FAS-dependent cell death in α-synuclein transgenic oligodendrocyte models of multiple system atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention

Analysis of recovered tourniquets from casualties of Operation Enduring Freedom and Operation New Dawn.

BACKGROUND: Tourniquet use recently became common in war, but knowledge gaps remain regarding analysis of recovered devices. The purpose of this study was to analyze tourniquets to identify opportunities for improved training. METHODS: We analyzed tourniquets recovered from deceased service members serving in support of recent combat operations by a team at Dover Air Force Base from 2010 to 2012. Device makes and models, breakage, deformation, band routing, and windlass turn numbers were counted. RESULTS: We recovered 824 tourniquets; 390 were used in care and 434 were carried unused. Most tourniquets were recommended by the Committee on Tactical Combat Casualty Care (Combat Application Tourniquet [CAT] or Special Operations Forces Tactical Tourniquet). The band was routed once through the buckle in 37% of used CATs, twice in 62%, and 1% had none. For tourniquets with data, the windlass turn number averaged 3.2 (range, 0-9). The CAT windlass turn number was associated positively with tourniquet deformation as moderate or severe deformation began at 2 turns, increased in likelihood stepwise with each turn, and became omnipresent at 7 or more. CONCLUSIONS: Tourniquet counts, band routings, windlass turn numbers, and deformation rates are candidate topics for instructors to refine training

Detection of elevated levels of soluble α-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies

A number of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy are characterized by the formation and intraneuronal accumulation of fibrillar aggregates of alpha-synuclein (alpha-syn) protein in affected brain regions. These and other findings suggest that the accumulation of alpha-syn in the brain plays an important role in the pathogenesis of these diseases. However, more recently it has been reported that early amyloid aggregates or 'soluble oligomers' are the pathogenic species that lead to neurodegeneration and neuronal cell death rather than the later 'mature fibrils'. In this study, we investigated the presence of alpha-syn oligomers in brain lysates prepared from frozen post-mortem brains of normal, Alzheimer's disease and DLB patients. The brain extracts were subjected to high speed centrifugation, to remove insoluble alpha-syn aggregates, followed by specific detection of soluble oligomers in the supernatants by employing FILA-1, an antibody that specifically binds to alpha-syn aggregates, but not to alpha-syn monomers, or to tau or beta-amyloid aggregates. Using this novel enzyme-linked immunosorbent assay (ELISA) method to quantify the amounts of alpha-syn oligomers in the brain extracts, our data clearly show an increase in the levels of soluble oligomers of alpha-syn in the DLB brains compared to those with Alzheimer's disease and the controls (P <0.0001). Our findings provide strong evidence to support the contention that elevated soluble oligomers of alpha-syn are involved in the pathogenesis of DLB. Furthermore, these findings establish FILA-1 as a very sensitive tool for the detection of oligomeric forms of alpha-syn in human brain lysates

FAS is upregulated in human MSA brain.

<p><b>A, </b><b>B,</b> Immunohistochemical staining for FAS in normal controls. In normal controls there were only occasional FAS-positive cells shown here in (A) the inferior temporal cortical white matter and (B) grey matter. Insert in (A) shows a FAS-positive cell resembling a microglia. <b>C–H,</b> Immunohistochemical staining for FAS in MSA cases. MSA tissue contained numerous FAS-positive glial cells shown here in the inferior temporal cortical (C) white matter and (D) grey matter. Arrows indicate FAS-positive oligodendrocytes, and arrowheads indicate FAS-positive astrocytes or microglia. Inserts in (C) and (D) shows enlarged view of boxed regions showing FAS-positive oligodendrocytes (judged by their nuclear morphology). The oligodendrocyte in (D) contains an inclusion. E, FAS-positive glial cells in the cerebellar white matter. Boxed region in (E) is enlarged in (F) showing oligodendrocytes. G, FAS-positive cells in the pons. Arrow indicates a FAS-positive glial cell resembling a GCI-bearing oligodendrocyte. Arrowheads indicate FAS-positive astrocytes and microglia. Boxed region in (G) is enlarged in (H). <b>I,</b> Brain tissue (precentral gyrus white matter) from human MSA (<i>n</i> = 8) and control cases (<i>n</i> = 10) was sequentially extracted for TBS and SDS soluble proteins and the SDS soluble fraction were used for further analysis. Four MSA and four control cases are shown. Proteins (20 μg) were resolved by SDS-PAGE and analyzed by immunoblotting using antibodies against α-syn, p25α and FAS. Actin was included as a loading control. The molecular sizes (kDa) of the presented bands are indicated to the left. There was a significant increase in SDS-soluble FAS protein in MSA cases compared to controls (<i>p</i> = 0.043).</p

