Reduction of APOE accounts for neurobehavioral deficits in fetal alcohol spectrum disorders.

A hallmark of fetal alcohol spectrum disorders (FASD) is neurobehavioral deficits that still do not have effective treatment. Here, we present that reduction of Apolipoprotein E (APOE) is critically involved in neurobehavioral deficits in FASD. We show that prenatal alcohol exposure (PAE) changes chromatin accessibility of Apoe locus, and causes reduction of APOE levels in both the brain and peripheral blood in postnatal mice. Of note, postnatal administration of an APOE receptor agonist (APOE-RA) mitigates motor learning deficits and anxiety in those mice. Several molecular and electrophysiological properties essential for learning, which are altered by PAE, are restored by APOE-RA. Our human genome-wide association study further reveals that the interaction of PAE and a single nucleotide polymorphism in the APOE enhancer which chromatin is closed by PAE in mice is associated with lower scores in the delayed matching-to-sample task in children. APOE in the plasma is also reduced in PAE children, and the reduced level is associated with their lower cognitive performance. These findings suggest that controlling the APOE level can serve as an effective treatment for neurobehavioral deficits in FASD.

Alternative splicing events as peripheral biomarkers for motor learning deficit caused by adverse prenatal environments.

Severity of neurobehavioral deficits in children born from adverse pregnancies, such as maternal alcohol consumption and diabetes, does not always correlate with the adversity's duration and intensity. Therefore, biological signatures for accurate prediction of the severity of neurobehavioral deficits, and robust tools for reliable identification of such biomarkers, have an urgent clinical need. Here, we demonstrate that significant changes in the alternative splicing (AS) pattern of offspring lymphocyte RNA can function as accurate peripheral biomarkers for motor learning deficits in mouse models of prenatal alcohol exposure (PAE) and offspring of mother with diabetes (OMD). An aptly trained deep-learning model identified 29 AS events common to PAE and OMD as superior predictors of motor learning deficits than AS events specific to PAE or OMD. Shapley-value analysis, a game-theory algorithm, deciphered the trained deep-learning model's learnt associations between its input, AS events, and output, motor learning performance. Shapley values of the deep-learning model's input identified the relative contribution of the 29 common AS events to the motor learning deficit. Gene ontology and predictive structure-function analyses, using Alphafold2 algorithm, supported existing evidence on the critical roles of these molecules in early brain development and function. The direction of most AS events was opposite in PAE and OMD, potentially from differential expression of RNA binding proteins in PAE and OMD. Altogether, this study posits that AS of lymphocyte RNA is a rich resource, and deep-learning is an effective tool, for discovery of peripheral biomarkers of neurobehavioral deficits in children of diverse adverse pregnancies.

Differentiation state and culture conditions impact neural stem/progenitor cell-derived extracellular vesicle bioactivity.

Extracellular vesicles (EVs) derived from neural progenitor/stem cells (NPSCs) have shown promising efficacy in a variety of preclinical models. However, NPSCs lack critical neuroregenerative functionality such as myelinating capacity. Further, culture conditions used in NPSC EV production lack standardization, limiting reproducibility challenging and potentially potency of the overall approach via lack of optimization. Here, we assessed whether oligodendrocyte precursor cells (OPCs) and immature oligodendrocytes (iOLs), which are further differentiated than NPSCs and which both give rise to mature myelinating oligodendrocytes, could yield EVs with neurotherapeutic properties comparable or superior to those from NPSCs. We additionally examined the effects of extracellular matrix (ECM) coating materials and the presence or absence of growth factors in cell culture on the ultimate properties of EVs. The data show that OPC EVs and iOL EVs performed similarly to NPSC EVs in cell proliferation and anti-inflammatory assays, but NPSC EVs performed better in a neurite outgrowth assay. Additionally, the presence of nerve growth factor (NGF) in culture was found to maximize NPSC EV bioactivity among the conditions tested. NPSC EVs produced under rationally-selected culture conditions (fibronectin + NGF) enhanced axonal regeneration and muscle reinnervation in a rat nerve crush injury model. These results highlight the need for standardization of culture conditions for neurotherapeutic NPSC EV production.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.

Deletion of the Thrombin Proteolytic Site in Neurofascin 155 Causes Disruption of Nodal and Paranodal Organization.

