Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples.

Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.

Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

Human axonal survival of motor neuron (a-SMN) protein stimulates axon growth, cell motility, C-C motif ligand 2 (CCL2), and insulin-like growth factor-1 (IGF1) production.

Spinal muscular atrophy is a fatal genetic disease of motoneurons due to loss of full-length survival of motor neuron protein, the main product of the disease gene SMN1. Axonal SMN (a-SMN) is an alternatively spliced isoform of SMN1, generated by retention of intron 3. To study a-SMN function, we generated cellular clones for the expression of the protein in mouse motoneuron-like NSC34 cells. The model was instrumental in providing evidence that a-SMN decreases cell growth and plays an important role in the processes of axon growth and cellular motility. In our conditions, low levels of a-SMN expression were sufficient to trigger the observed biological effects, which were not modified by further increasing the amounts of the expressed protein. Differential transcriptome analysis led to the identification of novel a-SMN-regulated factors, i.e. the transcripts coding for the two chemokines, C-C motif ligands 2 and 7 (CCL2 and CCL7), as well as the neuronal and myotrophic factor, insulin-like growth factor-1 (IGF1). a-SMN-dependent induction of CCL2 and IGF1 mRNAs resulted in increased intracellular levels and secretion of the respective protein products. Induction of CCL2 contributes to the a-SMN effects, mediating part of the action on axon growth and random cell motility, as indicated by chemokine knockdown and re-addition studies. Our results shed new light on a-SMN function and the underlying molecular mechanisms. The data provide a rational framework to understand the role of a-SMN deficiency in the etiopathogenesis of spinal muscular atrophy.

APP mutations in the Aß coding region are associated with abundant cerebral deposition of Aß38.

Aß is the main component of amyloid deposits in Alzheimer disease (AD) and its aggregation into oligomers, protofibrils and fibrils is considered a seminal event in the pathogenesis of AD. Aß with C-terminus at residue 42 is the most abundant species in parenchymal deposits, whereas Aß with C-terminus at residue 40 predominates in the amyloid of the walls of large vessels. Aß peptides with other C-termini have not yet been thoroughly investigated. We analysed Aß38 in the brains of patients with Aß deposition linked to sporadic and familial AD, hereditary cerebral haemorrhage with amyloidosis, or Down syndrome. Immunohistochemistry, confocal microscopy, immunoelectron microscopy, immunoprecipitation and the electrophoresis separation of low molecular weight aggregates revealed that Aß38 accumulates consistently in the brains of patients carrying APP mutations in the Aß coding region, but was not detected in the patients with APP mutations outside the Aß domain, in the patients with presenilin mutations or in subjects with Down syndrome. In the patients with sporadic AD, Aß38 was absent in the senile plaques, but it was detected only in the vessel walls of a small subset of patients with severe cerebral amyloid angiopathy. Our results suggest that APP mutations in the Aß coding region favour Aß38 accumulation in the brain and that the molecular mechanisms of Aß deposition in these patients may be different from those active in patients with familial AD associated with other genetic defects and sporadic AD.

Dry and survive: morphological changes during anhydrobiosis in a bdelloid rotifer.

Bdelloid rotifers are aquatic microinvertebrates able to cope with the loss of environmental water by entering dormancy, and are thus capable of living in temporary habitats. When water is evaporating, bdelloids contract into "tuns", silence metabolism and lose water from the body, a condition known as anhydrobiosis. Under controlled conditions, a bdelloid species (Macrotrachela quadricornifera) was made anhydrobiotic, and its morphology was studied by light, confocal and electron microscopy. A compact anatomy characterizes the anhydrobiotic rotifer, resulting in a considerable reduction of its body volume: the internal organs, precisely packed together, occupy the body cavity almost completely and the lumen of hollow organs disappears. Remarkable ultrastructural changes characterize the anhydrobiotic condition. The mitochondria are wholly surrounded by a ring of electron-dense particles, and the epidermal pores, open in the hydrated specimens, become gradually closed by structures similar to epithelial junctions. The cilia are densely packed: microtubules are still identifiable, but the axonemal organization appears disrupted. This is the first extensive comparative study on the morphological changes associated with the anhydrobiosis process in a rotifer, providing the basis for an improved understanding of the processes involved in this extreme adaptation.

