OBJECTIVE:Several neuromuscular disorders (NMDs) are characterized by progressive muscle damage and are marked by the elevation of circulating muscle proteins from activity-related injury. Despite a diverse array of genetic drivers, many NMDs share similar patterns of exercise intolerance and higher concentrations of muscle injury proteins relative to unaffected individuals. While the interplay between the nature of the muscle injury and the specific genetic driver is poorly understood, the similarities exhibited by various NMDs suggest that a common proteomic signature of muscle injury may exist. METHODS:We used an established exercise challenge and the SOMAscan proteomics platform to study the baseline and post-exercise proteomic profiles in a cross-sectional study of three different muscular dystrophies: Becker muscular dystrophy (BMD) and limb girdle muscular dystrophy types R9 and R12. RESULTS:Our Results Uncover a Common Signature of Circulating Proteins That Are Elevated in all Three Myopathies, Some of Which Are Further Elevated by Exercise in Becker Muscular Dystrophy and Limb Girdle Muscular Dystrophy Type R9, and Others That Are Not Responsive to Exercise. INTERPRETATION:Interestingly, these two signatures exhibit opposing trajectories with age in a larger cross-sectional cohort of BMD individuals. This research represents a first step toward defining an annotated protein signature coupled with activity-injury, a defining pathophysiological feature of many myopathies.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by the absence of dystrophin, which leads to mechanical instability of the muscle fiber membrane and a predisposition for cell membrane permeability and contraction-induced muscle injury. Deflazacort is an FDA-approved corticosteroid for treating DMD, and treatment of dystrophic mice with deflazacort reduces inflammation and improves muscle regeneration. Whether deflazacort protects from contraction-induced injury in mdx mice is unknown. To address this question, adult mdx mice were administered 1.2 mg/kg deflazacort daily by oral gavage for either 3 or 8-9 wk and compared with both vehicle-treated mdx mice and wild-type controls for various measures of susceptibility to injury. Both 3 and 8-9 wk of deflazacort treatment decreased Evans Blue dye (EBD) accumulation in vivo compared with vehicle-treated controls, but the reduction was substantially greater (58% vs. 26%) following shorter-term treatment. Furthermore, for dorsiflexor muscles evaluated in situ, 3 wk deflazacort treatment dramatically increased isometric force production, and the force decline induced by a single lengthening contraction was reduced more than 50% compared with vehicle-treated controls. Using ex vivo lumbrical muscle preparations, we found that levels of intercontraction calcium accumulation significantly correlated with force decline during repeated isometric contractions in all deflazacort-treated mice, and a trend for lower aberrant calcium accumulation was seen following 3 wk of treatment. Given that some protective effects were reduced or not present in a preclinical model of DMD with longer-term steroid treatment, these data provide important evidence for the beneficial use of short-term deflazacort.NEW & NOTEWORTHY Mechanical instability of muscle fiber membranes is a hallmark feature of Duchenne muscular dystrophy (DMD). Glucocorticoids are commonly prescribed in DMD; however, effects on contraction-induced injury and associated mechanisms remain unclear. Here, 3 wk deflazacort in mdx mice substantially reduced Evans Blue dye uptake and the lengthening contraction-induced force decline in vivo and resulted in a strong trend toward blunted calcium uptake during damaging contractions ex vivo. These findings indicate that short-term deflazacort protects from contraction-induced injury.

