Analysis of splicing abnormalities in the white matter of myotonic dystrophy type 1 brain using RNA sequencing.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by the genomic expansion of CTG repeats, in which RNA-binding proteins, such as muscleblind-like protein, are sequestered in the nucleus, and abnormal splicing is observed in various genes. Although abnormal splicing occurs in the brains of patients with DM1, its relation to central nervous system symptoms is unknown. Several imaging studies have indicated substantial white matter defects in patients with DM1. Here, we performed RNA sequencing and analysis of CTG repeat lengths in the frontal lobe of patients with DM1, separating the gray matter and white matter, to investigate splicing abnormalities in the DM1 brain, especially in the white matter. Several genes showed similar levels of splicing abnormalities in both gray and white matter, with an observable trend toward an increased number of repeats in the gray matter. These findings suggest that white matter defects in DM1 stem from aberrant RNA splicing in both gray and white matter. Notably, several of the genes displaying abnormal splicing are recognized as being dominantly expressed in astrocytes and oligodendrocytes, leading us to hypothesize that splicing defects in the white matter may be attributed to abnormal RNA splicing in glial cells.

Transcriptome alterations in myotonic dystrophy frontal cortex.

Myotonic dystrophy (DM) is caused by expanded CTG/CCTG repeats, causing symptoms in skeletal muscle, heart, and central nervous system (CNS). CNS issues are debilitating and include hypersomnolence, executive dysfunction, white matter atrophy, and neurofibrillary tangles. Here, we generate RNA-seq transcriptomes from DM and unaffected frontal cortex and identify 130 high-confidence splicing changes, most occurring only in cortex, not skeletal muscle or heart. Mis-spliced exons occur in neurotransmitter receptors, ion channels, and synaptic scaffolds, and GRIP1 mis-splicing modulates kinesin association. Optical mapping of expanded CTG repeats reveals extreme mosaicism, with some alleles showing >1,000 CTGs. Mis-splicing severity correlates with CTG repeat length across individuals. Upregulated genes tend to be microglial and endothelial, suggesting neuroinflammation, and downregulated genes tend to be neuronal. Many gene expression changes strongly correlate with mis-splicing, suggesting candidate biomarkers of disease. These findings provide a framework for mechanistic and therapeutic studies of the DM CNS.

Differences in splicing defects between the grey and white matter in myotonic dystrophy type 1 patients.

Myotonic dystrophy type 1 (DM1) is a multi-system disorder caused by CTG repeats in the myotonic dystrophy protein kinase (DMPK) gene. This leads to the sequestration of splicing factors such as muscleblind-like 1/2 (MBNL1/2) and aberrant splicing in the central nervous system. We investigated the splicing patterns of MBNL1/2 and genes controlled by MBNL2 in several regions of the brain and between the grey matter (GM) and white matter (WM) in DM1 patients using RT-PCR. Compared with amyotrophic lateral sclerosis (ALS, as disease controls), the percentage of spliced-in parameter (PSI) for most of the examined exons were significantly altered in most of the brain regions of DM1 patients, except for the cerebellum. The splicing of many genes was differently regulated between the GM and WM in both DM1 and ALS. In 7 out of the 15 examined splicing events, the level of PSI change between DM1 and ALS was significantly higher in the GM than in the WM. The differences in alternative splicing between the GM and WM may be related to the effect of DM1 on the WM of the brain.

Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation.

Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disease that is characterized by varying degrees of cerebellar dysfunction and Parkinsonism. The neuropathological hallmark of MSA is alpha-synuclein (AS)-positive glial cytoplasmic inclusions (GCIs). Although severe neuronal loss (NL) is also observed in MSA, neuronal inclusions (NIs) are rare compared to GCIs, such that the pathological mechanism of NL in MSA is unclear. GCIs and NIs are late-stage pathology features relative to AS oligomers and may not represent early pathological changes in MSA. To reveal the early pathology of MSA, it is necessary to examine the early aggregation of AS, i.e., AS oligomers. Here, we adopted a proximity ligation assay (PLA) to examine the distribution of AS oligomers in brain tissue samples from patients with MSA and other diseases. Surprisingly, MSA brains showed a widespread distribution and abundant accumulation of oligomeric AS in neurons as well as oligodendrocytes of the neocortex. In several regions, oligomeric AS signal intensity was higher in cases with MSA than in cases with Parkinson's disease. In contrast to previous studies, AS-PLA revealed abundant AS oligomer accumulation in Purkinje cells in MSA brains, identifying oligomeric AS accumulation as a possible cause of Purkinje cell loss. This wide distribution of AS oligomers in MSA brain neurons has not been described previously and indicates a pathological mechanism of NL in MSA.