α-synuclein expressing oligodendrocytes are sensitized to FAS-dependent toxicity.

<p><b>A, </b><i>In vitro</i> differentiation of (PLP)-α-syn tg oligodendrocytes. Oligodendrocyte progenitor cells were isolated from tg mouse forebrains and cultured for the indicated times. Cells were fixed for double-label immunostaining with antibodies against A2B5, NG2, O4, GalC and MBP (green; left panels) as well as monoclonal 15G7 against human α-syn (red; right panels). Differentiated cells (7 DIV) were immunostained with an antibody against p25α and visualized by phase contrast microscopy. Scale bars, 10 µm. <b>B,</b> Human α-syn and eGFP tg oligodendrocytes were treated for 24 h with DMSO (control), sFASL, mFASL or preincubated for 30 min with FAS-blocking antibody before challenge with mFASL. Cells were fixed and labeled with O4 and Hoechst. mFASL treatment induced oligodendrocyte cell death as determined by apoptotic nuclei with α-syn oligodendrocytes being significantly more sensitive to FAS mediated cell death than eGFP oligodendrocytes. Data represent mean ± SEM of total tg oligodendrocytes from three independent experiments. Student <i>t</i> test (<i>n</i> = 3): *<i>p</i><0.0001 compared with untreated cultures; #<i>p</i><0.001 compared with eGFP tg cultures exposed to the same challenge. Western blots prepared from lysates of wild-type and α-syn tg oligodendrocyte cultures were sequentially probed with monoclonal anti-FAS and anti-α-tubulin antibodies (insert).</p

FAS colocalizes with p25α and α-synuclein in human MSA brain.

<p>Double labeling immunofluorescence using FAS (red) and p25α (green) antibodies (A–F) and FAS (red) and α-syn (green) antibodies (G–L) in putamen of an MSA case. Protein colocalization is shown as yellow in merged images (C, F, I, L). A–C, Identification of p25α-positive/FAS-negative GCIs (arrows) in oligodendroglia. D–F, Colocalization of FAS and p25α within GCI-like structures but not in small punctate cytoplasmic granules. G–L, FAS and α-syn colocalized in 4% of the GCIs (J–L) but the majority of α-syn-positive GCIs did not colocalize FAS (G–I, arrows indicate α-syn-positive/FAS-negative inclusions).</p

α-synuclein dependent degeneration in OLN-93 cells requires FAS signaling and caspase-8 activation.

<p>OLN-t40-AS cells stably expressing human α-syn were treated with peptide aldehyde inhibitors (20 µM) against caspase-3 (Ac-DEVD-CHO), caspase-8 (Ac-IETD-CHO), caspase-9 (Ac-LEHD-CHO) and FAS-blocking antibody (ZB4) (1 µg/ml) 1 h prior to transfection with p25α. MT retraction was quantified my immunofluorescence microscopy 24 h after transfection. Bars represent the mean ± standard error of mean (SEM) from three independent experiments. Inhibition of caspase-3, caspase-8 and FAS but not caspase-9 caused a significant reduction in MT retraction as compared with the control cells (<i>p</i><0.05 with respect to untreated cells).</p