In the central nervous system, myelin is attached to the axon in the paranodal region by a trimolecular complex of Neurofascin155 (NF155) in the myelin membrane, interacting with Caspr1 and Contactin1 on the axolemma. Alternative splicing of a single Neurofascin transcript generates several different Neurofascins expressed by several cell types, but NF155, which is expressed by oligodendrocytes, contains a domain in the third fibronectinIII-like region of the molecule that is unique. The immunoglobulin 5-6 domain of NF155 is essential for binding to Contactin1, but less is known about the functions of the NF155-unique third fibronectinIII-like domain. Mutations and autoantibodies to this region are associated with several neurodevelopmental and demyelinating nervous system disorders. Here we used Crispr-Cas9 gene editing to delete a 9 bp sequence of NF155 in this unique domain, which has recently been identified as a thrombin binding site and implicated in plasticity of the myelin sheath. This small deletion results in dysmyelination, eversion of paranodal loops of myelin, substantial enlargement of the nodal gap, a complete loss of paranodal septate junctions, and mislocalization of Caspr1 and nodal sodium channels. The animals exhibit tremor and ataxia, and biochemical and mass spectrometric analysis indicates that while NF155 is transcribed and spliced normally, the NF155 protein is subsequently degraded, resulting in loss of the full length 155 kDa native protein. These findings reveal that this 9 bp region of NF155 in its unique third fibronectinIII-like domain is essential for stability of the protein.

Extracellular Vesicles as an Emerging Frontier in Spinal Cord Injury Pathobiology and Therapy.

Extracellular vesicles (EVs) are membrane-delimited particles that are secreted by nearly all cell types. EVs mediate crucial physiological functions and pathophysiological processes in the CNS. As carriers of diverse bioactive cargoes (e.g., proteins, lipids, and nucleic acids) that can be modified in response to external stimuli, EVs have emerged as pathological mediators following neurotrauma such as spinal cord injury (SCI). We discuss the roles of endogenous EVs in the CNS as well as crosstalk with peripheral EVs in relation to neurotrauma, with a particular focus on SCI. We then summarize the status of EV-based therapeutic advances in preclinical animal models for these conditions. Finally, we discuss new bioengineering strategies that are poised to enhance CNS-specific therapeutic capabilities of EVs.

Initial testing of an electronic application of the simplified modified Rankin Scale questionnaire (e-smRSq).

INTRODUCTION AND PURPOSE: To facilitate modified Rankin scale (mRS) assessments, we developed and tested a smartphone/web application of the simplified mRS questionnaire (e-smRSq). The e-smRSq guides raters towards a final score according to the smRSq algorithm, and offers hints for scoring based on the conventional mRS concepts. METHODS: Initially, three experienced mRS certified raters prepared 30 vignettes of unstructured stroke patient interviews, and determined consensus reference scores. Using the e-smRSq, 16 raters of varied professional backgrounds without mRS training scored the mRS for 24 randomly selected vignettes. Subsequently, 5 certified and 5 uncertified raters using the e-smRSq scored 23 mRS certification vignettes developed and used in the Strategies to Innovate Emergency Care Clinical Trials Network-Neurological Emergencies Treatment Trials (SIREN-NETT). Cohen's and Fleiss's kappa (κ), weighted kappa (κw), and intra-class correlation (ICC) compared rater scores with reference scores and assessed interrater reliability. RESULTS: For the 16 initial raters using the e-smRSq with 24 vignettes, the κ (Fleiss) was 0.62 and ICC 0.87 (CI 0.80-0.93). Comparing raters' scores with reference scores, Cohen's κ was 0.68 and κw 0.90. For the 10 subsequent raters using the e-smRSq on SIREN-NETT vignettes, κ (Fleiss) was 0.8 and ICC 0.95 (CI 0.91-0.97). Comparing all 10 raters scores with SIREN-NETT reference scores, Cohen's κ was 0.88 and κw 0.97. There was no significant difference between certified and uncertified raters. CONCLUSIONS: The e-smRSq appears to have good reproducibility and validity metrics among both certified and non-certified mRS raters, possibly owing to its simplicity. Further testing in stroke patients in warranted.

Mechanical thrombectomy in stroke - planning for service expansion using discrete event simulation.

Mechanical thrombectomy (MT) is a very effective, but highly time dependent, reperfusion technique in the management of acute ischaemic stroke caused by large artery occlusion. MT is provided by 24 neuroscience centres (NSCs) in the UK which receive patients directly ('mothership') and via transfer from district general hospitals (DGHs), the 'drip and ship' pathway. NSCs currently provide a within hours service but are working on service expansion to enable 24/7 availability. DGHs, too, will need to prepare for this service expansion to ensure good outcomes for their patients. We discuss options for service expansion in a DGH and regional stroke network in south-west England and use Sentinel Stroke National Audit Programme data and discrete event simulation to model and compare alternative workflow options to aid the planning process. We suggest that our modelled options could be considered by all NHS DGHs in their preparation for MT service expansion.

