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Critical Illness Neuromyopathy (CIPNM) is a complication in sepsis patients with still enigmatic disease mechanisms. We investigated a novel electrical stimulation method to better define neuromuscular dysfunction in patients with CIPNM.We studied 18 sepsis CIPNM patients on intensive care units, 13 at an early and 5 at a later disease stage, 7 sepsis control, and 8 neuropathy control patients. We applied slow conditioning electrical pulses at motor nerves and directly at the muscle to investigate a facilitation phenomenon (FP) of small or absent compound motor action potentials (CMAPs).Serial pulses induced a 2 to 490-fold increase in CMAP amplitudes in 17/18 Intensive Care Unit (ICU)-CIPNM patients (p 0.001). These effects were short lived and reproducible. Direct muscle stimulation in the tibialis anterior muscle resulted in up to 130-fold FP in 7/9 patients tested (p 0.01). In 4/5 post-ICU CIPNM patients FP was up to 10-fold. None of the 7 ICU sepsis control patients without CIPNM with similar disease severity and none of 8 neuropathy patients showed FP (p 0.001). On needle EMG only 5/16 ICU patients tested revealed spontaneous activity.Conditioning electrical stimulation detected a functional component of the disease process showing temporary improvement in sepsis-associated CIPNM.New test differentiates functional from degenerative pathology.

The pathogenesis of diabetic neuropathy is complex, and various pathogenic pathways have been proposed. A better understanding of the pathophysiology is warranted for developing novel therapeutic strategies. Here, we summarize recent evidence from experiments using animal models of type 1 and type 2 diabetes showing that low-grade intraneural inflammation is a facet of diabetic neuropathy. Our experimental data suggest that these mild inflammatory processes are a likely common terminal pathway in diabetic neuropathy associated with the degeneration of intraepidermal nerve fibers. In contrast to earlier reports claiming toxic effects of high-iron content, we found the opposite, i.e., nutritional iron deficiency caused low-grade inflammation and fiber degeneration while in normal or high non-heme iron nutrition no or only extremely mild inflammatory signs were identified in nerve tissue. Obesity and dyslipidemia also appear to trigger mild inflammation of peripheral nerves, associated with neuropathy even in the absence of overt diabetes mellitus. Our finding may be the experimental analog of recent observations identifying systemic proinflammatory activity in human sensorimotor diabetic neuropathy. In a rat model of type 1 diabetes, a mild neuropathy with inflammatory components could be induced by insulin treatment causing an abrupt reduction in HbA1c. This is in line with observations in patients with severe diabetes developing a small fiber neuropathy upon treatment-induced rapid HbA1c reduction. If the inflammatory pathogenesis could be further substantiated by data from human tissues and intervention studies, anti-inflammatory compounds with different modes of action may become candidates for the treatment or prevention of diabetic neuropathy.

Peripheral diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus. Previous studies showed an association between dietary iron load and inflammation in the development of PDN in a rat model of type 1 diabetes (T1D). Here we investigated the role of iron and neural inflammation in development of PDN in a animal model of obesity and type 2 diabetes (T2D). 3-month-old db/db mice were fed with a high, standard or low iron diet for 4 months. High iron chow lead to a significant increase in motor nerve conduction velocities compared to mice on standard and low iron chow. Direct beneficiary effects on lowering blood glucose and HbA1c concentrations were shown in the high iron treated diabetic mice. Numbers of pro-inflammatory M1 macrophages were reduced in nerve sections, and anti-inflammatory M2 macrophages were increased in db/db mice on high iron diet compared to other groups. These results confirm and extend our previous findings in STZ-diabetic rats by showing that dietary non-hem iron supplementation may partly prevent the development of PDN in opposition to iron restriction. The identification of these dietary iron effects on the metabolic and inflammatory mechanisms of PDN supports a role of dietary iron and leads us to suggest testing for iron levels in human diabetic patients.

Die Pathogenese der diabetischen Neuropathie ist komplex und bisher nicht eindeutig geklart. Ein besseres Verstandnis derselben ware jedoch Voraussetzung fur neue therapeutische Ansatze. Wir geben eine Ubersicht moglicher Pathomechanismen und daraus abgeleiteter Therapiestrategien unter Einbeziehung eigener Studienergebnisse. Neben Hyperglykamie stellen Adipositas und Dyslipidamie, insbesondere beim Typ-2-Diabetes, bedeutende Faktoren fur die Entwicklung einer diabetischen Neuropathie dar. Die heute meist angewandten Therapien einschlieslich der strengen Glukosekontrolle sind bisher noch unbefriedigend. Unter zu rascher HbA1c-Wert-Senkung (HbA1c: glykiertes Hamoglobin Typ A1c) muss zudem mit dem Auftreten einer therapieinduzierten Neuropathie im Sinne einer iatrogen induzierten Small-Fiber-Neuropathie gerechnet werden. Ergebnisse tierexperimenteller Arbeiten weisen darauf hin, dass intraneurale zellvermittelte Entzundungsvorgange eine gemeinsame Endstrecke in der Neuropathiepathogenese experimenteller Diabetesmodelle bilden. Die damit korrelierte Degeneration terminaler Nervenfasern wird durch systemischen Eisenmangel weiter verstarkt. Falls diese experimentellen Beobachtungen auf den Menschen ubertragbar sind, lassen sich neue Therapieansatze ableiten. Diese waren eine antiinflammatorische Behandlung mit verschiedenen Angriffspunkten, eine ausreichende Supplementierung von Eisen und die Behandlung des metabolischen Syndroms sowie der Adipositas durch Steigerung der korperlichen Aktivitat, die nach jungeren Studien auch antiinflammatorische Effekte haben soll und als nichtmedikamentoses Verfahren an Bedeutung gewinnen konnte.

