Perry Disease: Bench to Bedside Circulation and a Team Approach.

With technological applications, especially in genetic testing, new diseases have been discovered and new disease concepts have been proposed in recent years; however, the pathogenesis and treatment of these rare diseases are not as well established as those of common diseases. To demonstrate the importance of rare disease research, in this paper we focus on our research topic, Perry disease (Perry syndrome). Perry disease is a rare autosomal dominant neurodegenerative disorder clinically characterized by parkinsonism, depression/apathy, weight loss, and respiratory symptoms including central hypoventilation and central sleep apnea. The pathological classification of Perry disease falls under TAR DNA-binding protein 43 (TDP-43) proteinopathies. Patients with Perry disease exhibit DCTN1 mutations, which is the causative gene for the disease; they also show relatively uniform pathological and clinical features. This review summarizes recent findings regarding Perry disease from both basic and clinical perspectives. In addition, we describe technological innovations and outline future challenges and treatment prospects. We discuss the expansion of research from rare diseases to common diseases and the importance of collaboration between clinicians and researchers. Here, we highlight the importance of researching rare diseases as it contributes to a deeper understanding of more common diseases, thereby opening up new avenues for scientific exploration.

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.

Modeling Parkinson's Disease and Atypical Parkinsonian Syndromes Using Induced Pluripotent Stem Cells.

Parkinson's disease (PD) and atypical parkinsonian syndromes are age-dependent multifactorial neurodegenerative diseases, which are clinically characterized by bradykinesia, tremor, muscle rigidity and postural instability. Although these diseases share several common clinical phenotypes, their pathophysiological aspects vary among the disease categories. Extensive animal-based approaches, as well as postmortem studies, have provided important insights into the disease mechanisms and potential therapeutic targets. However, the exact pathological mechanisms triggering such diseases still remain elusive. Furthermore, the effects of drugs observed in animal models are not always reproduced in human clinical trials. By using induced pluripotent stem cell (iPSC) technology, it has become possible to establish patient-specific iPSCs from their somatic cells and to effectively differentiate these iPSCs into different types of neurons, reproducing some key aspects of the disease phenotypes in vitro. In this review, we summarize recent findings from iPSC-based modeling of PD and several atypical parkinsonian syndromes including multiple system atrophy, frontotemporal dementia and parkinsonism linked to chromosome 17 and Perry syndrome. Furthermore, we discuss future challenges and prospects for modeling and understanding PD and atypical parkinsonian syndromes.

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.

Critical role of collapsin response mediator protein-associated molecule CRAM for filopodia and growth cone development in neurons.

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.

A novel basic helix-loop-helix (bHLH) transcriptional repressor, NeuroAB, expressed in bipolar and amacrine cells in the chick retina.

Basic helix-loop-helix (bHLH) transcription factors are implicated in cell fate determination and differentiation in neurogenesis. We identified a novel chick bHLH transcription factor, NeuroAB. A phylogenetic tree prepared from bHLH sequences suggested that NeuroAB belongs to the BETA3 group in the Atonal-related protein family (ARPs). In situ hybridization and immunostaining indicated that NeuroAB is expressed predominantly in postmitotic bipolar cells and GABAergic amacrine cells in the retina. Reporter and DNA pull down assays indicated that NeuroAB functions as a transcriptional repressor by binding to the E-box sequence, and its activity is modulated by phosphorylation at a specific serine residue that fits the consensus phosphorylation site for glycogen synthase kinase 3beta (GSK3beta). Since members of the BETA3 group possess this consensus site, it is suggested that their activities are commonly regulated by GSK3beta or other kinases bearing the same substrate specificity. We found that the expression of GSK3beta is spatially and temporally regulated in the developing retina; its strong expression was observed in ganglion cells from E8 and a subset of amacrine cells from E12. These findings suggest that NeuroAB is involved in the maturation and maintenance of bipolar cells and GABAergic amacrine cells and regulation by GSK3beta plays an important role in retinogenesis.

Large-scale identification and characterization of genes with asymmetric expression patterns in the developing chick retina.

To understand the molecular basis of topographic retinotectal projection, an overall view of the asymmetrically expressed molecules in the developing retina is needed. We performed a large-scale screening using restriction landmark cDNA scanning (RLCS) in the embryonic day 8 (E8) chick retina. RLCS is a cDNA display system, in which a large number of cDNA species are displayed as two-dimensional spots with intensities reflecting their expression levels as mRNA. We searched for spots that gave different signal intensities between the nasal and temporal retinas or between the dorsal and ventral retinas, and detected about 200 spots that were preferential on one side in the retina. The asymmetric expression of each gene was verified by Northern blotting and in situ hybridization. By subsequent analyses using molecular cloning, DNA sequencing, and database searching, 33 asymmetric molecules along the nasotemporal (N-T) axis and 20 along the dorsoventral (D-V) axis were identified. These included transcription factors, secretory factors, transmembrane proteins, and intracellular proteins with various putative functions. Their expression profiles revealed by in situ hybridization are highly diverse and individual. Moreover, many of them begin to be expressed in the retina from the early developmental stages, suggesting that they are implicated in the establishment and maintenance of regional specificity in the developing retina. The molecular repertoire revealed by this work will provide candidates for future studies to elucidate the molecular mechanisms of topographic retinotectal map formation.

Axonal morphogenesis controlled by antagonistic roles of two CRMP subtypes in microtubule organization.

During development, cells undergo dynamic morphological changes by rearrangements of the cytoskeleton including microtubules. However, molecular mechanisms underlying the microtubule remodeling between orientated and disoriented formations are almost unknown. Here we found that novel subtypes of collapsin response mediator proteins (CRMP-As) and the originals (CRMP-Bs), which occur from the alternative usage of different first coding exons, are involved in this conversion of microtubule patterns. Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization.