TDP-43 differentially propagates to induce antero- and retrograde degeneration in the corticospinal circuits in mouse focal ALS models.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by TDP-43 inclusions in the cortical and spinal motor neurons. It remains unknown whether and how pathogenic TDP-43 spreads across neural connections to progress degenerative processes in the cortico-spinal motor circuitry. Here we established novel mouse ALS models that initially induced mutant TDP-43 inclusions in specific neuronal or cell types in the motor circuits, and investigated whether TDP-43 and relevant pathological processes spread across neuronal or cellular connections. We first developed ALS models that primarily induced TDP-43 inclusions in the corticospinal neurons, spinal motor neurons, or forelimb skeletal muscle, by using adeno-associated virus (AAV) expressing mutant TDP-43. We found that TDP-43 induced in the corticospinal neurons was transported along the axons anterogradely and transferred to the oligodendrocytes along the corticospinal tract (CST), coinciding with mild axon degeneration. In contrast, TDP-43 introduced in the spinal motor neurons did not spread retrogradely to the cortical or spinal neurons; however, it induced an extreme loss of spinal motor neurons and subsequent degeneration of neighboring spinal neurons, suggesting a degenerative propagation in a retrograde manner in the spinal cord. The intraspinal degeneration further led to severe muscle atrophy. Finally, TDP-43 induced in the skeletal muscle did not propagate pathological events to spinal neurons retrogradely. Our data revealed that mutant TDP-43 spread across neuro-glial connections anterogradely in the corticospinal pathway, whereas it exhibited different retrograde degenerative properties in the spinal circuits. This suggests that pathogenic TDP-43 may induce distinct antero- and retrograde mechanisms of degeneration in the motor system in ALS.

Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord.

Cerebrospinal fluid-contacting neurons (CSF-cNs) are enigmatic mechano- or chemosensory cells lying along the central canal of the spinal cord. Recent studies in zebrafish larvae and lampreys have shown that CSF-cNs control postures and movements via spinal connections. However, the structures, connectivity, and functions in mammals remain largely unknown. Here we developed a method to genetically target mouse CSF-cNs that highlighted structural connections and functions. We first found that intracerebroventricular injection of adeno-associated virus with a neuron-specific promoter and Pkd2l1-Cre mice specifically labeled CSF-cNs. Single-cell labeling of 71 CSF-cNs revealed rostral axon extensions of over 1800 µm in unmyelinated bundles in the ventral funiculus and terminated on CSF-cNs to form a recurrent circuitry, which was further determined by serial electron microscopy and electrophysiology. CSF-cNs were also found to connect with axial motor neurons and premotor interneurons around the central canal and within the axon bundles. Chemogenetic CSF-cNs inactivation reduced speed and step frequency during treadmill locomotion. Our data revealed the basic structures and connections of mouse CSF-cNs to control spinal motor circuits for proper locomotion. The versatile methods developed in this study will contribute to further understanding of CSF-cN functions in mammals.

Lesion Area in the Cerebral Cortex Determines the Patterns of Axon Rewiring of Motor and Sensory Corticospinal Tracts After Stroke.

The corticospinal tract (CST) is an essential neural pathway for reorganization that recovers motor functions after brain injuries such as stroke. CST comprises multiple pathways derived from different sensorimotor areas of the cerebral cortex; however, the patterns of reorganization in such complex pathways postinjury are largely unknown. Here we comprehensively examined the rewiring patterns of the CST pathways of multiple cerebral origins in a mouse stroke model that varied in size and location in the sensorimotor cortex. We found that spared contralesional motor and sensory CST axons crossed the midline and sprouted into the denervated side of the cervical spinal cord after stroke in a large cortical area. In contrast, the contralesional CST fibers did not sprout in a small stroke, whereas the ipsilesional axons from the spared motor area grew on the denervated side. We further showed that motor and sensory CST axons did not innervate the projecting areas mutually when either one was injured. The present results reveal the basic principles that generate the patterns of CST rewiring, which depend on stroke location and CST subtype. Our data indicate the importance of targeting different neural substrates to restore function among the types of injury.

Direct Comparison of Odor Responses of Homologous Glomeruli in the Medial and Lateral Maps of the Mouse Olfactory Bulb.

