Erez Eitan

Identification of preclinical Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood exosomes: A case-control study

Proteins pathogenic in Alzheimers disease (AD) were extracted from neurally derived blood exosomes and quantified to develop biomarkers for the staging of sporadic AD.

Telomere shortening in neurological disorders: an abundance of unanswered questions

Telomeres, ribonucleoprotein complexes that cap eukaryotic chromosomes, typically shorten in leukocytes with aging. Aging is a primary risk factor for neurodegenerative disease (ND), and a common assumption has arisen that leukocyte telomere length (LTL) can serve as a predictor of neurological disease. However, the evidence for shorter LTL in Alzheimers and Parkinsons patients is inconsistent. The diverse causes of telomere shortening may explain variability in LTL between studies and individuals. Additional research is needed to determine whether neuronal and glial telomeres shorten during aging and in neurodegenerative disorders, if and how LTL is related to brain cell telomere shortening, and whether telomere shortening plays a causal role in or exacerbates neurological disorders.

Extracellular vesicle-depleted fetal bovine and human sera have reduced capacity to support cell growth.

Background Fetal bovine serum (FBS) is the most widely used serum supplement for mammalian cell culture. It supports cell growth by providing nutrients, growth signals, and protection from stress. Attempts to develop serum-free media that support cell expansion to the same extent as serum-supplemented media have not yet succeeded, suggesting that FBS contains one or more as-yet-undefined growth factors. One potential vehicle for the delivery of growth factors from serum to cultured cells is extracellular vesicles (EVs). Methods EV-depleted FBS and human serum were generated by 120,000g centrifugation, and its cell growth–supporting activity was measured. Isolated EVs from FBS were quantified and characterized by nanoparticle tracking analysis, electron microscopy, and protein assay. EV internalization into cells was quantified using fluorescent plate reader analysis and microscopy. Results Most cell types cultured with EV-depleted FBS showed a reduced growth rate but not an increased sensitivity to the DNA-damaging agent etoposide and the endoplasmic reticulum stress–inducing chemical tunicamycin. Supplying cells with isolated FBS-derived EVs enhanced their growth. FBS-derived EVs were internalized by mouse and human cells wherein 65±26% of them interacted with the lysosomes. EV-depleted human serum also exhibited reduced cell growth–promoting activity. Conclusions EVs play a role in the cell growth and survival-promoting effects of FBS and human serum. Thus, it is important to take the effect of EV depletion under consideration when planning EV extraction experiments and while attempting to develop serum-free media that support rapid cell expansion. In addition, these findings suggest roles for circulating EVs in supporting cell growth and survival in vivo.

Novel telomerase‐increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis

Telomerase is expressed in the neonatal brain, in distinct regions of adult brain, and was shown to protect developing neurons from apoptosis. Telomerase reactivation by gene manipulation reverses neurodegeneration in aged telomerase‐deficient mice. Hence, we and others hypothesized that increasing telomerase expression by pharmaceutical compounds may protect brain cells from death caused by damaging agents. In this study, we demonstrate for the first time that the novel compound AGS‐499 increases telomerase activity and expression in the mouse brain and spinal cord (SC). It exerts neuroprotective effects in NMDA‐injected CD‐1 mice, delays the onset and progression of the amyotrophic lateral sclerosis (ALS) disease in SOD1 transgenic mice, and, after the onset of ALS, it increases the survival of motor neurons in the SC by 60%. The survival of telomerase‐expressing cells (i.e. motor neurons), but not telomerase‐deficient cells, exposed to oxidative stress was increased by AGS‐499 treatment, suggesting that the AGS‐499 effects are telomerase‐mediated. Therefore, a controlled and transient increase in telomerase expression and activity in the brain by AGS‐499 may exert neuroprotective effects.

Cargo proteins of plasma astrocyte-derived exosomes in Alzheimer's disease.

