances in understanding axon regeneration failure that have been made using genetically modified mice, focusing on the inhibitory influences in the CNS, and we illustrate the advantages of using the mouse as a surrogate organism to study axon regeneration and spinal cord repair