Introduction: Pathologic cardiac remodeling is a hallmark of hypertrophic cardiomyopathy (HCM). EDG-7500 is a novel oral, selective cardiac sarcomere modulator designed to slow the rate of contraction and speed the rate of relaxation without directly inhibiting myosin motor-head function. EDG-7500 is currently in a phase 2 clinical trial in HCM patients. Aims: To determine if EDG-7500 can prevent pathologic cardiac remodeling and disease progression in a mini-pig model of non-obstructive HCM (nHCM) caused by heterozygous MYH7 R403Q mutation. Approach: R403Q pigs were assigned to 2 groups; placebo control (CTRL; n=7-11) and EDG-7500 treated for 5-6 months (n=5-9), with untreated wild-type (WT; n=6-11) as reference. Cardiac magnetic resonance imaging (CMR) and terminal tissue collection occurred at 7-8 months of age. CMR sequences were collected in 12-16 parallel, short-axis views using a 3T scanner for calculation of left ventricular (LV) EF, volumes, T1-time, and LV mass. LV and left atrial (LA) mass also were measured postmortem. LV end diastolic pressure (EDP) and the end diastolic pressure-volume relationship (EDPVR) were measured in vivo via catheter. Myocardial atrial and brain natriuretic peptide (ANP and BNP) mRNA and α and β myosin heavy chain (MYHC) proteins were evaluated. Statistical significance was set at P≤0.05 (*denotes vs. CTRL) using one way ANOVA or linear regression with data reported as mean±SE. Results: EDG-7500 prevented increases in LV mass both in vivo (CMR; *45±3 vs. 61±6 in CTRL; WT, 46±3g) and postmortem without affecting LV end diastolic volume and EF. EDG-7500 attenuated: 1) increased LV EDP (*10±1 vs. 25±3 in CTRL; WT, 8±1mmHg); 2) decreased LV compliance (EDPVR *1.6±0.2 vs. 3.9±0.4 in CTRL; WT, 1.0±0.1mmHg/mL); and 3) increased LA mass (*12±1 vs. 22±2 in CTRL; WT, 7±1g). EDG-7500 also prevented increased T1-time (*971±31 vs. 1107±26 in CTRL; WT, 925±33msec), which was positively correlated to LV EDP (R=0.75). EDG-7500 prevented HCM-mediated increases in LV BNP (*16±6 vs. 46±13 in CTRL; WT, 1±0.2) and LA ANP mRNA (*3±1 vs.15±4 in CTRL; WT, 1±3), while preventing a pathologic isoform shift to more LA β-MYHC protein (*89%α:11%β vs. 75%α:25%β in CTRL; WT, 92%α:8%β). Conclusion: In a mini-pig model of nHCM, chronic EDG-7500 therapy prevents pathologic cardiac remodeling, decreased LV compliance, and disease progression without impairing resting systolic function. These findings support clinical investigation of EDG-7500 in nHCM.

This study explores the synthesis and application of artificial zymogens using protein-polymer hybrids to mimic the controlled enzyme activation observed in natural zymogens. Pro-trypsin (pro-TR) and pro-chymotrypsin (pro-CT) hybrids were engineered by modifying the surfaces of trypsin (TR) and chymotrypsin (CT) with cleavable peptide inhibitors utilizing surface-initiated atom transfer radical polymerization. These hybrids exhibited 70 and 90% reductions in catalytic efficiency for pro-TR and pro-CT, respectively, due to the inhibitory effect of the grafted peptide inhibitors. The activation of pro-TR by CT and pro-CT by TR resulted in 1.5- and 2.5-fold increases in enzymatic activity, respectively. Furthermore, the activated hybrids triggered an enzyme activation cascade, enabling amplification of activity through a dual pro-protease hybrid system. This study highlights the potential of artificial zymogens for therapeutic interventions and biodetection platforms by harnessing enzyme activation cascades for precise control of catalytic activity.

Assess the effect of treatment with EDG-5506 on biomarkers of muscle damage and function in adults with Becker Muscular Dystrophy.

Protein-polymer conjugates combine the unique properties of both proteins and synthetic polymers, making them important materials for biomedical applications. In this work, we synthesized and characterized protein-branched polymer bioconjugates that were precisely designed to retain protein functionality while preventing unwanted interactions. Using chymotrypsin as a model protein, we employed a controlled radical branching polymerization (CRBP) technique utilizing a water-soluble inibramer, sodium 2-bromoacrylate. The green-light-induced atom transfer radical polymerization (ATRP) enabled the grafting of branched polymers directly from the protein surface in the open air. The resulting bioconjugates exhibited a predetermined molecular weight, well-defined architecture, and high branching density. Conformational analysis by SEC-MALS validated the controlled grafting of branched polymers. Furthermore, enzymatic assays revealed that densely grafted polymers prevented protein inhibitor penetration, and the resulting conjugates retained up to 90% of their enzymatic activity. This study demonstrates a promising strategy for designing protein-polymer bioconjugates with tunable sieving behavior, opening avenues for applications in drug delivery and biotechnology.

