The p.(Leu97Ile) variant expands the genetic landscape of NEFL-associated Charcot\u2013Marie-tooth neuropathies

Brain axial and radial diffusivity changes with age and gender in healthy adults

The dysmyelinated axons of shiverer mice exhibit impaired conduction characteristics, similar to early postnatal axons before myelination, whereas the patterns of neuronal activity and connectivity are relatively comparable with those of wild-type myelinated axons. This unique dysmyelination pattern is exploited in the present study to determine the role of compact myelin in the loss and recovery of function following traumatic spinal cord injury (SCI). We applied in vivo diffusion tensor imaging (DTI) and post-mortem immunohistochemistry analysis to examine changes in myelin and axonal integrity, and evaluated these changes in concert with the analysis of locomotor function from 1 to 4 weeks following a mid-thoracic contusion injury in homozygous shiverer and heterozygous littermate mice. The DTI biomarkers, axial and radial diffusivities, are noninvasive indicators of axon and myelin integrity in response to SCI of both myelinated and dysmyelinated spinal cord. We show that myelin is critical for normal hind limb function in open field locomotion. However, when the functional outcome is limited during chronic SCI, the extent of recovery is associated with residual axonal integrity and independent of the extent of intact myelin at the lesion epicenter.

We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1-/- female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1flx/flx , retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1-/- optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxβ in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxβ displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1-/- mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1 Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1-/- optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE.SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

: The overall goal of this project is to determine the underlying mechanisms for the neurological symptoms associated with Gulf War Illness. The central hypothesis is that subthreshold exposures to organophosphates-OPs (defined as exposures not associated with acute signs of toxicity) may have adversely affected axonal transport and/or myelin integrity in affected individuals. We are studying two OPs, a representative insecticide that was used in the first gulf war, chlorpyrifos (CPF), and a representative, nerve agent, diisopropylfluorophosphate (DFP) in rats. The first two years of this proposal have been primarily dedicated to Specific Aim #1: which has been designed to evaluate OP effects on axonal transport in the living rat brain using manganese-enhanced magnetic resonance imaging (MEMRI) of the optic nerve axonal projections from the retina to the superior colliculus. The following procedures have been conducted to date (N=6): 1) baseline MRI scans; 2) daily injections of vehicle or chlorpyrifos (3.0-18.0 mg/kg) x 14 days; 2) a second MRI scan on the day following the last drug injection; 3) a third scan after a 4 week (OP-free) washout period. For each animal, a separate 6 hour and 24 hour scan was performed after Mn2+ eye injection. For this work a manuscript is currently under review for publication. In this work we also evaluated the effects of an acute (single) exposure to CPF. We have also completed the MR scanning portion for the DFP study and a preliminary analysis indicates similar results (persistent impairments of axonal transport). The experiments for specific aim #2 (devoted to the evaluation of OP-related effects on myelin with diffusion tensor imaging-(DTI) and histology are currently underway.

MORC2 mutations have been described as a rare cause of axonal Charcot-Marie-Tooth disease (CMT2Z). The aim of this work was to determine the frequency and distribution of these mutations throughout Spain, toprovide a comprehensivephenotypical description and, if possible, to establish a genotype-phenotype correlation. Retrospectively, data on patients diagnosed with CMT2Z in Spain werecollected and clinical, electrophysiological and muscle imaging information were analysed. Fifteen patients with CMT2Z were identified throughout Spain, seven of them belonging to a single kindred, whilst the rest were sporadic. The most common mutation was p.R252W, and four new mutations were identified. Eleven patients were categorized as having a scapuloperoneal phenotype, with asymmetric muscle weakness, early proximal upper limb involvement and frequent spontaneous muscular activity with distal sensory impairment and pes cavus, whilst two presented with a more classic length dependent sensory motor phenotype. This distinction was corroborated by the distribution of musclefatty infiltration in muscle imaging. Two other patients were classified as having a neurodevelopmental phenotype consisting in congenital or early onset, delay in motor milestones, and global developmental delay in one of them. Nerve conduction studies revealed an unequivocally axonal neuropathy with frequent spontaneous activity, and serum creatine kinase levels were increased in 50% of the patients. MORC2 mutations are a rare cause of CMT in Spain, but in-depth phenotyping reveals a recognizable phenotypic spectrum that will be clinically relevant for future identification of this disease.