Background A treatment-induced drop in HbA1c has been suggested to be a risk factor for TIND. Methods From 60 included patients with severe diabetes mellitus (HbA1c over 8.5) only 21 patients adhered to the study protocol over 1 year with a battery of autonomic nervous system tests scheduled before and after starting antidiabetic treatment. Results In patients with a drop of HbA1c greater than 2 per cent points only some neurophysiologic tests and lab values tended to deteriorate with a trend to improve at later time points along the study. None of these changes were statistically significant, most likely because the study failed to reach the planned number of patients. Conclusion Poor adherence to diabetes treatment and to following the study protocol were the assumed obstacles in our patient cohort selected for very high HbA1c levels. In future studies a multi-center trial and case numbers of up to 500 patients may be needed to account for drop outs in the range observed here. Moreover, the number of tests in each patient at each visit may have to be reduced and special educational group sessions are warranted to cope with the limited adherence. Trial registration Ethic Committee University of Leipzig 439/15-ek. Registered 22 April 2016

Sepsis-associated encephalopathy (SAE) is a frequent complication in patients with severe systemic infection resulting in acute brain dysfunction and incapacitating long-term sequelae. SAE includes delirium, premature death, post-traumatic stress disorder, and major long-term cognitive impairment. The underlying pathophysiology of SAE is largely unresolved and specific treatment options are missing. We induced the peritoneal contamination and infection (PCI) sepsis model in 769 mice and compared these with 259 control mice. We found that experimental sepsis causes synaptic pathology in the brain characterized by severely disordered synaptic plasticity with reduced long-term potentiation, changes in CA1 pyramidal neuron dendritic spines, and behavioral abnormalities indicating cognitive dysfunction. Using electrophysiology, we found reduced frequency of quantal and spontaneous excitatory postsynaptic currents whereas amplitudes of miniature, spontaneous, and evoked excitatory currents were increased, pointing towards a homeostatic synaptic scaling mechanism. Corresponding to dysfunctional excitatory synaptic function we discovered downregulation of genes related to neuronal and synaptic signaling in the brain, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), members of the transcription-regulatory EGR gene family, and the gene for dual-specificity phosphatase 6 (Dusp6). At the protein level, ARC expression and MAP kinase signaling in the brain were affected. For targeted rescue of dysfunctional synaptic signaling and plasticity, we overexpressed ARC in the hippocampus by microinjection of an adeno-associated virus containing a neuron-specific plasmid of the ARC transgene. Hereby we achieved recovery of defective synaptic plasticity in the hippocampal Schaffer collateral-CA1 pathway and improvement of memory dysfunction. Using a different rescue paradigm, PCI mice were subjected to enriched environment providing multiple activating stimuli. Enriched environment led to restoration of disordered long-term potentiation and memory, thus demonstrating the potential for activity-induced improvement. Together, we identified synaptic pathomechanisms of SAE after severe systemic infection and provide a conceptual approach to treat SAE-related disease mechanisms which may be applicable to patients afflicted with SAE.

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the peripheral demyelinating neuropathy was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands.Summary blurbA dimeric prion protein ligand activates Adgrg6 but fails to induce pro-myelination signaling upon chronic treatment in a mouse model of peripheral demyelination.

Some mutations affecting dynamin 2 (DNM2) can cause dominantly inherited Charcot–Marie–Tooth (CMT) neuropathy. Here, we describe the analysis of mice carrying the DNM2 K562E mutation which has been associated with dominant-intermediate CMT type B (CMTDIB). Contrary to our expectations, heterozygous DNM2 K562E mutant mice did not develop definitive signs of an axonal or demyelinating neuropathy. Rather, we found a primary myopathy-like phenotype in these mice. A likely interpretation of these results is that the lack of a neuropathy in this mouse model has allowed the unmasking of a primary myopathy due to the DNM2 K562E mutation which might be overshadowed by the neuropathy in humans. Consequently, we hypothesize that a primary myopathy may also contribute to the disease mechanism in some CMTDIB patients. We propose that these findings should be considered in the evaluation of patients, the determination of the underlying disease processes and the development of tailored potential treatment strategies., Human Molecular Genetics, 29 (8), ISSN:0964-6906, ISSN:1460-2083

Introduction The aim of this study was to determine the diagnostic usefulness of skin punch biopsies with emphasis on visualization and quantification of T-cells and macrophages in patients with polyneuropathies. Methods: We quantified inflammatory cells in skin samples (lower leg, upper thigh) in 187 patients and compared data with counts in their sural nerve biopsies and with skin biopsies from 32 healthy volunteers. Results: Vessel-bound T-cells and macrophages were increased in proximal and distal skin samples of neuropathy patients compared with controls (P

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands.

