Dose-dependent effect of myelin oligodendrocyte glycoprotein on visual function and optic nerve damage in experimental autoimmune encephalomyelitis.

Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprotein (MOG) to develop experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis. Typical EAE motor impairments were assessed daily and noninvasive visual evoked potentials (VEPs) were recorded at baseline and 5 weeks after immunization, with final histopathology of optic nerves (ONs). Immunized rats exhibited a relapsing-remitting clinical course. Both VEP and histological abnormalities were detected in a MOG dose-dependent gradient. Increasing MOG dosage augmented visual function impairment in EAE, which could be monitored with VEP recording to assess demyelination and axonal loss along ONs.

Subclinical anterior optic pathway involvement in early multiple sclerosis and clinically isolated syndromes.

Optical coherence tomography (OCT) is gaining increasing relevance in the assessment of patients with multiple sclerosis. Converging evidence point to the view that neuro-retinal changes, in eyes without acute optic neuritis, reflect inflammatory and neurodegenerative processes taking place throughout the CNS. The present study aims at exploring the usefulness of OCT as a marker of inflammation and disease burden in the earliest phases of the disease. Thus, a cohort of 150 consecutive patients underwent clinical, neurophysiological and brain MRI assessment as well as lumbar puncture as part of their diagnostic workup for a neurological episode suggestive of inflammatory CNS disorder; among those 32 patients had another previous misdiagnosed episode. For the present study, patients also received a visual pathway assessment (OCT, visual evoked potentials, visual acuity), measurement of CSF inflammatory markers (17 cytokines-chemokines, extracellular vesicles of myeloid origin), and dosage of plasma neurofilaments. Subclinical optic nerve involvement is frequently found in clinically isolated syndromes by visual evoked potentials (19.2%). OCT reveals ganglion cell layer asymmetries in 6.8% of patients; retinal fibre layer asymmetries, despite being more frequent (17.8%), display poor specificity. The presence of subclinical involvement is associated with a greater disease burden. Second, ganglion cell layer thinning reflects the severity of disease involvement even beyond the anterior optic pathway. In fact, the ganglion cell layer in eyes without evidence of subclinical optic involvement is correlated with Expanded Disability Status Scale, low contrast visual acuity, disease duration, brain lesion load, presence of gadolinium enhancing lesions, abnormalities along motor and somatosensory evoked potentials, and frequency of CSF-specific oligoclonal bands. Third, the inner nuclear layer thickens in a post-acute (1.1-3.7 months) phase after a relapse, and this phenomenon is counteracted by steroid treatment. Likewise, a longitudinal analysis on 65 patients shows that this swelling is transient and returns to normal values after 1 year follow-up. Notwithstanding, the clinical, MRI, serological and CSF markers of disease activity considered in the study are strictly associated with one another, but none of them are associated with the inner nuclear layer. Our findings challenge the current hypothesis that the inner nuclear layer is an acute phase marker of inflammatory activity. The present study suggests that instrumental evidence of subclinical optic nerve involvement is associated with a greater disease burden in clinically isolated syndrome. Neuro-retinal changes are present since the earliest phases of the disease and yield important information regarding the neurodegenerative and inflammatory processes occurring in the CNS.

The Danger of Walking with Socks: Evidence from Kinematic Analysis in People with Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is characterized by gait impairments and severely impacts the quality of life. Technological advances in biomechanics offer objective assessments of gait disabilities in clinical settings. Here we employed wearable sensors to measure electromyography (EMG) and body acceleration during walking and to quantify the altered gait pattern between people with progressive MS (PwPMS) and healthy controls (HCs). Forty consecutive patients attending our department as in-patients were examined together with fifteen healthy controls. All subjects performed the timed 10 min walking test (T10MW) using a wearable accelerator and 8 electrodes attached to bilateral thighs and legs so that body acceleration and EMG activity were recorded. The T10MWs were recorded under three conditions: standard (wearing shoes), reduced grip (wearing socks) and increased cognitive load (backward-counting dual-task). PwPMS showed worse kinematics of gait and increased muscle coactivation than controls at both the thigh and leg levels. Both reduced grip and increased cognitive load caused a reduction in the cadence and velocity of the T10MW, which were correlated with one another. A higher coactivation index at the thigh level of the more affected side was positively correlated with the time of the T10MW (r = 0.5, p < 0.01), Expanded Disability Status Scale (EDSS) (r = 0.4, p < 0.05), and negatively correlated with the cadence (r = -0.6, p < 0.001). Our results suggest that excessive coactivation at the thigh level is the major determinant of the gait performance as the disease progresses. Moreover, demanding walking conditions do not influence gait in controls but deteriorate walking performances in PwPMS, thus those conditions should be prevented during hospital examinations as well as in homecare environments.