Myelin growth is a tightly regulated process driven by multiple signals. ERK1/2-MAPK signaling is an important regulator of myelin thickness. Because, in demyelinating diseases, the myelin formed during remyelination fails to achieve normal thickness, increasing ERK1/2 activity in oligodendrocytes is of obvious therapeutic potential for promoting efficient remyelination. However, other studies have suggested that increased levels of ERK1/2 activity could, in fact, have detrimental effects on myelinating cells. Because the strength, duration, or timing of ERK1/2 activation may alter the biological outcomes of cellular responses markedly, here, we investigated the effect of modulating ERK1/2 activity in myelinating cells using transgenic mouse lines in which ERK1/2 activation was upregulated conditionally in a graded manner. We found enhanced myelin gene expression and myelin growth in the adult CNS at both moderate and hyperactivated levels of ERK1/2 when upregulation commenced during developmental myelination or was induced later during adulthood in quiescent preexisting oligodendrocytes, after active myelination is largely terminated. However, a late onset of demyelination and axonal degeneration occurred at hyperelevated, but not moderately elevated, levels regardless of the timing of the upregulation. Similarly, myelin and axonal pathology occurred with elevated ERK1/2 activity in Schwann cells. We conclude that a fine tuning of ERK1/2 signaling strength is critically important for normal oligodendrocyte and Schwann cell function and that disturbance of this balance has negative consequences for myelin and axonal integrity in the long term. Therefore, therapeutic modulation of ERK1/2 activity in demyelinating disease or peripheral neuropathies must be approached with caution. ERK1/2-MAPK activation in oligodendrocytes and Schwann cells is an important signal for promoting myelin growth during developmental myelination. Here, we show that, when ERK1/2 are activated in mature quiescent oligodendrocytes during adulthood, new myelin growth is reinitiated even after active myelination is terminated, which has implications for understanding the mechanism underlying plasticity of myelin in adult life. Paradoxically, simply increasing the "strength" of ERK1/2 activation changed the biological outcome from beneficial to detrimental, adversely affecting myelin and axonal integrity in both the CNS and PNS. Therefore, this study highlights the complexity of ERK1/2-MAPK signaling in the context of oligodendrocyte and Schwann cell function in the adult animal and emphasizes the need to approach potential therapeutic modulation of ERK1/2 activity with caution.

AXONAL DENSITY AND MYELIN INTEGRITY IN COGNITIVE DECLINE: A DIFFUSIONAL KURTOSIS IMAGING STUDY

Recent data indicate that patients after the Fontan procedure are at risk for significant liver dysfunction; however, the prevalence and extent of liver disease in the Fontan population remains unknown. Furthermore, limited data exist in regard to screening for liver disease in adult Fontan patients. We sought to determine the prevalence of liver disease in adult patients following the Fontan procedure using computed tomography (CT) and serum biomarkers of liver fibrosis. Adult Fontan patients underwent screening for liver disease as part of their annual evaluation. Screening consisted of laboratory evaluation and dual-phase liver CT scan. Laboratory evaluation included analysis of liver function, viral hepatitis serologies, and FibroSURE panel (LabCorp), a test that analyzes the results of serum biomarkers to provide a quantitative surrogate marker for liver fibrosis. Sixteen patients, mean age 30.3 (range 20-41) years, were enrolled in the study. Mean length of follow-up from time of Fontan palliation was 20.5 (range 11-33) years. No patients had serologic evidence of viral hepatitis or synthetic liver dysfunction. Twelve patients (75%) had abnormal FibroSURE scores, seven (44%) had elevated FibroSURE scores predictive of Metavir fibrosis stage F2 or greater on liver biopsy, and one (6%) had a FibroSURE score predictive of cirrhosis on biopsy. All 16 patients had abnormal radiologic liver findings identified on CT, including heterogeneous enhancement in 11 (69%), varices in six (38%), and liver nodules in five patients (31%). Length of time since Fontan surgery correlated significantly with an elevated FibroSURE score (P = .05) and having more CT scan abnormalities (P = .04). Liver fibrosis detected by serum biomarkers and dual phase CT scan is common in adult patients following the Fontan procedure. Further studies are needed to determine the long-term clinical significance of these findings.