Macroscopic and microscopic diversity of missplicing in the central nervous system of patients with myotonic dystrophy type 1.

Myotonic dystrophy type I (DM1) is a multiorgan disease caused by CTG-repeat expansion in the DMPK gene. Sequestration of the splicing factor MBNL1 results in aberrant splicing in many genes in DM1 skeletal muscle, whereas MBNL2 plays a leading role in missplicing in the central nervous system (CNS) of patients with DM1. Splicing misregulation of most MBNL2-regulated genes occurs in the temporal cortex but not in the cerebellum of autopsied patients with DM1. To understand the diversity at macroscopic and microscopic levels in CNS of patients with DM1. Using autopsied brain tissues, we examined alternative splicing ratios of MBNL2-regulated genes and expression levels of potential splicing factors. We found differences in splicing abnormalities among tested regions of the CNS from patients with DM1. In the frontal and temporal cortices and the hippocampus, many genes were aberrantly spliced, but severity differed among the brain regions. By contrast, there were no significant differences in the ratio of splicing variants for most of the genes in the cerebellar cortex and spinal cord between DM1 and control samples. We failed to find any change in the amount of potential factors (MBNL and CUGBP proteins and DMPK mRNA) which explain the modest missplicing in the cerebellum. LASER capture microdissection demonstrated splicing misregulation in the molecular layer of the cerebellum but not in the granular layer. This is the first study to reveal missplicing in a functional cell layer of DM1 and to compare splicing misregulation in a wide region of the CNS using statistical analysis.

Adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) and Nasu-Hakola disease: lesion staging and dynamic changes of axons and microglial subsets.

The brains of 10 Japanese patients with adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) encompassing hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) and pigmentary orthochromatic leukodystrophy (POLD) and eight Japanese patients with Nasu-Hakola disease (N-HD) and five age-matched Japanese controls were examined neuropathologically with special reference to lesion staging and dynamic changes of microglial subsets. In both diseases, the pathognomonic neuropathological features included spherically swollen axons (spheroids and globules), axon loss and changes of microglia in the white matter. In ALSP, four lesion stages based on the degree of axon loss were discernible: Stage I, patchy axon loss in the cerebral white matter without atrophy; Stage II, large patchy areas of axon loss with slight atrophy of the cerebral white matter and slight dilatation of the lateral ventricles; Stage III, extensive axon loss in the cerebral white matter and dilatation of the lateral and third ventricles without remarkable axon loss in the brainstem and cerebellum; Stage IV, devastated cerebral white matter with marked dilatation of the ventricles and axon loss in the brainstem and/or cerebellum. Internal capsule and pontine base were relatively well preserved in the N-HD, even at Stage IV, and the swollen axons were larger with a higher density in the ALSP. Microglial cells immunopositive for CD68, CD163 or CD204 were far more obvious in ALSP, than in N-HD, and the shape and density of the cells changed in each stage. With progression of the stage, clinical symptoms became worse to apathetic state, and epilepsy was frequently observed in patients at Stages III and IV in both diseases. From these findings, it is concluded that (i) shape, density and subsets of microglia change dynamically along the passage of stages and (ii) increase of IBA-1-, CD68-, CD163- and CD204-immunopositive cells precedes loss of axons in ALSP.