Visual evoked potentials can be reliably recorded using noninvasive epidermal electrodes in the anesthetized rat.

PURPOSE: Visual evoked potentials (VEPs) are a powerful tool to evaluate nervous conduction along the visual pathways, both in humans and in animal models. Traditionally, epidural screw electrodes are used to record VEPs in preclinical research. Here we tested the feasibility in the preclinical setting of the same noninvasive technique used for clinical VEP acquisition, by using epidermal cup electrodes with no surgical procedures. METHODS: Monocular flash VEPs were recorded bilaterally under sevoflurane anesthesia once a week for 6 weeks in 14 dark Agouti rats, 7 with implanted epidural screws and 7 with epidermal 6 mm Ø Ag/AgCl cups. RESULTS: VEP traces obtained with the two techniques were morphologically comparable. There were no significant differences in latency of the main visual component between screw-recorded VEPs (sVEPs) and cup-recorded VEPs (cVEPs). Amplitude values with epidermal cups were significantly lower than those with epidural screws. Both techniques provided latencies and amplitudes which were stable over time. Furthermore, with regard to latency both methods ensured highly repeatable measurements over time, with epidermal cups even providing slightly better results. On the other hand, considering amplitudes, cVEPs and sVEPs provided fairly acceptable repeatability. CONCLUSIONS: Epidermal cup electrodes can provide comparable results to those obtained with the "gold standard" epidural screws, while representing a simpler and less invasive technique to test nervous conduction along the visual pathways in the preclinical setting.

Optical coherence tomography with voxel-based morphometry: a new tool to unveil focal retinal neurodegeneration in multiple sclerosis.

Neurodegeneration is the main contributor to disability accumulation in multiple sclerosis. Previous studies in neuro-ophthalmology have revealed that neurodegeneration in multiple sclerosis also affects the neuro-retina. Optical coherence tomography has been used to measure thinning of retinal layers, which correlates with several other markers for axonal/neuronal loss in multiple sclerosis. However, the existing analytical tools have limitations in terms of sensitivity and do not provide topographical information. In this study, we aim to evaluate whether voxel-based morphometry can increase sensitivity in detecting neuroaxonal degeneration in the retina and offer topographical information. A total of 131 people with multiple sclerosis (41 clinically isolated syndrome, 53 relapsing-remitting and 37 progressive multiple sclerosis) and 50 healthy subjects were included. Only eyes with normal global peripapillary retinal nerve fibre layer thickness and no history of optic neuritis were considered. Voxel-based morphometry and voxel-wise statistical comparisons were performed on the following: (i) patients at different disease stages and 2) patients who experienced the first demyelination attack without subclinical optic neuritis, assessed by visual evoked potentials. Standard parameters failed to discern any differences; however, voxel-based morphometry-optical coherence tomography successfully detected focal macular atrophy of retinal nerve fibre layer and ganglion cell/inner plexiform layer, along with thickening of inner nuclear layer in patients who experienced the first demyelination attack (disease duration = 4.2 months). Notably, the atrophy pattern of the ganglion cell/inner plexiform layer was comparable across disease phenotypes. In contrast, the retinal nerve fibre layer atrophy spread from the optic nerve head to the fovea as the disease evolved towards the progressive phase. Furthermore, for patients who experienced the first neurological episode, the severity of retinal nerve fibre layer atrophy at entry could predict a second attack. Our results demonstrate that voxel-based morphometry-optical coherence tomography exhibits greater sensitivity than standard parameters in detecting focal retinal atrophy, even at clinical presentation, in eyes with no history of optic neuritis and with normal latency of visual evoked potentials. Thinning of the ganglion cell/inner plexiform layer primarily concentrated in nasal perifovea in all disease phenotypes, indicating selective vulnerability of retinal ganglion cells and their perifoveal axons. Conversely, the degree of retinal nerve fibre layer thinning seems to be related to the clinical course of multiple sclerosis. The findings suggest bidirectional neurodegeneration in the visual pathway. Voxel-based morphometry-optical coherence tomography shows potential as a valuable tool for monitoring neurodegeneration on a patient level and evaluating the efficacy of novel neuroprotective treatments.