Objectives: The goal of this study was to analyze safety and assess the efficacy of standard plasma exchange (PE) compared with immunoadsorption (IA) alone, or an alternating combination of both in deteriorating myasthenia gravis (MG). Methods: A total of 72 patients with MG who had received PE procedures for treatment of severe deterioration were retrospectively analyzed. They received either five cycles of PE (1–1.5 plasma volumes), or five cycles of IA in line with plasma separation, or a sequential alternating procedure of one cycle of PE followed by two cycles of IA, which was repeated once or more if needed. Results: A total of 19 patients received PE, 24 patients IA, and 29 the alternating combination therapy. All groups were equally distributed by sex and mean MG score before treatment. The number of treatment cycles and days on therapy did not differ between the groups. Mean MG scores at discharge were 3.0 (PE), 1.8 (IA) and 1.6 (combination) ( p = 0.028 for combination versus PE). Inpatient time was 30.7 days (PE), 22.3 days (IA) and 20.0 days in combination therapy ( p < 0.05 for combination versus PE). Side effects such as allergic reactions or hypocoagulability were significantly more frequent in the PE group (37% in PE versus 4% in IA and 3.6% in the alternating combination, p < 0.05). Conclusion: Semiselective IA in combination with PE, and to a lesser extent IA alone, was associated with a shorter hospital stay and more pronounced reduction of the MG score than PE.

Objective: To assess the therapy-related risk of malignancies in mitoxantrone-treated patients with multiple sclerosis. Methods: This retrospective observational cohort study included all mitoxantrone-treated patients with multiple sclerosis seen at our department between 1994 and 2007. We collected follow-up information on medically confirmed malignancies, life status, and cause of death, as of 2010. Malignancy rates were compared to the German national cancer registry matched for sex, age, and year of occurrence. Results: Follow-up was completed in 676 of 677 identified patients. Median follow-up time was 8.7 years (interquartile range 6.8–11.2), corresponding to 6,220 person-years. Median cumulative mitoxantrone dose was 79.0 mg/m 2 (interquartile range 50.8–102.4). Thirty-seven patients (5.5%) were diagnosed with a malignancy after mitoxantrone initiation, revealing a standardized incidence ratio of 1.50 (95% confidence interval [CI] 1.05–2.08). Entities included breast cancer (n = 9), colorectal cancer (n = 7), acute myeloid leukemia (n = 4, 0.6%), and others (each entity n = 1 or 2). The standardized incidence ratio of colorectal cancer was 2.98 (95% CI 1.20–6.14) and of acute myeloid leukemia 10.44 (95% CI 3.39–24.36). It was not increased for other entities including breast cancer. Multivariate Cox regression identified higher age at treatment initiation but neither cumulative mitoxantrone dose (>75 vs ≤75 mg/m 2 ) nor treatment with other immunosuppressive drugs or sex as a risk factor. Fifty-five patients had died, among them 12 of a malignancy and 43 reportedly of other causes. Conclusions: While the overall incidence of malignancies was only mildly increased, the risk of leukemia and colorectal cancer was heightened. If confirmed, posttherapy colonoscopy could become advisable.

Background Iron is an essential but potentially toxic metal in mammals. Here we investigated a pathogenic role of exogenous iron in peripheral diabetic neuropathy (PDN) in an animal model for type 1 diabetes. Methods Diabetes was induced by a single injection of streptozotocin (STZ) in 4-month-old Sprague–Dawley rats. STZ-diabetic rats and non-diabetic rats were fed with high, standard, or low iron diet. After three months of feeding, animals were tested. Results STZ-rats on standard iron diet showed overt diabetes, slowed motor nerve conduction, marked degeneration of distal intraepidermal nerve fibers, mild intraneural infiltration with macrophages and T-cells in the sciatic nerve, and increased iron levels in serum and dorsal root ganglion (DRG) neurons. While motor fibers were afflicted in all STZ-groups, only a low iron-diet led also to reduced sensory conduction velocities in the sciatic nerve. In addition, only STZ-rats on a low iron diet showed damaged mitochondria in numerous DRG neurons, a more profound intraepidermal nerve fiber degeneration indicating small fiber neuropathy, and even more inflammatory cells in sciatic nerves than seen in any other experimental group. Conclusions These results indicate that dietary iron-deficiency rather than iron overload, and mild inflammation may both promote neuropathy in STZ-induced experimental PDN.

Here, we investigated inflammatory signs of peripheral nerves in leptin-deficient obese ob/ob mice and the modulating effects of the exogenous iron load.Ob/ob and ob/+ control mice were fed with high, standard, or low iron diet for four months.We found intraepidermal nerve fiber degeneration in foot skin and low-grade neuropathic abnormalities including mildly slowed motor and compound sensory nerve conduction velocities and low-grade macrophage and T-cell infiltration without overt neuropathology in sciatic nerves of all ob/ob mice. Low dietary iron load caused more pronounced abnormalities than high iron load in ob/ob mice.Our data suggest that dietary non-heme iron deficiency may be a modulating factor in the pathogenesis of peripheral neuropathy in obese ob/ob mice with metabolic syndrome. Once the mechanisms can be further elucidated, how low dietary iron augments peripheral nerve degeneration and dysfunction via pro-inflammatory pathways and new therapeutic strategies could be developed.CMAP: compound muscle action potential; cSNCV: compound sensory nerve conduction velocity; IENFD: intraepidermal nerve fiber density; LDL: low-density lipoprotein; MetS: metabolic syndrome; MNCV: motor conduction velocity; NCV: nerve conduction velocity; PN: peripheral neuropathy; PNS: peripheral nervous system; STZ: streptozotocin; T2D: type 2 diabetes mellitus; TNF alpha: tumor necrosis factor alpha; WHO: World Health Organization.