Immunohistochemical characterization of CD33 expression on microglia in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by formation of multifocal bone cysts and development of leukoencephalopathy, caused by genetic mutations of either DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). Although increasing evidence suggests a defect in microglial TREM2/DAP12 function in NHD, the molecular mechanism underlying leukoencephalopathy with relevance to microglial dysfunction remains unknown. TREM2, by transmitting signals via the immunoreceptor tyrosine-based activation motif (ITAM) of DAP12, stimulates phagocytic activity of microglia, and ITAM signaling is counterbalanced by sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs)-mediated immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling. To investigate a role of CD33, a member of the Siglecs family acting as a negative regulator of microglia activation, in the pathology of NHD, we studied CD33 expression patterns in five NHD brains and 11 controls by immunohistochemistry. In NHD brains, CD33 was identified exclusively on ramified and amoeboid microglia accumulated in demyelinated white matter lesions but not expressed in astrocytes, oligodendrocytes, or neurons. However, the number of CD33-immunoreactive microglia showed great variability from case to case and from lesion to lesion without significant differences between NHD and control brains. These results do not support the view that CD33-expressing microglia play a central role in the development of leukoencephalopathy in NHD brains.

LC3, an autophagosome marker, is expressed on oligodendrocytes in Nasu-Hakola disease brains.

BACKGROUND: Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Neuropathologically, NHD exhibits profound loss of myelin and accumulation of axonal spheroids, accompanied by intense gliosis accentuated in the white matter of the frontal and temporal lobes. At present, the molecular mechanism responsible for development of leukoencephalopathy in NHD brains remains totally unknown. METHODS: By immunohistochemistry, we studied the expression of microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, in 5 NHD and 12 control brains. RESULTS: In all NHD brains, Nogo-A-positive, CNPase-positive oligodendrocytes surviving in the non-demyelinated white matter intensely expressed LC3. They also expressed ubiquitin, ubiquilin-1, and histone deacetylase 6 (HDAC6) but did not express Beclin 1 or sequestosome 1 (p62). Substantial numbers of axonal spheroids were also labeled with LC3 in NHD brains. In contrast, none of oligodendrocytes expressed LC3 in control brains. Furthermore, surviving oligodendrocytes located at the demyelinated lesion edge of multiple sclerosis (MS) did not express LC3, whereas infiltrating Iba1-positive macrophages and microglia intensely expressed LC3 in MS lesions. CONCLUSIONS: These results propose a novel hypothesis that aberrant regulation of autophagy might induce oligodendrogliopathy causative of leukoencephalopathy in NHD brains.

A novel ferritin light chain mutation in neuroferritinopathy with an atypical presentation.

Neuroferritinopathy or hereditary ferritinopathy is an inherited neurodegenerative disease caused by mutations in ferritin light chain (FTL) gene. The clinical features of the disease are highly variable, and include a movement disorder, behavioral abnormalities, and cognitive impairment. Neuropathologically, the disease is characterized by abnormal iron and ferritin depositions in the central nervous system. We report a family in which neuroferritinopathy begins with chronic headaches, later developing progressive orolingual and arm dystonia, dysarthria, cerebellar ataxia, pyramidal tract signs, and psychiatric symptoms. In the absence of classic clinical symptoms, the initial diagnosis of the disease was based on magnetic resonance imaging studies. Biochemical studies on the proband showed normal serum ferritin levels, but remarkably low cerebrospinal fluid (CSF) ferritin levels. A novel FTL mutation was identified in the proband. Our findings expand the genetic and clinical diversity of neuroferritinopathy and suggest CSF ferritin levels as a novel potential biochemical marker for the diagnosis of neuroferritinopathy.

Somatic instability of CTG repeats in the cerebellum of myotonic dystrophy type 1.

INTRODUCTION: We statistically analyzed somatic instability of the CTG expansion in the central nervous system and visceral organs in 7 patients with myotonic dystrophy type 1 and also report intracerebellar instability in 2 patients. METHODS: CTG repeat expansion was estimated in the samples from autopsied brains and visceral organs by Southern blot analysis. Pathological study was performed. Samples were taken from several sites in the cerebellum to examine intracerebellar instability. RESULTS: The CTG repeat expansion was shortest in the cerebellar cortex among all tissues examined. With regard to the intracerebellar difference, the shortest expansion was seen in the cortices of the hemisphere and vermis, whereas it was moderate in the dentate nucleus and longest in the white matter of the hemisphere and middle cerebellar peduncle. CONCLUSIONS: The shortest expansion might be attributable to packed granule cells in the cerebellar cortex. Further analysis of cell-specific methylation states might elucidate the enigma of somatic instability.

Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy.

The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs that disrupt splicing regulation by altering MBNL1 and CELF1 activities. While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated central nervous system (CNS) features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDA receptor (NMDAR) synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, misregulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq, and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly misregulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program.

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.

Phosphorylated Syk expression is enhanced in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adaptor complex on myeloid cells. The post-receptor signals are transmitted via rapid phosphorylation of the immunoreceptor tyrosine-based activating motif (ITAM) of DAP12, mediated by Src protein tyrosine kinases, followed by binding of phosphorylated ITAM to Src homology 2 (SH2) domains of spleen tyrosine kinase (Syk), resulting in autophosphorylation of the activation loop of Syk. To elucidate the molecular mechanism underlying the pathogenesis of NHD, we investigated Syk expression and activation in the frontal cortex and the hippocampus of three NHD and eight control brains by immunohistochemistry. In NHD brains, the majority of neurons expressed intense immunoreactivities for Syk and Y525/Y526-phosphorylated Syk (pSyk) chiefly located in the cytoplasm, while more limited populations of neurons expressed Src. The levels of pSyk expression were elevated significantly in NHD brains compared with control brains. In both NHD and control brains, substantial populations of microglia and macrophages expressed pSyk, while the great majority of reactive astrocytes and myelinating oligodendrocytes did not express pSyk, Syk or Src. These observations indicate that neuronal expression of pSyk was greatly enhanced in the cerebral cortex and the hippocampus of NHD brains, possibly via non-TREM2/DAP12 signaling pathways involved in Syk activation.

Immunohistochemical characterization of microglia in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adapter signaling complex expressed on osteoclasts, dendritic cells (DC), macrophages and microglia. Previous studies using knockout mice and mouse brain cell cultures suggest that a loss-of-function of DAP12/TREM2 in microglia plays a central role in the neuropathological manifestation of NHD. However, there exist no immunohistochemical studies that focus attention on microglia in NHD brains. To elucidate a role of microglia in the pathogenesis of NHD, we searched NHD-specific biomarkers and characterized their expression on microglia in NHD brains. Here, we identified allograft inflammatory factor 1 (AIF1, Iba1) and sialic acid binding Ig-like lectin 1 (SIGLEC1) as putative NHD-specific biomarkers by bioinformatics analysis of microarray data of NHD DC. We studied three NHD and eight control brains by immunohistochemistry with a panel of 16 antibodies, including those against Iba1 and SIGLEC1. We verified the absence of DAP12 expression in NHD brains and the expression of DAP12 immunoreactivity on ramified microglia in control brains. Unexpectedly, TREM2 was not expressed on microglia but expressed on a small subset of intravascular monocytes/macrophages in control and NHD brains. In the cortex of NHD brains, we identified accumulation of numerous Iba1-positive microglia to an extent similar to control brains, while SIGLEC1 was undetectable on microglia in all the brains examined. These observations indicate that human microglia in brain tissues do not express TREM2 and DAP12-deficient microglia are preserved in NHD brains, suggesting that the loss of DAP2/TREM2 function in microglia might not be primarily responsible for the neuropathological phenotype of NHD.

Neuropathology does not Correlate with Regional Differences in the Extent of Expansion of CTG Repeats in the Brain with Myotonic Dystrophy Type 1.