Oligodendrocyte involvement in Gulf War Illness.

Low level sarin nerve gas and other anti-cholinesterase agents have been implicated in Gulf War illness (GWI), a chronic multi-symptom disorder characterized by cognitive, pain and fatigue symptoms that continues to afflict roughly 32% of veterans from the 1990-1991 Gulf War. How disrupting cholinergic synaptic transmission could produce chronic illness is unclear, but recent research indicates that acetylcholine also mediates communication between axons and oligodendrocytes. Here we investigated the hypothesis that oligodendrocyte development is disrupted by Gulf War agents, by experiments using the sarin-surrogate acetylcholinesterase inhibitor, diisopropyl fluorophosphate (DFP). The effects of corticosterone, which is used in some GWI animal models, were also investigated. The data show that DFP decreased both the number of mature and dividing oligodendrocytes in the rat prefrontal cortex (PFC), but differences were found between PFC and corpus callosum. The differences seen between the PFC and corpus callosum likely reflect the higher percentage of proliferating oligodendroglia in the adult PFC. In cell culture, DFP also decreased oligodendrocyte survival through a non-cholinergic mechanism. Corticosterone promoted maturation of oligodendrocytes, and when used in combination with DFP it had protective effects by increasing the pool of mature oligodendrocytes and decreasing proliferation. Cell culture studies indicate direct effects of both DFP and corticosterone on OPCs, and by comparison with in vivo results, we conclude that in addition to direct effects, systemic effects and interruption of neuron-glia interactions contribute to the detrimental effects of GW agents on oligodendrocytes. Our results demonstrate that oligodendrocytes are an important component of the pathophysiology of GWI.

Treadmilling Model for Plasticity of the Myelin Sheath.

We propose a mechanism for myelin plasticity that would complement synaptic plasticity by adjusting conduction velocity for optimal spike-time arrival. In the proposed treadmilling model, myelin sheath thickness is a dynamic balance between the rates of new myelin deposited adjacent to the axon and removal of the outermost layer.

Regulation of myelin structure and conduction velocity by perinodal astrocytes.

The speed of impulse transmission is critical for optimal neural circuit function, but it is unclear how the appropriate conduction velocity is established in individual axons. The velocity of impulse transmission is influenced by the thickness of the myelin sheath and the morphology of electrogenic nodes of Ranvier along axons. Here we show that myelin thickness and nodal gap length are reversibly altered by astrocytes, glial cells that contact nodes of Ranvier. Thrombin-dependent proteolysis of a cell adhesion molecule that attaches myelin to the axon (neurofascin 155) is inhibited by vesicular release of thrombin protease inhibitors from perinodal astrocytes. Transgenic mice expressing a dominant-negative fragment of VAMP2 in astrocytes, to reduce exocytosis by 50%, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, and thinning of the myelin sheath in the optic nerve. These morphological changes alter the passive cable properties of axons to reduce conduction velocity and spike-time arrival in the CNS in parallel with a decrease in visual acuity. All effects were reversed by the thrombin inhibitor Fondaparinux. Similar results were obtained by viral transfection of tetanus toxin into astrocytes of rat corpus callosum. Previously, it was unknown how the myelin sheath could be thinned and the functions of perinodal astrocytes were not well understood. These findings describe a form of nervous system plasticity in which myelin structure and conduction velocity are adjusted by astrocytes. The thrombin-dependent cleavage of neurofascin 155 may also have relevance to myelin disruption and repair.

Using population-based routinely collected data from the Sentinel Stroke National Audit Programme to investigate factors associated with discharge to care home after rehabilitation.

OBJECTIVES: We investigated factors associated with Care Home (CH) discharge following stroke using routinely collected data in unselected patients and assessed the relevance of previous research findings to such patients seen in routine clinical practice. DESIGN: Retrospective analysis of data from the Sentinel Stroke National Audit Programme using univariate analysis and logistic regression. SETTING: A large acute and rehabilitation UK stroke unit with access to early supported discharge. SUBJECTS: All patients with stroke treated from 1 January 2014 to 1 January 2017. MAIN MEASURES: National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). RESULTS: Of 2584 patients (median age 78 years, interquartile range (IQR) 69-86; 50.6% male; 86.7% infarcts; median admission NIHSS 4, IQR 2-9), 401 (15.5%) died in hospital and 203 patients (7.9%) were permanently discharged to CH for the first time. Most had pre-discharge mRS scores of 4/5. Factors (odds ratios; 95% confidence intervals) associated with CH discharge included age (1.07; 1.05-1.10), incontinence (11.5; 7.13-19.25), dysphagia (2.13; 1.39-3.29), severe weakness (1.93; 1.28-2.92), pneumonia (1.68; 1.13-2.50), urinary tract infection (UTI) (1.70; 1.04-2.75) and depression (1.65; 1.00-2.72). In a subgroup of all patients with a pre-discharge mRS of 4/5, age (1.04; 1.02-1.06), incontinence (4.87; 2.39-11.02), UTI (2.0; 1.09-3.71) and pneumonia (1.59; 1.02-2.50) were the only factors associated with CH discharge. CONCLUSION: Potentially modifiable variables like incontinence, UTI and pneumonia were associated with CH discharge, particularly in the severely disabled.

