TMEM119-positive microglia were increased in the brains of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that has been reported to be affected by inflammatory cells, such as microglia and macrophages, through the concept of non-cell autonomous neuronal death. Resident microglia in the human brain and monocyte-derived macrophages (MoDM) infiltrating in tissues are difficult to distinguish. Therefore, the effects of microglia and MoDMs in ALS remain poorly understood. This study aimed to investigate the role of resident microglia and MoDMs in the pathogenesis of ALS using postmortem brain and spinal cord samples. The samples used for immunohistochemical analysis included 11 cases of sporadic ALS and 11 age-matched controls. We stained the cells with TMEM119 to detect resident microglia and CCR2 to detect MoDMs. In ALS cases, TMEM119-immunopositive resident microglia were abundant in the motor cortex and subcortical white matter (SWM) of the motor area, whereas CCR2-immunopositive MoDM was similar to control cases. In addition, the mean density of CD68-immunopositive cells in the SWM significantly correlated with the mean density of pTDP-43-positive GCIs. These results suggest that resident microglial activation plays an important role in the cerebral pathogenesis of ALS and may provide novel therapeutic strategies to target excessive activation of resident microglia in ALS.

[Severe consciousness disturbance after cord blood transplantation for relapsed T lymphoblastic lymphoma].

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) has a poor prognosis. Nelarabine has recently shown relatively good results in patients with relapsed or refractory T-ALL/LBL, but requires careful monitoring for neurological complications. A 50-year-old man with early recurrence of T-LBL after allogenic peripheral blood stem cell transplantation received nelarabine monotherapy and achieved complete remission after 1 cycle. He then received umbilical cord blood transplantation, and experienced sustained disturbance of consciousness. He later died of multiple organ failure, and autopsy suggested that nelarabine-induced leukoencephalopathy had caused the disturbance of consciousness. This case suggests that physicians should carefully monitor patients for neurological complications and consider imaging follow-up and consultation with a neurologist.

Evaluation of BCAS1-positive immature oligodendrocytes after cerebral ischemic stroke and SVD.

Ischemic cerebrovascular disease is an important cause of physical disability and dementia. Oligodendrocytes (OLGs), which differentiate from oligodendrocyte precursor cells (OPCs), are crucial for remyelination of the damaged brain and functional recovery. Breast carcinoma amplified sequence 1 (BCAS1) has recently been shown to be highly expressed in newly formed pre-myelinating oligodendrocytes (pre-mOLGs), while its expression level is reduced in mature OLGs. In this study, we analyzed BCAS1 expression by immunohistochemical analysis of human post-mortem brain tissue from six stroke patients (death within 2 months after stroke onset) and eight small vessel disease (SVD) patients. Control post-mortem brain tissue was from eight age-matched patients without any obvious central nervous system (CNS) pathology. The Olig2 expression in the area corresponding to the same section of the BCAS1-stained slice was analyzed to determine the total oligodendrocyte lineage. The percentage of differentiating OPCs in the oligodendrocyte lineage was calculated as the ratio of BCAS1+ to Olig2+ cells (BCAS1+/Olig2+). The stroke and SVD cases showed demyelination with decreased expression of myelin basic protein (MBP, a mature OLG marker). The stroke cases showed significantly increased numbers of early-stage BCAS1+ cells with an immature morphology and Olig2+ cells (pan-oligodendrocyte lineages) in the peri-infarct areas in both the cortex and white matter, but showed no increase in the number of late-stage BCAS1+ cells with a mature morphology. In contrast, the SVD cases showed no significant increase in Olig2+ and BCAS1+ cells. These results indicated that remyelination dysfunction could be attributed to insufficient maturation of OPCs in stroke and impaired recruitment of OPCs in SVD.

Utility of Skeletal Muscle CT in Diagnosing Spinal Muscular Atrophy Type 3 in a Patient Who Had Been Undiagnosed for 50 Years.

