Fibrous nanocellulose, crystalline nanocellulose, carbon nanotubes, and crocidolite asbestos elicit disparate immune responses upon pharyngeal aspiration in mice

With the rapid development of synthetic alternatives to mineral fibers, their possible effects on the environment and human health have become recognized as important issues worldwide. This study investigated effects of four fibrous materials, i.e. nanofibrillar/nanocrystalline celluloses (NCF and CNC), single-walled carbon nanotubes (CNTs), and crocidolite asbestos (ASB), on pulmonary inflammation and immune responses found in the lungs, as well as the effects on spleen and peripheral blood immune cell subsets. BALB/c mice were given NCF, CNC, CNT, and ASB on Day 1 by oropharyngeal aspiration. At 14 days post-exposure, the animals were evaluated. Total cell number, mononuclear phagocytes, polymorphonuclear leukocytes, lymphocytes, and LDH levels were significantly increased in ASB and CNT-exposed mice. Expression of cytokines and chemokines in bronchoalveolar lavage (BAL) was quite different in mice exposed to four particle types, as well as expression of antigen presentation-related surface proteins on BAL cells. The results revealed that pulmonary exposure to fibrous materials led to discrete local immune cell polarization patterns with a TH2-like response caused by ASB and TH1-like immune reaction to NCF, while CNT and CNC caused non-classical or non-uniform responses. These alterations in immune response following pulmonary exposure should be taken into account when testing the applicability of new nanosized materials with fibrous morphology

Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11

Objective Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work withZfyve26−/− mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction

Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

<div><p>Hereditary spastic paraplegias (HSPs) are a genetically diverse group of inherited neurological disorders (SPG1-72) with the cardinal feature of prominent lower-extremity spasticity due to a length-dependent axonopathy of corticospinal motor neurons. The most frequent form of autosomal dominant HSP results from mutations of the <i>SPG4</i> gene product spastin. This is an ATPase associated with diverse cellular activities (AAA) protein that binds to and severs microtubules. While spastin participates in crucial cellular processes such as cytokinesis, endosomal tubulation, and axon development, its role in HSP pathogenesis remains unclear. Spastin interacts in cells with the NA14 protein, a major target for auto-antibodies in Sjögren's syndrome (nuclear autoantigen 1; SSNA1). Our analysis of endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture revealed a clear distribution of both proteins to centrosomes, with NA14 localizing specifically to centrioles. Stable NA14 knockdown in cell lines dramatically affected cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increased axon outgrowth and branching, while also enhancing neuronal differentiation. We postulate that NA14 may act as an adaptor protein regulating spastin localization to centrosomes, temporally and spatially regulating the microtubule-severing activity of spastin that is particularly critical during the cell cycle and neuronal development.</p></div

NA14 and spastin localize to the centrosome and midbody during cytokinesis.

<p>(A) Merged images of endogenous NA14 (green) accumulated at the centrosome during interphase and at the midbodies during late cytokinesis, along with β-tubulin (red). (B) Endogenous spastin (green) localizes to the centrosome and the midbodies, as shown by co-staining for β-tubulin (red). Merged images are at the right. (C) HeLa cell lines stably expressing control shRNA (shCTL) or shRNAs against NA14 (sh3 shown) were immunostained for endogenous β-tubulin (green) and γ-tubulin (red), with merged images at the right. Bar: 10 µm. (D) Cell extracts from cell lines stably expressing the indicated shRNAs were immunoblotted for NA14. PLCγ1 (149 kDa) and β-tubulin levels were monitored to control for protein loading. # denotes a cross-reacting band. (E and F) Mitotic and multinucleated cells were quantified in control and NA14 shRNA stable cell lines (means ±SEM; <i>n</i> = 3, with 100 cells per experiment). Nuclei were identified by co-staining with DAPI. (G) Quantification of cell death in control and NA14 shRNA stable cells lines by measuring lactate dehydrogenase release from cells (means ±SEM; <i>n</i> = 3, with 100 cells per experiment). *<i>p</i><0.05; **<i>p</i><0.01.</p

PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified centrosomal P4.1-associated protein (CPAP), a human homologue of SAS-4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage-specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho-resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle

Wild-type NA14 but not NA14 (33–119) localizes to the centrosome.