Cholinergic signaling in myelination.

There is a long history of research on acetylcholine (ACh) function in myelinating glia, but a resurgence of interest recently as a result of the therapeutic potential of manipulating ACh signaling to promote remyelination, and the broader interest in neurotransmitter signaling in activity-dependent myelination. Myelinating glia express all the major types of muscarinic and nicotinic ACh receptors at different stages of development, and acetylcholinesterase and butyrylcholinesterase are highly expressed in white matter. This review traces the history of research on ACh signaling in Schwann cells, oligodendrocytes, and in the myelin sheath, and summarizes current knowledge on the intracellular signaling and functional consequences of ACh signaling in myelinating glia. Implications of ACh in diseases, such as Alzheimer's disease, multiple sclerosis, and white matter toxicity caused by pesticides are considered, together with an outline of major questions for future research. GLIA 2017;65:687-698.

Validation and comparison of two stroke prognostic models for in hospital, 30-day and 90-day mortality.

INTRODUCTION: We aimed to validate and compare two clinical prognostic models for mortality which include the National Institutes of Health Stroke Scale (NIHSS); the Age and NIHSS Score (ANS) and case mix model (CMM) of the Sentinel Stroke National Audit Program (SSNAP). The NIHSS on admission was also tested as a prognostic score. PATIENTS AND METHODS: Prospectively collected data from the SSNAP register for a cohort of patients (ischaemic and haemorrhagic stroke) admitted over 1 year to Gloucestershire Royal Hospital, England were accessed. The ANS and CMM were calculated and tested for in hospital, 30-day and 90-day mortality using calibration plots with Hosmer-Lemeshow tests, receiver operating characteristics curves and other measures of prognostic accuracy. RESULTS: Of 848 patients, 110 (12.9%) died in hospital, 112 (13.2%) at 30 days and 164 (19.2%) at 90 days. Calibration for all three scores was good, although Hosmer-Lemeshow test p values were <0.05 with the NIHSS alone for in hospital and 30-day deaths, suggesting deviation from good fit. The c-statistics for in hospital, 30-day and 90-day mortality were ANS (0.783, 0.782, 0.779) and CMM (0.783, 0.774, 0.758), respectively. The NIHSS alone showed fair discrimination but performed less well. A NIHSS score ≥6 was associated with significant mortality (p < 0.0001) in comparison to a score <6. CONCLUSION: A simple prognostic model containing age and admission NIHSS only, performed as well as a more complex score at predicting in hospital, 30-day and 90-day mortality. Admission NIHSS recording should be encouraged for stroke registries.

Evaluation of a single centre stroke service reconfiguration - the impact of transition from a combined (acute and rehabilitation) stroke unit to a hyperacute model of stroke care.

We reorganised the combined (acute and rehab) stroke unit (SU) at Gloucestershire Royal Hospital into a hyperacute stroke unit (HASU) and a rehab SU where patients are moved after spending about 72 hours on HASU. Continuous monitoring of physiological variables was introduced and consultant job plans were reorganised to provide a HASU physician of the week model with enhanced 7-day senior presence along with redistribution of junior medical staff. Sentinel Stroke National Audit Programme (SSNAP) data for 14 months preceding the reorganisation (n=1,049) and 14 months after (n=974) were accessed for outcomes. More patients were admitted directly to the HASU with favourable reductions in time to computerised tomography scanning and stroke consultant assessment after the change. There were significant reductions in length of stay, pneumonia and urinary tract infections at 7 days and a favourable shift in modified Rankin scores (odds ratio 1.60, 95% confidence interval 1.36-1.89, p<0.001) on discharge from hospital.

Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage.