Myelination requires extensive plasma membrane rearrangements, implying that molecules controlling membrane dynamics play prominent roles. The large GTPase dynamin 2 (DNM2) is a well-known regulator of membrane remodeling, membrane fission, and vesicular trafficking. Here, we genetically ablated Dnm2 in Schwann cells (SCs) and in oligodendrocytes of mice. Dnm2 deletion in developing SCs resulted in severely impaired axonal sorting and myelination onset. Induced Dnm2 deletion in adult SCs caused a rapidly-developing peripheral neuropathy with abundant demyelination. In both experimental settings, mutant SCs underwent prominent cell death, at least partially due to cytokinesis failure. Strikingly, when Dnm2 was deleted in adult SCs, non-recombined SCs still expressing DNM2 were able to remyelinate fast and efficiently, accompanied by neuropathy remission. These findings reveal a remarkable self-healing capability of peripheral nerves that are affected by SC loss. In the central nervous system, however, we found no major defects upon Dnm2 deletion in oligodendrocytes., eLife, 8, ISSN:2050-084X

Introduction: Sural nerve biopsy is an important means of establishing the diagnosis of inflammatory neuropathies. We investigated the diagnostic value of endoneurial edema. Methods: Diagnostic sural nerve biopsies from 42 patients with inflammatory and 28 patients with noninflammatory neuropathies were re-evaluated for the presence of endoneurial edema. Edema was assessed on hematoxylin-eosin stained paraffin and frozen sections and on azure II-methylene blue stained semithin sections. We determined the area of endoneurial edema on digitized images in relation to the entire endoneurial area of each fascicle. Results: Edema was more extensive in neuropathies with short disease duration (≤12 months) as compared to long duration (>12 months; P

Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.

See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

Neuromyelitis optica (NMO) is causally linked to autoantibodies (ABs) against aquaporin 4 (AQP4). Here, we focused on the pathogenic effects exclusively mediated by human ABs to AQP4 in vivo. We performed cell-free intrathecal (i.th.) passive transfer experiments in Lewis rats using purified patient NMO immunoglobulin G (IgG) and various recombinant human anti-AQP4 IgG-ABs via implanted i.th. catheters. Repetitive application of patient NMO IgG fractions and of recombinant human anti-AQP4 ABs induced signs of spinal cord disease. Magnetic resonance imaging (MRI) revealed longitudinal spinal cord lesions at the site of application of anti-AQP4 IgG. Somatosensory evoked potential amplitudes were reduced in symptomatic animals corroborating the observed functional impairment. Spinal cord histology showed specific IgG deposition in the grey and white matter in the affected areas. We did not find inflammatory cell infiltration nor activation of complement in spinal cord areas of immunoglobulin deposition. Moreover, destructive lesions showing axon or myelin damage and loss of astrocytes and oligodendrocytes were all absent. Immunoreactivity to AQP4 and to the excitatory amino acid transporter 2 (EAAT2) was markedly reduced whereas immunoreactivity to the astrocytic marker glial fibrillary acid protein (GFAP) was preserved. The expression of the NMDA-receptor NR1 subunit was down-regulated in areas of IgG deposition possibly induced by sustained glutamatergic overexcitation. Disease signs and histopathology were reversible within weeks after stopping injections. We conclude that in vivo application of ABs directed at AQP 4 can induce a reversible spinal cord disease in recipient rats by inducing distinct histopathological abnormalities. These findings may be the experimental correlate of “penumbra-like” lesions recently reported in NMO patients adjacent to effector-mediated tissue damage.

We performed electrophysiological studies in myelin protein mutant mice in order to characterize nerve conduction changes. We performed neurographic studies on the facial and sciatic nerves and needle electromyography (EMG). Mice homozygously deficient for the peripheral myelin protein 22 gene (Pmp22-/-) exhibited increased motor latencies, reduced nerve conduction velocities, and polyphasia of the M-response, which are the typical electrophysiological signs of dysmyelination. PMP22 +/- mice developed only mild conduction slowing at an old age and a mild reduction of the M-amplitude, which indicates mild axonal dysfunction. Mice overexpressing Pmp22 developed severe electrophysiological signs of dysmyelination. In myelin protein zero-deficient mice (P0 -/-), we found alterations similar to those found in Pmp22 -/- mice, whereas P0 +/- mice developed mildly increased sciatic nerve F-wave latencies only late in life, which indicates only mild dysmyelination. Connexin 32-deficient mice showed electrophysiological evidence of mild axonal damage. By EMG, we found the clinical and electrophysiological signs of neuromyotonia, that is, continuous spontaneous motor unit discharges, often in rhythmic patterns (myokymia), in P0 -/-, Pmp22 -/-, Trembler, Trembler-J, and Pmp22-overexpressing mice. This indicates abnormal impulse generation in these dysmyelinated nerves. In summary, our studies demonstrate nerve conduction changes in mice with myelin protein gene defects that are similar to those found in patients with Charcot-Marie-Tooth disorders. In addition, we identified new mouse models of hereditary neuromyotonia.

