Mafalda Cacciottolo

The APOE4 allele shows opposite sex bias in microbleeds and Alzheimer's disease of humans and mice

The apolipoprotein APOE4 allele confers greater risk of Alzheimers disease (AD) for women than men, in conjunction with greater clinical deficits per unit of AD neuropathology (plaques, tangles). Cerebral microbleeds, which contribute to cognitive dysfunctions during AD, also show APOE4 excess, but sex-APOE allele interactions are not described. We report that elderly men diagnosed for mild cognitive impairment and AD showed a higher risk of cerebral cortex microbleeds with APOE4 allele dose effect in 2 clinical cohorts (ADNI and KIDS). Sex-APOE interactions were further analyzed in EFAD mice carrying human APOE alleles and familial AD genes (5XFAD (+/-) /human APOE(+/+)). At 7 months, E4FAD mice had cerebral cortex microbleeds with female excess, in contrast to humans. Cerebral amyloid angiopathy, plaques, and soluble Aβ also showed female excess. Both the cerebral microbleeds and cerebral amyloid angiopathy increased in proportion to individual Aβ load. In humans, the opposite sex bias of APOE4 allele for microbleeds versus the plaques and tangles is the first example of organ-specific, sex-linked APOE allele effects, and further shows AD as a uniquely human condition.

Chaperone-mediated autophagy components are upregulated in sporadic inclusion-body myositis muscle fibres

Sporadic inclusion‐body myositis (s‐IBM) is an age‐associated degenerative muscle disease. Characteristic features are muscle‐fibre vacuolization and intramuscle‐fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone‐mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome‐associated membrane protein type 2A (LAMP2A) and the heat‐shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge.

Activation of the Unfolded Protein Response in Sporadic Inclusion Body Myositis But Not in Hereditary GNE Inclusion Body Myopathy

Abstract Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM),the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-&bgr; and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy caused by the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the endoplasmic reticulum (ER) lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was 1) increased activating transcription factor 4 (ATF4) protein and increased mRNA of its target C/EBP homologous protein; 2) cleavage of the ATF6 and increased mRNA of its target glucose-regulated protein 78; and 3) an increase of the spliced form of X-box binding protein 1 and increased mRNA of ER degradation–enhancing &agr;-mannosidase–like protein, target of heterodimer of cleaved ATF6 and spliced X-box binding protein 1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to similar pathologic phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle.

Dysferlin is a newly identified binding partner of AβPP and it co-aggregates with amyloid-β42 within sporadic inclusion-body myositis (s-IBM) muscle fibers.