Visual evoked potentials waveform analysis to measure intracortical damage in a preclinical model of multiple sclerosis.

Introduction: Visual evoked potentials (VEPs) are a non-invasive technique routinely used in clinical and preclinical practice. Discussion about inclusion of VEPs in McDonald criteria, used for Multiple Sclerosis (MS) diagnosis, increased the importance of VEP in MS preclinical models. While the interpretation of the N1 peak is recognized, less is known about the first and second positive VEP peaks, P1 and P2, and the implicit time of the different segments. Our hypothesis is that P2 latency delay describes intracortical neurophysiological dysfunction from the visual cortex to the other cortical areas. Methods: In this work, we analyzed VEP traces that were included in our two recently published papers on Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Compared with these previous publications other VEP peaks, P1 and P2, and the implicit time of components P1-N1, N1-P2 and P1-P2, were analyzed in blind. Results: Latencies of P2, P1-P2, P1-N1 and N1-P2 were increased in all EAE mice, including group without N1 latency change delay at early time points. In particular, at 7 dpi the P2 latency delay change was significantly higher compared with N1 latency change delay. Moreover, new analysis of these VEP components under the influence of neurostimulation revealed a decrease in P2 delay in stimulated animals. Discussion: P2 latency delay, P1-P2, P1-N1, and N1-P2 latency changes which reflect intracortical dysfunction, were consistently detected across all EAE groups before N1 change. Results underline the importance of analyzing all VEP components for a complete overview of the neurophysiological visual pathway dysfunction and treatment efficacy.

Transcranial direct current stimulation as a preventive treatment in multiple sclerosis? Preclinical evidence.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system, presenting with optic neuritis in about 20-30% of cases. Optic nerve demyelination, associated with delay of visual evoked potentials (VEPs), is also observed prior to motor signs in the preclinical MS model Experimental Autoimmune Encephalomyelitis (EAE). Transcranial direct current stimulation (tDCS), inducing polarity-dependent changes in neuronal excitability, is widely used to promote neuroplasticity in several neurological disorders. However, its potential effects on inflammation and demyelination are largely unknown. We tested the effectiveness of a preventive, 5-day tDCS treatment started 3 days post-immunization, in reducing the severity of VEP delays observed in early EAE. In mice undergoing cathodal tDCS (n = 6/26 eyes) VEPs were significantly less delayed compared with eyes from EAE-Sham (n = 24/32 eyes) and EAE-Anodal (n = 22/32 eyes). Optic nerve immunohistochemistry revealed a significantly lower cell density of microglia/macrophages, and less axonal loss in EAE-Cathodal vs EAE-Sham and EAE-Anodal, while the percent demyelination with Luxol-fast blue staining was comparable among EAE groups. Considering the latter result, immunofluorescence paranodal staining was performed, revealing a significantly higher number of complete paranode domains in EAE-Cathodal, closer to healthy mice, compared with EAE-Sham and EAE-Anodal groups. These results were reflected by the negative correlation between the number of complete paranode domains and VEP latency increase with respect to pre-immunization. Finally, cathodal tDCS was associated with a lower number, closer to healthy, of single paranodes in contrast to EAE-Sham. The effects of cathodal stimulation in preventing VEPs delays and optic nerve myelin damage were already observed in the pre-motor onset EAE stage, and were associated with a lower density of inflammatory cells. These findings suggest that tDCS may exert an anti-inflammatory effect with potential therapeutic application to be further explored in autoimmune demyelinating diseases.

Visual Evoked Potentials to Monitor Myelin Cuprizone-Induced Functional Changes.

