Proposal

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.