Proposal
To determine if C. pneumoniae exerts an affect
in the
cytochrome c apoptotic cascade in neuronal cells.
Laura Spearot
HYPOTHESIS: Our hypothesis is that neuronal
cells infected with C. pneumoniae can sustain persistent
infections rendering the neuronal cells resistant to
apoptosis. The anti-apoptotic pathway stimulated by
C. pneumoniae infection presumably involves blockade
of the mitochondrial/cytochrome c apoptotic pathway
as mitochondria appear to undergo stress and damage
in both C. pneumoniae infections and Alzheimerís
disease.
SPECIFIC AIM: To determine if C. pneumoniae
exerts an affect in the cytochrome c apoptotic cascade
in neuronal cells. We will experimentally induce apoptosis
in neuronal cells infected with C. pneumoniae and screen
for apoptotic markers such as annexinV, caspases, and
cytochrome c by immunocytochemistry and western analysis.
Neurodegenerative disorders such as Alzheimerís
disease (AD) are characterized by the chronic deterioration
of synaptic function and a progressive loss of cortical
neurons. Cytoskeletal changes occur within cortical
neurons such as the formation of paired helical filaments
(PHFs) into neurofibrillary tangles (NFTs), the signature
pathologies of AD. Accompanying these structures are
extracellular deposits of beta-amyloid, in particular
the beta-1-42 peptide that provides a nidus for the
formation of senile plaques.
Recently, apoptosis has been implicated
as a mechanism in the degeneration of selective neuronal
populations in AD. Several studies have shown that a
large percentage of cells contain DNA fragmentation
and an incomplete cell cycle activation in post-mitotic
neurons in AD brains. Other reports confirm that the
AD brain provides a pro-apoptotic environment, though
they see no evidence of the apoptotic process leading
to terminal completion. While a plethora of evidence
validates neurodegeneration as a major etiology in AD,
the initiating event(s) or stimulus has not yet been
identified in the sporadic form of this disease. One
plausible candidate is beta-1-42 peptide that has been
found to induce apoptosis-related changes in neurons
and is cytotoxic to neurons. Resident CNS cells such
as microglia, astroglia, and neuronal cells can generate
beta-1-42 peptides when stimulated by proinflammatory
molecules or by beta-1-42 itself. However, stimuli other
than beta-1-42 may trigger a proinflammatory response
in the brain that could result in the production or
processing of beta-1-42. These ìtriggersî
have been ill defined with regard to the pathogenesis
of sporadic AD.
In a previous study, we identified Chlamydia pneumoniae
in areas of neuropathology from brains of individuals
who had previously been diagnosed with sporadic AD.
Since the initial study, we have developed an animal
model using young non-transgenic BALB/c mice that have
been intranasally inoculated with C. pneumoniae and
found that these mice develop immunoreactive beta-1-42
plaques resembling those found in AD. More recently,
our in vitro studies suggest that neuronal cells are
less vulnerable to apoptosis when infected with C. pneumoniae.
Previous studies have shown that monocytes also are
resistant to apoptosis when infected with C. pneumoniae.
How C. pneumoniae inhibits the apoptotic process has
yet to be fully elucidated, though blockade of mitochondrial
cytochrome c release has been postulated to be one mechanism
by which C. pneumoniae exerts its anti-apoptotic activity.
Therefore, in these in vitro studies we will address
infection of neurons with C. pneumoniae, and the extent
to which the viability of the neurons is enhanced or
compromised during prolonged infections as a function
of apoptotic inhibition.
Summary
Effects on the apoptotic marker
Cytochrome c following a chlamydial infection in
neurons and astrocytes: Implications for Alzheimer’s
Disease
Laura Spearot1, Brian Balin2,
Denah Appelt3,
1 Bryn Mawr College, 101 Merion Ave. Bryn Mawr PA ,
2 Dept.of Pathology, Microbiology, & Immunology
3 Dept. of Neuroscience, Physiology & Pharmacology,
Center for Chronic Disorders of Aging Philadelphia College
of Osteopathic Medicine 4170 City Avenue, Philadelphia,
PA
Abstract:
Neurodegeneration has been well documented
in the CNS of Alzheimer individuals and evidence suggests
that apoptosis may be a contributing factor in the pathogenesis
of Alzheimer’s disease. Initiating events that
occur in apoptosis have been identified in Alzheimer
brains; however, completion of apoptotic processes is
not well understood. In earlier studies, Chlamydia pneumoniae
which is an intracellular respiratory pathogen, was
identified and isolated from brains of patients that
had been diagnosed with sporadic AD [1]. Our initial
hypothesis suggested that Chlamydia pneumoniae could
involve the apoptotic process. Chlamydia pneumoniae
has been found to inhibit apoptosis in neuronal [2]
and monocytes [3], although the precise apoptotic pathway
inhibited is ill defined Inhibition of apoptosis may
be one mechanism by which Chlamydia pneumoniae can sustain
an infection in the host to maintain an optimal intracellular
environment. This infection may influence the contradictory
findings of the completion of the apoptotic process
in the Alzheimer’s brain.
Our hypothesis is that astrocytes and
neuronal cells infected with Chlamydia pneumoniae can
sustain an infection rendering the cells resistant to
apoptosis. Since mitochondrial damage has been identified
in the pathogenesis of Alzheimer’s disease, the
focus of this study was to determine whether cytochrome
c, an electron carrier protein that is essential to
the mitochondrial repiratory process was affected following
a Chlamydia pneumoniae infection in astrocytes and neuronal
cells. Following damage to the mitochondria, cytochrome
c is translocated from the mitochondria to the cytoplasm
whereby cytochrome c activates the apoptotic process.