The visual system is one of the most accessible routes to study the central nervous system under pathological conditions, such as in multiple sclerosis (MS). Non-invasive visual evoked potential (VEP) and optical coherence tomography (OCT) were used to assess visual function and neuroretinal thickness in C57BL/6 taking 0.2% cuprizone for 7 weeks and at 5, 8, 12, and 15 days after returning to a normal diet. VEPs were significantly delayed starting from 4 weeks on cuprizone, with progressive recovery off cuprizone, becoming significant at day 8, complete at day 15. In contrast, OCT and neurofilament staining showed no significant axonal thinning. Optic nerve histology indicated that whilst there was significant myelin loss at 7 weeks on the cuprizone diet compared with healthy mice, at 15 days off cuprizone diet demyelination was significantly less severe. The number of Iba 1+ cells was found increased in cuprizone mice at 7 weeks on and 15 days off cuprizone. The combined use of VEPs and OCT allowed us to characterize non-invasively, in vivo, the functional and structural changes associated with demyelination and remyelination in a preclinical model of MS. This approach contributes to the non-invasive study of possible effective treatments to promote remyelination in demyelinating pathologies.

Intensive Neurorehabilitation and Gait Improvement in Progressive Multiple Sclerosis: Clinical, Kinematic and Electromyographic Analysis.

BACKGROUND: Gait deficit is a hallmark of multiple sclerosis and the walking capacity can be improved with neurorehabilitation. Technological advances in biomechanics offer opportunities to assess the effects of rehabilitation objectively. OBJECTIVE: Combining wireless surface electromyography and wearable inertial sensors to assess and monitor the gait pattern before and after an intensive multidisciplinary neurorehabilitation program (44 h/4weeks) to evaluate rehabilitation efficiency. METHODS: Forty people with progressive multiple sclerosis were enrolled. Wireless wearable devices were used to evaluate the gait. Instrumental gait analysis, clinical assessment, and patient report outcome measures were acquired before and after the neurorehabilitation. Spatiotemporal gait parameters, the co-activation index of lower limb muscles, and clinical assessments were compared pre- and post-treatment. RESULTS: Significant improvements after intensive neurorehabilitation were found in most of the clinical assessments, cadence, and velocity of the instrumental gait analysis, paralleled by amelioration of thigh co-activation on the less-affected side. Subjects with better balance performance and higher independence at baseline benefit more from the neurorehabilitation course. CONCLUSIONS: Significant improvements in gait performance were found in our cohort after an intensive neurorehabilitation course, for both quantitative and qualitative measures. Integrating kinematic and muscle activity measurements offers opportunities to objectively evaluate and interpret treatment effects.

Case Report: Off-Label Liraglutide Use in Children With Wolfram Syndrome Type 1: Extensive Characterization of Four Patients.

Aims: Wolfram syndrome type 1 is a rare recessive monogenic form of insulin-dependent diabetes mellitus with progressive neurodegeneration, poor prognosis, and no cure. Based on preclinical evidence we hypothesized that liraglutide, a glucagon-like peptide-1 receptor agonist, may be repurposed for the off-label treatment of Wolfram Syndrome type 1. We initiated an off-label treatment to investigate the safety, tolerability, and efficacy of liraglutide in pediatric patients with Wolfram Syndrome type 1. Methods: Pediatric patients with genetically confirmed Wolfram Syndrome type 1 were offered off-label treatment approved by The Regional Network Coordination Center for Rare Diseases, Pharmacological Research IRCCS Mario Negri, and the internal ethics committee. Four patients were enrolled; none refused nor were excluded or lost during follow-up. Liraglutide was administered as a daily subcutaneous injection. Starting dose was 0.3 mg/day. The dose was progressively increased as tolerated, up to the maximum dose of 1.8 mg/day. The primary outcome was evaluating the safety, tolerability, and efficacy of liraglutide in Wolfram Syndrome type 1 patients. Secondary endpoints were stabilization or improvement of C-peptide secretion as assessed by the mixed meal tolerance test. Exploratory endpoints were stabilization of neurological and neuro-ophthalmological degeneration, assessed by optical coherence tomography, electroretinogram, visual evoked potentials, and magnetic resonance imaging. Results: Four patients aged between 10 and 14 years at baseline were treated with liraglutide for 8-27 months. Liraglutide was well-tolerated: all patients reached and maintained the maximum dose, and none withdrew from the study. Only minor transient gastrointestinal symptoms were reported. No alterations in pancreatic enzymes, calcitonin, or thyroid hormones were observed. At the latest follow-up, the C-peptide area under the curve ranged from 81 to 171% of baseline. Time in range improved in two patients. Neuro-ophthalmological and neurophysiological disease parameters remained stable at the latest follow-up. Conclusions: We report preliminary data on the safety, tolerability, and efficacy of liraglutide in four pediatric patients with Wolfram Syndrome type 1. The apparent benefits both in terms of residual C-peptide secretion and neuro-ophthalmological disease progression warrant further studies on the repurposing of glucagon-like peptide-1 receptor agonists as disease-modifying agents for Wolfram Syndrome type 1.