Myotonic dystrophy (DM1) is known to be an adult-onset muscular dystrophy caused by the expansion of CTG repeats within the 3' untranslated region of the dystrophin myotonin protein kinase (DMPK) gene. The clinical features of DM1 include CNS symptoms, such as cognitive impairment and personality changes, the pathogenesis of which remains to be elucidated. We hypothesized that the distribution of neuropathological changes might be correlated with the extent of the length of the CTG repeats in the DMPK genes in DM1 patients. We studied the neuropathological changes in the brains of subjects with DM1 and investigated the extent of somatic instability in terms of CTG repeat expansion in the different brain regions of the same individuals by Southern blot analysis. The neuropathological changes included état criblé in the cerebral deep white matter and neurofibrillary tangles immunoreactive for phosphorylated tau in the hippocampus and entorhinal cortex, both of which were compatible with the subcortical dementia in DM1 patients. However, the length of the CTG repeats did not correlate with the regional differences in the extent of neuropathological changes. Our data suggested that pathomechanisms of dementia in DM1 might be more multifactorial rather than a toxic gain-of-function due to mutant RNA.

[Four siblings with becker muscular dystrophy (BMD) manifesting severe mental retardation].

A 33-year-old man with BMD manifesting severe mental retardation is reported. This patient has mild pseudohypertrophy in his calf muscles and showed an elevation of creatine kinase (CK) level in the serum (2215 IU/L). He was diagnosed as autistic at the age of three. His intellectual level was estimated to be two years old in social intelligence and four months old in speech ability at the age of 33. However his muscle strength remains within the normal range. All of his three siblings have similar symptoms, such as severe mental retardation and elevated CK level in the serum (1735-3641 IU/L) and lack apparent muscular weakness. Gene analyses by multiplex PCR and Southern blotting showed all of the siblings had the deletion of exon 4 in the dystrophin gene. Pathological findings of a muscle biopsy specimen showed a mild irregular dystrophin stain of the muscle surface membrane. This is a rare familial case of Becker muscular dystrophy manifesting severe mental retardation with scarce muscular weakness.

[Posterior spinal cord infarction presenting Brown-Séquard syndrome].

We report a 56 year-old-woman with spinal cord infarction. She experienced left-sided girdle pain without precipitating symptoms and she developed monoparesis of her left leg and urinary retention. She also presented the segmental loss of total sensations in the Th10-11 area of the left trunk, the disturbance of position and vibration senses in the left leg and the disturbance of pain and temperature senses in the right leg. T2-weighted MR imagings showed high signal intensity lesion in the left half of the spinal posterior column at Th9-10 vertebral levels. Somatosensory evoked potentials confirmed that the loss of position and vibration senses was unilateral. Though she became able to walk with canes two months later, her sensory disturbance showed no improvement. This is a rare case of unilateral posterior spinal cord infarction presenting Brown-Séquard syndrome.

[Transient left ventricular apical ballooning, "Takotsubo" cardiomyopathy, in an amyotrophic lateral sclerosis patient on long-term respiratory support].

A 52-year-old woman developed dysarthria and dysphagia in April 1997, then experienced progressive weakness in her arms and legs several months later, which led to a diagnosis of amyotrophic lateral sclerosis (ALS). In October 1998, the patient was placed on respiratory support and thereafter in a bedridden state. On December 6, 2004, the patient suddenly fell into cardiogenic shock. An echocardiographic examination demonstrated extensive akinesis of the left ventricle together with the hyperkinetic constraction of the cardiac base. The left ventricular akinesis completely returned to normal by December 13. Based on our these results and her clinical course, we made a diagnosis of "Takotsubo" cardiomyopathy. This is the first case reported to have developed this condition in an ALS patient on long-term respiratory support. Physicians should be aware of the potential risk of developing "Takotsubo" cardiomyopathy in respirator-dependent ALS patients.

[A case of esophageal achalasia followed by Parkinson's disease].

In 1992, a 63 year-old woman complained of dysphagia and chest pain, and was diagnosed with esophageal achalasia. Three years later, she developed resting tremor, cog-wheel rigidity, and retro-pulsion, and was diagnosed with Parkinson's disease and given appropriate medication. Several years later, intractable vomitting and aspiration pneumonia developed, and the lower esophageal sphincter was dilated using a pneumatic balloon dilator under gastroscopic guidance in 2004. That procedure improved her symptoms and the esophageal dilation was visualized on chest CT images. Herein, we report this rare case of esophageal achalasia followed by Parkinson's disease and discuss the relationship between the two diseases.