|
Characterization
of proteins important for fertility and cell death |
Principal Investigator : Chandrima Shaha Project Associates/Assistants Ph D
Students
Visiting
Fellow The
broad research interest of this laboratory is to understand the regulation of
cell-cell interactions and mechanisms of cell death. Current projects explore
pathways related to the survival of the spermatogenic cells with special
emphasis on processes leading to cellular apoptosis. The other major focus of
this research program exploits a unicellular model, the protozoan parasite to
answer questions related to the importance of apoptosis in parasite survival
and host-parasite interactions. The
overall objectives of our studies are to explore the mechanistics of apoptosis
induction in multicellular and unicellular model systems. A multicellular
model system using estrogen stimulated spermatogenic cell apoptosis is being
used to investigate the mechanisms of induction of apoptosis by this hormone
which has recently been implicated in control of spermatogenesis, thereby
fuelling the speculation that estrogen-like chemicals can adversely affect
spermatogenesis. A big question in apoptosis biology is whether apoptosis is
unique to multicellular organisms or if the process evolved prior to the
emergence of multicellularity. Our laboratory was one of the first to
demonstrate apoptotic death in promastigotes of Leishmania donovani, the
causative agent for Kala-Azar. We are continuing our studies to probe into the
conditions and mechanisms of apoptotic death in these protozoan parasites and
their hosts, using different model systems to demonstrate how these parasites
and their hosts respond to adverse conditions including exposure to anti-leishmanial
drugs. 1. Biology of spermatogenic cell survival and
function The
significant role that estrogens play in spermatogenesis has opened up an
exciting area of research in male reproductive biology. The realization that
estrogens are essential for proper maintenance of spermatogenesis, as well as
growing evidence pointing to the deleterious effects of estrogen-like
chemicals on male reproductive health, has made it imperative to dissect the
role estrogens play in the male. As detailed above, our previous studies
demonstrated the ability of estrogens to induce rat spermatogenic cell
apoptosis in vivo. This observation suggested two possibilities;
firstly, spermatogenic cell apoptosis could occur due to interference of
estrogen with the hypothalamo-pituitary axis or secondly, through direct
action on spermatogenic cells through estrogen receptors. Therefore, we used
an in vitro model to understand how estrogens can directly affect
spermatogenic cell physiology and survival. Estrogen was able to induce
spermatogenic cell apoptosis in vitro. To establish the role of
estrogen receptors on the effects observed on spermatogenic cells, we used two
estrogen receptor modulators, centchroman and tamoxifen during incubation with
estadiol. Arguably, if estrogen was exerting its action through its receptors
and not through any other indirect mechanisms, presence of estrogen receptor
modulators during exposure to estrogen should ideally inhibit estrogen action.
Both the modulators were able to increase survival of the spermatogenic cells
significantly as compared to estradiol only treatment showing that cell death
resulting from estrogen exposure occurred through interaction between estrogen
and its receptors. We showed the apoptotic nature of death by examining
apoptotic phenotypes like DNA fragmentation, DNA laddering, exposure of
phosphatidylserine and release of cytochrome c in the cytosol. We also
established that a transient mitochondrial hyperpolarization was observed as a
result of estrogen-receptor binding. An increase of hydrogen peroxide,
superoxide and nitric oxide was observed but experiments established that it
was superoxide and nitric oxide that were responsible for hyperpolarization
and not hydrogen peroxide. In summary, this work clearly identified a part of
the early biochemical changes that occur in spermatogenic cells during
estrogen exposure. 2. Biology of cell survival in protozoan
parasites Our
earlier studies have demonstrated the ability of promastigotes of L.donovani
to undergo apoptotic death in response to oxidative stress. Investigations
on promastigotes yield important information and they are a relevant model
system, but their utility is limited because the promastigote form is not a
clinically relevant stage although it is responsible for transmission of the
disease. The intracellular amastigotes encountered in the mammalian host are
actually the forms responsible for disease pathogenesis. Importantly,
promastigote and amastigote forms surviving in two disparate biological
environments are very different in terms of metabolic pathways and their