Olfactory sensory neurons (OSNs) expressing same-type odorant receptors typically project to a pair of glomeruli in the medial and lateral sides of the olfactory bulbs (OBs) in rodents. This multiple glomerular representation of homologous inputs is considered to have more important functional roles for odor information processing than the redundant backup system. However, a consensus idea is lacking and this hinders interpretation of the phenomenon. In addition, the shared and unique odorant response properties of the homologous glomeruli remain unclear because the majority of medial glomeruli are hidden in the septal OB, and thus it is difficult to directly compare them. OSNs, which express trace amine-associated odorant receptors (TAARs), were recently identified that project to a pair of glomeruli uniquely located in the dorsal OB. In this study, we measured the odorant-induced calcium responses of homologous pairs of TAAR glomeruli simultaneously in anesthetized mice and directly compared their response patterns. We found that they exhibited similar temporal response patterns and could not find differences in onset latency, rise time, decay time, or response amplitude. However, the medial glomeruli had significantly larger respiration-locked calcium fluctuations than the lateral glomeruli. This trend was observed with/without odorant stimulation in postsynaptic neurons of GABAergic, dopaminergic, and mitral/tufted cells, but not in presynaptic olfactory sensory axon terminals. This indicates that, at least in these TAAR glomeruli, the medial rather than the lateral OB map enhances the respiration-locked rhythm and transfers this information to higher brain centers.

Narrowly Confined and Glomerulus-Specific Onset Latencies of Odor-Evoked Calcium Transients in the Juxtaglomerular Cells of the Mouse Main Olfactory Bulb.

Odor information is transmitted from olfactory sensory neurons to principal neurons at the glomeruli of the olfactory bulb. The intraglomerular neuronal circuit also includes hundreds of interneurons referred to as juxtaglomerular (JG) cells. Stimulus selectivity is well correlated among many JG cells that are associated with the same glomerulus, consistent with their highly homogeneous sensory inputs. However, much less is known about the temporal aspects of their activity, including the temporal coordination of their odor-evoked responses. As many JG cells within a glomerular module respond to the same stimulus, the extent to which their activity is temporally aligned will affect the temporal profile of their population inhibitory inputs. Using random-access high-speed two-photon microscopy, we recorded the odor-evoked calcium transients of mouse JG cells and compared the onset latency and rise time among neurons putatively associated with the same and different glomeruli. Whereas the overall onset latencies of odor-evoked transients were distributed across a ∼150 ms time window, those from cells putatively associated with the same glomerulus were confined to a much narrower window of several tens of milliseconds. This result suggests that onset latency primarily depends on the associated glomerulus. We also observed glomerular specificity in the rise time. The glomerulus-specific temporal pattern of odor-evoked activity implies that the temporal patterns of inputs from the intraglomerular circuit are unique to individual glomerulus-odor pairs, which may contribute to efficient shaping of the temporal pattern of activity in the principal neurons.

The osteopontin-CD44 axis in hepatic cancer stem cells regulates IFN signaling and HCV replication.

Osteopontin (OPN) is involved in cell proliferation, migration, inflammation, and tumor progression in various tissues. OPN induces stemness by interacting with CD44, but the functional relevance of OPN-mediated interferon (IFN) signaling and hepatitis C virus (HCV) replication in stem cell populations remains unclear. In this study, we investigated the effect of OPN on HCV replication and IFN signaling in cancer stem cells (CSCs) positive for epithelial cell adhesion molecule (EpCAM) and CD44. We show that the EpCAM+/CD44+ CSCs show marked HCV replication when compared to EpCAM-/CD44- cells. In addition, OPN significantly enhances this HCV replication in EpCAM+/CD44+ CSCs and markedly suppresses IFN-stimulated gene expression. The GSK-3ß inhibitor BIO increases the EpCAM+/CD44+ CSC population and OPN expression and impairs IFN signaling via STAT1 degradation. Taken together, our data suggest that OPN enhances HCV replication in the EpCAM+/CD44+ CSCs, while it also negatively regulates the IFN signaling pathway via inhibition of STAT1 phosphorylation and degradation. Therefore, OPN may represent a novel therapeutic target for treating HCV-related hepatocellular carcinoma.

