Lin, Wen‐Lang

TAR DNA‐binding protein of 43 kDa (TDP‐43) is a nuclear protein involved in transcriptional repression and alternative splicing. It has been shown to be a component of ubiquitin‐positive, tau‐ and á‐synuclein‐negative inclusions in neuronal soma and nuclei as well as neurites of frontotemporal lobar degeneration (FTLD‐U) and in motor neurons of amyotrophic lateral sclerosis (ALS). Recently, it has been co‐localized with Lewy bodies in Lewy body disease (LBD). In this study we use immunogold electron microscopy to study the ultrastructural features of TDP‐43‐labeled structures. The results showed that in all FTLD‐U, ALS and LBD, TDP‐43 was localized to filaments coated with granular material. Both coated and uncoated regions of the filaments were labeled. In FTLD‐U a subset of neurites filled with densely packed filaments were also labeled. The filaments have a diameter of 10–17 nm. Neurofilament and glial filaments were not labeled by TDP‐43. The data suggests that TDP‐43 is a constituent protein in filamentous inclusions in neurodegenerative diseases. Supported by NIH grant AG17216.

Abstract Introduction The cytoprotective PTEN‐induced kinase 1 (PINK1)‐parkin RBR E3 ubiquitin protein ligase (PRKN) pathway selectively labels damaged mitochondria with phosphorylated ubiquitin (pS65‐Ub) for their autophagic removal (mitophagy). Because dysfunctions of mitochondria and degradation pathways are early features of Alzheimer's disease (AD), mitophagy impairments may contribute to the pathogenesis. Methods Morphology, levels, and distribution of the mitophagy tag pS65‐Ub were evaluated by biochemical analyses combined with tissue and single cell imaging in AD autopsy brain and in transgenic mouse models. Results Analyses revealed significant increases of pS65‐Ub levels in AD brain, which strongly correlated with granulovacuolar degeneration (GVD) and early phospho‐tau deposits, but were independent of amyloid beta pathology. Single cell analyses revealed predominant co‐localization of pS65‐Ub with mitochondria, GVD bodies, and/or lysosomes depending on the brain region analyzed. Discussion Our study highlights mitophagy alterations in AD that are associated with early tau pathology, and suggests that distinct mitochondrial, autophagic, and/or lysosomal failure may contribute to the selective vulnerability in disease.

Frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions (FTLD‐U) is the most common cause of frontal lobe dementia. Recently, the TAR DNA binding protein (TDP‐43) has been described as a marker for FTLD‐U. TDP‐43 has been reported to be specific for neuronal inclusions in FTLD‐U and not other degenerative disorders such as Alzheimer's disease (AD). The reported specificity of TDP‐43 makes it possible to detect FTLD‐U in the setting of other neurodegenerative diseases. On this assumption, we determined the frequency of FTLD‐U in AD. Additionally, since FTLD‐U is often associated with hippocampal sclerosis (HpScl), a process characterized by neuronal loss in Sommer's sector of the hippocampus, we also determined the frequency of FTLD‐U in HpScl. A series of 74 AD and 20 HpScl cases were screened with TDP‐43 immunohistochemistry. Immunoreactivity for TDP‐43 was detected in 13 of 20 (65%) HpScl cases and 42 of 74 (57%) AD cases, including 33 of 44 (75%) cases of AD with HpScl and 9 of 30 (30%) cases of AD without HpScl. The specificity of TDP‐43 was assessed by double immunolabeling for tau at the light and electron microscopic level. The frequency of tau and TDP‐43 co‐localization was 13%. At the EM level TDP‐43 immunoreactivity was present in cytosolic granular material, but some tau‐positive filaments were also labeled. The results suggest that FTLD‐U occurs frequently in HpScl and AD.

Progranulin is a mediator of development, tumorigenesis and wound repair. Mutations in the gene for progranulin cause frontotemporal dementia. Little is known about the role of progranulin in the nervous system. The cleavage products of progranulin, granulins, were first isolated from granule‐enriched fractions of human and equine neutrophils and from rat bone marrow. Immunohistochemistry of granulin has been reported in macrophages or monocytes. Here we use a post‐embedding immunogold labeling method to ultrastructurally localize progranulin in mouse and human tissue. Bone marrow from mouse femoral bone was fixed in 4% paraformaldehyde. Human choroid plexus and a brain abscess were obtained from autopsy brains fixed in formalin. Tissues were dehydrated in ethanols, infiltrated and embedded in LR White resin. Thin sections were collected on Formvar‐coated nickel grids and incubated with antibodies to mouse or human progranulin, followed by gold‐conjugated secondary antibodies. In all tissues progranulin was localized to secretory granules and vesicles in neutrophils and macrophages. Labeled granules had morphological features of secretory granules. They were membrane‐bound vesicles with pale to optically dense contents and ranged from 200 to 500 microns in diameter. This is the first report of the ultrastructural localization of progranulin to mouse and human neutrophils and macrophages in normal and inflammatory conditions, including neuroinflammatory disorders.

Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells.

AbstractMutations in microtubule‐associated protein tau gene (MAPT) cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP‐17). Here, we describe a patient with FTDP‐17 and a novel missense mutation in exon 13 of MAPT, p.E372G. We compare clinicopathologic features of this patient to two previously unreported patients with another exon 13 mutation, p.G389R. The patient with the p.E372G mutation was a 40‐year‐old man with behavioral variant frontotemporal dementia (bvFTD), who subsequently developed agrammatic speech and parkinsonism. One of the FTDP‐17 patients with p.G389R mutation presented at age 24 with agrammatic variant of primary progressive aphasia, and subsequently behavioral dysfunction. The other presented at age 53 with bvFTD, followed by agrammatic speech and corticobasal syndrome. Neuropathologic features of FTDP‐17 due to p.E372G were similar to those of p.G389R, including tau‐immunoreactive Pick body‐like neuronal inclusions and swollen, tapering thread‐like processes in white matter immunoreactive for 3‐repeat and 4‐repeat tau. Biochemical analysis of insoluble tau showed similar isoform compositions in p.E372G and p.G389R. Functional studies of the p.E372G mutation showed marked increase in tau filament formation and its reduced ability to promote microtubule assembly. Together these findings indicate that p.E372G is a pathogenic MAPT mutation that causes FTDP‐17 similar to p.G389R.