The Cyanotoxin 2,4-DAB Reduces Viability and Causes Behavioral and Molecular Dysfunctions Associated with Neurodegeneration in Larval Zebrafish

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s disease. While the cyanotoxin β-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA, including 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether 2,4-DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB; however, only modulated startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB’s effects, we analyzed the protein profile of larval zebrafish exposed to 500 µM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

Clinicopathological Characteristics and Prognostic Factors in Axial Chondroblastomas: A Retrospective Analysis of 61 Cases and Comparison with Extra-Axial Chondroblastomas

Background The clinical characteristics and prognostic factors of axial chondroblastoma (ACB) are still poorly understood. Purpose To characterize clinicopathological characteristics in a large ACB cohort and investigate their correlation with survival. We also sought to compare these results with extra-axial CB (EACB). Methods Our institution's local database was retrospectively reviewed and included a total of 132 CB patients, including 61 ACB patients and 71 EACB patients. Immunohistochemistry was used to assess the expression levels of Vimentin (Vim), S100, and cytokeratin (CK) on tumor cells in 132 tissue specimens. Results Overall, ACB and EACB had similar characteristics, except for older age and tumor size, as well as higher Vim expression, incidence of surrounding tissue invasion and postoperative sensory or motor dysfunction. Whereas wide resection and absence of invasion of surrounding tissues were consistently associated with favorable survival in the ACB and EACB cohorts in univariate analysis, most parameters showed differential prognostic significance between the 2 groups. Significant prognostic factors for local recurrence-free survival in multivariate analysis included the type of resection and chicken-wire calcification in the ACB cohort. Multivariate analysis of overall survival demonstrated that the type of resection was a significant predictor in the ACB cohort, whereas the type of resection and postoperative sensory or motor dysfunction were predictive of overall survival in the EACB group. Conclusion These data suggest that there may be distinct biological behaviors between ACB and EACB and may provide useful information to better understand the prognostic characteristics of patients with ACB and to improve outcome prediction in patients with ACB.

Schwannoma Gene Therapy via Adeno-Associated Viral Vector Delivery of Apoptosis-Associated Speck-like Protein Containing CARD (ASC): Preclinical Efficacy and Safety

Schwannomas are tumors derived from Schwann-lineage cells, cells that protect and support myelinated nerves in the peripheral nervous system. They are typically slow-growing, encapsulated and benign. These tumors develop along peripheral, spinal and cranial nerves causing pain, sensory-motor dysfunction and death. Primary treatment for schwannoma is operative resection which can be associated with significant morbidity. Pharmacotherapy is largely restricted to bevacizumab, which has minimal or no efficacy for many patients and can be associated with treatment-limiting adverse effects. Given the suffering and morbidity associated with schwannoma and the paucity of therapeutic options, there is an urgent need for safe and effective therapies for schwannomas. We previously demonstrated that adeno-associated virus serotype 1 (AAV1) vector mediated delivery of the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) under the control of the P0 promoter, produced a prolonged reduction in tumor volume and tumor-associated pain in human xenograft and mouse syngeneic schwannoma models. Here, we present data essential for the translation of our AAV1-P0-ASC schwannoma gene therapy to clinical trials. We determine the minimum effective dose of AAV1-P0-hASC required to induce an anti-tumor effect in the xenograft human-schwannoma model. We also show that the presence of preexisting AAV1 immunity does not alter the antitumor efficacy of AAV-P0-mASC in a syngeneic mouse schwannoma model. Furthermore, the maximum deliverable intratumoral dose of AAV1-P0-ASC was not associated with neuronal toxicity in immunocompetent mice. Taken together, these safety and efficacy data support the translation of the AAV1-P0-ASC schwannoma gene therapy strategy to clinical trials.

Correlation Between Proprioceptive Impairment and Motor Deficits After Stroke: A Meta-Analysis Review