A 69-year-old woman presented with progressive limb weakness lasting 50 years. She denied any congenital disorders or a family history of neuromuscular disease. At ages 29, 46, and 58 years, she underwent hospitalization and evaluations including electromyogram (EMG) and muscle biopsy, but the results were inconclusive. As a result, she received a tentative diagnosis of myopathy of unknown etiology. However, at the age of 69 years, a computed tomography (CT) scan of her skeletal muscles revealed severe involvement of the triceps brachii, iliopsoas, and gastrocnemius muscles, along with preservation of the biceps brachii, gluteus maximus, and tibialis anterior muscles, which was consistent with spinal muscular atrophy (SMA). Finally, genetic testing revealed the deletion of the survival of the motor neuron 1 (SMN1) gene, confirming the diagnosis of SMA type 3. As our case suggests, SMA patients with prolonged disease duration could be underdiagnosed even after EMG and muscle biopsy. A skeletal CT scan could be useful for the diagnosis of SMA patients compared with MRI.

Safety and tolerability of bosutinib in patients with amyotrophic lateral sclerosis (iDReAM study): A multicentre, open-label, dose-escalation phase 1 trial.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of motor neurons, and development of effective medicines is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified the Src/c-Abl inhibitor bosutinib, which is approved for the treatment of chronic myelogenous leukemia (CML), as a candidate for the molecular targeted therapy of ALS. Methods: An open-label, multicentre, dose-escalation phase 1 study using a 3 + 3 design was conducted in 4 hospitals in Japan to evaluate the safety and tolerability of bosutinib in patients with ALS. Furthermore, the exploratory efficacy was evaluated using Revised ALS Functional Rating Scale (ALSFRS-R), predictive biomarkers including plasma neurofilament light chain (NFL) were explored, and single-cell RNA sequencing of iPSC-derived motor neurons was conducted. Patients, whose total ALSFRS-R scores decreased by 1-3 points during the 12-week, received escalating doses starting from 100 mg quaque die (QD) up to 400 mg QD based on dose-limiting toxicity (DLT) occurrence, and all participants who received one dose of the study drug were included in the primary analysis. This trial is registered with ClinicalTrials.gov, NCT04744532, as Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic Lateral Sclerosis Medicine (iDReAM) study. Findings: Between March 29, 2019 and May 7, 2021, 20 patients were enrolled, 13 of whom received bosutinib treatment and 12 were included in the safety and efficacy analyses. No DLTs were observed up to 300 mg QD, but DLTs were observed in 3/3 patients of the 400 mg QD cohort. In all patients receiving 100 mg-400 mg, the prevalent adverse events (AEs) were gastrointestinal AEs in 12 patients (92.3%), liver function related AEs in 7 patients (53.8%), and rash in 3 patients (23.1%). The safety profile was consistent with that known for CML treatment, and ALS-specific AEs were not observed. A subset of patients (5/9 patients) was found to respond well to bosutinib treatment over the 12-week treatment period. It was found that the treatment-responsive patients could be distinguished by their lower levels of plasma NFL. Furthermore, single-cell RNA sequencing of iPSC-derived motor neurons revealed the pathogenesis related molecular signature in patients with ALS showing responsiveness to bosutinib. Interpretation: This is the first trial of a Src/c-Abl inhibitor, bosutinib, for patients with ALS. The safety and tolerability of bosutinib up to 300 mg, not 400 mg, in ALS were described, and responsiveness of patients on motor function was observed. Since this was an open-label trial within a short period with a limited number of patients, further clinical trials will be required. Funding: AMED and iPS Cell Research Fund.

Conformational change of RNA-helicase DHX30 by ALS/FTD-linked FUS induces mitochondrial dysfunction and cytosolic aggregates.

