Autoimmune synaptopathies.

Autoantibodies targeting proteins at the neuromuscular junction are known to cause several distinct myasthenic syndromes. Recently, autoantibodies targeting neurotransmitter receptors and associated proteins have also emerged as a cause of severe, but potentially treatable, diseases of the CNS. Here, we review the clinical evidence as well as in vitro and in vivo experimental evidence that autoantibodies account for myasthenic syndromes and autoimmune disorders of the CNS by disrupting the functional or structural integrity of synapses. Studying neurological and psychiatric diseases of autoimmune origin may provide new insights into the cellular and circuit mechanisms underlying a broad range of CNS disorders.

Glycine receptor autoantibodies disrupt inhibitory neurotransmission.

Chloride-permeable glycine receptors have an important role in fast inhibitory neurotransmission in the spinal cord and brainstem. Human immunoglobulin G (IgG) autoantibodies to glycine receptors are found in a substantial proportion of patients with progressive encephalomyelitis with rigidity and myoclonus, and less frequently in other variants of stiff person syndrome. Demonstrating a pathogenic role of glycine receptor autoantibodies would help justify the use of immunomodulatory therapies and provide insight into the mechanisms involved. Here, purified IgGs from four patients with progressive encephalomyelitis with rigidity and myoclonus or stiff person syndrome, and glycine receptor autoantibodies, were observed to disrupt profoundly glycinergic neurotransmission. In whole-cell patch clamp recordings from cultured rat spinal motor neurons, glycinergic synaptic currents were almost completely abolished following incubation in patient IgGs. Most human autoantibodies targeting other CNS neurotransmitter receptors, such as N-methyl-d-aspartate (NMDA) receptors, affect whole cell currents only after several hours incubation and this effect has been shown to be the result of antibody-mediated crosslinking and internalization of receptors. By contrast, we observed substantial reductions in glycinergic currents with all four patient IgG preparations with 15 min of exposure to patient IgGs. Moreover, monovalent Fab fragments generated from the purified IgG of three of four patients also profoundly reduced glycinergic currents compared with control Fab-IgG. We conclude that human glycine receptor autoantibodies disrupt glycinergic neurotransmission, and also suggest that the pathogenic mechanisms include direct antagonistic actions on glycine receptors.

Redefining progressive encephalomyelitis with rigidity and myoclonus after the discovery of antibodies to glycine receptors.

PURPOSE OF REVIEW: This review highlights the recent discovery of antibodies to glycine receptor (GlyR-Ab) and discusses the relationship between these antibodies and neurological disorders. RECENT FINDINGS: Since the initial description in 2008 of antibodies to glycine receptors (GlyR-Abs) in a patient with progressive encephalomyelitis with rigidity and myoclonus (PERM), these antibodies have been found in PERM and in some patients with a variety of stiff person spectrum (SPS) or related disorders. Patients with GlyR-Abs often improve with aggressive immunotherapy, and antibody titres correlate with disease severity. Around 25% of patients have another autoimmune condition and 10-20% have an underlying malignancy. GlyR-Abs bind to extracellular determinants, are mainly Immunoglobulin G1 subclass and induce GlyR internalization in Human embryonic kidney 293 cells, suggesting pathogenicity. The spectrum of neurological disease associated with GlyR-Abs has not been fully characterized, and lower titres may not be syndrome specific, but GlyR-Abs, like antibodies to other neuronal cell-surface antigens, define immunotherapy-responsive disease and are likely to be pathogenic. This distinguishes them from the glutamic acid decarboxylase antibodies that can also be found at high titres in patients with classical stiff person syndrome which is more often chronic and relatively resistant to immunological treatments. SUMMARY: Irrespective of the clinical features, GlyR-Abs are helpful in the diagnosis of patients who very often have a subacute, progressive and life-threatening disorder which shows a favourable response to immunotherapy.

ß-Propeller protein-associated neurodegeneration: a new X-linked dominant disorder with brain iron accumulation.