Human Molecular Genetics, 14 (23), ISSN:0964-6906, ISSN:1460-2083

Introduction Nerve conduction studies provide insights into the functional consequences of axonal and myelin pathology in peripheral neuropathies. We investigated whether isoflurane inhalation anesthesia alters F-wave latencies and F-persistence in the sciatic nerve of adult rats. Methods Ten rats were investigated at 3 different isoflurane concentrations followed by ketamine-xylazine injection anesthesia. To assess F-wave latencies, a stimulation paradigm was chosen to minimize H-reflex masking of F-waves. Results F-wave persistence rates were reduced with 3.5% isoflurane concentration at 4 and 10 Hz supramaximal stimulation and marginally reduced with 2.5% isoflurane when compared with ketamine-xylazine. F-wave amplitudes decreased progressively with rising stimulus frequency in all types of anesthesia and most at 3.5% isoflurane concentration. Conclusions The type of anesthesia and the stimulus repetition rate have an impact on some F-wave parameters. Higher isoflurane concentrations and repetition rates are not recommended in experimental studies using rat neuropathy models where F-waves are of interest. Muscle Nerve 50:257–261, 2014

Spinal muscular atrophy with respiratory distress type 1 is a neuromuscular disorder characterized by progressive weakness and atrophy of the diaphragm and skeletal muscles, leading to death in childhood. No effective treatment is available. The neuromuscular degeneration (Nmd(2J)) mouse shares a crucial mutation in the immunoglobulin mu-binding protein 2 gene (Ighmbp2) with spinal muscular atrophy with respiratory distress type 1 patients and also displays some basic features of the human disease. This model serves as a promising tool in understanding the complex mechanisms of the disease and in exploring novel treatment modalities such as insulin-like growth factor 1 (IGF1) which supports myogenic and neurogenic survival and stimulates differentiation during development. Here we investigated the treatment effects with polyethylene glycol-coupled IGF1 and its mechanisms of action in neurons and muscles. Polyethylene glycol-coupled IGF1 was applied subcutaneously every second day from post-natal Day 14 to post-natal Day 42 and the outcome was assessed by morphology, electromyography, and molecular studies. We found reduced IGF1 serum levels in Nmd(2J) mice 2 weeks after birth, which was normalized by polyethylene glycol-coupled IGF1 treatment. Nmd(2J) mice showed marked neurogenic muscle fibre atrophy in the gastrocnemius muscle and polyethylene glycol-coupled IGF1 treatment resulted in muscle fibre hypertrophy and slowed fibre degeneration along with significantly higher numbers of functionally active axonal sprouts. In the diaphragm with predominant myogenic changes a profound protection from muscle fibre degeneration was observed under treatment. No effects of polyethylene glycol-coupled IGF1 were monitored at the level of motor neuron survival. The beneficial effects of polyethylene glycol-coupled IGF1 corresponded to a marked activation of the IGF1 receptor, resulting in enhanced phosphorylation of Akt (protein kinase B) and the ribosomal protein S6 kinase in striated muscles and spinal cord from Nmd(2J) mice. Based on these findings, polyethylene glycol-coupled IGF1 may hold promise as a candidate for future treatment trials in human patients with spinal muscular atrophy with respiratory distress type 1.

The role of CD8⁺ T cells in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is still unclear. We describe here significantly reduced disease activity of EAE both in Lewis rats depleted of CD8⁺ T cells by monoclonal antibodies and CD8 knockout rats, which was accompanied by reduced leukocyte infiltration into the spinal cord. We detected myelin basic protein (MBP)-specific CD8⁺ T cells in peripheral lymphoid organs of CD8-depleted animals which, however, failed to differentiate into interferon-γ-producing effector cells. Our results indicate that CD8⁺ T cells interact with myelin-specific CD8⁺ T cells early in EAE enabling them to differentiate into pathogenic effector cells.

Stiff person syndrome (SPS) is an autoimmune CNS disorder characterized by muscle rigidity, spasms and anxiety. The majority of patients have high-titer autoantibodies (ab) against glutamate decarboxylase (GAD65). A pathogenic role of SPS-associated IgG with ab against GAD65 has been shown for anxiety-like behavior but not for the core motor signs. We repetitively injected the purified IgG fraction of an SPS patient with severe motor impairment but without anxious comorbidity containing high titers of anti-GAD65 ab (SPS-IgG) into the lateral ventricle (i.c.v.) or intrathecally (i.th.) at the spinal level in experimental rats. We analyzed the effects on motor and anxiety-like behavior. Non-SPS human IgG fractions served as controls. Animals injected i.c.v. with SPS-IgG showed stiffness-like behavior with impaired walking ability and reduced grip strength of the upper limbs as well as postural and sensorimotor dysfunction. Testing for anxiety-like behavior revealed no significant differences between SPS and control IgG-treated rats. IgG deposits were found only in rats treated with SPS-IgG and were localized predominantly in CNS structures involved in motor control including globus pallidus, internal capsule, striatum and anterior thalamus. Double immunofluorescence staining revealed that predominantly GABAergic interneurons were positive for i.c.v. injected SPS-IgG. Rats injected i.th. with SPS-IgG did not present obvious motor symptoms and had a normal synaptic transmission at the spinal level. We conclude that SPS-like motor dysfunction can be induced in rats by passive transfer of IgG from an SPS-patient with high titer of anti-GAD65 ab. GABAergic dysfunction in supraspinal motor pathways rather than in the spinal cord may lead to motor deficits observed in the rats contrasting observations made in SPS with amphiphysin antibodies.

