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.

Roles of microglia/macrophage and antibody in cell sheet transplantation in the central nervous system.

BACKGROUND: We previously established a human mesenchymal stem cell (MSC) line that was modified to express trophic factors. Transplanting a cell sheet produced from this line in an amyotrophic lateral sclerosis mouse model showed a beneficial trend for mouse life spans. However, the sheet survived for less than 14 days, and numerous microglia and macrophages were observed within and adjacent to the sheet. Here, we examined the roles of microglia and macrophages as well as acquired antibodies in cell sheet transplantation. METHODS: We observed the effects of several MSC lines on macrophages in vitro, that is, phenotype polarization (M1 or M2) and migration. We then investigated how phenotypic polarization affected MSC survival using antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). We also confirmed the role of complement on cytotoxicity. Lastly, we selectively eliminated microglia and macrophages in vivo to determine whether these cells were cytoprotective to the donor sheet. RESULTS: In vitro co-culture with MSCs induced M2 polarization in macrophages and facilitated their migration toward MSCs in vitro. There was no difference between M1 and M2 phenotypes on ADCC and ADCP. Cytotoxicity was observed even in the absence of complement. Eliminating microglia/macrophage populations in vivo resulted in increased survival of donor cells after transplantation. CONCLUSIONS: Acquired antibodies played a role in ADCC and ADCP. MSCs induced M2 polarization in macrophages and facilitated their migration toward MSCs in vitro. Despite these favorable characteristics of microglia and macrophages, deletion of these cells was advantageous for the survival of donor cells in vivo.

CGG expansion in NOTCH2NLC is associated with oculopharyngodistal myopathy with neurological manifestations.

Oculopharyngodistal myopathy (OPDM) is a rare hereditary muscle disease characterized by progressive distal limb weakness, ptosis, ophthalmoplegia, bulbar muscle weakness and rimmed vacuoles on muscle biopsy. Recently, CGG repeat expansions in the noncoding regions of two genes, LRP12 and GIPC1, have been reported to be causative for OPDM. Furthermore, neuronal intranuclear inclusion disease (NIID) has been recently reported to be caused by CGG repeat expansions in NOTCH2NLC. We aimed to identify and to clinicopathologically characterize patients with OPDM who have CGG repeat expansions in NOTCH2NLC (OPDM_NOTCH2NLC). Note that 211 patients from 201 families, who were clinically or clinicopathologically diagnosed with OPDM or oculopharyngeal muscular dystrophy, were screened for CGG expansions in NOTCH2NLC by repeat primed-PCR. Clinical information and muscle pathology slides of identified patients with OPDM_NOTCH2NLC were re-reviewed. Intra-myonuclear inclusions were evaluated using immunohistochemistry and electron microscopy (EM). Seven Japanese OPDM patients had CGG repeat expansions in NOTCH2NLC. All seven patients clinically demonstrated ptosis, ophthalmoplegia, dysarthria and muscle weakness; they myopathologically had intra-myonuclear inclusions stained with anti-poly-ubiquitinated proteins, anti-SUMO1 and anti-p62 antibodies, which were diagnostic of NIID (typically on skin biopsy), in addition to rimmed vacuoles. The sample for EM was available only from one patient, which demonstrated intranuclear inclusions of 12.6 ± 1.6 nm in diameter. We identified seven patients with OPDM_NOTCH2NLC. Our patients had various additional central and/or peripheral nervous system involvement, although all were clinicopathologically compatible; thus, they were diagnosed as having OPDM and expanding a phenotype of the neuromyodegenerative disease caused by CGG repeat expansions in NOTCH2NLC.

[A case of sporadic late-onset nemaline myopathy associated with myasthenia gravis positive for anti-titin antibody and anti-Kv1.4 antibody].