Introduction: EDG-7500 is a cardiac sarcomere modulator that has been shown to slow early LV contraction and to improve diastolic filling, without directly inhibiting cardiac myosin. To assess the acute hemodynamic effects of EDG-7500 in a model of depressed systolic and diastolic LV function, we studied dogs with pacing-induced LV dysfunction before and after EDG-7500 administration. Methods: Beagle dogs (n = 7) underwent chronic RV pacing (180 to 240 ppm), and were studied, in sinus rhythm, at two levels of induced-dysfunction: mid-range EF (mEF, targeting 50%), and reduced EF (rEF, targeting 40%). At each level, animals were evaluated by echocardiography before and after administration of 0.3 mg/kg IV EDG-7500. Healthy beagle dogs served as disease controls (n = 7). Results: Post-pacing (Paced) dogs had depressed EF (mEF: 50 ± 4 and rEF: 40 ± 4 vs. 74 ± 1%, P < 0.05) and short-axis global circumferential strain (mEF: -23 ± 2 and rEF: -14 ± 2 vs. -33 ± 1%, P < 0.05), and elevated NT-proBNPs (mEF: 1,049 ± 319 and rEF: 4,236 ± 722 vs. 302 ± 27 pmol/L, P < 0.05) when compared vs. healthy dogs. Paced dogs had slower early diastolic mitral annular velocity (mean e’, mEF: 11.0 ± 0.9 and rEF: 8.4 ± 0.8 vs. 14.2 ± 0.7 cm/s, P < 0.05) and elevated E/e’ (mEF: 7.4 ± 0.7 and rEF: 8.4 ± 0.9 vs. 6.0 ± 0.2 n/u, P < 0.05), at similar heart rates. Acute EDG-7500 increased e’ (mean e’, mEF: +9 ± 2 and rEF: +12 ± 3%, P<0.05) and decreased E/e’ (mEF: -7 ± 2% and rEF: -9 ± 2%, P<0.05), with no detectable effects on EF (mEF: +3 ± 6% and rEF: -5 ± 3%, P=NS). There were no adverse clinical observations. Conclusions: Treatment with EDG-7500 was well tolerated in dogs with tachycardia-induced mid-range (mEF) and reduced LV systolic function (rEF). In both cases, EDG-7500 improved echocardiographic indices of early ventricular filling and filling pressures, without observable effect on systolic performance. This data supports exploration of the effects of EDG-7500 in patient populations with LV diastolic dysfunction and mild to moderately reduced cardiac systolic performance.

Introduction: Hypertrophic Cardiomyopathy (HCM) is a myocardial disease characterized by LV hypertrophy, hyperdynamic contraction, and diastolic dysfunction that results in impaired exercise capacity. In HCM, excess acto-myosin crossbridge formation during diastole increases myocardial stiffness leading to hindered ventricular filling and limited cardiac reserve. EDG-7500, a novel sarcomere regulator that slows the rate of contraction but does not directly inhibit the myosin motor head, may offer salutary effects in HCM with minimal reduction in systolic performance. Here, this hypothesis was tested in the setting of the pathological HCM beta-myosin heavy chain (MYH7) R403Q mutation. Methods: In vitro, force dynamics were evaluated in human IPSC-derived engineered heart tissues (EHT) with the MYH7 R403Q mutation compared to isogenic controls (WT). The biomechanical effects of EDG-7500 were evaluated in permeabilized fibers from tissues isolated from minipigs carrying the MYH7 R403Q mutation. The in vivo cardiac responses to EDG-7500 were studied via echocardiography and invasive hemodynamics in the R403Q minipigs. In a subset of pigs, β-adrenergic receptor (β-AR) dependent cardiac output reserve was studied. Results: R403Q mutant EHT preserved peak force (PF) relative to WT but impaired relaxation kinetics (RT90: +19±4 ms, 171±4 vs. 152±2 ms in WT, P<0.05). EDG-7500 (0.3uM) accelerated relaxation kinetics in R403Q EHTs (RT90: -8±3ms, 180±4 to 172±2 ms, P<0.05) without affecting PF. RT90 improvement occurred at 6-fold lower concentrations than systolic inhibition (PF IC20: 1.95 ± 0.70 μM). In vivo, EDG-7500 improved early LV filling, accelerating e’ (6.9 ± 0.8 to 8.6 ± 0.6 cm/s, P < 0.05) and decreased atrial volume (-22%, P < 0.05) while enhancing β-AR dependent cardiac output recruitment; in R403Q pigs, EDG-7500 plus dobutamine led to higher cardiac outputs relative to only dobutamine (3.1±0.3 vs. 2.6±0.3 L/min, P<0.05). Conclusions: EDG-7500, a novel cardiac sarcomere regulator, improved diastolic function in EHT and animals with the HCM-pathogenic MYH7 R403Q mutation, improving β-AR dependent cardiac output reserve. This novel profile could be valuable for the treatment of patients with HCM and diseases of diastolic dysfunction.

