Neuronal guidance genes in health and diseases.

Neurons migrate from their birthplaces to the destinations, and extending axons navigate to their synaptic targets by sensing various extracellular cues in spatiotemporally controlled manners. These evolutionally conserved guidance cues and their receptors regulate multiple aspects of neural development to establish the highly complex nervous system by mediating both short- and long-range cell-cell communications. Neuronal guidance genes (encoding cues, receptors, or downstream signal transducers) are critical not only for development of the nervous system but also for synaptic maintenance, remodeling, and function in the adult brain. One emerging theme is the combinatorial and complementary functions of relatively limited classes of neuronal guidance genes in multiple processes, including neuronal migration, axonal guidance, synaptogenesis, and circuit formation. Importantly, neuronal guidance genes also regulate cell migration and cell-cell communications outside the nervous system. We are just beginning to understand how cells integrate multiple guidance and adhesion signaling inputs to determine overall cellular/subcellular behavior and how aberrant guidance signaling in various cell types contributes to diverse human diseases, ranging from developmental, neuropsychiatric, and neurodegenerative disorders to cancer metastasis. We review classic studies and recent advances in understanding signaling mechanisms of the guidance genes as well as their roles in human diseases. Furthermore, we discuss the remaining challenges and therapeutic potentials of modulating neuronal guidance pathways in neural repair.

Behavioral profile in a Dctn1G71A knock-in mouse model of Perry disease.

Perry disease (Perry syndrome) is a rare, rapidly progressive, autosomal dominant neurodegenerative disease characterized by parkinsonism, depression/apathy, weight loss, and respiratory symptoms including central hypoventilation. It is caused by missense mutations (e.g. p.G71A) in the DCTN1 gene. We previously generated transgenic mice that expressed human DCTN1G71A mutant protein under the control of Thy1 promoter. These mice exhibited apathy-like behavior and parkinsonism. However, it is possible that this phenotype was due to a gene-dosage imbalance or transgene insertion position. To circumvent these potential caveats, we have generated a knock-in mouse model carrying a p.G71A mutation in Dctn1. Heterozygous Dctn1G71A and wild-type littermates were subjected to a battery of behavioral analyses. Furthermore, immunohistochemistry for tyrosine hydroxylase (TH) was performed on brain sections of these mice, and TH signal intensity in substantia nigral neurons was quantified. Dctn1G71A mice were immobile for longer than wild-type mice of the same age and sex in the tail-suspension test, revealing depressive characteristics. In addition, the beam-walking test and pole test detected motor deficits in Dctn1G71A female mice. Finally, immunostaining revealed a decrease in TH immunoreactivity in neurons of the substantia nigra in the Dctn1G71A mice. Collectively, heterozygous Dctn1G71A mice showed depression-like behavior, motor deficits, and a functional reduction in substantia nigral neurons, as judged by TH immunostaining, thereby exhibiting multiple features of Perry disease. Hence, this mouse model will be useful in elucidating pathological mechanisms of Perry disease and for developing novel therapeutic strategies against it.

DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.

A crucial role for Arf6 in the response of commissural axons to Slit.

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation.

Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

Behavioral defects in a DCTN1G71A transgenic mouse model of Perry syndrome.

Perry syndrome is a rare neurodegenerative disease characterized by parkinsonism, depression/apathy, weight loss, and central hypoventilation. Our previously-conducted genome-wide association scan and subsequent studies identified nine mutations in DCTN1, the largest protein subunit of the dynactin complex, in patients with Perry syndrome. These included G71A in the microtubule-binding cytoskeleton-associated protein Gly-rich domain of p150Glued. The dynactin complex is essential for function of the microtubule-based cytoplasmic retrograde motor dynein. To test the hypothesis that the G71A mutation in the DCTN1 gene is sufficient to cause Perry syndrome, we generated DCTN1G71A transgenic mice. These mice initially developed normally, but young animals showed decreased exploratory activity and aged animals showed impaired motor coordination. These behavioral defects parallel apathy-like symptoms and parkinsonism encountered in Perry syndrome. TDP-43 aggregates were not detected in the substantia nigra and cerebral cortex of the transgenic mice, although pathological aggregates of TDP-43 have been considered a major neuropathological feature of Perry syndrome. Our study reveals that a single mutation in the DCTN1 gene recapitulates symptoms of Perry syndrome patients, and provides evidence that DCTN1G71A transgenic mice represent a novel rodent model of Perry syndrome.

The BH3-only SNARE BNip1 mediates photoreceptor apoptosis in response to vesicular fusion defects.

Intracellular vesicular transport is important for photoreceptor function and maintenance. However, the mechanism underlying photoreceptor degeneration in response to vesicular transport defects is unknown. Here, we report that photoreceptors undergo apoptosis in a zebrafish ß-soluble N-ethylmaleimide-sensitive factor attachment protein (ß-SNAP) mutant. ß-SNAP cooperates with N-ethylmaleimide-sensitive factor to recycle the SNAP receptor (SNARE), a key component of the membrane fusion machinery, by disassembling the cis-SNARE complex generated in the vesicular fusion process. We found that photoreceptor apoptosis in the ß-SNAP mutant was dependent on the BH3-only protein BNip1. BNip1 functions as a component of the syntaxin-18 SNARE complex and regulates retrograde transport from the Golgi to the endoplasmic reticulum. Failure to disassemble the syntaxin-18 cis-SNARE complex caused BNip1-dependent apoptosis. These data suggest that the syntaxin-18 cis-SNARE complex functions as an alarm factor that monitors vesicular fusion competence and that BNip1 transforms vesicular fusion defects into photoreceptor apoptosis.