Current knowledge of the pathogenic mechanisms involved in Guillain-Barre syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) supports an autoimmune etiology. Some of the cellular and humoral immune responses that contribute to the development of GS and CIDP resemble those implicated in multiple sclerosis (MS). Also, the role of persistent axonal damage in these diseases has recently been revisited, similar to the situation in MS. Currently axonal damage is best understood in GBS associated with preceding infections. In all other subtypes, histopathological studies speak to the heterogeneity of disease mechanisms in GBS and CIDP. Several immunotherapies are available to treat GBS or CIDP. Intravenous immunoglobulin and plasma exchange are of comparable therapeutic efficacy for the acute phase of both diseases, whereas prednisone (or analogues) and immunosuppressive drugs are effective only to treat CIDP, which usually requires long-term therapy. Although some approaches have been made to support axonal regeneration in the model of experimental autoimmune neuritis, currently no promising specific therapeutic concept is available which can be transferred to treat the human disease.

Isolated generalized dystonia is a central motor network disorder characterized by twisted movements or postures. The most frequent genetic cause is a GAG deletion in the Tor1a (DYT1) gene encoding torsinA with a reduced penetrance of 30-40 % suggesting additional genetic or environmental modifiers. Development of dystonia-like movements after a standardized peripheral nerve crush lesion in wild type (wt) and Tor1a+/- mice, that express 50 % torsinA only, was assessed by scoring of hindlimb movements during tail suspension, by rotarod testing and by computer-assisted gait analysis. Western blot analysis was performed for dopamine transporter (DAT), D1 and D2 receptors from striatal and quantitative RT-PCR analysis for DAT from midbrain dissections. Autoradiography was used to assess the functional DAT binding in striatum. Striatal dopamine and its metabolites were analyzed by high performance liquid chromatography. After nerve crush injury, we found abnormal posturing in the lesioned hindlimb of both mutant and wt mice indicating the profound influence of the nerve lesion (15x vs. 12x relative to control) resembling human peripheral pseudodystonia. In mutant mice the phenotypic abnormalities were increased by about 40 % (p

Neuromyelitis Optica Spectrum Disorders (NMOSD) are associated with autoantibodies (ABs) targeting the astrocytic aquaporin-4 water channels (AQP4-ABs). These ABs have a direct pathogenic role by initiating a variety of immunological and inflammatory processes in the course of disease. In a recently-established animal model, chronic intrathecal passive-transfer of immunoglobulin G from NMOSD patients (NMO-IgG), or of recombinant human AQP4-ABs (rAB-AQP4), provided evidence for complementary and immune-cell independent effects of AQP4-ABs. Utilizing this animal model, we here tested the effects of systemically and intrathecally applied pooled human immunoglobulins (IVIg) using a preventive and a therapeutic paradigm. In NMO-IgG animals, prophylactic application of systemic IVIg led to a reduced median disease score of 2.4 on a 0–10 scale, in comparison to 4.1 with sham treatment. Therapeutic IVIg, applied systemically after the 10th intrathecal NMO-IgG injection, significantly reduced the disease score by 0.8. Intrathecal IVIg application induced a beneficial effect in animals with NMO-IgG (median score IVIg 1.6 vs. sham 3.7) or with rAB-AQP4 (median score IVIg 2.0 vs. sham 3.7). We here provide evidence that treatment with IVIg ameliorates disease symptoms in this passive-transfer model, in analogy to former studies investigating passive-transfer animal models of other antibody-mediated disorders.

We report a novel in vivo mouse model system to study regeneration of injured motor nerve and spatiotemporal pattern of denervation in experimental nerve diseases. The lateral thoracic nerve (LTN), as a pure motor nerve, innervates the cutaneous maximus muscle (CMM) by some of the shortest and the longest motor nerve fibers in the mouse body. Its branches and nerve terminals can be imaged in whole mount preparations. Here we describe the branching pattern of the LTN and its innervation of the CMM, and characterize degeneration and regeneration over time after a LTN crush by morphological and electrophysiological analyses. We demonstrate the utility of this model in a well-established neurotoxicity paradigm and in a genetic disease model of the peripheral neuropathy. Furthermore, this system enables punch biopsies that allow repeated and multi-location examinations for LTN regeneration and CMM reinnervation over time. The presence of the LTN and the CMM in a variety of species and its easy accessibility suggests that this in vivo model system offers considerable promise for future nerve degeneration and regeneration research.

Stiff person syndrome with auto-antibodies against amphiphysin is characterized by muscular stiffness, spasms, and anxiety which is a less appreciated core symptom. Here, we report that intrathecal application of purified immunoglobulin G-antibodies against amphiphysin from one patient induce anxiety behavior in rats. Immunostaining demonstrated binding of anti-amphiphysin antibodies to brain structures which are associated with anxiety disorders, such as the amygdala. We propose that antibody-mediated amphiphysin deficiency may account for anxiety behavior in stiff person syndrome via presynaptic dysregulation of GABAergic pathways.