Background: Hypertrophic Cardiomyopathy (HCM) is a disease where excess acto-myosin crossbridge formation results in hyperdynamic contraction, LV hypertrophy, and diastolic dysfunction due to increased myocardial stiffness. Conventional negative inotropes alleviate, to some degree, the systolic alteration in HCM, but do not improve LV filling. EDG-7500 is a small molecule that preferentially decreases diastolic tension and slows the velocity of myocardial force generation by regulating, but not inhibiting, cardiac myosin. This study assessed pharmacodynamic responses to EDG-7500. Methods: Myofilament biomechanics and sarcomere structure were evaluated on LV permeabilized pig fibers. In vivo, dogs were instrumented for arterial pressure and LV pressure-volume recordings. Data were obtained with vehicle, EDG-7500 (n = 5; 0.3 mg/kg IV), or metoprolol (2-4 mg/kg IV, n = 3); systemic and LV hemodynamics and load-independent function were examined. *:P<0.05 for EDG-7500 vs. vehicle Results: X-ray diffraction studies showed EDG-7500 preserved the myosin head population competent to form crossbridges as well as the recruitment of reserve myosin-heads during calcium activation. EDG-7500 (1μM) slowed crossbridge formation, with a limited reduction in maximal force (-6±2%) and decreased diastolic tension. At 0.3 mg/kg, EDG-7500 slowed early contraction (PEP: +10±1%*) and reduced the peak rate of LV pressure development (+dP/dtmax: -8 ± 1%*) with minimal reductions in peak end systolic elastance (Emax: -4±1%*). Systemic mean arterial pressure and cardiac output were unchanged. EDG-7500 increased LV end-diastolic volume (+5 ± 1%*) and preserved LV end-diastolic pressure (EDP: -8 ± 3%, 9 ± 1 to 8 ± 1 mmHg). A downward and rightward shift of the pressure-volume relationship indicated improved LV compliance. β-AR blockade did not elicit this pro-compliant effect (EDP: +26 ± 12%). Conclusions: Cardiac sarcomere regulation with EDG-7500 has a unique cardiovascular profile characterized by the overall preservation of the myosin motor-head population and a slowing of early cardiac contraction and improved LV distensibility. This cardiovascular profile could have salutary effects in patients with HCM or patients with impaired diastolic function.

Skeletal muscle function and regenerative capacity decline during aging, yet factors driving these changes are incompletely understood. Muscle regeneration requires temporally coordinated transcriptional programs to drive myogenic stem cells to activate, proliferate, fuse to form myofibers, and to mature as myonuclei, restoring muscle function after injury. We assessed global changes in myogenic transcrip-tion programs distinguishing muscle regeneration in aged mice from young mice by comparing pseudotime trajectories from single -nu-cleus RNA sequencing of myogenic nuclei. Aging-specific differences in coordinating myogenic transcription programs necessary for restoring muscle function occur following muscle injury, likely contributing to compromised regeneration in aged mice. Differences in pseudotime alignment of myogenic nuclei when comparing aged with young mice via dynamic time warping revealed pseudotempo-ral differences becoming progressively more severe as regeneration proceeds. Disruptions in timing of myogenic gene expression pro-grams may contribute to incomplete skeletal muscle regeneration and declines in muscle function as organisms age.

Neuromuscular Disorders 33 (2023) S66-S192 increasing dystrophin protein levels by suppressing DTM expression.Here we test this hypothesis in bmx mice, the first mouse model of BMD generated through deletion of murine exons 45 through 47.This mutation recreates a common BMD genotype which frequently presents clinically with more severe pathology with earlier onset of cardiomyopathy.We find daily oral treatment with vamorolone or prednisolone improves bmx strength through grip strength and hang time assays.Examining histopathology, both drugs reduce fiber size and decrease the percentage of centrally nucleated fibers.Importantly, vamorolone shows improved safety by avoiding the induction of anxiety behavior and stunted growth, key side effects that are apparent in prednisolone treatment.Intriguingly, we also find vamorolone increases dystrophin protein in both skeletal muscle and heart.This data indicates vamorolone, which is nearing approval for DMD after its initial development in mdx mice, also shows efficacy in a mouse model of BMD and therefore warrants clinical investigation in BMD patients.

