Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actinbinding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation. Molecular & Cellular Proteomics

IntroductionMyofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology.ResultsSkeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy.ConclusionsOur findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0280-0) contains supplementary material, which is available to authorized users.

Desminopathy is a subtype of myofibrillar myopathy caused by desmin mutations and characterized by protein aggregates accumulating in muscle fibers. The aim of this study was to assess the protein composition of these aggregates. Aggregates and intact myofiber sections were obtained from skeletal muscle biopsies of five desminopathy patients by laser microdissection and analyzed by a label-free spectral count-based proteomic approach. We identified 397 proteins with 22 showing significantly higher spectral indices in aggregates (ratio >1.8, p < 0.05). Fifteen of these proteins not previously reported as specific aggregate components provide new insights regarding pathomechanisms of desminopathy. Results of proteomic analysis were supported by immunolocalization studies and parallel reaction monitoring. Three mutant desmin variants were detected directly on the protein level as components of the aggregates, suggesting their direct involvement in aggregate-formation and demonstrating for the first time that proteomic analysis can be used for direct identification of a disease-causing mutation in myofibrillar myopathy. Comparison of the proteomic results in desminopathy with our previous analysis of aggregate composition in filaminopathy, another myofibrillar myopathy subtype, allows to determine subtype-specific proteomic profile that facilitates identification of the specific disorder.

The vascular endothelial growth factor (VEGF) is a homodimeric disulfide bound glycoprotein that promotes endothelial growth, accompanied by higher vascular permeability, and therefore represents an important factor for angiogenesis and vascularization. In addition, VEGF also has a neurotrophic and neuroprotective impact on glial and neuronal cells within the CNS and PNS. Recently, we have shown that VEGF increases somato- and dendritogenesis in neonatal, but not in mature CNS neurons [1], and leads to axonal growth cone guidance during embryonic development of the PNS [2, 3]. We assume that microRNAs are involved in the neuronal plasticity by altering expression patterns of corresponding VEGF receptors [4]. Therefore, this review focuses on microRNAs and their impact on the regulation of neuronal development at the posttranscriptional level within the CNS and PNS. Besides this, recent data about the regenerative impact of VEGF in the CNS and PNS are discussed, with a close look at the expression of VEGF and its corresponding miRNAs in these neuronal structures.

Amyotrophic lateral sclerosis is a devastating motor neuron disease and to this day not curable. While 5-10% of patients inherit the disease (familiar ALS), up to 95% of patients are diagnosed with the sporadic form (sALS). ALS is characterized by the degeneration of upper motor neurons in the cerebral cortex and of lower motor neurons in the brainstem and spinal cord. The wobbler mouse resembles almost all phenotypical hallmarks of human sALS patients and is therefore an excellent motor neuron disease model. The motor neuron disease of the wobbler mouse develops over a time course of around 40 days and can be divided into three phases: p0, presymptomatic; p20, early clinical; and p40, stable clinical phase. Recent findings suggest an essential implication of the NAD-producing enzyme Nmnat2 in neurodegeneration as well as maintenance of healthy axons. Here, we were able to show a significant downregulation of both gene and protein expression of Nmnat2 in the spinal cord of the wobbler mice at the stable clinical phase. The product of the enzyme NAD is also significantly reduced, and the values of the reactive oxygen species are significantly increased in the spinal cord of the wobbler mouse at p40. Thus, the deregulated expression of Nmnat2 appears to have a great influence on the cellular stress in the spinal cord of wobbler mice.

Vascular endothelial growth factor (VEGF) is well known as the growth factor with wide-ranging functions even in the central nervous system (CNS). Presently, most attention is given to the investigation of its role in neuronal protection, growth and maturation processes, whereby most effects are mediated through VEGF receptor 2 (VEGFR-2). The purpose of our current study is to provide new insights into the impact of VEGF on immature and mature Purkinje cells (PCs) in accordance with maturity and related receptor expression. Therefore, to expand our knowledge of VEGF effects in PCs development and associated VEGFR-2 expression, we used cultivated organotypic cerebellar slice cultures in immunohistochemical or microinjection studies, followed by confocal laser scanning microscopy (CLSM) and morphometric analysis. Additionally, we incorporated in our study the method of laser microdissection, followed by quantitative polymerase chain reaction (qPCR). For the first time we could show the age-dependent VEGF sensitivity of PCs with the largest promoting effects being on dendritic length and cell soma size in neonatal and juvenile stages. Once mature, PCs were no longer susceptible to VEGF stimulation. Analysis of VEGFR-2 expression revealed its presence in PCs throughout development, which underlined its mediating functions in neuronal cells.

Amyotrophic lateral sclerosis (ALS) is one of the most common incurable motor neuron disorders in adults. The majority of all ALS cases occur sporadically (sALS). Symptoms of ALS are caused by a progressive degeneration of motor neurons located in the motor cortex and spinal cord. The question arises why motor neurons selectively degenerate in ALS, while other cells and systems appear to be spared the disease. Members of the intrinsic apoptotic pathway are frequent targets of altered microRNA expression. Therefore, microRNAs and their effects on cell survival are subject of controversial debates. In this study, we investigated the expression of numerous members of the intrinsic apoptotic cascade by qPCR, western blot, and immunostaining in two different regions of the CNS of wobbler mice. Further we addressed the expression of miR-29b-3p targeting BMF, Bax, and, Bak, members of the apoptotic pathway. We show a tissue-specific differential expression of BMF, Bax, and cleaved-Caspase 3 in wobbler mice. An opposing regulation of miR-29b-3p expression in the cerebellum and cervical spinal cord of wobbler mice suggests different mechanisms regulating the intrinsic apoptotic pathway. Based on our findings, it could be speculated that miR-29b-3p might regulate antiapoptotic survival mechanisms in CNS areas that are not affected by neurodegeneration in the wobbler mouse ALS model.

Glioblastoma multiforme (GBM) is the most common primary brain tumor. It is highly aggressive with an unfavorable prognosis for the patients despite therapies including surgery, irradiation, and chemotherapy. One important characteristic of highly vascularized GBM is the strong expression of vascular endothelial growth factor (VEGF). VEGF has become a new target in the treatment of GBM, and targeted therapies such as the VEGF-receptor blocker axitinib are in clinical trials. Most studies focus on VEGF-induced angiogenesis, but only very few investigations analyze autocrine or paracrine effects of VEGF on the tumor cells. In this study, we examined the impact of VEGF, irradiation, and axitinib on cell proliferation and cell motility in human GBM cell lines U-251 and U-373. VEGF receptor 2 was shown to be expressed within both cell lines by using PCR and immunochemistry. Moreover, we performed 24-h videography to analyze motility, and a viability assay for cell proliferation. We observed increasing effects of VEGF and irradiation on cell motility in both cell lines, as well as strong inhibiting effects on cellular motility by VEGF-receptor blockade using axitinib. Moreover, axitinib diminished irradiation induced accelerating effects. While VEGF stimulation or irradiation did not affect cell proliferation, axitinib significantly decreased cell proliferation in both cell lines. Therefore, the impairment of VEGF signaling might have a crucial role in the treatment of GBM.