Transcript modello per invio relazione di metà e fine periodo

MODELLO PER INVIO RELAZIONE DI METÀ E FINE PERIODO
NOME E COGNOME: Eleonora Cipollari
UNIVERSITÀ: Università degli Studi di Parma
DIPARTIMENTO (in caso di borsa per soggiorno all’estero specificare l’ente presso cui si è svolta
la ricerca): Division of Translational Medicine and Human Genetics, Perelman School of Medicine
at the University of Pennsylvania
TUTOR (in caso di borsa per soggiorno all’estero specificare il tutor dell’ente presso cui si è
svolta la ricerca): Professor Dr. Daniel J. Rader, Dr. Nicholas N. Lyssenko
TIPOLOGIA DI BORSA RICEVUTA: Borsa di ricerca SIF per soggiorno all’estero
TIPOLOGIA DI RELAZIONE (es.: metà periodo o finale): Relazione di metà periodo
TITOLO DELLA RELAZIONE: Cholesterol efflux capacity of cerebrospinal fluid: development of a
novel metric and assessment of variability in a small cohort of subjects
RELAZIONE:
Alterations in lipid metabolism is a common feature underlying atherosclerotic
cardiovascular disease (ASCVD), cognitive decline in dementia and neurodegenerative diseases (1).
A hallmark of ASCVD is accumulation of cholesteryl ester-laden macrophages (called ‘foam’ cells)
in the media intima of arteries (2). The physiological process that counteracts macrophage
accumulation of excess cholesterol starts with cholesterol efflux to interstitial fluid for cholesterol
transport onto plasma lipid-poor apolipoproteins and high-density lipoproteins (HDL (3).
Cholesterol efflux to HDL occurs via both passive and transport-mediated mechanisms and reflects
the critical first step of macrophage reverse cholesterol transport (mRCT) in plasma and lymph to
the liver for ultimate fecal excretion (4).
Since the inheritance of the apolipoprotein E (apoE) ε4 allele was identified as one of the
risk factor for developing Alzheimer’s disease (AD), brain cholesterol has gained increasing
attention for its possible role in AD pathogenesis (5). In human brain, different cells synergistically
work in maintaining cholesterol homeostasis and nervous optimal functions. Most adult neurons
are not competent in synthesizing de novo cholesterol and, as net cholesterol acceptors,
continuously rely on cholesterol derived from glial cells to sustain an adequate membrane pool for
axon transmission and healthy synaptic function. In neurons, B-(BACE) and -secretase, the two
enzymes responsible for amyloid precursor protein (APP) processing, co-localize with cholesterolDa inviare a: Società Italiana di Farmacologia – e-mail: [email protected]; [email protected]
rich lipid rafts of the plasma membrane and changes in cholesterol accumulation have been
shown to promote toxic A cell incorporation and production (6). Astrocytes and microglia, acting
as cholesterol exporters, synthesize cholesterol in situ and secrete it within ApoE particles, which
are responsible for cholesterol supply to neurons (7). High membrane cholesterol in astrocytes
and neurons has been shown to increase Ca-mediated cell death in presence of toxic A (8).
Moreover, in primary microglial cells, the reduction of intracellular cholesterol levels due to ApoEmediated cholesterol efflux is critical to regulate the level of intracellular A degradation (9, 10).
A part of metabolism to diffusional 24-S-hydroxycholesterol, a major pathway in neurons
and the only route available in glial cells for cell cholesterol disposal is efflux to cerebrospinal fluid
(CSF) (11, 12). Therefore, our current understanding of Alzheimer’s disease suggests that
cholesterol efflux may play a role in the etiology of this condition by as many as three different
mechanisms. Intriguingly and similarly to lipids in the circulation, brain cholesterol is carried on
apolipoproteins and HDL particles in CSF. CSF originates by selective permeability of blood
components at the choroid plexuses, it bathes the brain parenchyma and then drains into the
systemic lymphatic circulation via the perineural space of the olfactory nerve or dural lymph
vessels (13, 14).
Consequently, it is possible that, as well as the plasma ability to remove cholesterol from
macrophage is crucial to avoid progression of ASCVD, the capacity of CSF to remove cholesterol
from neurons, astrocytes and microglia is relevant to prevent Aβ toxicity and maintain proper
brain homeostasis. In particular, the aim of this study is to adapt a well-established assay that
measures HDL cholesterol efflux capacity (HDL-CEC), a metric inversely associated with ASCVD (15,
16), to measure cerebrospinal fluid cholesterol efflux capacity (CSF-CEC) for pathophysiological
investigation of Alzheimer’s disease.
In the first stage of the project, we selected and characterized cell lines that will be used as
reference model for the CSF-CEC assay. We have selected SH-SY5Y human neural cells, A172
human astrocyte cells and murine microglial N9 cells. These thee cell lines are widely used in the
scientific community and are thought to reflect molecular biology of the corresponding primary
cells most closely among all the available immortalized cells. Characteristics of selected brainrelated cell lines are compared to those of the J774 mouse macrophage cells that are in standard
use for HDL cholesterol efflux capacity quantification.
The expression of cholesterol efflux proteins have been determined at basal and
upregulated levels by Western Blot. The three cell types express ABCA1, ABCG1 and SR-B1, (Fig.1),
albeit at lower levels than J774 cells. The cell lines have also been characterized for the secretion
of apolipoprotein AI (apoA-I) and apoE and for the the ability to efflux cholesterol by the ABCA1dependent and ABCA1-independent pathways to different acceptors (apoAI, HDL and plasma). The
cells do not express apolipoprotein AI (apoA-I) or apoE, except for N9 cells, which express apoE at
low levels (data not shown). The cells release cholesterol to exogenously added apoA-I and HDL,
albeit again at lower levels than J774 cells (Fig. 2B, C). A172 and N9 cells, better than SH-SY5Y cells,
were able to differentiate between plasma with high and low CEC (Fig. 3). Preliminary results on
cholesterol efflux from these cells to CSF show a much more robust response. Next steps of the
project will include the optimization of assay parameters, the assessment of variability in CSF-CEC
Da inviare a: Società Italiana di Farmacologia – e-mail: [email protected]; [email protected]
and the identification CSF factors that are correlated with CSF-CEC in a small cohort of anonymized
individuals.
Figure 1
Figure 2
Figure 3
Da inviare a: Società Italiana di Farmacologia – e-mail: [email protected]; [email protected]
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Da inviare a: Società Italiana di Farmacologia – e-mail: [email protected]; [email protected]