Identification of novel synaptic components by transcriptome profiling of the murine neuromuscular junction

The neuromuscular junction (NMJ) has been studied for over a century, yet we still do not have a complete picture of all its structural and functional components, knowledge of which is paramount in devising treatment strategies for neuromuscular diseases. Previous microarray-based approaches aimed at elucidating novel NMJ components were hindered by technological limitations. Recent technological advancements propelled next-generation RNA-sequencing with its wider dynamic range to the forefront of transcriptome-level gene expression profiling. We utilized laser-capture microdissection to isolate myonuclei underlying the NMJ combined with RNA-sequencing and successfully generated NMJ gene expression profiles of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles and identified a large number of potential novel NMJ genes. The expression levels of canonical NMJ genes were nearly identical between the EDL and SOL, which suggests that the core NMJ gene program might be well conserved between different skeletal muscle types. We used in vivo muscle electroporation to overexpress one of our candidate genes, the transcription factor T-box 21 (TBX21), in the tibialis anterior (TA) muscle and observed an increased density of postsynaptic acetylcholine receptors. TBX21 may thus represent a novel transcription factor contributing to the regulation of the NMJ gene program, with a role in postsynaptic sensitivity. We also generated NMJ gene expression profiles of the TA muscle of 10-month-old (“young”) and 30-month-old (“old”) mice to investigate the effect of aging on the NMJ gene program. Strikingly, the NMJ gene program was remarkably stable, with nearly identical expression levels of canonical NMJ genes between young and old mice. This implies that age-related perturbations of the NMJ are likely caused by external factors, such as accumulated myofiber damage and changes in nerve input, rather than by gradual dysregulation of the NMJ gene program with increasing age. Our findings argue against the hypothesis that aging leads to a broad deterioration of the NMJ gene program that would contribute to perturbations of NMJ structure and function. Furthermore, functional annotation analysis of our different NMJ gene expression datasets strongly indicates the importance of an extensive number of hitherto unknown glycoproteins, as well as of posttranslational modifications, especially glycosylations, at the synaptic basal lamina. We highlight a set of candidate genes that encode for enzymes putatively involved in these processes at the NMJ, and which are potentially involved in the pathophysiology of neuromuscular diseases such as congenital myasthenic syndromes. This thesis expands our understanding of the complexity of the NMJ and lays the foundation for further research that will functionally characterize novel synaptic components and provide the basis for novel therapeutic treatment strategies

Revising Type-2 Computation and Degrees of Discontinuity

By the sometimes so-called MAIN THEOREM of Recursive Analysis, every computable real function is necessarily continuous. Weihrauch and Zheng (TCS'2000), Brattka (MLQ'2005), and Ziegler (ToCS'2006) have considered different relaxed notions of computability to cover also discontinuous functions. The present work compares and unifies these approaches. This is based on the concept of the JUMP of a representation: both a TTE-counterpart to the well known recursion-theoretic jump on Kleene's Arithmetical Hierarchy of hypercomputation: and a formalization of revising computation in the sense of Shoenfield. We also consider Markov and Banach/Mazur oracle-computation of discontinuous fu nctions and characterize the computational power of Type-2 nondeterminism to coincide with the first level of the Analytical Hierarchy.Comment: to appear in Proc. CCA'0

Universality, optimality, and randomness deficiency

A Martin-Löf test UU is universal if it captures all non-Martin-Löf random sequences, and it is optimal if for every ML-test VV there is a c∈ωc∈ω such that ∀n(Vn+c⊆Un)∀n(Vn+c⊆Un). We study the computational differences between universal and optimal ML-tests as well as the effects that these differences have on both the notion of layerwise computability and the Weihrauch degree of LAYLAY, the function that produces a bound for a given Martin-Löf random sequence's randomness deficiency. We prove several robustness and idempotence results concerning the Weihrauch degree of LAYLAY, and we show that layerwise computability is more restrictive than Weihrauch reducibility to LAYLAY. Along similar lines we also study the principle RDRD, a variant of LAYLAY outputting the precise randomness deficiency of sequences instead of only an upper bound as LAYLAY