Genetic mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). Although mitochondrial dysfunction and stress granule have been crucially implicated in FUS proteinopathy, the molecular basis remains unclear. Here, we show that DHX30, a component of mitochondrial RNA granules required for mitochondrial ribosome assembly, interacts with FUS, and plays a crucial role in ALS-FUS. WT FUS did not affect mitochondrial localization of DHX30, but the mutant FUS lowered the signal of mitochondrial DHX30 and promoted the colocalization of cytosolic FUS aggregates and stress granule markers. The immunohistochemistry of the spinal cord from an ALS-FUS patient also confirmed the colocalization, and the immunoelectron microscope demonstrated decreased mitochondrial DHX30 signal in the spinal motor neurons. Subcellular fractionation by the detergent-solubility and density-gradient ultracentrifugation revealed that mutant FUS also promoted cytosolic mislocalization of DHX30 and aggregate formation. Interestingly, the mutant FUS disrupted the DHX30 conformation with aberrant disulfide formation, leading to impaired mitochondrial translation. Moreover, blue-native gel electrophoresis revealed an OXPHOS assembly defect caused by the FUS mutant, which was similar to that caused by DHX30 knockdown. Collectively, our study proposes DHX30 as a pivotal molecule in which disulfide-mediated conformational change mediates mitochondrial dysfunction and cytosolic aggregate formation in ALS-FUS.

An autopsy case of adult-onset neuronal intranuclear inclusion disease with perivascular preservation in cerebral white matter.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by appearance of eosinophilic hyaline intranuclear inclusions. While the main symptoms of adult-onset NIID are dementia or limb weakness, some patients present with encephalitic episodes and transient neurological symptoms. The pathophysiology of these acute, transient symptoms, however, remains unknown. Here, we describe an autopsy case of adult-onset NIID with progressive dementia and transient hemiparesis. The patient was a 70-year-old man without a relevant family history, and initially presented with progressive dementia. He then exhibited transient left hemiparesis at 75 years of age and died of ureteral cancer at 77 years of age. Neuropathological examination revealed the presence of multiple areas of focal spongiosis in the subcortical white matter and patchy myelin pallor of the white matter, as in previous reports. However, perivascular areas were preserved even in the damaged white matter. In addition, dense glial fibrillary acidic protein (GFAP)-immunoreactive astrocytic processes were observed in these areas. [Correction added on 23 January 2022, after first online publication: the preceding sentence has been corrected to improve readability.] GFAP immunohistochemistry revealed decreased density and morphological abnormalities of astrocytes in the affected white matter. These pathological findings might reflect blood-brain barrier impairment and dysregulation of blood flow, which may be related to the pathophysiology of the acute, transient symptoms observed in NIID.

Monoclonal gammopathy of renal significance (MGRS)-related AL amyloidosis complicated by amyloid myopathy: a case report.

BACKGROUND: Lately, monoclonal gammopathy of renal significance (MGRS) has been defined as a group of renal disorders that are strongly associated with monoclonal protein, including amyloid immunoglobulin light chain (AL) amyloidosis. Amyloid myopathy is rare (1.5% of all patients with amyloidosis) and the prognosis is poor. Furthermore, only approximately 20% of patients with amyloid myopathy are reported to have renal involvement, indicating a lack of data in the literature. CASE PRESENTATION: Here, we report a rare case of MGRS-related AL amyloidosis complicated by amyloid myopathy that presented with muscle weakness in the upper and lower limbs, neck and fingers, and nephrotic syndrome. Blood, urine, and bone marrow examination revealed monoclonal gammopathy of undetermined significance (MGUS) (Bence Jones protein-lambda). Muscle biopsy of the vastus lateralis muscle demonstrated amyloid proteins in the sarcolemma and in the blood vessel walls on Congo red staining, suggesting amyloid myopathy, and tiny inclusions in fibers on modified Gomori trichrome stain. Although we thought they were reminiscent of nemaline bodies, we could not confirm the nature of this structure. Renal biopsy demonstrated amyloid proteins in the mesangial region, part of the capillary walls, and the blood vessel walls on direct fast scarlet staining. As these amyloid proteins were positive for p-component staining and negative for amyloid A staining, ß2-microglobulin, and pre-albumin, and as lambda light chains were positive in the mesangial region, we diagnosed the patient with MGRS-related AL amyloidosis. Although he was treated with melphalan and dexamethasone, his symptoms did not improve. CONCLUSIONS: AL amyloidosis involving the kidneys and muscles has a poor prognosis, and a delayed diagnosis of amyloid myopathy is common because of its rarity and frequent misdiagnosis, which increases organ function deterioration. Therefore, early detection, therapeutic intervention, and careful follow-up are crucial.