<p>(A) Endogenous NA14 and HA-NA14 from stable cell lines (green) were co-stained with pericentrin antibodies (red) and visualized using confocal microscopy. Hatched boxes are enlarged in the lower right-hand corner insets. (B) HA-NA14 (33–119) stably expressed in HEK293T cells (red) was co-stained with pericentrin antibodies (green). Relative fluorescence intensities for the indicated linear regions in merged images (boxed area enlarged in lower left-hand corner inset) were measured using Zeiss LSM710 software and graphed. AU, arbitrary units. Scale bars: 10 µm. (C) Relative numbers of HeLa cells accumulating the indicated endogenous or recombinant NA14 proteins in the centrosome were quantified (means ±SEM; <i>n</i> = 3, with 100 cells per trial).</p

Morphological analysis of control and NA14-overexpressing cortical neurons.

<p>(A) Representative neurons at developmental stages I, II, and III were co-stained for tau-1 (red) and MAP-2 (green). Merged images are at the right. Scale bar: 40 µm. (B) Pie graphs showing the percentages of cortical neurons in stages I, II, and III in each experimental group (<i>n</i>>100). More neurons remained in stages I and II in untransfected and NA14 (33–119) expressing cultures than in HA-NA14 expressing neurons. (C) β-tubulin staining (black) reveals processes of transfected and control cultured neurons at 3 and 6DIV. Scale bar: 40 µm. (D and E) Quantifications of primary axon length as well as number of primary axon branches in cortical neurons in primary culture (means ±SEM; <i>n</i> = 3, with 30–60 neurons per trial). (F) Numbers of dendrites per cell are shown graphically (means ±SD; <i>n</i> = 3, with 30–60 neurons per trial). *<i>p</i><0.05, ***<i>p</i><0.001.</p

NA14 localizes to the centrosome and interacts with the M87 isoform of spastin.

<p>(A) Top, Schematic diagrams showing the domain organizations of spastin isoforms generated through use of 2 different translation start codons (exon 4 splice cassettes are also indicated). Bottom, HeLa cells were transfected with Myc-tagged spastin M1 or spastin M87 and immunoblotted (IB) with Myc-tag antibody. Migrations of molecular weight standards (in kDa) are indicated at the left. (B) Myc-spastin isoforms M1 and M87 (green) were co-stained with pericentrin antibodies (red) and visualized by confocal microscopy. Scale bars: 10 µm. (C) Myc-spastin isoforms M1 and M87 (red) were co-stained with pericentrin antibodies (green) and visualized by confocal microscopy. Relative fluorescence intensities for the indicated linear regions in merged images (boxed areas enlarged in insets at the bottom left) were measured using Zeiss LSM710 software and graphed (right). Scale bar: 10 µm. (D) Relative numbers of HeLa cells accumulating the indicated endogenous or recombinant spastin proteins in the centrosome were quantified (means ±SEM; <i>n</i> = 3, with 100 cells per trial). (E) HeLa cells were transfected with Myc–spastin M1 or M87 as indicated, immunoprecipitated (IP) with NA14 antibodies or non-immune IgG, and immunoblotted (IB) with Myc-epitope antibodies. The input represents 5% of the starting material. Migrations of molecular weight standards (in kDa) are indicated at the left, and the spastin isoform to the right.</p

NA14 accumulates at the base of axons in cultured cortical neurons at 6DIV.

<p>(A) Mixed cortical neurons at 1, 3 and 6DIV were immunostained for endogenous NA14 (green) and γ-tubulin (red); NA14 localizes to the centrosome at 6DIV. Scale bars: 40 µm. Relative fluorescence intensities for the indicated linear regions in merged images were measured using Zeiss LSM710 software and graphed (at the right). AU, arbitrary units. (B) Neurons at 1, 3 and 6DIV were immunostained for endogenous NA14 (red), neurofilaments (gray) and pericentrin (green). The merged images are indicated. Scale bars: 40 µm.</p