Loss of TMEM106B potentiates lysosomal and FTLD-like pathology in progranulin-deficient mice

Single nucleotide polymorphisms (SNPs) in TMEM106B encoding the lysosomal type II transmembrane protein 106B increase the risk for frontotemporal lobar degeneration (FTLD) of GRN (progranulin gene) mutation carriers. Currently, it is unclear if progranulin (PGRN) and TMEM106B are synergistically linked and if a gain or a loss of function of TMEM106B is responsible for the increased disease risk of patients with GRN haploinsufficiency. We therefore compare behavioral abnormalities, gene expression patterns, lysosomal activity, and TDP-43 pathology in single and double knockout animals. Grn-/- /Tmem106b-/- mice show a strongly reduced life span and massive motor deficits. Gene expression analysis reveals an upregulation of molecular signature characteristic for disease-associated microglia and autophagy. Dysregulation of maturation of lysosomal proteins as well as an accumulation of ubiquitinated proteins and widespread p62 deposition suggest that proteostasis is impaired. Moreover, while single Grn-/- knockouts only occasionally show TDP-43 pathology, the double knockout mice exhibit deposition of phosphorylated TDP-43. Thus, a loss of function of TMEM106B may enhance the risk for GRN-associated FTLD by reduced protein turnover in the lysosomal/autophagic system

MAPK Signaling Determines Anxiety in the Juvenile Mouse Brain but Depression-Like Behavior in Adults

MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype

Sarcoma classification by DNA methylation profiling

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications

The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons

Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons.status: publishe

Neuronal morphology of granular neurons in dentate gyrus of Braf^cko mice.

<p>(<b>A</b>) Camera lucida drawings of representative granule neurons of Braf^cko controls and mutants. (<b>B</b>) Total and mean length of dendrites was significantly reduced in granular neurons of <i>Braf</i> deficient mice. (<b>C,D</b>) Spine density and tortuosity were not altered due to <i>Braf</i> knockout. (error bars: 95% confidence interval; n.s.: not significant, *: <i>P</i><0.05) (<b>E,F</b>) Sholl analysis of the dendritic branching revealed an overall reduction of complexity of the granular neurons. Number of intersections (<b>E</b>) and dendritic length (<b>F</b>) were significantly reduced in the distance of 80–120 and 140–160 µm (intersections) and 90–120 and 150–160 µm (dendritic length) from soma, respectively (T: tendency, *: <i>P</i><0.05).</p

Molecular analysis and emotional behavior of Braf^cko mice.

<p>(<b>A</b>) Immunohistochemical detection of BRAF of Braf^cko mice revealed depletion of the protein in the hippocampus (shown) and the cortex (not shown). (<b>B</b>) Western blot analysis of hippocampal samples of Braf^cko mice validates the depletion of BRAF protein and the subsequent inactivation of ERK/MAPK signaling. While total levels of ERK1/2 in Braf^cko mice were unaffected, a strong reduction, but no complete depletion of Phospho-ERK1 and -ERK2 was observed in the hippocampus of mutants. The remaining ERK1/2 activity in Braf^cko forebrain regions likely results from non-glutamatergic neuronal or glial cells that express BRAF, but which do not express the CamkIIa-Cre transgene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035035#pone.0035035.s002" target="_blank">Figure S2I</a>). (<b>C</b>) Elevated plus maze: Mutant mice spent significantly more time in the open arms of the maze. (<b>D</b>) Light/dark box: <i>Braf</i> mutants spent more time in the aversive lit compartment. (<b>E,F</b>) No changes in depression-like behavior were observed in Braf^cko mutants in the forced swim test (<b>E</b>) and the tail suspension test (<b>F</b>). (n.s.: not significant, ***: <i>P</i><0.001).</p

Daily voluntary wheel running activity in <i>Braf</i> mutants and controls.

<p>(<b>A</b>) Representative actograms of Braf^cko and control mice. The Braf^cko mouse (lower panel) showed a much more fragmented pattern in a LD 1∶23 experiment compared to its floxed littermate (upper panel). Vertical white bar represents daily light phase. (<b>B</b>) Activity during resting phase is significantly increased in Braf^cko mice. (<b>C</b>) Period length analysis revealed no differences between floxed (23.78±0.04) and Braf^cko mice (23.87±0.04). (<b>D</b>) Mean daily voluntary wheel running activity over 17 days was decreased in Braf^cko mice (7.51±0.23) compared to floxed (9.63±0.22). (n.s.: not significant, ***: <i>P</i><0.001).</p

GABA_A receptor-mediated inhibition is not enhanced in hippocampi of Braf^cko mice.

<p>(<b>A</b>) Extracellular recording of population spikes in CA1 pyramidal cell layer evoked by electrical stimulation of the Schaffer collateral/commissural pathway. Peak amplitudes of population spikes were normalized before application of GABA_A receptor antagonist, picrotoxin (100 µM). Inset depicts individual population spikes recorded at like-numbered time points of the graph in a control slice. Arrow indicates electrical stimulation of afferent pathway. (<b>B</b>) Whole-cell recording of IPSCs from CA1 pyramidal cells. Application of diazepam (3 µM) enhanced IPSC amplitude and slowed decay (inset). Effects of diazepam were quantified with respect to half width, peak amplitude, and area of IPSC. Histograms did not reveal any differences between genotypes.</p

Bioinformatic prediction of CREB1 and ETS/SRF target genes.

<p>Differentially downregulated genes from Braf^cko mice were analyzed for the presence of evolutionary conserved CREB1 and ETS/SRF target sites. Nine genes were found representing putative functional ERK/MAPK pathway effectors (for more detailed results, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035035#pone.0035035.s007" target="_blank">Table S3</a>). (n.t.: not tested).</p