BCAS1-positive immature oligodendrocytes are affected by the α-synuclein-induced pathology of multiple system atrophy.

Multiple system atrophy (MSA) is pathologically characterized by the presence of fibrillar α-synuclein-immunoreactive inclusions in oligodendrocytes. Although the myelinating process of oligodendrocytes can be observed in adult human brains, little is known regarding the presence of α-synuclein pathology in immature oligodendrocytes and how their maturation and myelination are affected in MSA brains. Recently, breast carcinoma amplified sequence 1 (BCAS1) has been found to be specifically expressed in immature oligodendrocytes undergoing maturation and myelination. Here, we analyzed the altered dynamics of oligodendroglial maturation in both MSA brains and primary oligodendroglial cell cultures which were incubated with α-synuclein pre-formed fibrils. The numbers of BCAS1-expressing oligodendrocytes that displayed a matured morphology negatively correlated with the density of pathological inclusions in MSA brains but not with that in Parkinson's disease and diffuse Lewy body disease. In addition, a portion of the BCAS1-expressing oligodendrocyte population showed cytoplasmic inclusions, which were labeled with antibodies against phosphorylated α-synuclein and cleaved caspase-9. Further in vitro examination indicated that the α-synuclein pre-formed fibrils induced cytoplasmic inclusions in the majority of BCAS1-expressing oligodendrocytes. In contrast, the majority of BCAS1-non-expressing mature oligodendrocytes did not develop inclusions on day 4 after maturation induction. Furthermore, exposure of α-synuclein pre-formed fibrils in the BCAS1-positive phase caused a reduction in oligodendroglial cell viability. Our results indicated that oligodendroglial maturation and myelination are impaired in the BCAS1-positive phase of MSA brains, which may lead to the insufficient replacement of defective oligodendrocytes. In vitro, the high susceptibility of BCAS1-expressing primary oligodendrocytes to the extracellular α-synuclein pre-formed fibrils suggests the involvement of insufficient oligodendroglial maturation in MSA disease progression and support the hypothesis that the BCAS1-positive oligodendrocyte lineage cells are prone to take up aggregated α-synuclein in vivo.

Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic lateral sclerosis Medicine (iDReAM) study: protocol for a phase I dose escalation study of bosutinib for amyotrophic lateral sclerosis patients.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive and severe neurodegenerative disease caused by motor neuron death. There have as yet been no fundamental curative medicines, and the development of a medicine for ALS is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified an Src/c-Abl inhibitor, bosutinib, as a candidate molecular targeted therapy for ALS. The objectives of this study are to evaluate the safety and tolerability of bosutinib for the treatment of patients with ALS and to explore the efficacy of bosutinib on ALS. This study is the first clinical trial of administered bosutinib for patients with ALS. METHODS AND ANALYSIS: An open-label, multicentre phase I dose escalation study has been designed. The study consists of a 12-week observation period, a 1-week transitional period, a 12-week study treatment period and a 4-week follow-up period. After completion of the transitional period, subjects whose total ALS Functional Rating Scale-Revised (ALSFRS-R) score decreased by 1-3 points during the 12-week observation period receive bosutinib for 12 weeks. Three to six patients with ALS are enrolled in each of the four bosutinib dose levels (100, 200, 300 or 400 mg/day) to evaluate the safety and tolerability under a 3+3 dose escalation study design. Dose escalation and maximum tolerated dose are determined by the safety assessment committee comprising oncologists/haematologists and neurologists based on the incidence of dose-limiting toxicity in the first 4 weeks of the treatment at each dose level. A recommended phase II dose is determined by the safety assessment committee on completion of the 12-week study treatment in all subjects at all dose levels. The efficacy of bosutinib is also evaluated exploratorily using ALS clinical scores and biomarkers. ETHICS AND DISSEMINATION: This study received full ethical approval from the institutional review board of each participating site. The findings of the study will be disseminated in peer-reviewed journals and at scientific conferences. TRIAL REGISTRATION NUMBER: UMIN000036295; Pre-results, JMA-IIA00419; Pre-results.