Introduction: Proprioceptive impairment is a common symptom after stroke. Clarifying how proprioception correlates with motor function after stroke may be helpful in optimizing proprioception-augmented movement training. Previous studies have shown inconsistent findings. A meta-analysis is an optimal method to explore the correlation and identify the factors contributing to these inconsistencies.Objective: To explore the correlation between proprioception and motor function after stroke through a meta-analysis, taking into account characteristics of the measurements used in these studies.Methods: We searched multiple databases until November 2021 for eligible studies that measured both proprioception and motor functions in persons with stroke and reported their correlation or data for correlation analysis. A meta-analysis of the correlations was performed. The subgroup analysis and meta-regression were further conducted to investigate potential factors contributing to the heterogeneity of correlation strength, based on the participants' characteristics, proprioception, and motor function measures.Results: In total, 28 studies comprising of 1,829 participants with stroke were included in the meta-analysis. The overall correlation between proprioception and motor function was significant (r = 0.267, p < 0.05), but there was heterogeneity across studies (I2 = 45%, p < 0.05). The results of the subgroup analysis showed proprioception of the axial segment in weight-bearing conditions (r = 0.443, p < 0.05) and upper limb without weight-bearing (r = 0.292, p < 0.05) had a stronger correlation with motor function than proprioception of the lower limb without weight-bearing. The proprioception measured through ipsilateral matching (r = 0.412, p < 0.05) showed a stronger correlation with motor function than through contralateral matching. The International Classification of Functioning, Disability, and Health (ICF) domains of motor function, movement function (r = 0.338, p < 0.05), activity performance (r = 0.239, p < 0.05), and independence (r = 0.319, p < 0.05) showed a stronger correlation with proprioception than with other domains.Conclusion: There is a significant correlation between proprioception and motor dysfunction after stroke. The proprioception measured in the axial segment under weight-bearing conditions or measured with ipsilateral matching, and motor function, specifically in the ICF domains of movement function, activity performance, and independence showed a positive contribution to the association between proprioception and motor function. The correlation does not imply causation and might be underestimated by attributes of current tests for proprioception and motor function. Further studies are needed to clarify the cause-effect relationship.

Testing of the therapeutic efficacy and safety of AMPA receptor RNA aptamers in an ALS mouse model

In motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients, the RNA editing at the glutamine/arginine site of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors is defective or incomplete. As a result, AMPA receptors containing the abnormally expressed, unedited isoform of GluA2 are highly Ca2+-permeable, and are responsible for mediating abnormal Ca2+ influx, thereby triggering motor neuron degeneration and cell death. Thus, blocking the AMPA receptor–mediated, abnormal Ca2+ influx is a potential therapeutic strategy for treatment of sporadic ALS. Here, we report a study of the efficacy and safety of two RNA aptamers targeting AMPA receptors on the ALS phenotype of AR2 mice. A 12-wk continuous, intracerebroventricular infusion of aptamers to AR2 mice reduced the progression of motor dysfunction, normalized TDP-43 mislocalization, and prevented death of motor neurons. Our results demonstrate that the use of AMPA receptor aptamers as a novel class of AMPA receptor antagonists is a promising strategy for developing an ALS treatment approach.

Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed. Methods Wild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression. Results We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection. Conclusions Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

Development of novel clinical examination scales for the measurement of disease severity in Creutzfeldt-Jakob disease

ObjectiveTo use a robust statistical methodology to develop and validate clinical rating scales quantifying longitudinal motor and cognitive dysfunction in sporadic Creutzfeldt-Jakob disease (sCJD) at the bedside.MethodsRasch analysis was used to iteratively construct interval scales measuring composite cognitive and motor dysfunction from pooled bedside neurocognitive examinations collected as part of the prospective National Prion Monitoring Cohort study, October 2008–December 2016.A longitudinal clinical examination dataset constructed from 528 patients with sCJD, comprising 1030 Motor Scale and 757 Cognitive Scale scores over 130 patient-years of study, was used to demonstrate scale utility.ResultsThe Rasch-derived Motor Scale consists of 8 items, including assessments reliant on pyramidal, extrapyramidal and cerebellar systems. The Cognitive Scale comprises 6 items, and includes measures of executive function, language, visual perception and memory. Both scales are unidimensional, perform independently of age or gender and have excellent inter-rater reliability. They can be completed in minutes at the bedside, as part of a normal neurocognitive examination. A composite Examination Scale can be derived by averaging both scores. Several scale uses, in measuring longitudinal change, prognosis and phenotypic heterogeneity are illustrated.ConclusionsThese two novel sCJD Motor and Cognitive Scales and the composite Examination Scale should prove useful to objectively measure phenotypic and clinical change in future clinical trials and for patient stratification. This statistical approach can help to overcome obstacles to assessing clinical change in rapidly progressive, multisystem conditions with limited longitudinal follow-up.