Neurodegenerative disorders with high iron in the basal ganglia encompass an expanding collection of single gene disorders collectively known as neurodegeneration with brain iron accumulation. These disorders can largely be distinguished from one another by their associated clinical and neuroimaging features. The aim of this study was to define the phenotype that is associated with mutations in WDR45, a new causative gene for neurodegeneration with brain iron accumulation located on the X chromosome. The study subjects consisted of WDR45 mutation-positive individuals identified after screening a large international cohort of patients with idiopathic neurodegeneration with brain iron accumulation. Their records were reviewed, including longitudinal clinical, laboratory and imaging data. Twenty-three mutation-positive subjects were identified (20 females). The natural history of their disease was remarkably uniform: global developmental delay in childhood and further regression in early adulthood with progressive dystonia, parkinsonism and dementia. Common early comorbidities included seizures, spasticity and disordered sleep. The symptoms of parkinsonism improved with l-DOPA; however, nearly all patients experienced early motor fluctuations that quickly progressed to disabling dyskinesias, warranting discontinuation of l-DOPA. Brain magnetic resonance imaging showed iron in the substantia nigra and globus pallidus, with a 'halo' of T1 hyperintense signal in the substantia nigra. All patients harboured de novo mutations in WDR45, encoding a beta-propeller protein postulated to play a role in autophagy. Beta-propeller protein-associated neurodegeneration, the only X-linked disorder of neurodegeneration with brain iron accumulation, is associated with de novo mutations in WDR45 and is recognizable by a unique combination of clinical, natural history and neuroimaging features.

ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons.

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9ß1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and ß1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and ß1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of ß1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

Endogenous patterns of activity are required for the maturation of a motor network.

Many parts of the nervous system become active before development is complete, including the embryonic spinal cord. Remarkably, although the subject has been debated for over a century (Harrison, 1904), it is still unclear whether such activity is required for normal development of motor circuitry. In Drosophila, embryonic motor output is initially poorly organized, and coordinated crawling-like behavior gradually emerges over the subsequent phase of development. We show that reversibly blocking synaptic transmission during this phase severely delays the first appearance of coordinated movements. When we interfere with the pattern of neuronal firing during this period, coordination is also delayed or blocked. We conclude that there is a period during which endogenous patterns of neuronal activity are required for the normal development of motor circuits in Drosophila.

Comparison of bifunctional chelates for (64)Cu antibody imaging.

PURPOSE: Improved bifunctional chelates (BFCs) are needed to facilitate efficient (64)Cu radiolabeling of monoclonal antibodies (mAbs) under mild conditions and to yield stable, target-specific agents. The utility of two novel BFCs, 1-Oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA), for mAb imaging with (64)Cu were compared to the commonly used S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA). METHODS: The BFCs were conjugated to trastuzumab, which targets the HER2/neu receptor. (64)Cu radiolabeling of the conjugates was optimized. Receptor binding was analyzed using flow cytometry and radioassays. Finally, PET imaging and biodistribution studies were done in mice bearing either HER2/neu-positive or HER2/neu-negative tumors. RESULTS: (64)Cu-Oxo-DO3A- and PCTA-trastuzumab were prepared at room temperature in >95% radiochemical yield (RCY) in <30 min, compared to only 88% RCY after 2 h for the preparation of (64)Cu-DOTA-trastuzumab under the same conditions. Cell studies confirmed that the immunoreactivity of the mAb was retained for each of the bioconjugates. In vivo studies showed that (64)Cu-Oxo-DO3A- and PCTA-trastuzumab had higher uptake than the (64)Cu-DOTA-trastuzumab at 24 h in HER2/neu-positive tumors, resulting in higher tumor to background ratios and better tumor images. By 40 h all three of the (64)Cu-BFC-trastuzumab conjugates allowed for clear visualization of the HER2/neu-positive tumors but not the negative control tumor. CONCLUSION: The antibody conjugates of PCTA and Oxo-DO3A were shown to have superior (64)Cu radiolabeling efficiency and stability compared to the analogous DOTA conjugate. In addition, (64)Cu-PCTA and Oxo-DO3A antibody conjugates may facilitate earlier imaging with greater target to background ratios than the analogous (64)Cu-DOTA antibody conjugates.

Antibody-mediated central nervous system diseases.

Antibody-mediated central nervous system diseases are a relatively new area of clinical neuroscience with growing impact. Their recognition has challenged the dogma of the blood-brain barrier preventing antibody access into the central nervous system. The antibodies discovered so far are mainly against neurotransmitter receptors (e.g. N-methyl-d-aspartate and glycine receptors) and ion channel-associated proteins (leucine-rich glioma inactivated protein 1 and contactin-associated protein 2) and are expressed on the surface of neuronal synapses and elsewhere. The disorders are reversible with immunotherapies that reduce antibody levels. Although rare, the identification of these disorders in clinical practice has made central nervous system autoimmune diseases a consideration in the differential diagnoses of many clinical presentations. There is still much to learn about the aetiology of the diseases and the mechanisms by which the antibodies act, the neuronal and glial changes that follow antibody-attack, and the compensatory changes that may be required to ensure good recovery.

Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS.

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Successful use of acarbose to manage post-prandial glycaemia in two patients with type 1 diabetes on continuous subcutaneous insulin infusion.