Midline crossing and Slit responsiveness of commissural axons require USP33.

Commissural axons cross the ventral midline of the neural tube in a Slit-dependent manner. The underlying molecular mechanisms remain unclear. We found that the deubiquitinating enzyme USP33 interacts with the Robo1 receptor. USP33 was essential for midline crossing by commissural axons and for their response to Slit. Our results reveal a previously unknown role for USP33 in vertebrate commissural axon guidance and in Slit signaling.

Deubiquitinating enzyme USP33/VDU1 is required for Slit signaling in inhibiting breast cancer cell migration.

Slit regulates migration of not only neurons, but also nonneuronal cells, such as leukocytes and cancer cells. Slit effect on cancer cell migration has not been well-characterized. In this study, we used several different assays to examine Slit effect on breast cancer cell migration in vitro. We show that ubiquitin-specific protease 33 (USP33)/VDU1, originally identified as a von Hippel-Lindau tumor suppressor (VHL) protein-interacting deubiquitinating enzyme, binds to the Robo1 receptor, and that USP33 is required for Slit responsiveness in breast cancer cells. Slit induces redistribution of Robo1 from intracellular compartments to the plasma membrane in a USP33-dependent manner. Slit impairs directional migration of breast cancer cells without affecting their migration speed. This inhibitory effect is Robo-mediated and USP33-dependent. These data uncover a previously unknown function of USP33 and reveal a new player in Slit-Robo signaling in cancer cell migration.

Inhibition of TRPC5 channels by Ca2+-binding protein 1 in Xenopus oocytes.

The transient receptor potential canonical type 5 (TRPC5) channel is a member of the channels that has been implicated in neurite extension and growth cone morphology of hippocampal neurons. Although homomeric TRPC5 channels are activated following stimulation of G(q/11)-coupled receptors, the exact mechanism for this activation remains unresolved. Using two-electrode voltage clamp recordings, we show that the activity of TRPC5 channels expressed in Xenopus oocytes is dependent on the presence of Ca2+ at the extracellular as well as the cytoplasmic side of the plasma membrane. TRPC5 was activated by the stimulation of coexpressed M5 muscarinic receptors or by ionomycin. The TRPC5 activity was detectable with the presence of submillimolar levels of extracellular Ca2+, but it was eliminated by the injection of 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid into the oocytes. Lanthanum could substitute for extracellular Ca2+ to support TRPC5 activity. Coexpression of Ca2+-binding protein 1 (CaBP1), but not calmodulin (CaM), inhibited the TRPC5 activity, without affecting the cell surface expression of TRPC5 proteins. Using in vitro binding assays, we demonstrated direction interactions between CaBP1 and TRPC5. The CaBP1-binding sites at the C terminus of TRPC5 are closely localized, but not identical, to CaM-binding sites. We conclude that TRPC5 is a Ca2+-regulated channel, and its activity is negatively controlled by CaBP1.

A Purkinje cell specific GoLoco domain protein, L7/Pcp-2, modulates receptor-mediated inhibition of Cav2.1 Ca2+ channels in a dose-dependent manner.

L7/Pcp-2 is a GoLoco domain protein encoded by a Purkinje cell dendritic mRNA. Although biochemical interactions of GoLoco proteins with Galpha(o) and Galpha(i) are well documented, little is known about effector function modulation resulting from these interactions. The P-type Ca2+ channels might be physiological effectors of L7 because (1) they are the major voltage-dependent Ca2+ channels (VDCC) that modulate Purkinje cell output and (2) they are regulated by G(i/o) proteins. As a first step towards validating this hypothesis and to further understand the possible physiological effect of L7 protein and its two isoforms, we have coexpressed Ca(v)2.1 channels and kappa-opioid receptors (KORs) with varying amounts of L7A or L7B in Xenopus oocytes and measured ionic currents by two-electrode voltage clamping. Without receptor activation L7 did not alter the Ca2+ channel activity. With tonic and weak activation of the receptors, however, the Ca2+ channels were inhibited by 40-50%. This inhibition was enhanced by low, but dampened by high, expression levels of L7A and L7B and differences were observed between the two isoforms. The enhancing effect of L7 was occluded by overexpression of Gbetagamma, whereas the disinhibition was antagonized by overexpression of Galpha(o). We propose that L7 differentially affects the Galpha and Gbetagamma arms of receptor-induced G(i/o) signaling in a concentration-dependent manner, through which it increases the dynamic range of regulation of P/Q-type Ca2+ channels by G(i/o) protein-coupled receptors. This provides a framework for designing further experiments to determine how dendritic local fluctuations in L7 protein levels might influence signal processing in Purkinje cells.

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3.

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.