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The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.
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Células Amacrinas , Adhesión Celular , Endocitosis , Fosfohidrolasa PTEN , Retina , Vía de Señalización Wnt , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Retina/metabolismo , Ratones , Células Amacrinas/metabolismo , Ratones Noqueados , Transporte de Proteínas , Proteínas Wnt/metabolismo , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Stable patients with multiple sclerosis (MS) may discontinue treatment, but the risk of disease activity is unknown. Serum neurofilament light chain (sNfL) and serum glial fibrillary acidic protein (sGFAP) are biomarkers of subclinical disease activity and may help risk stratification. In this study, sNfL and sGFAP levels in stable patients were evaluated before and after treatment discontinuation to determine association with disease activity. METHODS: This observational study included patients enrolled in the Comprehensive Longitudinal Investigation in MS at the Brigham and Women's Hospital who discontinued treatment after >2 years disease activity-free. Two serum samples within 2 years, before and after treatment stop, were sent for sNfL and sGFAP measurements by single-molecule array. Biannual neurologic examinations and yearly MRI scans determined disease activity by 3 time-to-event outcomes: 6-month confirmed disability worsening (CDW), clinical attacks, and MRI activity (new T2 or contrast-enhancing lesions). Associations between each outcome and log-transformed sNfL and sGFAP levels pretreatment stop and posttreatment stop and the percent change were estimated using multivariable Cox regression analysis adjusting for age, disability, disease duration, and duration from attack before treatment stop. RESULTS: Seventy-eight patients (92% female) discontinued treatment at a median (interquartile range) age of 48.5 years (39.0-55.7) and disease duration of 12.3 years (7.5-18.8) and were followed up for 6.3 years (4.2-8.5). CDW occurred in 27 patients (35%), new attacks in 19 (24%), and new MRI activity in 26 (33%). Higher posttreatment stop sNfL level was associated with CDW (adjusted hazard ratio (aHR) 2.80, 95% CI 1.36-5.76, p = 0.005) and new MRI activity (aHR 3.09, 95% CI 1.42-6.70, p = 0.004). Patients who had >100% increase in sNfL level from pretreatment stop to posttreatment stop had greater risk of CDW (HR 3.87, 95% CI 1.4-10.7, p = 0.009) and developing new MRI activity (HR 4.02, 95% CI 1.51-10.7, p = 0.005). Patients who had >50% increase in sGFAP level also had greater risk of CDW (HR 5.34, 95% CI 1.4-19.9, p = 0.012) and developing new MRI activity (HR 5.16, 95% CI 1.71-15.6, p = 0.004). DISCUSSION: Stable patients who discontinue treatment may be risk stratified by sNfL and sGFAP levels measured before and after discontinuing treatment. Further studies are needed to validate findings and determine whether resuming treatment in patients with increasing biomarker levels reduces risk of subsequent disease activity.
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Esclerosis Múltiple , Humanos , Femenino , Persona de Mediana Edad , Masculino , Esclerosis Múltiple/diagnóstico por imagen , Esclerosis Múltiple/tratamiento farmacológico , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Biomarcadores , Imagen por Resonancia MagnéticaRESUMEN
An 18-protein multiple sclerosis (MS) disease activity (DA) test was validated based on associations between algorithm scores and clinical/radiographic assessments (N = 614 serum samples; Train [n = 426; algorithm development] and Test [n = 188; evaluation] subsets). The multi-protein model was trained based on presence/absence of gadolinium-positive (Gd+) lesions and was also strongly associated with new/enlarging T2 lesions, and active versus stable disease (composite of radiographic and clinical evidence of DA) with improved performance (p < 0.05) compared to the neurofilament light single protein model. The odds of having ≥1 Gd+ lesions with a moderate/high DA score were 4.49 times that of a low DA score, and the odds of having ≥2 Gd+ lesions with a high DA score were 20.99 times that of a low/moderate DA score. The MSDA Test was clinically validated with improved performance compared to the top-performing single-protein model and can serve as a quantitative tool to enhance the care of MS patients.