susceptibility to anti-leishmanial compounds. Therefore, observations with
intracellular amastigotes addressing questions related to parasite survival
are more relevant and could lay the foundation for rational strategies of drug
development. However, reports on mechanisms of apoptosis-like death inside the
macrophages are scarce. The capability of the amastigotes to survive
within the host cell parasitophorous vacuoles as non-motile amastigotes
determine disease pathogenesis, but the mechanism of elimination of the
parasites from these vacuoles are not well understood. By using the anti-leishmanial
drug potassium antimony tartrate, we demonstrate that upon drug exposure,
intracellular Leishmania donovani amastigotes undergo apoptotic death
characterized by nuclear DNA fragmentation and externalization of
phosphatidylserine. Changes upstream of DNA fragmentation included generation
of reactive oxygen species like superoxide, nitric oxide and H2O2
that were primarily concentrated in the parasitophorous vacuoles. In the
presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis (4-benzoic
acid) porphyrin chloride, or an inhibitor of inducible nitric oxide synthase,
a diminution of reactive oxygen species generation and improvement of
amastigote survival was observed suggesting a close link between drug induced
oxidative stress and amastigote death. Changes downstream to reactive oxygen
species increase involved elevation of intracellular Ca2+
concentrations in both the parasite and the host that was preventable by
antioxidants. Flufenamic acid, a non-specific cation channel blocker decreased
the elevation of Ca2+
in both the cell types and reduced amastigote death, thus establishing a
connection between elevated Ca2+
and parasite clearance. This influx of Ca2+
was preceded by a fall in the amastigote mitochondrial membrane potential.
Therefore, this study projects the importance of non-specific cation channels
as important modulators of antimonial efficacy and lends credence to the
suggestion that within the host cell, apoptosis is the preferred mode of death
for the parasites. Continuing
our studies with promastigotes as a model system, to understand the mechanism
of oxidative stress induced apoptotic death through a mitochondrial mechanism,
we explored if inhibition of mitochondrial respiratory chain would lead to
parasite death. Currently, the biochemical changes consequent to respiratory
chain inhibition and their relationship to cell death in Leishmania spp.
remain elusive. Inhibitors of respiratory chain complexes I, II and III were
able to induce apoptotic death of the blood stream form of Leishmania
donovani. Complex I inhibition resulted in mitochondrial hyperpolarization
that was preceeded by increased superoxide
production. Limitation of electron
transport by thenoyltrifluoroacetone and antimycin A, inhibitors of complexes
II and III respectively resulted in dissipation of mitochondrial membrane
potential that was sensitive to cyclosporin A, a blocker of mitochondrial
permeability transition pore. Further studies conducted with
thenoyltrifluoroacetone showed maximal generation of hydrogen peroxide with a
moderate elevation of superoxide levels. Complex III inhibition provoked
superoxide generation only. Interference with complex II but not complexes I
and III increased intracellular Ca2+.
A tight link between Ca2+
and reactive oxygen species was
demonstrated by antioxidant induced diminution of Ca2+
increase. However, chelation of
extracellular Ca2+
could not abrogate the early increase of
reactive oxygen species providing evidence that Ca2+
elevation was downstream to reactive
oxygen species generation. Ca2+
influx occurred through non-selective
cation and L-type channels and Na+/
Ca2+ exchanger
like pathways. Antioxidant like glutathione and Ca2+
channel blockers reduced apoptotic
death. This study provides a new possibility that concurrent inhibition of
respiratory chain complex II with pentamidine administration increases
cytotoxicity of the drug. This increased cytotoxicity was connected to a four
fold elevation in intracellular Ca2+
that was pooled only from intracellular
sources. Therefore, inhibition of complexes I, II and III lead to apoptosis
and complex II inhibition in parallel with pentamidine administration enhance
drug efficacy.
Publications Original peer-reviewed articles 1.
Sudhandiran G and Shaha C (2003) Antimonial induced increase in intracellular
Ca2+ through non-selective cation channels in the host and the parasite is
responsible for apoptosis of intracellular Leishmania donovani
amastigotes. J Biol Chem 278:25120-25132. 2.
Mehta A and Shaha C (2003) Apoptotic death in Leishmania donovani
promastigotes in response to respiratory chain inhibition: complex II
inhibition results in increased pentamidine cytotoxicity. J Biol Chem
(in press). |