Volume 10, Number 1, 2007
© Mary Ann Liebert, Inc.
Evidence that Aging and Amyloid Promote
Microglial Cell Senescence
Barry E. Flanary,1Nicole W. Sammons,1Cuong Nguyen,2
Douglas Walker,3and Wolfgang J. Streit1
Advanced age and presence of intracerebral amyloid deposits are known to be major risk fac-
tors for development of neurodegeneration in Alzheimer’s disease (AD), and both have been
associated with microglial activation. However, the specific role of activated microglia in AD
pathogenesis remains unresolved. Here we report that microglial cells exhibit significant telo-
mere shortening and reduction of telomerase activity with normal aging in rats, and that in
humans there is a tendency toward telomere shortening with presence of dementia. Human
brains containing high amyloid loads demonstrate a significantly higher degree of microglial
dystrophy than nondemented, amyloid-free control subjects. Collectively, these findings show
that microglial cell senescence associated with telomere shortening and normal aging is ex-
acerbated by the presence of amyloid. They suggest that degeneration of microglia is a fac-
tor in the pathogenesis of AD.
tem (CNS) constituting the brain’s endogenous
system of immunocompetent cells.1,2Micro-
glial cells are activated quickly in acute CNS
injury situations when they undergo mitosis to
increase their numbers.3,4This acute neuroin-
flammatory response, which correlates well
with the postaxotomy recovery of motoneu-
rons and subsequent axonal regeneration, sup-
ports the view that microglia are constitutively
neuroprotective cells.5,6On the other hand, po-
tentially detrimental, chronic microglial neu-
ICROGLIA REPRESENT A MAJOR glial cell pop-
ulation within the central nervous sys-
roinflammation and neurotoxicity have often
been discussed as factors in the pathogenesis of
neurodegenerative disorders, particularly Alz-
heimer’s disease (AD) where intracerebral
amyloid deposits are thought to induce and
sustain prolonged microglial activation accom-
panied by the production of microglial neuro-
toxins.7–11However, postmortem observations
have produced evidence showing that some of
the ostensibly activated microglia in aged and
diseased human brain also exhibit signs of cell
death and cytoplasmic degeneration,12–16the
latter having been termed microglial dystro-
phy.12These observations document that mi-
croglia themselves are subject to structural de-
1Department of Neuroscience, University of Florida College of Medicine, McKnight Brain Institute, Gainesville,
2Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine,
3Laboratory of Neuroinflammation, Sun Health Research Institute, Sun City, Arizona.
terioration, and they raise the possibility that
microglial degeneration may precede neuro-
degeneration and therefore contribute to it
through diminishing neuroprotection.
Telomeres, the physical ends of eukaryotic
chromosomes, naturally shorten during each
cell division because of the inability of DNA
polymerase to completely replicate linear DNA
molecules.17Certain cells can maintain their
telomeres utilizing telomerase, an enzyme ca-
pable of relengthening telomeres.18However,
in somatic cells, telomeres continually shorten
with age and division,19and cells eventually
exhaust their replicative potential, become in-
capable of further division,20and ultimately en-
ter replicative senescence, which is a nondi-
viding state characterized by critically short
telomeres,21and substantial changes in cell
function and gene expression.22,23The shortest
telomere length, not the average, is thought to
be responsible for maintaining chromosome
stability, cell viability, and determining when
a cell will enter senescence.21Recent work from
this laboratory examining rat microglia telom-
eres and telomerase activity has shown that
telomere shortening occurs over time in vitro
and that mechanisms of telomere maintenance
are active during microglial activation and pro-
liferation in vivo24,25supporting the idea that
microglia, as the only mature cell type in the
CNS capable of significant cell division, are
subject to replicative senescence.26,27In the cur-
rent study, we have sought more direct evi-
dence in support of microglial cell senescence
focusing to incorporate the role of amyloid pro-
tein in this process. The findings reported here
provide strong support for a novel view on AD
pathogenesis that takes into account both ag-
ing and amyloid as important factors in a com-
mon pathway that involves progressive dam-
age to the brain’s immune system.
MATERIALS AND METHODS
FACS isolation of rat microglia
Male Fisher-344 Brown Norway hybrid rats
were exsanguinated while they were under
deep sodium pentobarbital anesthesia (50
mg/kg body weight) using ice-cold phosphate-
buffered saline (PBS). The brains were removed
and placed in PBS on ice. Dissection and isola-
tion of cerebral cortex from the chilled brains
was performed immediately after removal.
Cortex tissue was collected from 3- and 30-
month-old rats (three animals per group). Tis-
sue was processed according to established iso-
lation protocols.28Fluorescence-activated cell
sort (FACS) analysis was performed with a
FACSVantage SE cell sorter and CellQuest soft-
ware (BD Biosciences/Becton Dickinson, San
Jose, CA). Monoclonal antibodies used to iso-
late microglia during FACS analysis were flu-
orescein isothiocyanate (FITC)-conjugated anti-
rat CD45 (leukocyte common antigen), and
PE-conjugated anti-rat CD11b/c (CR3 comple-
ment; BD Biosciences/Pharmingen, San Diego,
CA), with microglia being identified as the
CD11b/c-high and CD45-low cell popula-
Culturing of rat microglia
Microglia were isolated from newborn
Sprague-Dawley rat brains. The cerebral cor-
tices of neonatal rats (?3 days) were stripped
of meninges and minced with a sterile scalpel
blade in a 35 ? 10 mm dish containing filter-
sterilized 37°C solution D (0.137 M NaCl, 0.2 M
NaH2PO4, 0.2 M KH2PO4, 5.4 mM KCl, 5 mM
dextrose [glucose], 58.5 mM sucrose, 0.25
?g/mL Fungizone [Gibco, Carlsbad, CA], and
1 ? 106U penicillin/streptomycin in sterile
water). The tissue fragments/cell suspension
were incubated in 37°C solution D containing
1.0% trypsin (Invitrogen, Carlsbad, CA) for 30
minutes at 37°C on a bidirectional tilting plat-
form. An equal volume of Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal
bovine serum (FBS; Gibco) and 1% peni-
cillin/streptomycin (complete medium) was
added to quench the trypsin reaction. The
mixed brain cell suspension was then passed
through a 130 ?m Nitex filter (Tetko, Briarcliff
Manor, NY) and centrifuged (4000 rpm
[2,900g], 10 minutes). The resulting pellet was
resuspended in 10 mL of complete medium,
passed through a 40-?m Nitex filter, and plated
on poly-L-lysine (0.01 g/L) (Sigma-Aldrich, St.
Louis, MO) coated, solution D-rinsed, 175 cm2
flasks at a density of 1.5 brains per flask. The
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Address reprint requests to:
Wolfgang J. Streit, Ph.D.
Department of Neuroscience
University of Florida College of Medicine
McKnight Brain Institute
Gainesville, FL 32610-0244
Received July 11, 2006
Accepted November 14, 2006
FLANARY ET AL.