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Esclerosis Múltiple , Humanos , Imagen por Resonancia Magnética , Proteínas Sanguíneas , Gadolinio , AlgoritmosRESUMEN
BACKGROUND: Early risk-stratification in multiple sclerosis (MS) may impact treatment decisions. Current predictive models have identified that clinical and imaging characteristics of aggressive disease are associated with worse long-term outcomes. Serum biomarkers, neurofilament (sNfL) and glial fibrillary acidic protein (sGFAP), reflect subclinical disease activity through separate pathological processes and may contribute to predictive models of clinical and MRI outcomes. METHODS: We conducted a retrospective analysis of the Comprehensive Longitudinal Investigation of Multiple Sclerosis at the Brigham and Women's Hospital (CLIMB study), where patients with multiple sclerosis are seen every 6 months and undergo Expanded Disability Status Scale (EDSS) assessment, have annual brain MRI scans where volumetric analysis is conducted to calculate T2-lesion volume (T2LV) and brain parenchymal fraction (BPF), and donate a yearly blood sample for subsequent analysis. We included patients with newly diagnosed relapsing-remitting MS and serum samples obtained at baseline visit and 1-year follow-up (both within 3 years of onset), and were assessed at 10-year follow-up. We measured sNfL and sGFAP by single molecule array at baseline visit and at 1-year follow-up. A predictive clinical model was developed using age, sex, Expanded Disability Status Scale (EDSS), pyramidal signs, relapse rate, and spinal cord lesions at first visit. The main outcome was odds of developing of secondary progressive (SP)MS at year 10. Secondary outcomes included 10-year EDSS, brain T2LV and BPF. We compared the goodness-of-fit of the predictive clinical model with and without sNfL and sGFAP at baseline and 1-year follow-up, for each outcome by area under the receiver operating characteristic curve (AUC) or R-squared. RESULTS: A total 144 patients with median MS onset at age 37.4 years (interquartile range: 29.4-45.4), 64% female, were included. SPMS developed in 25 (17.4%) patients. The AUC for the predictive clinical model without biomarker data was 0.73, which improved to 0.77 when both sNfL and sGFAP were included in the model (P = 0.021). In this model, higher baseline sGFAP associated with developing SPMS (OR=3.3 [95%CI:1.1,10.6], P = 0.04). Adding 1-year follow-up biomarker levels further improved the model fit (AUC = 0.79) but this change was not statistically significant (P = 0.15). Adding baseline biomarker data also improved the R-squared of clinical models for 10-year EDSS from 0.24 to 0.28 (P = 0.032), while additional 1-year follow-up levels did not. Baseline sGFAP was associated with 10-year EDSS (ß=0.58 [95%CI:0.00,1.16], P = 0.05). For MRI outcomes, baseline biomarker levels improved R-squared for T2LV from 0.12 to 0.27 (P<0.001), and BPF from 0.15 to 0.20 (P = 0.042). Adding 1-year follow-up biomarker data further improved T2LV to 0.33 (P = 0.0065) and BPF to 0.23 (P = 0.048). Baseline sNfL was associated with T2LV (ß=0.34 [95%CI:0.21,0.48], P<0.001) and 1-year follow-up sNfL with BPF (ß=-2.53% [95%CI:-4.18,-0.89], P = 0.003). CONCLUSIONS: Early biomarker levels modestly improve predictive models containing clinical and MRI variables. Worse clinical outcomes, SPMS and EDSS, are associated with higher sGFAP levels and worse MRI outcomes, T2LV and BPF, are associated with higher sNfL levels. Prospective study implementing these predictive models into clinical practice are needed to determine if early biomarker levels meaningfully impact clinical practice.
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Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple , Humanos , Femenino , Adulto , Masculino , Esclerosis Múltiple/diagnóstico , Estudios Retrospectivos , Estudios Prospectivos , Proteína Ácida Fibrilar de la Glía , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Esclerosis Múltiple Crónica Progresiva/metabolismo , BiomarcadoresRESUMEN
Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1 SA 6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1 SA 6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1 SA 6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1 SA 6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1 SA 6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1 SA 6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1 SA 6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1 SA 6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1 SA 6 vs. Ascl1, and position Ascl1 SA 6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.
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Background: There is an emerging evidence of the role of the microbiome in neurological diseases. Endotoxin is a component of gram-negative bacteria and thought to be one of the possible signals between the gut microbiota and the immune system. Previous studies explored the blood levels of endotoxin using an endpoint chromogenic assay. Methods: We validated and compared the analytical performance of two kinetic assays for the quantification of endotoxin in serum: (1) the Limulus Amebocyte Lysate (LAL) Kinetic-QCL assay and (2) the turbidimetric LAL Pyrogent-5000 assay. We used the best-performing validated assay to measure the endotoxin level in 20 patients with multiple sclerosis (MS) and eight healthy controls. Results: The Pyrogent-5000 and QCL assay achieved similar performance in regard to spike recovery and linear dilution; however, the Pyrogent-5000 had a better signal to noise in the calibrator curve. By using the Pyrogent-5000 assay, we found that serum samples from MS patients and healthy controls have a similar level of endotoxin; hence, we did not find evidence to support a penetration of endotoxin in the blood of MS patients. Our findings do not exclude a role of endotoxin in mediating signals from the gut microbiota in MS patients directly at the gut-blood barrier where numerous antigen-presenting cells are actively sensing metabolites and bacterial products.
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OBJECTIVE: Serum neurofilament light (sNfL) is a promising new biomarker in multiple sclerosis (MS). We explored the relationship between sNfL and health outcomes and resource use in MS patients. METHODS: MS patients with serum samples and health-outcome measurements collected longitudinally between 2011 and 2016 were analyzed. sNfL values were evaluated across age and gender. Data were analyzed using correlation with log-transformed sNfL values. RESULTS: A total of 304 MS patients with a mean age of 32.9 years, average EDSS of 1.6 (SD = 1.5) and baseline sNfL of 8.8 (range 1.23-78.3) pg/mL were studied. Baseline sNFL values increased with age and were higher in females. Baseline sNfL correlated with baseline Multiple Sclerosis Quality of Life physical composite (mean = 49.4 (9.1), P = 0.035) and baseline EDSS (P = 0.002). Other PRO measures at baseline did not show a significant relationship with baseline sNfL. Average of baseline and follow-up sNfL correlated with MSQoL physical-role limitations (mean = 48.9 (10.8), P = 0.043) and social-functioning (mean = 52.3 (7), P = 0.034) at 24-month follow-up. We found a trend for numerically higher sNfL levels in nonpersistent patients compared to those who were persistent to treatment (11.13 vs. 8.53 pg/mL, P = 0.093) measured as average of baseline and 24-month values. Baseline NfL was associated with number of intravenous steroid infusions (mean = 0.2; SD = 3.0, P = 0.013), whereas the average of baseline and 12 months NfL values related to inpatient stays at 12 months (mean = 0.2; SD = 3.0 P = 0.053). CONCLUSION: Serum NfL is a patient-centric biomarker that correlated with MS patient health-outcomes and healthcare utilization measures in a real-world cohort.