Abstract Background Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in‐frame, partial loss‐of‐function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA‐approved drugs. BMD, however, is understudied and underserved—there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de‐risk potential therapeutics before first‐in‐human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. Methods We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X‐linked) mice, which express an in‐frame ~40 000 bp deletion of exons 45–47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. Results Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild‐type (WT) mice, despite a substantial improvement in pathology over dystrophin‐null mdx52 mice. bmx mice show impaired motor function in grip strength (−39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (−25%, P = 0.0036). Additionally, muscle‐specific serum CK is increased >60‐fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4‐fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20–50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. Conclusions The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Artificial-intelligence tools that enable companies to share data about drug candidates while keeping sensitive information safe can unleash the potential of machine learning and cutting-edge lab techniques, for the common good.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

We are entering an era in which therapeutic proteins are assembled using building block-like strategies, with no standardized schema to discuss these formats. Existing nomenclatures, like AbML, sacrifice human readability for precision. Therefore, considering even a dozen such formats, in combination with hundreds of possible targets, can create confusion and increase the complexity of drug discovery. To address this challenge, we introduce Verified Taxonomy for Antibodies (VERITAS). This classification and nomenclature scheme is extensible to multispecific therapeutic formats and beyond. VERITAS names are easy to understand while drawing direct connections to the structure of a given format, with or without specific target information, making these names useful to adopt in scientific discourse and as inputs to machine learning algorithms for drug development.

Organophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme?s activity and enhancing its stability. Biotherapeutics have achieved global economic success due to their high specificity towards their drug targets, providing exceptional safety and efficiency. The ongoing shift away from small molecule drugs towards biotherapeutics heightens the need to further improve the pharmacokinetics of these biological drugs. Three pervasive obstacles that limit the therapeutic capacity of biotherapeutics are proteolytic degradation, circulating half-life, and the development of anti-drug antibodies. These challenges can culminate in limited efficiency and consequently warrant the need for higher drug doses and more frequent administration. We have explored the coupling of biotherapeutics to long-lived and biocompatible red blood cells (RBCs) to address limited pharmacokinetics. Butyrylcholinesterase (BChE), for example, provides prophylactic protection against organophosphate nerve agents (OPNAs), yet the short circulation life of the drug requires extraordinary doses. Herein, we report the rapid and tunable chemical engineering of BChE to RBC membranes to create a cell-based delivery system that retains the enzyme activity and enhances stability. In a three-step process that first pre-modifies BChE with a cell-reactive polymer chain, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to the surface of each RBC without diminishing the bioscavenging capacity of the enzyme. Critically, this membrane-engineering approach was cell-tolerated with minimal hemolysis observed. These results provide strong evidence for the ability of engineered RBCs to serve as an enhanced biotherapeutic delivery vehicle. Statement of Significance Organophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme's activity and enhancing its stability. (c) 2021 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Traumatic brain injury (TBI) lesions are known to evolve over time, but the duration and consequences of cerebral remodelling are unclear. Degenerative mechanisms occurring in the chronic phase after TBI could constitute "tertiary" lesions related to the neurological outcome.The objective of this prospective study of severe TBI was to longitudinally evaluate the volume of white and grey matter structures and white matter integrity with 2 time-point multimodal MRI.Longitudinal MRI follow-up was obtained for 11 healthy controls (HCs) and 22 individuals with TBI (mean [SD] 60 [15] months after injury) along with neuropsychological assessments. TBI individuals were classified in the "favourable" recovery group (Glasgow Outcome Scale Extended [GOSE] 6–8) and "unfavourable" recovery group (GOSE 3–5) at 5 years. Variation in brain volumes (3D T1-weighted image) and white matter integrity (diffusion tensor imaging [DTI]) were quantitatively assessed over time and used to predict neurological outcome.TBI individuals showed a marked decrease in volumes of whole white matter (median -11.4% [interquartile range -5.8; -14.6]; p < 0.001) and deep grey nuclear structures (-17.1% [-10.6; -20.5]; p < 0.001). HCs did not show any significant change over the same time period. Median volumetric loss in several brain regions was higher with GOSE 3–5 than 6–8. These lesions were associated with lower fractional anisotropy and higher mean diffusivity at baseline. Volumetric variations were positively correlated with normalized fractional anisotropy and negatively with normalized mean diffusivity at baseline and follow-up. A computed predictive model with baseline DTI showed good accuracy to predict neurological outcome (area under the receiver operating characteristic curve 0.82 [95% confidence interval 0.81–0.83])We characterised the striking atrophy of deep brain structures after severe TBI. DTI imaging in the subacute phase can predict the occurrence and localization of these tertiary lesions as well as long-term neurological outcome.Trial registration: ClinicalTrials.gov: NCT00577954. Registered on October 2006.