Functional evolution of visual involvement in experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple sclerosis (MS). C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein exhibit chronic disease course, together with optic neuritis, consisting of demyelination/axonal loss of the optic nerve. OBJECTIVES: To characterize functional and structural visual damages in two different phases of EAE: pre- and post-motor onset. METHODS: Visual alterations were detected with Visual Evoked Potential (VEP), Electroretinogram (ERG) and Optical Coherence Tomography (OCT). Optic nerve histology was performed at 7 (pre-motor onset) or 37 (post-motor onset) days post-immunization (dpi). RESULTS: At 7 dpi, optic nerve inflammation was similar in EAE eyes with and without VEP latency delay. Demyelination was detected in EAE eyes with latency delay (p < 0.0001), while axonal loss (p < 0.0001) and ERG b-wave amplitude (p = 0.004) were decreased in EAE eyes without latency delay compared to Healthy controls. At 37 dpi, functional and structural optic nerve damage were comparable between EAE groups, while a decrease of ERG amplitude and NGCC thickness were found in EAE eyes with VEP latency delay detected post-motor onset. CONCLUSIONS: Thanks to non-invasive methods, we studied the visual system in a MS model, which could be useful for developing specific therapeutic strategies to target different disease phases.

Neuro-Retina Might Reflect Alzheimer's Disease Stage.

BACKGROUND: Alzheimer's disease (AD) pathological hallmarks were found in retinas of AD patients. Several studies showed a significant reduction of neuro-retina thickness measured through optical coherence tomography (OCT) in AD patients, but possible correlations between retina morphology, cognition, and cerebrospinal fluid (CSF) AD biomarkers (Aß42, t-tau, and p-tau) have been poorly investigated so far. OBJECTIVE: In the present cross-sectional study, we measured the thickness of neuro-retinal layers through OCT searching for possible correlations with patients' cognitive performances and CSF AD biomarkers. METHODS: 137 consecutive subjects [43 with AD, 37 with mild cognitive impairment (MCI), and 57 healthy controls (HC)], received an OCT scan acquisition to measure the peripapillary retinal nerve fiber layer (RNFL) thickness. In a subsample of 21 AD, 18 MCI, and 18 HC, the macular volume of ganglion cell layer (GCL), inner plexiform layer (IPL), and inner nuclear layer was computed. A comprehensive neuropsychological assessment and CSF AD biomarkers' concentrations were available in AD and MCI patients. RESULTS: Peripapillary RNFL, global, and in superior quadrant was significantly thinner in AD and MCI patients when compared to HC, while macular GCL volume was significantly reduced only in AD. RNFL thickness in nasal and inferior quadrants was correlated with single CSF AD biomarker concentrations, but no differences were found in retina morphology depending on the presence of a CSF profile typical for AD. Memory performances were positively associated with GCL and IPL volume. CONCLUSION: Our findings might propose OCT as a reliable and easy to handle tool able to detect neuro-retinal atrophy in AD in relation with cognitive performances.

Non-invasive visual evoked potentials to assess optic nerve involvement in the dark agouti rat model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein.