A 66-year-old woman who had myasthenia gravis (MG) admitted for type II respiratory failure and right heart failure. Although she had neither ptosis, eye movement disorder, nor diplopia, she had orbital muscles weakness, reduction of gag reflex, dysarthria, dysphagia, and mild proximal muscle weakness. Blood tests showed anti-striated muscle antibodies (anti-titin antibody and anti-Kv1.4 antibody). A muscle biopsy of the left biceps showed a marked variation in fiber size, mild mononuclear cell infiltration was seen surrounding blood vessels in perimysium and nemaline bodies in some fibers. Immunohistochemical stains showed many muscle fibers express HLA-ABC. The patient was diagnosed as sporadic late-onset nemaline myopathy (SLONM) with MG, and treated by tacrolimus. After treatment, her respiratory function gradually improved and she discharged. In the case of atypical MG, measurement of anti-striated muscle antibody or muscle biopsy should be considered.

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.

Dynamics of host and graft after cell sheet transplantation: Basic study for the application of amyotrophic lateral sclerosis.

Stem cells offer great hope for the therapy of neurological disorders. Using a human artificial chromosome (HAC), we generated modified mesenchymal stem cells (MSCs), termed HAC-MSC that express 3 growth factors and 2 marker proteins including luciferase, and previously demonstrated that intrathecal administration of HAC-MSCs extended the lifespan in a mouse model of amyotrophic lateral sclerosis (ALS). However, donor cells disappeared rapidly after transplantation. To overcome this poor survival, we transplanted the HAC-MSCs as a sheet structure which retained the extracellular matrix. We investigated, here, whether cell sheet showed a longer survival than intrathecal administration. Also, the therapeutic effects on ALS model mice were examined. In vivo imaging showed that luciferase signals increased immediately after transplantation up to 7 days, and these signals were sustained for up to 14 days. In contrast, following intrathecal administration, signals were drastically decreased by day 3. Moreover, cell sheet transplantation successfully prolonged the survival of donor HAC-MSCs. Cell sheet transplantation increased the level of p-Akt at the graft area. Pathologically, none of the donor cells differentiated into neurons, astrocytes or microglial cells. When the cell sheet was transplanted into ALS model mice, there was an encouraging trend in the delayed onset of symptoms and increased lifespan. If each group was subdivided into rapid and slow progressors based on cut-off values for respective median survival, the survival of rapid progressors differed significantly between groups (treated vs. sham-operated = 145.4 ±â€¯1.4 vs. 139.2 ±â€¯1.2). The effect of HAC-MSC sheet transplantation still has a temporally narrow therapeutic window. Further improvement could be achieved by optimization of the transplantation conditions, e.g. co-transplantation of HAC-MSCs with endothelial progenitor cells.

Effect of caffeine on long-term potentiation-like effects induced by quadripulse transcranial magnetic stimulation.

Caffeine, an adenosine receptor antagonist, is known to affect sleep-awake cycles, the stress response, and learning and memory. It has been suggested that caffeine influences synaptic plasticity, but the effects of caffeine on synaptic plasticity in the human brain remain unexplored. The present study aimed to investigate the effects of caffeine on long-term potentiation (LTP)-like effects in the primary motor cortex of healthy humans. Twelve healthy participants (six women and six men; mean age: 44.8 ± 1.5 years) underwent quadripulse magnetic stimulation with an inter-stimulus interval of 5 ms (QPS5) to induce LTP-like effects, 2 h after administration of either a caffeine (200 mg) or placebo tablet in a double-blind crossover design. We recorded motor-evoked potentials (MEPs) before and after QPS5. The degree of MEP enhancement was compared between the placebo and caffeine conditions. Neither active nor resting motor thresholds were influenced by caffeine administration. Following caffeine administration, the degree of potentiation significantly decreased in "significant responders", whose average MEP ratios were greater than 1.24 in the placebo condition. The observed reduction in potentiation following caffeine administration is consistent with the A2A receptor antagonistic effect of caffeine. This is the first report of an effect of caffeine on neural synaptic plasticity in the human brain, which is consistent with the caffeine-induced plasticity reduction observed in primate studies. Because we studied only a small number of subjects, we cannot firmly conclude that caffeine reduces LTP in humans. The present results will, however, be helpful when considering further or new clinical uses of caffeine.

Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.

BACKGROUND: Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. RESULTS: We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. CONCLUSIONS: These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.

Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.

Epilepsy is a common neurological disorder, and mutations in genes encoding ion channels or neurotransmitter receptors are frequent causes of monogenic forms of epilepsy. Here we show that abnormal expansions of TTTCA and TTTTA repeats in intron 4 of SAMD12 cause benign adult familial myoclonic epilepsy (BAFME). Single-molecule, real-time sequencing of BAC clones and nanopore sequencing of genomic DNA identified two repeat configurations in SAMD12. Intriguingly, in two families with a clinical diagnosis of BAFME in which no repeat expansions in SAMD12 were observed, we identified similar expansions of TTTCA and TTTTA repeats in introns of TNRC6A and RAPGEF2, indicating that expansions of the same repeat motifs are involved in the pathogenesis of BAFME regardless of the genes in which the expanded repeats are located. This discovery that expansions of noncoding repeats lead to neuronal dysfunction responsible for myoclonic tremor and epilepsy extends the understanding of diseases with such repeat expansion.

Focal lesion in upper part of brachial plexus can be detected by magnetic cervical motor root stimulation.

BACKGROUND: The utilities of magnetic cervical motor root stimulation are well known for lesions in the lower part of the brachial plexus, but not for lesions in the other parts. OBJECTIVE: The aim of paper is to show the utilities of magnetic cervical motor root stimulation for lesions in the upper part of the brachial plexus. METHODS: We analyzed the brachial plexus using both electrical stimulation at Erb's point and magnetic cervical motor root stimulation in a patient with brachial plexopathy caused by tumor invasion. RESULTS: On the fourth day after onset, magnetic cervical motor root stimulation revealed abnormal findings in the upper part of the brachial plexus. Two weeks after onset, needle electromyography supported the existence of the focal lesion. CONCLUSION: Magnetic cervical motor root stimulation is useful in detecting abnormal findings in the upper part of the brachial plexus, even at the acute phase.

A case of atypical amyloid polyneuropathy with predominant upper-limb involvement with the diagnosis unexpectedly found at lung operation.

We present a patient of familial amyloid polyneuropathy (FAP) with predominant upper-limb involvement, the pattern of which resembled a mononeuropathy multiplex pattern. Sural nerve biopsy failed to diagnose the disorder, but lung partial resection performed later for other diagnostic purposes suggested FAP. A rare mutation in the transthyretin gene (S50R) was subsequently confirmed. Diagnostic challenges of FAP with atypical clinical presentations, including difficulties in pathological diagnosis, are discussed with a review of the literature.

Magnetic stimulation of the cauda equina in the spinal canal with a flat, large round coil.

Magnetic round coil stimulation over the spinal enlargement activates the spinal nerves at the neuro-foramina level. However, activation of the cauda equina in the spinal canal has never been described in the literature. This study, for which 40 healthy subjects were recruited, activated the cauda equina using a round 20-cm-diameter coil designated as a Magnetic Augmented Translumbosacral Stimulation (MATS) coil. Magnetic stimulation placing the edge of the coil over the L1 and L3 spinous processes elicited compound muscle action potentials (CMAPs) from the abductor hallucis muscle. The CMAPs were compared with those elicited through high-voltage electrical stimulation. The CMAP latencies to L1 level MATS coil stimulation were not significantly different from those evoked by electrical stimulation at the same level. The CMAP latencies to L3 level MATS coil stimulation were varied in each subject. In fact, the L1 level MATS coil stimulation is considered to activate the cauda equina at the root exit site from the conus medullaris; the L3 level MATS coil stimulation activates some mid-part of the cauda equina or the distal cauda equina by spreading current. The MATS coil facilitates evaluation of spinal nerve conduction in the cauda equina.

Very fast oscillations evoked by median nerve stimulation in the human thalamus and subthalamic nucleus.

