Tomokatsu Yoshida

Knockdown of the Drosophila fused in sarcoma (FUS) homologue causes deficient locomotive behavior and shortening of motoneuron terminal branches.

Mutations in the fused in sarcoma/translated in liposarcoma gene (FUS/TLS, FUS) have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). FUS is an RNA-binding protein that is normally localized in the nucleus, but is mislocalized to the cytoplasm in ALS, and comprises cytoplasmic inclusions in ALS-affected areas. However, it is still unknown whether the neurodegeneration that occurs in ALS is caused by the loss of FUS nuclear function, or by the gain of toxic function due to cytoplasmic FUS aggregation. Cabeza (Caz) is a Drosophila orthologue of human FUS. Here, we generated Drosophila models with Caz knockdown, and investigated their phenotypes. In wild-type Drosophila, Caz was strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with a presynaptic marker, suggesting that Caz physiologically functions in neuronal cell bodies and/or their axons. Fly models with neuron-specific Caz knockdown exhibited reduced climbing ability in adulthood and anatomical defects in presynaptic terminals of motoneurons in third instar larvae. Our results demonstrated that decreased expression of Drosophila Caz is sufficient to cause degeneration of motoneurons and locomotive disability in the absence of abnormal cytoplasmic Caz aggregates, suggesting that the pathogenic mechanism underlying FUS-related ALS should be ascribed more to the loss of physiological FUS functions in the nucleus than to the toxicity of cytoplasmic FUS aggregates. Since the Caz-knockdown Drosophila model we presented recapitulates key features of human ALS, it would be a suitable animal model for the screening of genes and chemicals that might modify the pathogenic processes that lead to the degeneration of motoneurons in ALS.

White Matter Loss in the Splenium of the Corpus callosum in a Case of Posterior Cortical Atrophy: A Diffusion Tensor Imaging Study

There have been several functional imaging studies using PET and SPECT to investigate posterior cortical atrophy (PCA). These studies have suggested dysfunction of corticocortical connections which is consistent with the occipitoparietal stream. However, there are no reports suggesting disturbance of the white matter that interconnects the temporal, parietal and occipital cortices. We measured fractional anisotropy (FA) in the genu and splenium of the corpus callosum and created color maps using diffusion tensor imaging (DTI), which is a relatively new MRI technique that allows visualization of the directionality of water diffusion, in a patient with PCA and compared these findings with those in 5 typical Alzheimer disease (AD) patients. The PCA patient was a 75-year-old man presenting with progressive complex visual disorder who satisfied the clinical diagnostic criteria for PCA. In 5 typical AD patients, the FA index in the splenium was higher than that in the genu; however, in the PCA patient, the FA index in the splenium was significantly lower than that in the genu. A DTI-based color map of the PCA patient showed reduction of anisotropy and fiber volume in the splenium. These findings suggest that the splenium of the corpus callosum secondarily degenerated due to neuronal degeneration of the temporal, parietal and occipital cortices and suggest that reduction of the FA in the splenium is one of the characteristics of PCA.

Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS

In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) is the Drosophila ortholog of human FUS. Previously, we established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons (MNs) at neuromuscular junctions; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause MN degeneration similar to that seen in FUS-related ALS. Here, we aimed to explore molecules that affect these ALS-like phenotypes of our Drosophila models with eye-specific and neuron-specific Caz-knockdown. We examined several previously reported ALS-related genes and found genetic links between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the MN-degeneration phenotype caused by Caz-knockdown. Conversely, the overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. Our data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. Our results indicate that therapeutic agents that up-regulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of MNs in ALS.

Clinical aspects and pathology of Alexander disease, and morphological and functional alteration of astrocytes induced by GFAP mutation