composed of Aβ42 [1], and in both diseases Aβ42 was proposed to be pathogenically important [1, 7]. Accordingly, we asked whether dysferlin abnormalities, including accumulation within Aβ42-immuno-positive aggregates, might occur in s-IBM myofibers. Immunofluorescence using a well-characterized antidysferlin monoclonal antibody (Hamlet1, Novocastra) revealed, in all 5 s-IBM muscle biopsies studied, that dysferlin was prominently reduced in the muscle fiber sarcolemma, in contrast to the 4 age-matched controls that had normal sarcolemmal distribution of dysferlin (Fig. 1a–d, f). In addition, most of the s-IBM-vacuolated and/or otherwise abnormal muscle fibers had intra-myofiber dysferlin-immunoreactive “plaque-like” cytoplasmic aggregates (Fig. 1b–d, f). None of 8 disease-control biopsies (4 ALs, 4 peripheral neuropathies) had similar dysferlinimmunoreactive aggregates (not shown). Immunoblots performed from those same s-IBM and control biopsies did not reveal any significant differences in the amount of total dysferlin (Fig. 2a, a’). These data suggest that the sarcolemmal absence of dysferlin might relate to its abnormal distribution into the cytoplasm, perhaps secondary to a dysferlin trapping into the visible cytoplasmic protein aggregates, and probably sub-microscopic forms thereof. Double immunofluorescence, using an anti-Aβ42-specific polyclonal antibody (Calbiochem, La Jolla) plus a monoclonal anti-dysferlin antibody, revealed that virtually all dysferlin-immunoreactive aggregates co-localized with Aβ42-immunoreactive aggregates, both being usually “plaque-like” and of various size and distribution (Fig. 1d–g). Very rarely, there were dysferlin only aggregates. To verify whether immunohistochemical co-localization of dysferlin and Aβ42 reflects their physical association, we performed co-immunoprecipitation/immunoblotting using the anti-dysferlin and 6e10 Dysferlin is a 237 kDa type II trans-membrane protein involved in plasmalemmal repair and resealing after injury, and in T-tubule construction and calcium homeostasis [reviewed in 2]. Although in skeletal and cardiac muscle dysferlin is localized mainly to plasmalemma and cytoplasmic vesicles, recently, it was also localized to T-tubules and other myofiber structures [2]. DYSF gene mutations cause three main clinical phenotypes of autosomal recessive myopathies, called dysferlinopathies [2]. Unlike normal human muscle biopsies in which dysferlin is immunohistochemically present in the muscle fiber sarcolemma, in dysferlinopathies, dysferlin is absent from the sarcolemma, and is either absent or prominently reduced per immunoblots of muscle biopsies and monocytes [2, 5]. Beyond muscle diseases, in Alzheimer disease (AD) brain, dysferlin was abnormally accumulated in dystrophic neurites within amyloid-β (Aβ) plaques [6]. s-IBM, a common aging-associated degenerative myopathy, is pathogenically multifaceted [reviewed in 1]. s-IBM biopsied muscle fibers exhibit a unique complex phenotype, characterized by vacuolated muscle fibers containing congophilic multiprotein aggregates [1]. s-IBM muscle fiber degeneration shares several pathomolecular aspects with AD brain, including accumulations of Aβ and phosphorylated tau, the latter in the form of paired-helical filaments [reviewed in 1]. similarly to AD brain, s-IBM muscle fiber-accumulated Aβ is mainly

Diurnal variation in the proinflammatory activity of urban fine particulate matter (PM 2.5 ) by in vitro assays

Background: Ambient particulate matter (PM) smaller than 2.5 µm in diameter (PM 2.5) undergoes diurnal changes in chemical composition due to photochemical oxidation. In this study we examine the relationships between oxidative activity and inflammatory responses associated with these diurnal chemical changes. Because secondary PM contains a higher fraction of oxidized PM species, we hypothesized that PM 2.5 collected during afternoon hours would induce a greater inflammatory response than primary, morning PM 2.5. Methods: Time-integrated aqueous slurry samples of ambient PM 2.5 were collected using a direct aerosol-into-liquid collection system during defined morning and afternoon time periods. PM 2.5 samples were collected for 5 weeks in the late summer (August-September) of 2016 at a central Los Angeles site. Morning samples, largely consisting of fresh primary traffic emissions (primary PM), were collected from 6-9am (am-PM 2.5), and afternoon samples were collected from 12-4pm (pm-PM 2.5), when PM composition is dominated by products of photochemical oxidation (secondary PM). The two diurnally phased PM 2.5 slurries (am- and pm-PM 2.5) were characterized for chemical composition and BV-2 microglia were assayed in vitro for oxidative and inflammatory gene responses. Results: Contrary to expectations, the am-PM 2.5 slurry had more proinflammatory activity than the pm-PM 2.5 slurry as revealed by nitric oxide (NO) induction, as well as the upregulation of proinflammatory cytokines IL-1β, IL-6, and CCL2 (MCP-1), as assessed by messenger RNA production. Conclusions: The diurnal differences observed in this study may be in part attributed to the greater content of transition metals and water-insoluble organic carbon (WIOC) of am-PM 2.5 (primary PM) vs. pm-PM 2.5 (secondary PM), as these two classes of compounds can increase PM 2.5 toxicity.