[An autopsy case after endovascular thrombectomy for cardioembolic stroke due to nonbacterial thrombotic endocarditis].

A 73-year-old women visited emergency department because of sudden right hemiplegia. She had a history of duodenum papilla cancer terminal stage and multiple liver metastasis. On admission, diffusion weighted images revealed high intensity area at left middle cerebral artery territory. In addition, 3D-TOF MRA depicted proximal part of the left internal carotid artery. We performed endovascular thrombectomy because low platelet count met contraindication of intravenous recombinant tissue plasminogen activator therapy. Although we could get partial recanalization of middle cerebral artery occlusion after thrombectomy, the patient eventually died due to multiple organ failure. Autopsy findings showed white thrombus on mitral valve and also left middle cerebral artery occluded by similar white thrombus without infective findings. The patient was finally diagnosed with nonbacterial thrombotic endocarditis due to white thrombus on the mitral valve. We should select appropriate mechanical thrombectomy devices with a case of cerebral infarction due to nonbacterial thrombotic endocarditis because its thrombus is often white thrombus and would be hard.

Chronic cerebral hypoperfusion upregulates leptin receptor expression in astrocytes and tau phosphorylation in tau transgenic mice.

Alzheimer's disease (AD) is the most common type of dementia in aging adults. Increasing evidence has revealed that vascular risk factors influence the midlife development of AD and that diet-induced obesity accelerates tau phosphorylation in tau transgenic mice and increases the level of serum leptin receptor (leptin-R). Leptin-R is upregulated in the peri-infarct cortices after acute cerebral ischemia. Leptin may be protective against the development of AD as it can inactivate GSK-3ß through the phosphorylation of Ser-9, leading to the reduction of tau phosphorylation. Using tau transgenic mice, the present study examined whether chronic cerebral hypoperfusion affects leptin-R signaling and tau phosphorylation. Eight-month-old tau transgenic mice (T44) overexpressing the shortest human tau isoform were subjected to chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) using microcoils or sham surgery. Their brains were analyzed four weeks later to evaluate the expression of phosphorylated tau and leptin-R via immunohistochemistry and Western blot analysis. In addition, expression of leptin-R was examined in the rat primary astrocyte cultures subjected to prolonged chemical hypoxic stress, as well as in autopsied brains. BCAS upregulated leptin-R expression and promoted the expression of phosphorylated tau in T44 Tg mice. In primary astrocyte cultures, leptin-R was upregulated under hypoxic conditions via the phosphorylated AKT/pAKT pathway, possibly suppressing the expression of caspase 3. Leptin-R was also strongly expressed in autopsied brains with AD and cerebrovascular diseases. These results collectively indicate that chronic cerebral hypoperfusion promotes leptin-R signaling and tau phosphorylation.

Phosphorylated NF-κB subunit p65 aggregates in granulovacuolar degeneration and neurites in neurodegenerative diseases with tauopathy.

Granulovacuolar degeneration (GVD) was originally reported in Alzheimer's disease (AD) and later found in aging brains. Pathologically, GVD is thought to be associated with the development of tauopathy, but the precise mechanism remains unknown. Previous studies have suggested that GVD contains proteins associated with an inflammatory signal. In this study, we examined phosphorylated p65 (pp65), which is the activated form of a subunit of nuclear factor-kappa B (NF-κB), in the hippocampus of 21 autopsied cases, including AD, amyotrophic lateral sclerosis cases with optineurin mutation (ALS-OPTN), and a variety of other neurodegenerative disorder cases and normal controls. In all cases, GVDs were immunopositive for pp65. The density of pp65-positive GVDs statistically correlated with that of casein kinase 1 delta (CK1δ), which is known as GVD marker. pp65 was also detected in neurites in AD and ALS-OPTN. The number of neurons with pp65-immunoreactive GVD was significantly higher in the AD group than in the non-AD group. Double immunostaining showed the colocalization of CK1δ and pp65. pp65-positive GVD was found in a neuron with AT8-positive neurofibrillary tangles. Moreover, pp65 was also found in neurites that were immunostained with phosphorylated tau, phosphorylated α-synuclein, or TDP-43 (transactivation response element DNA-binding protein 43 kDa). Therefore, the activation of the NF-κB pathway may be related to the pathology of GVD formation and dementia with tauopathy, including AD and ALS-OPTN. We propose that pp65 is useful as a GVD marker, and that the NF-κB pathway could be a therapeutic target not only for AD, but for age-related neurodegenerative diseases in general.