Alexander disease (AxD) is pathologically characterized by the presence of Rosenthal fibers (RF), which are made up of GFAP, αB‐crystallin and heat shock protein 27, in the cytoplasm of perivascular and subpial astrocyte endfeet. Since GFAP mutation has been confirmed in reported cases of AxD, clinical or experimental research is being conducted on the relationship between GFAP mutation and the onset pathology as well as the clinical form. We conducted a nationwide survey and a clinical study, and classified AxD into three types: cerebral AxD (type 1), which primarily has an infantile onset with presence of seizures, psychomotor developmental retardation, macrocephaly, and abnormalities in the superior frontal cerebral white matter observed in a brain MRI; bulbospinal AxD (type 2), which primarily has an adult onset with presence of muscle weakness, hyperreflexia, bulbar or pseudobulbar symptoms, signal abnormalities, and atrophy observed in an MRI of the medulla oblongata and upper cervical spinal cord; and an intermediate form (type 3) which has the characteristics of both. A research on GFAP mutations and aggregate formation concluded that GFAP mutations decreased the solubility of GFAP. According to our cell model experiment, the formation of mutant GFAP aggravates depending on the site of the GFAP mutation. Furthermore, there is a possibility that polymorphism in the GFAP promoter gene regulates the degree to which GFAP is expressed; it may have an effect on clinical heterogeneity. Recent research using cell and animal models suggests that the pathology of AxD involves not only mere functional abnormalities in intermediate filaments but also functional abnormalities in astrocytes as well as in neurons. Clarification of the glia–neuron interactions will prove the disease to be very interesting.

Glial fibrillary acidic protein mutations in adult‐onset Alexander disease: clinical features observed in 12 Japanese patients

Yoshida T, Sasayama H, Mizuta I, Okamoto Y, Yoshida M, Riku Y, Hayashi Y, Yonezu T, Takata Y, Ohnari K, Okuda S, Aiba I, Nakagawa M. Glial fibrillary acidic protein mutations in adult‐onset Alexander disease: clinical features observed in 12 Japanese patients. 
Acta Neurol Scand: 2011: 124: 104–108.
© 2010 John Wiley & Sons A/S.

The functional alteration of mutant GFAP depends on the location of the domain: morphological and functional studies using astrocytoma-derived cells

AbstractTo clarify the functional effects of mutant glial fibrillary acidic protein (GFAP), we examined the expression patterns of mutant GFAPs (V87G, R88C, and R416W) in astrocytoma-derived cells and performed migration assay. The morphological change was found in mutant GFAP cells, although the number of changes was small. On migration assay, the migration rate in cells with the V87G or R88C mutation, which are located in the helical rod domain in GFAP, was significantly higher than those of wild-type and R416W. These findings suggest that the functional abnormalities of astrocytes might be induced prior to aggregation of GFAP in Alexander disease and that the functional alteration depends on the location of the domain.

Novel GFAP mutation in patient with adult-onset Alexander disease presenting with spastic ataxia.