Critical role of JSAP1 and JLP in axonal transport in the cerebellar Purkinje cells of mice.

JNK/stress-activated protein kinase-associated protein 1 (JSAP1) and JNK-associated leucine zipper protein (JLP) are structurally related scaffolding proteins that are highly expressed in the brain. Here, we found that JSAP1 and JLP play functionally redundant and essential roles in mouse cerebellar Purkinje cell (PC) survival. Mice containing PCs with deletions in both JSAP1 and JLP exhibited PC axonal dystrophy, followed by gradual, progressive neuronal loss. Kinesin-1 cargoes accumulated selectively in the swollen axons of Jsap1/Jlp-deficient PCs. In addition, autophagy inactivation in these mice markedly accelerated PC degeneration. These findings suggest that JSAP1 and JLP play critical roles in kinesin-1-dependent axonal transport, which prevents brain neuronal degeneration.

JSAP1 and JLP are required for ARF6 localization to the midbody in cytokinesis.

The ADP-ribosylation factor 6 (ARF6) GTPase is important in cytokinesis and localizes to the midbody. However, the mechanism and regulation of ARF6's recruitment to the midbody are largely unknown. Here, we investigated the functions of two binding partners of active ARF6, c-Jun NH2 -terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) and JNK-associated leucine zipper protein (JLP), by gene knockout and rescue experiments in mouse embryonic fibroblasts. Depleting both JSAP1 and JLP impaired ARF6's localization to the midbody and delayed cytokinesis. These defects were almost completely rescued by wild-type JSAP1 or JLP, but not by JSAP1 or JLP mutants that were unable to interact with active ARF6 or with the kinesin heavy chain (KHC) of kinesin-1. In transfected cells, a constitutively active form of ARF6 associated with KHC only when co-expressed with wild-type JSAP1 or JLP and not with a JSAP1 or JLP mutant. These findings suggest that JSAP1 and JLP, which might be paralogous to each other, are critical and functionally redundant in cytokinesis and control ARF6 localization to the midbody by forming a tripartite complex of JSAP1/JLP, active ARF6, and kinesin-1.

The scaffold protein JLP plays a key role in regulating ultraviolet B-induced apoptosis in mice.

The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2 -terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation.

Human microRNA-1245 down-regulates the NKG2D receptor in natural killer cells and impairs NKG2D-mediated functions.

BACKGROUND: NKG2D is an activating receptor expressed by natural killer and T cells, which have crucial functions in tumor and microbial immunosurveillance. Several cytokines have been identified as modulators of NKG2D receptor expression. However, little is known about NKG2D gene regulation. In this study, we found that microRNA 1245 attenuated the expression of NKG2D in natural killer cells. DESIGN AND METHODS: We investigated the potential interactions between the 3'-untranslated region of the NKG2D gene and microRNA as well as their functional roles in the regulation of NKG2D expression and cytotoxicity in natural killer cells. RESULTS: Transforming growth factor-ß1, a major negative regulator of NKG2D expression, post-transcriptionally up-regulated mature microRNA-1245 expression, thus down-regulating NKG2D expression and impairing NKG2D-mediated immune responses in natural killer cells. Conversely, microRNA-1245 down-regulation significantly increased the expression of NKG2D expression in natural killer cells, resulting in more efficient NKG2D-mediated cytotoxicity. CONCLUSIONS These results reveal a novel NKG2D regulatory pathway mediated by microRNA-1245, which may represent one of the mechanisms used by transforming growth factor-ß1 to attenuate NKG2D expression in natural killer cells.

Role of plasma membrane localization of the scaffold protein JSAP1 during differentiation of cerebellar granule cell precursors.

