Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild typ

Proteome profiling of s-nitrosylated synaptosomal proteins by isobaric mass tags.

peer reviewed• Protocol for quantitative proteomics of nitrosylation on synaptosomal proteins. • Identification of endogenous nitrosylation independent of induction by NO donors. • Use of iodoTMT sixplex mass tags for stable labeling, enrichment, identification, and multiplex quantitation. • Applicable on low amounts of sample material of mouse and human brain tissue.[Figure: see text

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.This article is published as Elashry, Abdelnaser M., Samer S. Habash, Paramasivan Vijayapalani, Nahal Brocke-Ahmadinejad, Roman Blümel, Arun Seetharam, Heiko Schoof, and Florian MW Grundler. "Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii." Scientific Reports 10 (2020): 3315. DOI: 10.1038/s41598-020-60186-0. Posted with permission.</p

Quantitative proteomics of synaptosome S

peer reviewedIncreasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2-/- mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies