Judy S. Liu

Expression of Inducible Nitric Oxide Synthase and Nitrotyrosine in Multiple Sclerosis Lesions

Nitric oxide generated by the inducible form of nitric oxide synthase (iNOS) may contribute to the pathogenesis of multiple sclerosis (MS). In this report, we studied postmortem tissues of MS patients for the expression of iNOS by in situ hybridization and immunocytochemistry. Immunocytochemistry for nitrotyrosine, a putative footprint for peroxynitrite formation was also performed. In acute MS lesions, intense reactivity for iNOS mRNA and protein was detected in reactive astrocytes throughout the lesion and in adjacent normal appearing white matter. Staining of macrophages, inflammatory cell infiltrates, and endothelial cells was variable from case to case, but generally detected only in acute lesions. In chronic MS lesions reactive astrocytes at the lesion edge were positive for iNOS whereas the lesion center was nonreactive. Normal appearing white matter demonstrated little reactivity, as did tissues from noninflamed control brains. Staining for nitrotyrosine was also detected in acute but not chronic MS lesions, and displayed a diffuse parenchymal, membranous, and perivascular pattern of immunoreactivity. These results support the conclusion that iNOS is induced in multiple cell types in MS lesions and that astrocyte-derived nitric oxide could be important in orchestrating inflammatory responses in MS, particularly at the blood-brain barrier.

Both Doublecortin and Doublecortin-Like Kinase Play a Role in Cortical Interneuron Migration

Type I lissencephaly, a genetic disease characterized by disorganized cortical layers and gyral abnormalities, is associated with severe cognitive impairment and epilepsy. Two genes, LIS1 and doublecortin (DCX), have been shown to be responsible for a large proportion of cases of type I lissencephaly. Both genes encode microtubule-associated proteins that have been shown to be important for radial migration of cortical pyramidal neurons. To investigate whether DCX also plays a role in cortical interneuron migration, we inactivated DCX in the ganglionic eminence of rat embryonic day 17 brain slices using short hairpin RNA. We found that, when DCX expression was blocked, the migration of interneurons from the ganglionic eminence to the cerebral cortex was slowed but not absent, similar to what had previously been reported for radial neuronal migration. In addition, the processes of DCX-deficient migrating interneurons were more branched than their counterparts in control experiments. These effects were rescued by DCX overexpression, confirming the specificity to DCX inactivation. A similar delay in interneuron migration was observed when Doublecortin-like kinase (DCLK), a microtubule-associated protein related to DCX, was inactivated, although the morphology of the cells was not affected. The importance of these genes in interneuron migration was confirmed by our finding that the cortices of Dcx, Dclk, and Dcx/Dclk mutant mice contained a reduced number of such cells in the cortex and their distribution was different compared with wild-type controls. However, the defect was different for each group of mutant animals, suggesting that DCX and DCLK have distinct roles in cortical interneuron migration.

Molecular Genetics of Neuronal Migration Disorders

Cortical malformations associated with defects in neuronal migration result in severe developmental consequences including intractable epilepsy and intellectual disability. Genetic causes of migration defects have been identified with the advent and widespread use of high-resolution MRI and genetic techniques. Thus, the full phenotypic range of these genetic disorders is becoming apparent. Genes that cause lissencephaly, pachygyria, subcortical band heterotopia, and periventricular nodular heterotopias have been defined. Many of these genes are involved in cytoskeletal regulation including the function of microtubules (LIS1, TUBA1A,TUBB3, and DCX) and of actin (FilaminA). Thus, the molecular pathways regulating neuronal migration including the cytoskeletal pathways appear to be defined by human mutation syndromes. Basic science, including cell biology and animal models of these disorders, has informed our understanding of the pathogenesis of neuronal migration disorders and further progress depends on the continued integration of the clinical and basic sciences.

The emerging role of the tubulin code: From the tubulin molecule to neuronal function and disease

Across different cell types and tissues, microtubules are assembled from highly conserved dimers of α‐ and β‐tubulin. Despite their highly similar structures, microtubules have functional heterogeneity, generated either by the expression of different tubulin genes, encoding distinct isotypes, or by posttranslational modifications of tubulin. This genetically encoded and posttranslational generated heterogeneity of tubulin—the “tubulin code”—has the potential to modulate microtubule structure, dynamics, and interactions with associated proteins. The tubulin code is therefore believed to regulate microtubule functions on a cellular and sub‐cellular level. This review highlights the importance of the tubulin code for tubulin structure, as well as on microtubule dynamics and functions in neurons. It further summarizes recent developments in the understanding of mutations in tubulin genes, and how they are linked to neurodegenerative and neurodevelopmental disorders. The current advances in the knowledge of the tubulin code on the molecular and the functional level will certainly lead to a better understanding of how complex signaling events control microtubule functions, especially in cells of the nervous system.