Promising treatments are being developed to promote functional recovery after spinal cord injury (SCI). Magnetic resonance imaging, specifically Diffusion Tensor Imaging (DTI) has been shown to non-invasively measure both axonal and myelin integrity following traumatic brain and SCI. A novel data-driven model-selection algorithm known as Diffusion Basis Spectrum Imaging (DBSI) has been proposed to more accurately delineate white matter injury. The objective of this study was to investigate whether DTI/DBSI changes that extend to level of the cerebral peduncle and internal capsule following a SCI could be correlated with clinical function. A prospective non-randomized cohort of 23 patients with chronic spinal cord injuries and 17 control subjects underwent cranial diffusion weighted imaging, followed by whole brain DTI and DBSI computations. Region-based analyses were performed on cerebral peduncle and internal capsule. Three subgroups of patients were included in the region-based analysis. Tract-Based Spatial Statistics (TBSS) was also applied to allow whole-brain white matter analysis between controls and all patients. Functional assessments were made using International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) as modified by the American Spinal Injury Association (ASIA) Scale. Whole brain white matter analysis using TBSS finds no statistical difference between controls and all patients. Only cervical ASIA A/B patients in cerebral peduncle showed differences from controls in DTI and DBSI results with region-based analysis. Cervical ASIA A/B SCI patients had higher levels of axonal injury and edema/tissue loss as measured by DBSI at the level of the cerebral peduncle. DTI Fractional Anisotropy (FA), Axial Diffusivity (AD) and Radial Diffusivity (RD) was able to detect differences in cervical ASIA A/B patients, but were non-specific to pathologies. Increased water fraction indicated by DBSI non-restricted isotropic diffusion fraction in the cerebral peduncle, explains the simultaneously increased DTI AD and DTI RD values. Our results further demonstrate the utility of DTI to detect disruption in axonal integrity in white matter, yet a clear shortcoming in differentiating true axonal injury from inflammation/tissue loss. Our results suggest a preservation of axonal integrity at the cortical level and has implications for future regenerative clinical trials.

G.P.88 Peripheral neuropathies caused by mutations in the myelin protein zero

ABSTRACTBackgroundINF2 mutations cause focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth disease (CMT). Accurate genetic diagnosis is critical, as INF2‐related FSGS is typically resistant to immunotherapy yet rarely recurs after transplantation, and its associated neuropathy can mimic treatable immune‐mediated disorders such as chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).MethodsWe performed a multicenter study investigating 3329 Japanese patients with inherited peripheral neuropathies/CMT who underwent gene panel sequencing or whole‐exome analysis between 2007 and 2024. Clinical data, including electrophysiological assessments, were obtained from the patients' medical records.ResultsWe identified six pathogenic INF2 variants in eight patients, all of which were located within the diaphanous inhibitory domain. Structural modeling revealed clustering of variants near the diaphanous autoregulatory domain‐binding pocket, which is critical for INF2 autoinhibition. Clinically, all cases were sporadic, with a median age at neurological onset of 9 years. All patients exhibited lower limb weakness, and 6/8 (75%) had sensory disturbances. All patients also developed kidney dysfunction, with 7/8 (88%) progressing to end‐stage renal disease at a median age of 15 years. Furthermore, all patients showed demyelinating neuropathy, and 2/8 (25%) received immunotherapy due to suspected immune‐mediated neuropathy.ConclusionAlthough INF2 variants are a rare cause of CMT in Japan, they should be considered in pediatric patients with demyelinating neuropathy and early‐onset proteinuria, even in the absence of a family history. Blood and urine tests assessing renal dysfunction can provide guidance for appropriate genetic testing.