Excessive Daytime Sleepiness Is Associated With Non-motor Symptoms of Multiple System Atrophy: A Cross-Sectional Study in China

Objectives: Excessive daytime sleepiness (EDS) in multiple system atrophy (MSA) has received scant attention in the literature, thus the present cross-sectional study aimed to investigate the prevalence of EDS and its potential risk factors among Chinese patients with MSA.Methods: A total of 66 patients with MSA (60.6% males) were consecutively recruited. Eighteen patients (27.3%, 13 men) with Epworth Sleepiness Scale score >10 were defined as having EDS. Demographic, motor [Unified Multiple-System Atrophy (UMSARS)] and non-motor symptoms [Non-Motor Symptoms Scale (NMSS)], and sleep parameters [polysomnography (PSG)] were compared between patients with MSA with and without EDS. A logistic regression analysis was used to calculate the risk factors of EDS in patients with MSA.Results: There were no significant differences in age, sex, MSA onset age, disease duration, MSA sub-type, and motor symptom severity between MSA patients with and without EDS. However, compared with the MSA patients without EDS, their counterparts with EDS had higher scores of NMSS (65.3 ± 23.1 vs. 43.4 ± 25.3, P = .0002), Hamilton Anxiety (HAMA) [15.3 (10.3–20.0) vs. 9.5 (3.0–15.0), P = 0.006], Hamilton Depression (HAMD) [13.7 (12.5–17.8) vs. 9.0 (4.0–13.0), P = 0.015], and Fatigue Severity Scale (FSS) [29.8 (17.3–47.8) vs. 18.7 (10.3–21.8), P = 0.040]. Conversely, the patients with EDS had lower score of Mini-Mental State Examination (MMSE) [23.3 (20.3–27.0) vs. 25.7 (22.0–29.0), P = 0.023]. Similarly, there was a significantly lower percentage of N3 sleep (%) [0.3 (0–0) vs. 2.0 (0–0), P = 0.007] and a higher apnea-hypopnea index (AHI/h) [30.5 (14.5–47.8) vs. 19.3 (5.0–28.7), P = 0.034] in patients with EDS. After adjusting for age, sex, disease duration, MSA sub-type, and UMSARS score, the odds ratio (OR) (95% CI) of EDS was higher while increasing scores in FSS [1.06 (1.02–1.11)], HAMA [1.16 (1.04–1.28)], HAMD [1.13 (1.02–1.25)], NMSS [1.04 (1.01–1.07)], and AHI [1.03 (1.00–1.10)]. The OR of EDS was lower while the MMSE score was increasing [0.85 (0.72–1.00)].Conclusions: The presence and severity of EDS may be significantly associated with the non-motor dysfunction, including fatigue, anxiety, depression, cognitive dysfunction, and sleep-related breathing disorder, but not with the motor dysfunction in MSA.

Endoplasmic Reticulum Stress-Associated Neuronal Death and Innate Immune Response in Neurological Diseases

Neuronal death and inflammatory response are two common pathological hallmarks of acute central nervous system injury and chronic degenerative disorders, both of which are closely related to cognitive and motor dysfunction associated with various neurological diseases. Neurological diseases are highly heterogeneous; however, they share a common pathogenesis, that is, the aberrant accumulation of misfolded/unfolded proteins within the endoplasmic reticulum (ER). Fortunately, the cell has intrinsic quality control mechanisms to maintain the proteostasis network, such as chaperone-mediated folding and ER-associated degradation. However, when these control mechanisms fail, misfolded/unfolded proteins accumulate in the ER lumen and contribute to ER stress. ER stress has been implicated in nearly all neurological diseases. ER stress initiates the unfolded protein response to restore proteostasis, and if the damage is irreversible, it elicits intracellular cascades of death and inflammation. With the growing appreciation of a functional association between ER stress and neurological diseases and with the improved understanding of the multiple underlying molecular mechanisms, pharmacological and genetic targeting of ER stress are beginning to emerge as therapeutic approaches for neurological diseases.

Saikokeishikankyoto extract alleviates muscle atrophy in KKAy mice

Sarcopenic obesity is associated with increased visceral fat and decreased muscle mass, resulting in decreased insulin sensitivity, increased production of inflammatory cytokines, and oxidative stress. In this study, we first evaluated the effects of herbal medicines on the transcriptional activity of the Sirtuin 1 (sirt1) promoter in vitro as an indicator of their therapeutic effect. Our data suggested that hot water Saikokeishikankyoto (SKK) extracts increased sirt1 transcriptional activity in vitro, identifying it as a candidate therapeutic for evaluation in the KKAy type 2 diabetic obesity mouse model. These in vivo evaluations revealed that SKK treatment increased the wet weight and muscle fiber content in cross sections of the gastrocnemius muscle (GA) and restored motor function in these animals. In addition, SKK treatment reduced tumor necrosis factor-α (TNFα) expression in the sera and suppressed Atrogin1 and MuRF1 transcription in the GA samples. This treatment also increased sirt1 expression in these tissues. These results suggest that SKK inhibits skeletal muscle atrophy and improves motor function in KKAy mice by suppressing inflammation. In actual clinical practice, SKK is expected to inhibit muscle atrophy and improve motor dysfunction in sarcopenic obesity. Graphical abstract