Doublecortin (Dcx) family proteins regulate filamentous actin structure in developing neurons

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins α-actinin-1 and α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.

Genetic and phenotypic diversity of NHE6 mutations in Christianson syndrome

Recently, Christianson syndrome (CS) has been determined to be caused by mutations in the X‐linked Na+/H+ exchanger 6 (NHE6). We aimed to determine the diagnostic criteria and mutational spectrum for CS.

Doublecortin-Like Kinases Promote Neuronal Survival and Induce Growth Cone Reformation via Distinct Mechanisms

After axotomy, neuronal survival and growth cone re-formation are required for axon regeneration. We discovered that doublecortin-like kinases (DCLKs), members of the doublecortin (DCX) family expressed in adult retinal ganglion cells (RGCs), play critical roles in both processes, through distinct mechanisms. Overexpression of DCLK2 accelerated growth cone re-formation in vitro and enhanced the initiation and elongation of axon re-growth after optic nerve injury. These effects depended on both the microtubule (MT)-binding domain and the serine-proline-rich (S/P-rich) region of DCXs in-cis in the same molecules. While the MT-binding domain is known to stabilize MT structures, we show that the S/P-rich region prevents F-actin destabilization in injured axon stumps. Additionally, while DCXs synergize with mTOR to stimulate axon regeneration, alone they can promote neuronal survival possibly by regulating the retrograde propagation of injury signals. Multifunctional DCXs thus represent potential targets for promoting both survival and regeneration of injured neurons.

Polarity in migrating neurons is related to a mechanism analogous to cytokinesis

Migrating neurons are bipolar, with a leading process and a trailing process [1]. The proximal region of the leading process displays a concentration of F-actin that contributes to the advance of the soma and the centrosome [2-7]. Here, we show that kinesin-6, a microtubule-based motor protein best known for its role in cytokinesis, also concentrates in this region. Depletion of kinesin-6 results in multipolar neurons that either are stationary or continuously change their direction of movement. In such neurons, F-actin no longer concentrates in a single process. During cytokinesis, kinesin-6 forms a complex with a Rho-family GTPase-activating protein called MgcRacGAP to signal to the actin cytoskeleton so that cortical movements are concentrated in the cleavage furrow [8-13]. During neuronal migration, MgcRacGap also concentrates in the proximal region of the leading process, and inhibition of its activity results in a phenotype similar to kinesin-6 depletion. We conclude that neuronal migration utilizes a cytoskeletal pathway analogous to cytokinesis, with kinesin-6 signaling through MgcRacGap to the actin cytoskeleton to constrain process number and restrict protrusive activity to a single leading process, thus resulting in a bipolar neuron able to move in a directed fashion.

Doublecortin (DCX) mediates endocytosis of neurofascin independently of microtubule-binding

Doublecortin on X chromosome (DCX) is one of two major genetic loci underlying human lissencephaly, a neurodevelopmental disorder with defects in neuronal migration and axon outgrowth. DCX is a microtubule-binding protein, and much work has focused on its microtubule-associated functions. DCX has other reported binding partners, including the cell adhesion molecule neurofascin, but the functional significance of the DCX–neurofascin interaction is not understood. Neurofascin localizes strongly to the axon initial segment in mature neurons, where it plays a role in assembling and maintaining other axon initial segment components. During development, neurofascin likely plays additional roles in axon guidance and in GABAergic synaptogenesis. We show here that DCX can modulate the surface distribution of neurofascin in developing cultured rat neurons and thereby the relative extent of accumulation between the axon initial segment and soma and dendrites. Mechanistically, DCX acts via increasing endocytosis of neurofascin from soma and dendrites. Surprisingly, DCX increases neurofascin endocytosis apparently independently of its microtubule-binding activity. We additionally show that the patient allele DCXG253D still binds microtubules but is deficient in promoting neurofascin endocytosis. We propose that DCX acts as an endocytic adaptor for neurofascin to fine-tune its surface distribution during neuronal development.

Differential Neuronal Susceptibility and Apoptosis in Congenital ZIKV Infection

To characterize the mechanism of Zika virus (ZIKV)‐associated microcephaly, we performed immunolabeling on brain tissue from a 20‐week fetus with intrauterine ZIKV infection. Although ZIKV demonstrated a wide range of neuronal and non‐neuronal tropism, the infection rate was highest in intermediate progenitor cells and immature neurons. Apoptosis was observed in both infected and uninfected bystander cortical neurons, suggesting a role for paracrine factors in induction of neuronal apoptosis. Our results highlight differential neuronal susceptibility and neuronal apoptosis as potential mechanisms in the development of ZIKV‐associated microcephaly, and may provide insights into the design and best timing of future therapy. Ann Neurol 2017;82:121–127