We collected the evidence for potential biomarkers in nerve biopsies that might be of use in diagnosis, assessment, or treatment response in chronic inflammatory demyelinating polyneuropathies (CIDPs). We performed a literature search in PubMed from 1965 to May 2010 using the key words (["chronic inflammatory polyneuropathy" or "polyradiculoneuritis" or {"chronic and neuritis"}] and "nerve biopsy") and searched manually within these references for relevant publications related to the subject. Twenty references gave information about potential biomarkers for CIDP. Evidence of demyelination alone is not specific for CIDP, but may support the diagnosis in the context of a typical clinical pattern. Although the total numbers of inflammatory cells do not distinguish well between CIDP and non-inflammatory neuropathies, the pattern of macrophage clusters around endoneurial vessels may be a simple marker of inflammation with good sensitivity and specificity. Immunohistochemistry for matrix metalloproteinase-9 may be useful for the distinction of inflammatory and non-inflammatory neuropathies. Microarrays which give a complex pattern of up- and downregulated genes also show promise for developing a biomarker. Immunohistochemistry on sural nerve biopsies has the potential to distinguish inflammatory from non-inflammatory neuropathies. More research is needed to establish the diagnostic validity of specific markers and of gene expression studies and to test whether they can distinguish between subtypes of inflammatory neuropathies.

The integrity of peripheral nerves relies on communication between axons and Schwann cells. The axonal signals that ensure myelin maintenance are distinct from those that direct myelination and are largely unknown. Here we show that ablation of the prion protein PrP(C) triggers a chronic demyelinating polyneuropathy (CDP) in four independently targeted mouse strains. Ablation of the neighboring Prnd locus, or inbreeding to four distinct mouse strains, did not modulate the CDP. CDP was triggered by depletion of PrP(C) specifically in neurons, but not in Schwann cells, and was suppressed by PrP(C) expression restricted to neurons but not to Schwann cells. CDP was prevented by PrP(C) variants that undergo proteolytic amino-proximal cleavage, but not by variants that are nonpermissive for cleavage, including secreted PrP(C) lacking its glycolipid membrane anchor. These results indicate that neuronal expression and regulated proteolysis of PrP(C) are essential for myelin maintenance.

The blood-brain barrier (BBB) serves as a border limiting access of immunoglobulins from the circulation into the brain. This becomes relevant when studying the pathogenesis of antibody-mediated autoimmune CNS disorders. Here, we characterized the BBB dysfunction in a model of mild experimental adoptive transfer autoimmune encephalomyelitis (AT-EAE). We show that large molecules can readily penetrate the BBB between days 3 and 7 after EAE-induction. This model may be valuable for studying putative pathogenic effects of immunoglobulins in the central nervous system.

Substantial therapeutic progress has been made in myasthenia gravis (MG) even before the era of molecular medicine. Here we characterize modern treatment algorithms that are adapted to disease severity and introduce the principle of escalating treatment strategies for MG. In very mild cases and in some ocular forms of MG, treatment with acetylcholinesterase inhibitors may be sufficient, at least temporarily, but commonly some kind of immunologically active treatment is needed. In generalized MG, a wide array of immunosuppressive treatments has been established through observational studies, some prospective, but most of them have never been tested in a double-blind, prospective and randomized trial. Within the immunologically active drugs, glucocorticosteroids (GCS) and the immunosuppressive drug azathioprine (Aza) have been studied the longest. Aza is still the standard base-line treatment, in particular in cases where high doses of GCS would be needed to maintain remission. If Aza is not tolerated, several alternatives are available including cyclosporine A (Cic A), mycophenolate mofetil, cyclophosphamide, and methotrexate, all of them off-label in most western countries. Tacrolimus is under investigation. More severe cases may profit from drug combinations in which compounds with more rapidly acting drugs (GCS, Cic A) are combined with others showing a more delayed action (Aza). All such combination therapies need to be supervised by an experienced neuroimmunological center because of potentially serious adverse reactions. Serial measurements of anti-acetylcholine receptor antibodies, once these are elevated, is a useful adjunct for monitoring long-term treatment success and may help in weaning from higher to lower doses or to single drugs rather than combinations. For very severe and treatment-resistant cases, co-treatment with intravenous immunoglobulins or different modalities of plasmapheresis may be considered on the short term while the humanized monoclonal anti-CD 20 antibody (rituximab) is a candidate for the long term. In highly refractory cases also immuno-ablation via high-dose cyclophosphamide, followed by hematologic trophic factors such as G-CSF, has been tried successfully. Future developments may include other immunologically active monoclonal antibodies (e.g., anti-CD 52, Campath-1). Up to 10% of patients with MG are associated with a malignant thymoma, often referred to as paraneoplastic MG, as detected by CT scan or MRI, and these patients require thymomectomy and sometimes postsurgical chemotherapy and radiation treatment. In nonthymoma patients with generalised MG, including older children and adults up to the 5th decade, a complete transsternal thymectomy is recommended based on available open trials and expert opinion, preferentially during the first year of disease. Endoscopic surgery may also be effective. Before surgery, pretreatment with immunosuppressive medication or plasmapheresis is usually recommended to ameliorate MG and subsequently reduce perioperative morbidity and mortality which is now near zero in experienced centers. Myasthenic crisis is the life-threatening exacerbation of MG and is best treated by plasmapheresis, mostly combined with immunoadsorption techniques. Intravenous immunoglobulins are a reasonable alternative, but a shortage in supplies and high prices limit its use.