Adult-onset Alexander disease is rare and clinically characterized by slowly progressive signs of brainstem and spinal cord involvement. Missense mutations in the gene encoding the glial fibrillary acidic protein (GFAP) have been identified as a genetic basis for Alexander disease. We here report a Japanese patient with adult-onset Alexander disease with a novel GFAP mutation. A 36-year-old man of Japanese descent, a child of nonconsanguineous parents, with a 10-year history of slowly progressive gait disturbance, was referred to us. His early motor and intellectual development were normal. Neurological examination revealed rhythmic ocular nystagmoid movement, dysarthria, truncal and limb ataxia, increased muscle stretch reflex with bilateral Babinski sign, and spasticity in his lower extremities. Palatal myoclonus was not noted. He was ambulatory, but his gait was unsteady owing to ataxia and spasticity in the lower extremities. Brain MRI demonstrated a marked atrophy of the medulla oblongata and cervical spinal cord, and a mild atrophy of the cerebellar hemisphere (Fig. 1A). Fluid attenuation inversion recovery (FLAIR) images revealed abnormal hyperintensities in cerebellar dentate nucleus (Fig. 1B) and the periventricular white matter (Fig. 1C). Molecular genetic analysis of GFAP was performed using the patient’s genomic DNA after obtaining written informed consent. Sequence analysis revealed a heterozygous 302T > C substitution in exon 1 of GFAP, leading to an L101P substitution. The L101P substitution is located in the C-terminal end of the 1A rod domain of GFAP occurring in a highly conserved amino acid residue across species (Fig. 1D). The sequence change was confirmed by restriction fragment length polymorphism (RFLP) using enzyme digestion by BcgI in the patient and 100 normal control subjects (Fig. 1E). To obtain biochemical evidence of pathogenecity of the novel GFAP L101P mutant, we transfected wild-type and mutant GFAP, and examined the solubility of the GFAP protein. Samples were sequentially extracted with different stringent buffers and subjected to western blot analysis (see Supplementary methods). The well-characterized mutant R416W GFAP was largely recovered from the detergent-resistant S2 fraction because of the decreased solubility of mutant GFAP (Fig. 1F, lane 8) as previously reported. In this assay, wild-type GFAP was predominantly detected in the soluble S1 fraction (Fig. 1F, lane 2). In contrast, the mutant L101P GFAP was largely observed in the detergent-resistant S2 fraction (Fig. 1F, lane 7). Transfected cells were further analyzed for GFAP assembly by confocal microscopy (see Supplementary methods). Whereas wild-type GFAP displayed cytoplasmic distribution with a filamentous network, the L101P mutant yielded an irregular dot-like structure largely lacking the filamentous structure (Supp. Info. Fig.). In this study, we identified a novel GFAP mutation in a Japanese patient with adult-onset Alexander disease presenting with slowly progressive spastic ataxia. The parents of the patient are unaffected; hence, the mutation seems to arise de novo in the patient as in most cases of Alexander disease. Indeed, the mother did not carry the mutation. Unfortunately, DNA sample was unavailable from the father, who has recently died of heart disease. GFAP is a member of the intermediate filament family with a conserved central helical rod domain flanked by the head and tail domains (Fig. 1D). The L101P mutation detected in the patient is located in the coil 1A rod domain, which is considered to play an essential role in filament formation. Mutations of the a-helix regions in the rod domain are considered to alter the charge and hydrophobic interactions within coiled coils. Thus, mutations in the domain may affect the solubility of GFAP, which is supported by our biochemical experiments using cells expressing mutant GFAP. To date, more than 10 GFAP missense mutations associated with adult-onset Alexander disease have been found, with nearly all occurring in the rod domain. The genotype–phenotype correlation in Alexander disease has been poorly understood particularly in adult-onset cases, probably owing to the very small number of patients. Alexander disease in our patient is clinically characterized by slowly progressive spastic ataxia with bulbar signs without palatal myoclonus. In patients with adult-onset Alexander disease, bulbar symptoms, gait ataxia, and spasticity are common clinical features, whereas ocular motor abnormalities, autonomic dysfunctions, and palatal myoclonus have been reported with varying frequency. Our patient exhibited atrophy of the medulla and spinal cord, and abnormal hyperintensities of the periventricular white matter and cerebellar dentate nucleus on FLAIR images. The characteristic atrophy of the medulla and spinal cord is invariably present in adult-onset Alexander disease. In contrast, leukoencephalopathy and abnormal signal intensities of the cerebellum are not always observed in adultonset cases. The question of why missense mutations in the same critical domain of GFAP result in such different clinical phenotypes and MRI findings in Alexander disease is intriguing and deserves further attention and elucidation.

The process of inducing GFAP aggregates in astrocytoma-derived cells is different between R239C and R416W mutant GFAP. A time-lapse recording study

Alexander disease (ALX) is a rare neurodegenerative disease caused by the gene mutations encoding glial fibrillary acidic protein (GFAP). The formation of aggregates in the cytoplasm of astrocytes, which mainly consists of GFAP, is characteristic of ALX. To examine the dynamic process of aggregates between the different domains of GFAP, we performed time-lapse recording on two different mutant GFAP. R239C and R416W GFAP mutations located in the rod domain and tail domain, respectively, were transfected into astrocytoma-derived cells, and their real-time dynamics were observed using time-lapse recording. Our time-lapse recording study indicated that the process of inducing aggregates would be different between R239C and R416W. In GFP-R239C cells, 32.4% first appeared as aggregates, and clusters of aggregates in the cytoplasm tended to move inward and form amorphous aggregates. On the other hand, 82.0% of GFP-R416W cells first showed disrupted GFAP, with a bubble-like or ring-like structure; however, most cells maintained their structure and were capable of cell division. Our result indicates that the mechanism of GFAP aggregation depends on the domain in which the point mutation is located. A different approach to ALX therapy should be considered according to the domain of GFAP.

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.

A case of cerebral aquaporinopathy

A 35-year-old woman was hospitalized due to impaired consciousness. Magnetic resonance imaging (MRI) revealed multiple parenchymal lesions in supra and infratentorial brain regions, which were considered responsible for her declining consciousness level. She was treated with intravenous methylprednisolone. Neurological symptoms improved and she was discharged. She was readmitted 14 months later due to intractable hiccups. A follow-up brain MRI revealed an abnormal signal near the area postrema in the dorsal medulla. Serum aquaporin-4 antibody levels were positive, but there were no visual manifestations or myelitis. Spinal MRI was negative for longitudinally extended transverse myelitis throughout the clinical course.