Development and characterization of agonistic antibodies targeting the Ig-like 1 domain of MuSK

Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise

Food restriction does not relieve PTSD-like anxiety

We used the inescapable foot shock paradigm (IFS) in rats as an animal model for post-traumatic stress disorder (PTSD). Previously we showed that exercise reversed the enhanced stress sensitivity induced by IFS. From literature it is known that food restriction has antidepressant and anxiolytic effects. Since both treatments influence energy expenditure, we questioned whether food restriction reduces anxiety in the IFS model via a comparable, NPY dependent mechanism as enrichment. Anxiety of IFS-exposed animals was measured as change in locomotion and freezing after sudden silence in an open field test, before and after two weeks of food restriction. In addition a forced swim test (FST) was performed. Next, using qPCR, the expression of neuropeptide Y (NPY) and the neuropeptide Y1 receptor (Y1 receptor) was measured in the amygdala. Food restriction increased locomotion and decreased freezing behavior both in control and IFS animals. These effects were small. IFS-induced anxiety was not abolished after two weeks of food restriction. IFS did not influence immobility or the duration of swimming in the FST of animals fed ad libitum. However, food restriction increased swimming and decreased the duration of immobility in IFS-exposed animals. Y1 receptor expression in the basolateral amygdala decreased after both IFS and food restriction. Although food restriction seems to induce a general anxiolytic effect, it does not operate via enhanced Y1 receptor expression and has no effect on the more pathogenic anxiety induced by IFS

Food restriction does not relieve PTSD-like anxiety

We used the inescapable foot shock paradigm (IFS) in rats as an animal model for post-traumatic stress disorder (PTSD). Previously we showed that exercise reversed the enhanced stress sensitivity induced by IFS. From literature it is known that food restriction has antidepressant and anxiolytic effects. Since both treatments influence energy expenditure, we questioned whether food restriction reduces anxiety in the IFS model via a comparable, NPY dependent mechanism as enrichment. Anxiety of IFS-exposed animals was measured as change in locomotion and freezing after sudden silence in an open field test, before and after two weeks of food restriction. In addition a forced swim test (FST) was performed. Next, using qPCR, the expression of neuropeptide Y (NPY) and the neuropeptide Y1 receptor (Y1 receptor) was measured in the amygdala. Food restriction increased locomotion and decreased freezing behavior both in control and IFS animals. These effects were small. IFS-induced anxiety was not abolished after two weeks of food restriction. IFS did not influence immobility or the duration of swimming in the FST of animals fed ad libitum. However, food restriction increased swimming and decreased the duration of immobility in IFS-exposed animals. Y1 receptor expression in the basolateral amygdala decreased after both IFS and food restriction. Although food restriction seems to induce a general anxiolytic effect, it does not operate via enhanced Y1 receptor expression and has no effect on the more pathogenic anxiety induced by IFS

MuSK myasthenia gravis monoclonal antibodies: Valency dictates pathogenicity

ObjectiveTo isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.MethodsWe generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.ResultsThe isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.ConclusionsPatient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic

Distribution and Load of Amyloid-beta Pathology in Parkinson Disease and Dementia with Lewy Bodies

Parkinson disease (PD), Parkinson disease with dementia (PDD), and Dementia with Lewy bodies (DLB) differ clinically with regard to the presence and timing of dementia. In this postmortem study, we evaluated whether the burden and distribution pattern of amyloid-β (Aβ) pathology differs among these disease entities. We assessed Aβ phases and neuritic plaque scores in 133 patients fulfilling clinical diagnostic criteria for PD, PDD, and DLB, and determined the presence and load of Aβ pathology in 5 cortical and 4 subcortical regions in a subset of patients (n = 89) using a multispectral imaging system. Aβ phases and neuritic plaque scores were higher in DLB versus PDD (both p < 0.001) and in PDD vs PD patients (p = 0.020 and 0.022, respectively). Aβ pathology was more often observed in the entorhinal cortex, amygdala and putamen in DLB versus PDD patients; Aβ load was higher in both cortical and subcortical regions. PDD patients had more frequent Aβ pathology in temporal cortex and higher Aβ load in cortical regions and striatum versus PD patients. Our findings suggest that the load and extent of Aβ pathology may contribute to cognitive dysfunction in PDD and the early-stage severe dementia in DLB

Development and characterization of agonistic antibodies targeting the Ig-like 1 domain of MuSK

Abstract Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise

Enrichment of serum IgG4 in MuSK myasthenia gravis patients

Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disease belonging to a growing group of IgG4 autoimmune diseases (IgG4-AIDs), in which the majority of pathogenic autoantibodies are of the IgG4 subclass. The more prevalent form of MG with acetylcholine receptor (AChR) antibodies is caused by IgG1-3 autoantibodies. A dominant role for IgG4 in autoimmune disease is intriguing due to its antiinflammatory characteristics. It is unclear why MuSK autoantibodies are predominantly IgG4. We hypothesized that MuSK MG patients have a general predisposition to generate IgG4 responses, therefore resulting in high levels of circulating IgG4. To investigate this, we quantified serum Ig isotypes and IgG subclasses using nephelometric and turbidimetric assays in MuSK MG and AChR MG patients not under influence of immunosuppressive treatment. Absolute serum IgG1 was increased in both MuSK and AChR MG patients compared to healthy donors. In addition, only MuSK MG patients on average had significantly increased and enriched serum IgG4. Although more MuSK MG patients had elevated serum IgG4, for most the IgG4 serum levels fell within the normal range. Correlation analyses suggest MuSK-specific antibodies do not solely explain the variation in IgG4 levels. In conclusion, although serum IgG4 levels are slightly increased, the levels do not support ubiquitous IgG4 responses in MuSK MG patients as the underlying cause of dominant IgG4 MuSK antibodies