In our studies, apoptosis was experimentally induced
by staurosporine in astrocytes and neuronal cells that
were both uninfected SK-N-MC cells and cells and infected
with Chlamydia pneumoniae for 72hrs. Cytochrome c production
was analyzed by immunofluorescent microscopy utilizing
an antibody specific to cytochrome c. Our results suggest
that Chlamydia pneumoniae infected neuronal cell differentially
activate cytochrome c as compared to infected astrocytes.
In both infected neurons and astrocytes induction of
apoptosis with staurosporine did not appear to induce
the apoptotic event. Thus our data appear to indicate
that an infection of both cell types blocks the apoptotic
induction with staurosporine which may be independent
of the cytochrome c pathway.
Introduction:
Neurodegenerative disorders such as Alzheimer’s
disease (AD) are characterized by the chronic deterioration
of synaptic function and a progressive loss of cortical
neurons. Cytoskeletal changes occur within cortical
neurons such as the formation of paired helical filaments
(PHFs) into neurofibrillary tangles (NFTs), the signature
pathologies of AD. Accompanying these structures are
extracellular deposits of b-amyloid, in particular the
A b -1-42 peptide that provides a nidus for the formation
of senile plaques.
Recently, apoptosis has been implicated
as a mechanism in the degeneration of selective neuronal
populations in AD. Several studies have shown that a
large percentage of cells contain DNA fragmentation
and an incomplete cell cycle activation in post-mitotic
neurons in AD brains [4]. Other reports confirm that
the AD brain provides a pro-apoptotic environment, though
they see no evidence of the apoptotic process leading
to terminal completion[5]. While a plethora of evidence
validates neurodegeneration as a major etiology in AD,
the initiating event(s) or stimulus has not yet been
identified in the sporadic form of this disease. One
plausible candidate is A b -1-42 peptide that has been
found to induce apoptosis-related changes in neurons
and is cytotoxic to neurons [5]. Resident CNS cells
such as microglia, astroglia, and neuronal cells can
generate A b -1-42 peptides when stimulated by proinflammatory
molecules or by Ab -1-42, itself. However, stimuli other
than A b -1-42 may trigger a proinflammatory response
in the brain that could result in the production or
processing of A b -1-42. These “triggers”
have been ill defined with regard to the pathogenesis
of sporadic AD.
In a previous study, Chlamydia pneumoniae
was identified in areas of neuropathology from brains
of individuals who had previously been diagnosed with
sporadic AD [1]. More recently, in vitro studies suggest
that neuronal cells are less vulnerable to apoptosis
when infected with C. pneumoniae [2]. Previous studies
have shown that monocytes also are resistant to apoptosis
when infected with C. pneumoniae [3]. How C. pneumoniae
infection affects the apoptotic process has yet to be
fully elucidated though mitochondria appear to undergo
stress and damage in both C. pneumoniae infections and
Alzheimer’s disease. Cytochrome c release from
mitochondria has been postulated to be one mechanism
by which C. pneumoniae may alter the apoptotic mechanism.
Therefore, our in vitro studies have addressed infection
of neurons and astrocytes with C. pneumoniae. We are
specifically addressing the extent to which the viability
of these cells are enhanced or compromised during an
infection with C. pneumoniae as well as the infection
affects on cytochrome c apoptotic cascade.
Conclusions:
Induction of apoptosis with staurosporine
did not appear to induce the apoptotic event in both
infected neurons and astocytes. Chlamydia pneumoniae
infected neuronal cell differentially activate cytochrome
c as compared to infected astrocytes. Therefore an infection
of both neurons and astocytes blocks the apoptotic induction
with staurosporine which may be independent of the cytochrome
c pathway.
References:
1. Balin, B.J., H.C. Gerard, E.J. Arking, D.M. Appelt,
P.J. Branigan, J.T. Abrams, J.A. Whittum-Hudson, and
A.P. Hudson, Identification and localization of Chlamydia
pneumoniae in the Alzheimer's brain. Med Microbiol Immunol
(Berl), 1998. 187(1): p. 23-42.
2. Appelt, DM, Roupas, M., Way, D., Bell,
MG., Albert., E., Hammond, CJ., Balin, BJ.. Inhibition
of Apoptosis in Neuronal cells Infected with Chlamydophila
(Chlamydia) pneumoniae BMC Neuroscience. *submitted
2007
3. Geng, Y., R.B. Shane, K. Berencsi,
E. Gonczol, M.H. Zaki, D.J. Margolis, G. Trinchieri,
and A.H. Rook, Chlamydia pneumoniae inhibits apoptosis
in human peripheral blood mononuclear cells through
induction of IL-10. J Immunol, 2000. 164(10): p. 5522-9.
4. Cotman, C.W., Apoptosis decision cascades
and neuronal degeneration in Alzheimer's disease. Neurobiol
Aging, 1998. 19(1 Suppl): p. S29-32.
5. Raina, A.K., A. Hochman, H. Ickes, X. Zhu, O. Ogawa,
A.D. Cash, S. Shimohama, G. Perry, and M.A. Smith, Apoptotic
promoters and inhibitors in Alzheimer's disease: Who
wins out? Prog Neuropsychopharmacol Biol Psychiatry,
2003. 27(2): p. 251-4.
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