Post-prandial hyperglycaemia is a particular problem for some patients with diabetes despite administering continuous subcutaneous insulin infusion (CSII) to deliver insulin flexibly. We describe two cases of patients on CSII with persistent post-prandial hyperglycaemia despite varying insulin doses and timing. Treatment with acarbose improved their glycaemic control.

The development of motor coordination in Drosophila embryos.

We used non-invasive muscle imaging to study the onset of motor activity and emergence of coordinated movement in Drosophila embryos. Earliest movements are myogenic, and neurally controlled muscle contractions first appear with the onset of bursting activity 17 hours after egg laying. Initial episodes of activity are poorly organised and coordinated crawling sequences only begin to appear after a further hour of bursting. Thus, network performance improves during this first period of activity. The embryo continues to exhibit bursts of crawling-like sequences until shortly before hatching, while other reflexes also mature. Bursting does not begin as a reflex response to sensory input but appears to reflect the onset of spontaneous activity in the motor network. It does not require GABA-mediated transmission, and, by using a light-activated channel to excite the network, we demonstrate activity-dependent depression that may cause burst termination.

Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells.

BACKGROUND: Placental transfer of nutrients and secretion of hormones is essential for normal fetal development. AIM OF THE STUDY: To determine whether biotin supply affects biotin homeostasis, proliferation rates, and progesterone secretion in placenta cells. METHODS: JAr choriocarcinoma cells were cultured in media containing deficient (25 pmol/L), physiological (250 pmol/L), or pharmacological concentrations (10,000 pmol/L) of biotin for three weeks; markers for biotin homeostasis, proliferation, and hormone secretion were quantified. RESULTS: Biotin concentrations in culture media correlated negatively with expression of the biotin transporter SMVT, as judged by cellular transport rates of biotin, abundance of SMVT protein, and transcriptional activity of SMVT reporter-gene constructs. Notwithstanding this homeostatic mechanism, biotin concentrations in media correlated positively with activities of biotin-dependent propionyl-CoA carboxylase, abundance of biotinylated carboxylases, and with biotinylation of histones. Biotin deficiency was associated with decreased rates of thymidine uptake into JAr cells [pmol thymidine/( 10(6) cells x 24 h)]: 1.6 +/- 0.1 (25 pmol/L biotin) versus 2.3 +/- 0.2 (250 pmol/L biotin) versus 3.7 +/- 0.4 (10,000 pmol/L biotin), suggesting that cell proliferation depends on biotin. Secretion of progesterone was reduced in biotin-deficient cells; this effect was caused by reduced generation of new cells in deficient media rather than by an immediate effect of biotin on progesterone secretion at the singlecell-level. CONCLUSIONS: This study provides evidence that choriocarcinoma cells cannot maintain normal activities of biotin-dependent metabolic pathways if biotin concentrations in culture media are low. It is uncertain whether activities of biotin-dependent pathways in placenta affect fetal development in vivo.

Genome-wide expression profiling reveals genes associated with amphotericin B and fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans.

OBJECTIVES: The aim of this study was to identify changes in the gene expression profile of Candida albicans associated with the acquisition of experimentally induced resistance to amphotericin B and fluconazole. METHODS: C. albicans SC5314 was passed in increasing concentrations of amphotericin B to generate isolate SC5314-AR. Susceptibility testing by Etest revealed SC5314-AR to be highly resistant to both amphotericin B and fluconazole. The gene expression profile of SC5314-AR was compared with that of SC5314 using DNA microarray analysis. Sterol composition was determined for both strains. RESULTS: Upon examination of MICs of antifungal compounds, it was found that SC5314-AR was resistant to both amphotericin B and fluconazole. By microarray analysis a total of 134 genes were found to be differentially expressed, that is up-regulated or down-regulated by at least 50%, in SC5314-AR. In addition to the cell stress genes DDR48 and RTA2, the ergosterol biosynthesis genes ERG5, ERG6 and ERG25 were up-regulated. Several histone genes, protein synthesis genes and energy generation genes were down-regulated. Sterol analysis revealed the prevalence of sterol intermediates eburicol and lanosterol in SC5314-AR, whereas ergosterol was the predominant sterol in SC5314. CONCLUSION: Along with changes in expression of these ergosterol biosynthesis genes was the accumulation of sterol intermediates in the resistant strain, which would account for the decreased affinity of amphotericin B for membrane sterols and a decreased requirement for lanosterol demethylase activity in membrane sterol production. Furthermore, other genes are implicated as having a potential role in the polyene and azole antifungal resistant phenotype.