Comparative Approach to Define Increased Regulatory T Cells in Different Cancer Subtypes by Combined Assessment of CD127 and FOXP3

In recent years an increase of functional CD4+CD25+ regulatory T cells (Treg cells) has been established for patients with solid tumors, acute leukemias, and lymphomas. We have reported an expanded pool of CD4+CD25high Treg cells in patients with chronic lymphatic leukemia (CLL), multiple myeloma (MM) as well as its premalignant precursor monoclonal gammopathy of undetermined significance (MGUS). In healthy individuals, low-level expression of CD127 on T cells in addition to the expression of FOXP3 has been associated with Treg cells. Here, we demonstrate that the expanded FOXP3+ T-cell population in patients with colorectal cancer, CLL, MGUS, MM, follicular lymphoma, and Hodgkin's disease are exclusively CD127low Treg cells and were strongly suppressive. A significant portion of CD127lowFOXP3+ Treg cells expressed only low levels of CD25 suggesting that the previously reported expansion of CD25+ Treg cells underestimates the true expansion. The assessment of CCR7 and CD45RA expression on the expanded CD4+CD127lowFOXP3+ Treg cells revealed an increase of both naïve as well as central and effector memory Treg cells in peripheral blood. Our data strongly support superiority of combined CD127 and FOXP3 analysis in comparison to CD25 and FOXP3 assessment for further quantification of Treg cells in malignant diseases

BDNF is a mediator of glycolytic fiber-type specification in mouse skeletal muscle

Brain-derived neurotrophic factor (BDNF) influences the differentiation, plasticity, and survival of central neurons and likewise, affects the development of the neuromuscular system. Besides its neuronal origin, BDNF is also a member of the myokine family. However, the role of skeletal muscle-derived BDNF in regulating neuromuscular physiology in vivo remains unclear. Using gain- and loss-of-function animal models, we show that muscle-specific ablation of BDNF shifts the proportion of muscle fibers from type IIB to IIX, concomitant with elevated slow muscle-type gene expression. Furthermore, BDNF deletion reduces motor end plate volume without affecting neuromuscular junction (NMJ) integrity. These morphological changes are associated with slow muscle function and a greater resistance to contraction-induced fatigue. Conversely, BDNF overexpression promotes a fast muscle-type gene program and elevates glycolytic fiber number. These findings indicate that BDNF is required for fiber-type specification and provide insights into its potential modulation as a therapeutic target in muscle diseases

Current Guidelines for Obesity Prevention in Childhood and Adolescence

Objective: Current guidelines for prevention of obesity in childhood and adolescence are discussed. Methods: A literature search was performed in Medline via PubMed, and appropriate studies were analyzed. Results: Programs to prevent childhood obesity have so far remained mainly school-based and effects have been limited. Analyses by age group show that prevention programs have the best results in younger children (Conclusion: Behavior-oriented prevention programs showed only limited long-term effects. Certain groups at risk for the development of obesity are not reached effectively by current programs. Although universally valid conclusions cannot be drawn given the heterogeneity of available studies, clearly combining behavior-based programs with community-based prevention to counteract an ‘obesogenic environment' is crucial for sustainable success of future obesity prevention programs

In vivo Expansion of Naïve CD4+CD25high FOXP3+ Regulatory T Cells in Patients with Colorectal Carcinoma after IL-2 Administration

Regulatory T cells (Treg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of Treg cells was established. In IL-2 treated cancer patients a further Treg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional Treg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve Treg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve Treg cells indicate IL-2 dependent thymic generation of naïve Treg cells as a mechanism leading to increased frequencies of Treg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine Treg cells after IL-2 administration. These results point to a more complex regulation of Treg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve Treg cells