A high incidence of asymptomatic neuromuscular disease was found. More than half showed EMG evidence of a `myopathy' and of denervation. The commonest syndrome was a neuromyopathy with myopathic units and spontaneous muscle activity, especially fasciculations and nerve fibre loss but with normal or near normal nerve conduction. This is thought to be the result of a neuronal or axonal disorder.

Altered myelination and axonal integrity in adults with childhood lead exposure: A diffusion tensor imaging study

By the time patients with congenital heart disease (CHD) reach adolescence and early adulthood, rhythm status has often become an active issue, if not the central issue, in their cardiac management. For some, arrhythmias are intrinsic to the structural malformation itself, as is the case with Wolff-Parkinson-White syndrome in the setting of Ebstein’s anomaly, or atrioventricular (AV) block in the setting of “congenitally corrected” transposition of the great arteries (L-TGA). For most other CHD patients, arrhythmias represent an acquired condition related to the unique myocardial substrate created by surgical scars in conjunction with cyanosis and abnormal pressure/volume loads of long duration. This review will attempt to address the major rhythm difficulties faced by adults with CHD, concentrating on electrophysiological features that distinguish CHD from more conventional forms of adult heart disease. A listing of specific arrhythmias and commonly associated congenital defects is provided in Table 1 to serve as an outline for the discussion. View this table: TABLE 1. Specific Arrhythmias and Associated Defects in Adults With Congenital Heart Disease As of 2001, there were estimated to be 800 000 adults with CHD living in the United States,1 a number that has grown steadily as more survivors of childhood surgery reach maturity. Of these, roughly 45% are considered to have mild forms of CHD (eg, atrial septal defect, valvar pulmonary stenosis), 40% are classified as having moderate disease (eg, tetralogy of Fallot, Ebstein’s anomaly), and 15% are considered to have complex disease (eg, single ventricle, transposition of the great arteries). Although arrhythmias can develop within any of the 3 subgroups, the incidence is highest for patients in the moderate and severe categories. Tetralogy of Fallot is an instructive example of moderate CHD with a large arrhythmia burden. As many as one third of patients with repaired tetralogy develop symptomatic atrial tachycardias by …

Toxic compounds which interfere with axonal integrity and transport are many and varied. Among the first to be utilized were the spindle inhibitors such as colchicine and the vinca alkaloids (e.g. vincristine, vinblastine). These compounds, together with podophyllotoxin and the maytansinoids (maytansine and maytanprine), possess a special affinity for tubulin, block the formation of axonal microtubules and, perhaps for these reasons, disrupt axonal transport. They also induce an accumulation of l0-nm neurofilaments following direct application to nervous tissue, although the basis for this effect is unknown (reviewed in [5]). Systemic administration of such agents (e.g. vincristine) produces a distal, sensori-motor neuropathy in man [2]. A similar type of neuropathy also accompanies systemic treatment with agents which have no known adverse effects on microtubules. Some of these compounds, such as pyridinethione and p-bromophenylacetylurea, induce an abnormality in fast anterograde transport and/or retrograde return of fast transported materials, as well as an accumulation of tubulo-vesicular profiles, prior to degeneration of distal axons [10]. Other agents apparently inactive on microtubules, such as the hexacarbons (e.g. 2-hexanone, 2,5-hexanedione, 2,4-hexanediol), acrylamide and carbon disulfide, induce early distal axonal changes in the form of multifocal accumulations of 10-nm neurofilaments, together with varying amounts of smooth endoplasmic reticulum, vesicles and mitochondria [17, 21 ]. A similar pattern of neurofila mentous giant axonal degeneration is seen in proximal axons of animals systemically intoxicated with ββ’-iminodipropionitrile (IDPN), an agent which impairs anterograde transport of neurofilament triplet proteins while producing little distal nerve-fiber breakdown [6], Some of these agents (2,5-hexanedione, IDPN, acrylamide) induce similar changes in the axonal cytoskeleton following direct application to peripheral nerves, and are useful in exploring the relationship between perturbations of axonal transport and the onset of nerve-fiber breakdown. This paper describes recent advances in our understanding of these questions which have come from the use of 2,5-hexanedione (2,5-HD) as an experimental probe.