Oligodendrocytes derive from progenitors (OPCs) through the interplay of epigenomic and transcriptional events. By integrating high-resolution methylomics, RNA-sequencing, and multiple transgenic lines, this study defines the role of DNMT1 in developmental myelination. We detected hypermethylation of genes related to cell cycle and neurogenesis during differentiation of OPCs, yet genetic ablation of Dnmt1 resulted in inefficient OPC expansion and severe hypomyelination associated with ataxia and tremors in mice. This phenotype was not caused by lineage switch or massive apoptosis but was characterized by a profound defect of differentiation associated with changes in exon-skipping and intron-retention splicing events and by the activation of an endoplasmic reticulum stress response. Therefore, loss of Dnmt1 in OPCs is not sufficient to induce a lineage switch but acts as an important determinant of the coordination between RNA splicing and protein synthesis necessary for myelin formation.

Validation of ABCD2 scores ascertained by referring clinicians: a retrospective transient ischaemic attack clinic cohort study.

INTRODUCTION: Transient ischaemic attack (TIA) services routinely use ABCD2 scores ascertained by referring clinicians to triage patients. Most ABCD2 validation studies have used ABCD2 scores calculated by stroke-specialist investigators and not referring clinicians. This study aimed to assess the usefulness of referring clinicians' ABCD2 scores in predicting strokes. METHODS: A retrospective study of a TIA clinic cohort from Gloucester, UK, followed up for 4 years from 2010 to 2012. ABCD2 scores were dichotomised to high risk-ABCD2≥4 and low risk-ABCD2<4. Outcomes of interest were subsequent stroke and stroke or TIA. Survival analysis was used determine the cumulative probability of these outcomes and to identify if ABCD2 risk category was associated with stroke. RESULTS: Of 1067 (284 high risk, 783 low risk) patients, 49.6% were classified by the clinic stroke physicians as TIA/minor stroke and 50.4% as mimics. Follow-up was for a median of 34.9 (IQR 27.7-41.6) months with 56 strokes and 106 strokes/TIA. The number of strokes by 7 days, 90 days and 48 months, respectively, were: high risk 0, 2 and 20 and low risk 2, 6 and 36 (p=0.21). Unadjusted HR for subsequent stroke was 1.41 (95% CI 0.82 to 2.46) in the high-risk group compared with the low-risk group and HR adjusted for the diagnosis of TIA/stroke was 1.2 (95% CI 0.69 to 2.08). CONCLUSIONS: ABCD2 scores recorded by referring clinicians did not identify patients at high risk of subsequent stroke, suggesting that the score should not be used for TIA clinic triage.

Diagnosis of TIA (DOT) score--design and validation of a new clinical diagnostic tool for transient ischaemic attack.

BACKGROUND: The diagnosis of Transient Ischaemic Attack (TIA) can be difficult and 50-60% of patients seen in TIA clinics turn out to be mimics. Many of these mimics have high ABCD2 scores and fill urgent TIA clinic slots inappropriately. A TIA diagnostic tool may help non-specialists make the diagnosis with greater accuracy and improve TIA clinic triage. The only available diagnostic score (Dawson et al) is limited in scope and not widely used. The Diagnosis of TIA (DOT) Score is a new and internally validated web and mobile app based diagnostic tool which encompasses both brain and retinal TIA. METHODS: The score was derived retrospectively from a single centre TIA clinic database using stepwise logistic regression by backwards elimination to find the best model. An optimum cutpoint was obtained for the score. The derivation and validation cohorts were separate samples drawn from the years 2010/12 and 2013 respectively. Receiver Operating Characteristic (ROC) curves and area under the curve (AUC) were calculated and the diagnostic accuracy of DOT was compared to the Dawson score. A web and smartphone calculator were designed subsequently. RESULTS: The derivation cohort had 879 patients and the validation cohort 525. The final model had seventeen predictors and had an AUC of 0.91 (95% CI: 0.89-0.93). When tested on the validation cohort, the AUC for DOTS was 0.89 (0.86-0.92) while that of the Dawson score was 0.77 (0.73-0.81). The sensitivity and specificity of the DOT score were 89% (CI: 84%-93%) and 76% (70%-81%) respectively while those of the Dawson score were 83% (78%-88%) and 51% (45%-57%). Other diagnostic accuracy measures (DOT vs. Dawson) include positive predictive values (75% vs. 58%), negative predictive values (89% vs. 79%), positive likelihood ratios (3.67 vs. 1.70) and negative likelihood ratios (0.15 vs. 0.32). CONCLUSION: The DOT score shows promise as a diagnostic tool for TIA and requires independent external validation before it can be widely used. It could potentially improve the triage of patients assessed for suspected TIA.