Recurrent Epstein-Barr Virus-positive (EBV+) Primary Central Nervous System Lymphoma (PCNSL) in a Patient with Clinical Features of Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS).

Primary central nervous system lymphoma (PCNSL) and chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) can share clinical features and may be indistinguishable, even after brain biopsy. We encountered a case of Epstein-Barr virus-positive (EBV+) PCNSL recurrence in a patient with clinical features of CLIPPERS, and repeat brain biopsy was required to reach the correct diagnosis. Four years after the initial diagnosis and treatment of PCNSL, "peppering" punctate enhanced lesions with transient steroid responsiveness were detected during brain magnetic resonance imaging (MRI). A second brain biopsy supported a diagnosis of CLIPPERS, while a third biopsy confirmed the diagnosis of recurrent PCNSL.

NLRP3 Inflammasome-Related Proteins Are Upregulated in the Putamen of Patients With Multiple System Atrophy.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia, and autonomic dysfunction. Microglial infiltration is an important mediator in MSA. The nucleotide-binding domain, leucine-rich repeats-containing family, pyrin domain-containing-3 (NLRP3) inflammasome complex, comprising NLRP3, apoptotic speck protein containing a caspase recruitment domain (ASC), and cysteine aspartic acid protease 1 (Caspase 1), regulates microglial inflammation in several neurodegenerative diseases. However, its role in MSA remains unknown. This study aimed to investigate the role of the NLRP3 inflammasome in MSA. Immunohistochemical staining of postmortem brains from 11 cases of MSA, 5 of Parkinson disease, and 6 age-matched controls were assessed. The relationships among α-synuclein deposition, microglial infiltration, and NLRP3 inflammasome-related proteins (NLRP3, ASC, and Caspase 1) were quantitatively analyzed. Double-labeling immunofluorescence staining confirmed colocalization of NLRP3 inflammasome-related proteins and Cluster of Differentiation 68. We demonstrated that the density of microglia expressing NLRP3 inflammasome-related proteins was increased in the putamina of MSA cases and was significantly related to the deposition of phosphorylated α-synuclein-positive glial cytoplasmic inclusions, tyrosine hydroxylase-positive fiber loss, and gliosis of glial fibrillary acidic protein-positive astrocytes. Our study suggests that the NLRP3 inflammasome is significantly upregulated and correlates with the neurodegenerative process in MSA.

Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.

Amyotrophic Lateral Sclerosis after Receiving the Human Papilloma Virus Vaccine: A Case Report of a 15-year-old Girl.

We herein report a 15-year-old girl who developed rapid progressive muscle weakness soon after the third injection of a bivalent human papilloma virus (HPV) vaccine. Although immunotherapies were performed for possible vaccine-related disorders, she died of respiratory failure 14 months after the onset of the disease. A genetic analysis identified a heterozygous p.P525L mutation of the fused in sarcoma (FUS) gene, and a histopathological analysis was also consistent with FUS-associated amyotrophic lateral sclerosis (ALS) without any evidence of neuroinflammation. We concluded the diagnosis to be FUS-ALS, although we cannot completely rule out the possibility that the vaccination worked as a trigger.

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72 Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis.

Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-κB suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-κB suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-α-mediated NF-κB activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-α-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-κB are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-κB activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS.

CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS.

The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.