Experimental autoimmune encephalomyelitis (EAE) is the primary disease model of multiple sclerosis (MS), one of the most diffused neurological diseases characterized by fatigue, muscle weakness, vision loss, anxiety and depression. EAE can be induced through injection of myelin peptides to susceptible mouse or rat strains. In particular, EAE elicited by the autoimmune reaction against myelin oligodendrocyte glycoprotein (MOG) presents the common features of human MS: inflammation, demyelination and axonal loss. Optic neuritis affects visual pathways in both MS and in several EAE models. Neurophysiological evaluation through visual evoked potential (VEP) recording is useful to check visual pathway dysfunctions and to test the efficacy of innovative treatments against optic neuritis. For this purpose, we investigate the extent of VEP abnormalities in the dark agouti (DA) rat immunized with MOG, which develops a relapsing-remitting disease course. Together with the detection of motor signs, we acquired VEPs during both early and late stages of EAE, taking advantage of a non-invasive recording procedure that allows long follow-up studies. The validation of VEP outcomes was determined by comparison with ON histopathology, aimed at revealing inflammation, demyelination and nerve fiber loss. Our results indicate that the first VEP latency delay in MOG-EAE DA rats appeared before motor deficits and were mainly related to an inflammatory state. Subsequent VEP delays, detected during relapsing EAE phases, were associated with a combination of inflammation, demyelination and axonal loss. Moreover, DA rats with atypical EAE clinical course tested at extremely late time points, manifested abnormal VEPs although motor signs were mild. Overall, our data demonstrated that non-invasive VEPs are a powerful tool to detect visual involvement at different stages of EAE, prompting their validation as biomarkers to test novel treatments against MS optic neuritis.

Optical Coherence Tomography Reveals Retinal Neuroaxonal Thinning in Frontotemporal Dementia as in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) and frontotemporal dementia (FTD) are leading causes of cognitive decline. Optical coherence tomography (OCT) allows the measurement of thickness of retinal neuroaxonal layers. While in AD and mild cognitive impairment (MCI), retinal nerve fiber layer (RNFL) thinning is frequently reported, less information is available on ganglion cell layer-inner plexiform layer (GCL-IPL). Data on FTD are lacking. OBJECTIVE: To obtain cross-sectional information on RNFL and GCL-IPL thickness among MCI, AD, FTD, and healthy controls (HC), and their correlations with dementia severity. METHODS: Peripapillary OCT scans were obtained in 27 MCI, 39 AD, 17 FTD, 49 HC using high-definition Heidelberg Spectral-domain OCT, with RNFL and GCL-IPL thickness measurement. Statistical analysis tested group effects and correlation with gender, disease duration and severity (Mini-Mental State Examination, MMSE). RESULTS: RNFL showed a significant group effect [F(4,132) = 3.786, p = 0.006], being reduced versus controls in MCI (p = 0.033), moderate AD (p = 0.025), and FTD (p < 0.001), and versus mild AD in FTD (p = 0.042). GCL-IPL showed a significant group effect as well [F(4,121) = 5.104, p < 0.001], with reduction in moderate AD versus HC (p < 0.001), MCI (p = 0.037), and mild AD (p = 0.009); in FTD versus HC (p = 0.002) and mild AD (p = 0.038). In AD, GCL-IPL correlated with MMSE (r = 0.487, p = 0.003), without significant effects of age, gender, or disease duration. CONCLUSION: Retinal neuroaxonal thinning occurs in MCI/AD consistently with previous reports, as well as in FTD. Correlation with disease severity in AD suggests that retinal and brain neurodegeneration may occur in parallel to some extent, and prompts larger studies aimed at providing surrogate endpoints for clinical trials in AD.

Identifying robust communities and multi-community nodes by combining top-down and bottom-up approaches to clustering.

Biological functions are carried out by groups of interacting molecules, cells or tissues, known as communities. Membership in these communities may overlap when biological components are involved in multiple functions. However, traditional clustering methods detect non-overlapping communities. These detected communities may also be unstable and difficult to replicate, because traditional methods are sensitive to noise and parameter settings. These aspects of traditional clustering methods limit our ability to detect biological communities, and therefore our ability to understand biological functions. To address these limitations and detect robust overlapping biological communities, we propose an unorthodox clustering method called SpeakEasy which identifies communities using top-down and bottom-up approaches simultaneously. Specifically, nodes join communities based on their local connections, as well as global information about the network structure. This method can quantify the stability of each community, automatically identify the number of communities, and quickly cluster networks with hundreds of thousands of nodes. SpeakEasy shows top performance on synthetic clustering benchmarks and accurately identifies meaningful biological communities in a range of datasets, including: gene microarrays, protein interactions, sorted cell populations, electrophysiology and fMRI brain imaging.