Efficient intercellular transfer of RNAs, proteins, and lipids as protected exosomal cargo has been demonstrated in the CNS, but distinct physiologic and pathologic roles have not been well defined for this pathway. The capacity to isolate immunochemically human plasma neuron‐derived exosomes (NDEs), containing neuronspecific cargo, has permitted characterization of CNS‐derived exosomes in living humans. Constituents of the amyloid β‐peptide (Aβ)42‐generating system now are examined in 2 distinct sets of human neural cells by quantification in astrocyte‐derived exosomes (ADEs) and NDEs, enriched separately from plasmas of patients with Alzheimers disease (AD) or frontotemporal dementia (FTD) andmatched cognitively normal controls. ADE levels of β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE‐1), γ‐secretase, soluble Aβ42, soluble amyloid precursor protein (sAPP)β, sAPPα, glial‐derived neurotrophic factor (GDNF), P‐T181‐tau, and P‐S396‐tau were significantly (3‐ to 20‐fold) higher than levels in NDEs for patients and controls. BACE‐1 levels also were a mean of 7‐fold higher in ADEs than in NDEs from cultured rat type‐specific neural cells. Levels of BACE‐1 and sAPPβ were significantly higher and of GDNF significantly lower in ADEs of patients with AD than in those of controls, but not significantly different in patients with FTD than in controls. Abundant proteins of the Aβ42 peptide‐generating system in ADEs may sustain levels in neurons. ADE cargo proteins may be useful for studies of mechanisms of cellular interactions and effects of BACE‐1 inhibitors in AD.—Goetzl, E. J., Mustapic, M., Kapogiannis, D., Eitan, E., Lobach, I. V., Goetzl, L., Schwartz, J. B., Miller, B. L. Cargo proteins of plasma astrocyte‐derived exosomes in Alzheimers disease. FASEB J. 30, 3853–3859 (2016) www.fasebj.org

Impact of lysosome status on extracellular vesicle content and release

Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells.

Are there roles for brain cell senescence in aging and neurodegenerative disorders

The term cellular senescence was introduced more than five decades ago to describe the state of growth arrest observed in aging cells. Since this initial discovery, the phenotypes associated with cellular senescence have expanded beyond growth arrest to include alterations in cellular metabolism, secreted cytokines, epigenetic regulation and protein expression. Recently, senescence has been shown to play an important role in vivo not only in relation to aging, but also during embryonic development. Thus, cellular senescence serves different purposes and comprises a wide range of distinct phenotypes across multiple cell types. Whether all cell types, including post-mitotic neurons, are capable of entering into a senescent state remains unclear. In this review we examine recent data that suggest that cellular senescence plays a role in brain aging and, notably, may not be limited to glia but also neurons. We suggest that there is a high level of similarity between some of the pathological changes that occur in the brain in Alzheimer’s and Parkinson’s diseases and those phenotypes observed in cellular senescence, leading us to propose that neurons and glia can exhibit hallmarks of senescence previously documented in peripheral tissues.

Aging enhances release of exosomal cytokine mRNAs by Aβ1-42-stimulated macrophages

Amyloid‐β1‐42 (Aβ) peptide effects on human models of central nervous system (CNS)‐patrolling macrophages (Ms) and CD4 memory T‐cells (CD4‐Tms) were investigated to examine immune responses to Aβ in Alzheimers disease. Aβ and lipopolysaccharide (LPS) elicited similar M cytokine and exosomal mRNA (ex‐mRNA) responses. Aβ‐ and LPS‐stimulated Ms from 20 ≥65‐yr‐old subjects generated significantly more IL‐1, TNF‐α, and IL‐6, but not IL‐8 or IL‐12, and significantly more ex‐mRNAs for IL‐6, TNF‐α, and IL‐12, but not for IL‐8 or IL‐1, than Ms from 20 matched 21‐ to 45‐yr‐old subjects. CD4‐Tm generation of IL‐2, IL‐4, and IFN‐γ and, for young subjects, IL‐10, but not IL‐6, evoked by Aβ was significantly lower than with anti‐T‐cell antigen receptor antibodies (Abs). Abs significantly increased all CD4‐Tm ex‐mRNAs, but only IL‐2 and IL6 ex‐mRNAs were increased by Aβ. There were no significant differences between cytokine and ex‐mRNA responses of CD4‐Tms from the old compared to the young subjects. M‐derived serum exosomes from the old subjects had significantly higher IL‐6 and IL‐12 ex‐mRNA levels than those from the young subjects, whereas there were no differences for CD4‐Tm‐derived serum exosomes. An Aβ level relevant to neurodegeneration elicited broad M cytokine and ex‐mRNA responses that were significantly greater in the old subjects, but only narrow and age‐independent CD4‐Tm responses.—Mitsuhashi, M., Taub, D. D., Kapogiannis, D., Eitan, E., Zukley, L., Mattson, M. P., Ferrucci, L., Schwartz, J. B., Goetzl, E. J., Aging enhances release of exosomal cytokine mRNAs by Aβ1‐42‐stimulated macrophages. FASEB J. 27, 5141–5150 (2013). www.fasebj.org

Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes

Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.

Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes

Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimers and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum, or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EVs enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner.