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Summary of
Research Interests
Experimental Approaches
We examine the effects of chemokines and viral proteins using
different experimental approaches including traditional biochemical/molecular
biology techniques and novel imaging and proteomics approaches,
as briefly described in the next paragraphs. Rat and human
central neurons are used as a model for the study of chemokine
receptors naturally expressed by neurons. Alternative models
include neuronal human cell lines expressing specific chemokine
receptors. In addition, post-mortem brain tissue samples for
control and HIV-infected individuals are used for the analyses
of specific pathways of interest ex vivo.
Expression and function of chemokine
receptors in rat hippocampal neurons: Neurons express
functional chemokine receptors, such as CXCR4 and CX3CR1,
which are coupled to intracellular calcium mobilization and
survival pathways.
Primary neuronal and glial cultures:
Rat cultures: : Central neurons from different
brain areas (i.e. hippocampus, cerebral cortex and cerebellum)
are routinely used in the lab. Hippocampal or cortical neurons
are grown in a bilaminar cell culture system - an excellent
model for studying signal transduction mechanisms in pure
populations of neurons. In this system, neurons are co-cultured
in close juxtaposition to a glial feeder-layer, which supports
their growth and differentiation. This model allows us to
study the effect of chemokines and/or gp120 on neurons in
a pure neuronal population as well as in a mixed
neuronal-glial population. We have extensively used these
cultures for biochemical and molecular biological studies,
as well as electrophysiological and imaging analyses. As
mentioned in the preliminary data section, these neurons
express a variety of chemokine receptors - including CXCR4,
CCR5 and CX3CR1 – which are coupled to important survival
pathways, and we have characterized several physiological
and pathological effects of chemokine receptors in neurons.
Human cultures: Chemokine receptors are highly conserved
among species and human and rat chemokine receptors share
most of their physiological characteristics and behave very
similarly, particularly for our parameters of interest.
Nevertheless, to support the data obtained with rat cultures
and to determine the significance of our observations in
the HIV neuropathology, we also test the effect of chemokines
and HIV envelope proteins in human neurons and, alternatively,
human neuronal cell lines. We use cultured neurons that
derive from neural precursors cells (HNPC). Primary HNPC
are cultured by the vendor, cryopreserved as secondary cultures
of purified immature neurons or glia and delivered frozen
to us. Neuronal differentiation is assessed by staining
for neurofilament, MAP-2, and b-tubulin 3. These cells are
cultured in an optimized medium required to promote and/or
maintain their differentiation for few days or weeks before
the experiments. Preliminary experiments on untreated human
neurons (7 DIV - 11DIV) showed that they express chemokine
receptors and that Rb/pRb localization resembles what is
normally found in rat neurons. Astrocytes and microglia
from the same commercial source are used for the experiments
involving glia.
Rb and pRb localization in human neurons:
Endogenous expression and phosphorylation of Rb in human neuronal
cultures, identified by expression of neuronal-specific markers
(β-Tubulin and NSE). These neurons also express the chemokine
receptor CXCR4.
Brain Tissue Samples
Human brain tissue samples from HIV-infected patients are
obtained from the National
NeuroAIDS Tissue Consortium. These include fixed (10%
formalin) and paraffin embedded specimens from unidentified
subjects of either sex, age 21-66. Sections from various parts
of the brain (i.e. frontal cortex, hippocampus) have been
provided along with the available information on neurological
deficits, neuropathological diagnoses, viral load, CD4 counts,
and antiretroviral treatment.
Cellular/molecular biology and proteomics:
SDF-1α increases DNA binding activity of the transcription
factor NF-κβ as indicated by Electro Mobility
Shift Assays. |
Survival, immunocytochemistry, immunoblots and shift assays:
For survival experiments neurons are generally separated from
the glia just before exposure to gp120 and/or other substances.
Immunocytochemistry studies are performed on both pure and
mixed neuronal cultures. Nuclear fluorescent dyes, TUNEL assay,
Annexin V binding to phosphatidyilserine residues or caspase
activation assays are used for the quantitative detection
of cell death and in situ apoptosis. Western blot analyses
and electro mobility shift assays are routinely used to determine
expression/phosphorylation of target proteins and DNA binding
activity of transcription factors.
Calcium-imaging studies: Fura-2 based Ca-imaging and
single cell microfluorimetry are used to evaluate calcium
responses from individual cells using the image acquisition
and analysis software Metafluor. Our current imaging rig is
equipped with an Olympus microscope (IX70), a Roper Scientific
CCD camera, a DG4 light switcher as well as a filter wheel
and an Eppendorf microinjector. This set up also has “uncaging”
capabilities.
Transfections: Calcium phosphate transfections and
other techniques are used to transfect neuronal and non-neuronal
cells. We have recently optimized our protocol for cortical
and hippocampal neurons and used it for single cell experiments,
i.e. survival and immunocytochemistry - in which enhanced
fluorescent proteins (EGFP) are co-transfected to identify
neurons expressing exogenous DNA - as well as in population
studies with reporter genes (luciferase-based assays). Although
the efficiency of transfection of primary neuronal cultures
is generally low with this method, alternative procedures
include the use of poly-cationic molecules (i.e. PEI) and
viral vectors (lentiviruses and adenoviruses). Using the latter
method we have been able to transfect up to 80-90% of hippocampal
neurons. In addition, a number of new reagents for transfection
of primary cells, including neurons, are now commercially
available and can be used as alternative strategies.
Expression of EGFP-Rb in human astrocytes: Cells were
transfected with full length Rb (A) or a truncated form or
Rb (B) lacking the nuclear localization sequence. These constructs
are being used to determine the effect of chemokines on subcellular
localization of Rb in neuronal and non-neuronal cells.
SELDI-TOF MS: This technology is available to us through
the A.J.
Drexel Institute of Basic and Applied Protein Science
and our collaboration with Dr.
Chaiken. Surface-enhanced laser-desorption/ionization
time-of flight mass spectrometry (SELDI-TOF MS) will be used
for studies of differential protein profiling and for the
analysis of protein-protein interaction or DNA-protein interaction.
Retained proteins are detected by the ProteinChip Biology
System II (Ciphergen) and analyzed by the ProteinChip software.
Chemical or biological protein chip arrays will be used for
affinity capture of protein samples, depending on the experimental
requirements. By using an anionic chip, we began studying
the expression profiles of cells treated with SDF-1α
and detected over-expression of a 12kD protein, which remains
to be identified. On-chip proteolysis and peptide mapping
will be used to determine the nature of the proteins of interest.
Standard purification protocols, including selection media
used in the chips, and LC-MS-MS will be adopted.
Preliminary Identification of 12 kDa Protein in cell treated
with SDF-1α by mass spectrometry.
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