We previously reported that the scaffold protein c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions in cerebellar granule cell precursors (GCPs) to promote their cell-cycle exit and differentiation. In this study, we used immunocytochemistry to examine the subcellular distribution of JSAP1 in proliferating cultured GCPs. We found that when stimulated with fibroblast growth factor-2 (FGF-2), a factor that promotes GCP differentiation through JNK and extracellular signal-regulated kinase (ERK) signaling, JSAP1 translocated to the plasma membrane and colocalized with activated JNK and ERK. In transfected cells expressing a constitutively activated FGF receptor (FGFR), JSAP1 and the activated FGFR colocalized at the plasma membrane with not only activated but also unphosphorylated and inactive JNK and ERK. These colocalizations did not occur when a mutant JSAP1 lacking the JNK-binding domain was substituted for wild-type JSAP1. Biochemical analyses of transfected cells showed that activated FGFR increased JSAP1's affinity for JNK and ERK and that JSAP1 enhanced FGFR-induced JNK and ERK activation. Collectively, these results suggest that when stimulated by FGFR, JSAP1 translocates to the plasma membrane, where it recruits JNK and ERK and facilitates their activation, leading to the differentiation of cerebellar GCPs.

The scaffold protein JSAP1 regulates proliferation and differentiation of cerebellar granule cell precursors by modulating JNK signaling.

Cerebellar granule cell precursors (GCPs) proliferate in the outer part of the external granular layer (EGL). They begin their differentiation by exiting the cell cycle and migrating into the inner part of the EGL. Here we report that JSAP1, a scaffold protein for JNK signaling pathways, is expressed predominantly in the post-mitotic GCPs of the inner EGL. JSAP1 knockdown or treatment with a JNK inhibitor enhances the proliferation of cultured GCPs, but the overexpression of wild-type JSAP1 leads to increased proportions of p27(Kip1)- and NeuN-positive cells, even with saturating concentrations of Sonic hedgehog (Shh), a potent GCP mitogen. However, these differentiation-promoting effects on GCPs are attenuated significantly in cells overexpressing a mutant JSAP1 that lacks the JNK-binding domain. Together, these data suggest that JSAP1 antagonizes the mitogenic effect of Shh on GCPs and promotes their exit from the cell cycle and differentiation, by modulating JNK activity.

Ablation of the scaffold protein JLP causes reduced fertility in male mice.

The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the G(alpha13) and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa.

Neural-specific ablation of the scaffold protein JSAP1 in mice causes neonatal death.

We previously identified c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffolding factor for JNK intracellular signaling pathways. Targeted gene-disruption studies have shown that JSAP1-null mice are unable to breathe and die shortly after birth. Although neural defects might be responsible for their death, there has been no convincing evidence for this. Here we first generated genetically engineered mice carrying a loxP-flanked (floxed) jsap1 gene. To evaluate the validity of this deletion as a jsap1 conditional knockout (KO), we created mice in which the same exon was deleted in all cell lineages, and compared their phenotypes with those of the jsap1 conventional KO mice reported previously. The two KO lines showed indistinguishable phenotypes, i.e., neonatal death and morphological defects in the telencephalon, indicating that the conditional deletion was a true null mutation. We then introduced the floxed jsap1 deletion mutant specifically into the neural lineage, and found that the jsap1 conditional KO mice showed essentially the same phenotypes as the JSAP1-null mice. These results strongly suggest that the neonatal death of jsap1-deficient mice is caused by defects in the nervous system.

Expression and distribution of JNK/SAPK-associated scaffold protein JSAP1 in developing and adult mouse brain.

The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.

Impairment of cardiomyogenesis in embryonic stem cells lacking scaffold protein JSAP1.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, is important in embryonic stem (ES) cells during neurogenesis. In that study, we also observed the altered expression of mesodermal marker genes, which indicated that JSAP1 is involved in the differentiation of mesodermal lineages. Here, we investigated the function of JSAP1 in cardiomyocyte development using JSAP1-null ES cells, and found that cardiomyogenesis was impaired in the JSAP1-null mutant. The JSAP1 deficiency resulted in lower gene expression of the cardiac transcription factor Nkx2.5 and contractile proteins. In contrast, the mutant showed a significantly higher expression of mesoderm-related markers other than those of the cardiomyocyte lineage. Together, these results suggest that JSAP1 may be important for the differentiation of the mesodermal lineages, functioning as a positive factor for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into other lineages.

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration.

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.

Structure, regulation, and function of micro1 in the sea urchin Hemicentrotus pulcherrimus.

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear beta-catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of beta-catenin is highly suggestive. We demonstrated that micro1 is a direct target of beta-catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.