In this prospective study, intraepidermal nerve fiber densities (IENFD) and subepidermal nerve plexus densities (SENPD) were quantified by immunostaining in skin punch biopsies from the distal calf in 99 patients with clinical symptoms of painful sensory neuropathy and from 37 age-matched healthy volunteers. The clinical diagnosis was based on history and abnormal thermal thresholds on quantitative sensory testing (QST). In patients with neuropathy, IENFD and SENPD were reduced to about 50% of controls. Elevated warm detection thresholds on QST correlated with IENFD but not with SENPD. Using receiver-operating characteristic (ROC) curve analysis of IENFD values, the diagnostic sensitivity for detecting neuropathy was 0.80 and the specificity 0.82. For SENPD, sensitivity was 0.81 and specificity 0.88. With ROC analysis of both IENFD and SENPD together, the diagnostic sensitivity was further improved to 0.92. The combined examination of IENFD and SENPD is a highly sensitive and specific diagnostic tool in patients suspected to suffer from painful sensory neuropathies but with normal values on clinical neurophysiological studies.

Human Molecular Genetics, 16 (24), ISSN:0964-6906, ISSN:1460-2083

Pain is a common symptom in peripheral neuropathies. The factors determining why some peripheral neuropathies are painful and others are not are incompletely understood. Pro-inflammatory cytokines have been implicated to play a crucial role in the generation of pain.To investigate whether cytokine profiles differ between patients with painful or painless neuropathy.In this prospective study, we analyzed blood mRNA and protein levels of the pro-inflammatory cytokines interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4 and IL-10 in 32 patients with painful neuropathy, 20 patients with painless neuropathy, and 38 healthy control subjects, using quantitative real-time PCR and ELISA.Patients with a painful neuropathy had about twofold higher IL-2 mRNA (p = 0.001) and TNF mRNA (p0.0001) and protein levels (p = 0.009) than healthy control subjects and about twofold higher IL-2 and TNF mRNA (p = 0.03; p = 0.001) and protein levels (p = 0.04; p = 0.04) than patients with painless neuropathy. In contrast, mRNA levels of the anti-inflammatory cytokine IL-10 were about twofold higher in patients with painless neuropathy than in patients with painful neuropathy (p = 0.001) and controls (p = 0.004). IL-4 protein levels were 20-fold higher in patients with painless neuropathy (p0.0001) and 17-fold higher in patients with painful neuropathy (p0.0001) than in healthy control subjects.A pro-inflammatory cytokine profile seems to be associated with pain in the setting of a peripheral neuropathy, corroborating findings in animal models with experimental painful neuropathies. This may have implications for future treatment strategies.

Familial amyotrophic lateral sclerosis (ALS) is frequently associated with mutations in the SOD1 gene. We identified a rapidly progressive disease in a patient with an inherited ALS. The identified heterozygous T>A exchange in position 1067 in the SOD1 gene results in an amino acid substitution of lysine for asparagine at position 86 (N86K) of the SOD1 protein. The family history suggested that this autosomal dominantly inherited mutation may be associated with rapidly progressive disease.

Giant cell arteritis (GCA) is the most common systemic vasculitis in elderly individuals. Diagnosis is confirmed by temporal artery (TA) biopsy, although biopsy results are often negative. Despite the use of corticosteroids, disease may progress. Identification of causal agents will improve outcomes. Biopsy-positive GCA is associated with TA infection by varicella-zoster virus (VZV).To analyze VZV infection in TAs of patients with clinically suspected GCA whose TAs were histopathologically negative and in normal TAs removed post mortem from age-matched individuals.A cross-sectional study for VZV antigen was performed from January 2013 to March 2015 using archived, deidentified, formalin-fixed, paraffin-embedded GCA-negative, GCA-positive, and normal TAs (50 sections/TA) collected during the past 30 years. Regions adjacent to those containing VZV were examined by hematoxylin-eosin staining. Immunohistochemistry identified inflammatory cells and cell types around nerve bundles containing VZV. A combination of 17 tertiary referral centers and private practices worldwide contributed archived TAs from individuals older than 50 years.Presence and distribution of VZV antigen in TAs and histopathological changes in sections adjacent to those containing VZV were confirmed by 2 independent readers.Varicella-zoster virus antigen was found in 45 of 70 GCA-negative TAs (64%), compared with 11 of 49 normal TAs (22%) (relative risk [RR] = 2.86; 95% CI, 1.75-5.31; P .001). Extension of our earlier study revealed VZV antigen in 68 of 93 GCA-positive TAs (73%), compared with 11 of 49 normal TAs (22%) (RR = 3.26; 95% CI, 2.03-5.98; P .001). Compared with normal TAs, VZV antigen was more likely to be present in the adventitia of both GCA-negative TAs (RR = 2.43; 95% CI, 1.82-3.41; P .001) and GCA-positive TAs (RR = 2.03; 95% CI, 1.52-2.86; P .001). Varicella-zoster virus antigen was frequently found in perineurial cells expressing claudin-1 around nerve bundles. Of 45 GCA-negative participants whose TAs contained VZV antigen, 1 had histopathological features characteristic of GCA, and 16 (36%) showed adventitial inflammation adjacent to viral antigen; no inflammation was seen in normal TAs.In patients with clinically suspected GCA, prevalence of VZV in their TAs is similar independent of whether biopsy results are negative or positive pathologically. Antiviral treatment may confer additional benefit to patients with biopsy-negative GCA treated with corticosteroids, although the optimal antiviral regimen remains to be determined.