Very fast oscillations (VFOs; 500-1,500 Hz) are associated with sensory-evoked potentials (SEPs), but their origin is unknown. To characterize the origins of VFOs, we studied 35 patients with deep brain stimulation (DBS) electrodes [15 with thalamic and 20 with the subthalamic nucleus (STN) electrodes]. We recorded median nerve stimulation-evoked SEPs from the thalamus and STN with microelectrodes during stereotactic surgery and from the contacts of the DBS electrodes postoperatively. We also examined the firing of individual neurons in thalamus in relation to the VFOs. In the thalamus, VFOs with frequencies around 1,000 Hz were superimposed on slow potentials. Both slow and fast SEP components showed phase reversals in the somatosensory thalamus [ventralis caudalis (Vc)]. Median nerve poststimulus time histograms showed that single thalamic neurons fired at preferred times at intervals between 0.8 to 1.2 ms that were synchronous with the VFOs, although the neurons fired only once or a few times per trial. In the STN, low-amplitude SEPs with VFOs were observed at a latency similar to the thalamic SEPs. The VFOs from STN probably represent volume conduction, possibly from the medial lemniscus. We conclude that the thalamic VFOs are generated within Vc and that they induce time-locked firing in a network of neurons.

Unique form of propriospinal myoclonus as a possible complication of an enteropathogenic toxin.

Propriospinal myoclonus is an uncommon form of spinal myoclonus propagated, presumably, by slowly conducting polysynaptic intraspinal pathways. Although most patients demonstrate no clear etiology, a variety of disorders have been linked to this abnormal movement, including trauma, multiple sclerosis, tumors, and infectious disorders such as herpes zoster, human immunodeficiency virus, and Lyme disease. We describe 2 young male patients from the same town in Northern Ontario, Canada, exposed to an outbreak of Escherichia coli O157:H7 from contaminated municipal water, who developed identical clinical and electrophysiological features suggestive of a rhythmic form of propriospinal myoclonus with activity alternating between abdominal and paraspinal muscles. A toxin-mediated microvascular thrombosis is proposed as a possible pathogenic mechanism underlying this novel association.

Functional connectivity revealed by single-photon emission computed tomography (SPECT) during repetitive transcranial magnetic stimulation (rTMS) of the motor cortex.

OBJECTIVE: In the present study, we studied effects of 1 Hz repetitive transcranial magnetic stimulation (rTMS) over the left primary motor cortex (M1) on regional cerebral blood flow (rCBF) using single-photon emission computed tomography (SPECT). METHODS: SPECT measurements were carried out under two experimental conditions: real and sham stimulation. In sham stimulation, to exclude other components besides currents in the brain in rTMS, we applied sound and electrical stimulation to the skin of the head. 99mTc-ethyl cysteinate dimer was injected during the real or sham stimulation. Images were analyzed with the statistical parametric mapping software (SPM99). Relative differences in adjusted rCBF between two conditions were determined by a voxel-by-voxel paired t test. RESULTS: 1 Hz rTMS at an intensity of 1.1 x active motor threshold evoked increase of rCBF in the contralateral (right) cerebellar hemisphere. Reduction of rCBF was observed in the contralateral M1, superior parietal lobule (most probably corresponding to PE area in the monkey) (Rizzolatti G, Luppino G, Matelli M. Electroenceph clin Neurophysiol 1998;106:283-296), inferior parietal lobule (PF area in the monkey (Rizzolatti et al., 1998)), dorsal and ventral premotor areas (dPM, vPM) and supplementary motor area (SMA). CONCLUSIONS: Increase of rCBF in the contralateral cerebellum must reflect facilitatory connection between the motor cortex and contralateral cerebellum. Reduced rCBF in the contralateral M1 may be produced by transcallosal inhibitory effect of the left motor cortical activation. CBF decrease in the right PM, SMA and parietal cortex may reflect some secondary effects. Low frequency rTMS at an intensity of around threshold for active muscles can evoke rCBF changes. SIGNIFICANCE: We demonstrated that rCBF changes could be elicited even by low frequency rTMS at such a low intensity as the threshold for an active muscle. Combination of rTMS and SPECT is one of powerful tools to study interareal connection within the human brain.