Neuropathology of the recessive A673V APP mutation: Alzheimer disease with distinctive features.

Mutations of three different genes, encoding ß-amyloid precursor protein (APP), presenilin 1 and presenilin 2 are associated with familial Alzheimer's disease (AD). Recently, the APP mutation A673V has been identified that stands out from all the genetic defects previously reported in these three genes, since it causes the disease only in the homozygous state (Di Fede et al. in Science 323:1473-1477, 2009). We here provide the detailed neuropathological picture of the proband of this family, who was homozygous for the APP A673V mutation and recently came to death. The brain has been studied by histological and immunohistochemical techniques, at the optical and ultrastructural levels. Cerebral Aß accumulation and tau pathology were severe and extensive. Peculiar features were the configuration of the Aß deposits that were of large size, mostly perivascular and exhibited a close correspondence between the pattern elicited by amyloid stainings and the labeling obtained with immunoreagents specific for Aß40 or Aß42. Moreover, Aß deposition spared the neostriatum while deeply affecting the cerebellum, and therefore was not in compliance with the hierarchical topographical sequence of involvement documented in sporadic AD. Therefore, the neuropathological picture of familial AD caused by the APP recessive mutation A673V presents distinctive characteristics compared to sporadic AD or familial AD inherited as a dominant trait. Main peculiar features are the morphology, structural properties and composition of the Aß deposits as well as their topographic distribution in the brain.

SREBP1 drives Keratin-80-dependent cytoskeletal changes and invasive behavior in endocrine-resistant ERα breast cancer.

Approximately 30% of ERα breast cancer patients relapse with metastatic disease following adjuvant endocrine therapies. The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain undergoes epigenetic reprogramming in aromatase inhibitors (AI)-resistant cells, leading to Keratin-80 (KRT80) upregulation. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 1 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion and cellular stiffening, collectively promoting cancer cell invasion. Shearwave elasticity imaging performed on prospectively recruited patients confirms KRT80 levels correlate with stiffer tumors. Immunohistochemistry showed increased KRT80-positive cells at relapse and, using several clinical endpoints, KRT80 expression associates with poor survival. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment.

Author Correction: SREBP1 drives keratin-80-dependent cytoskeletal changes and invasive behavior in endocrine-resistant ERα breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Surviving starvation: changes accompanying starvation tolerance in a bdelloid rotifer.

Bdelloid rotifers survive desiccation and starvation by halting activity and entering a kind of dormancy. To understand the mechanisms of survival in the absence of food source, we studied the anatomical and ultrastructural changes occurring in a bdelloid species, Macrotrachela quadricornifera Milne 1886, after starvation for different periods. The starved rotifers present a progressive reduction of body size accompanied with a consistent reduction of the volume of the stomach syncytium, where lipid inclusions and digestive vacuoles tend to fade with prolonged starvation. Similar reduction occurs in the vitellarium gland, in which yolk granules progressively decrease in number and size. The changes observed in the syncytia of the stomach and the vitellarium suggest that during starvation M. quadricornifera uses resources diverted from the stomach syncytium first and from the vitellarium syncytium later, resources that are normally allocated to reproduction. The fine structure of starved bdelloids is compared with that of anhydrobiotic bdelloids, revealing that survival during either forms of dormancy is sustained by different physiological mechanisms.

New mutations in MAPT gene causing frontotemporal lobar degeneration: biochemical and structural characterization.

Frontotemporal lobar degeneration (FTLD) can be sporadic or familial. The genes encoding the microtubule-associated protein tau (MAPT) and progranulin (GRN) are the most relevant genes so far known causing the hereditary forms. Following genetic screening of patients affected by FTLD, we identified 2 new MAPT mutations, P364S and G366R, the former in a sporadic case. In the study we report the clinical and genetic features of the patients carrying these mutations, and the functional effects of the mutations, analyzed in vitro in order to investigate their pathogenic character. Both mutations resulted in reduced ability of tau to promote microtubule polymerization; the P364S protein variant also showed a high propensity to aggregate into filaments. These results suggest a high probability that these mutations are pathogenic. Our findings highlight the importance of genetic analysis also in sporadic forms of FTLD, and the role of in vitro studies to evaluate the pathologic features of new mutations.