The Toxicologist - Society of Toxicology

carried to the brain in primates during experimental autoimmune encephalitis
(EAE), the animal model of MS. However, the role of PGN or its cytosolic sensor
(NOD2) in the progression of EAE is unknown. Both NOD2-/- and RICK-/-
mice were protected from the progression of EAE. The peripheral activation of
myelin oligodendrocyte glycoprotein (MOG)-specific T cells was similar in WT,
NOD2-/- and RICK-/- mice. However, the number of CD4 T cells and MOGspecific
T cells in the central nervous system (CNS) at the peak of disease severity
was slightly reduced in NOD2-/- mice and significantly lower in RICK-/- mice.
Additionally, the activation of antigen-presenting cells in the CNS was slightly reduced
in NOD2-/- mice and significantly reduced in RICK-/- mice. The results in
NOD2-/- mice are moderate likely due to microglia being activated by NOD1.
The reduced activation of microglia in RICK-/- mice results in less re-priming of T
cells once they infiltrate the CNS, resulting in their death. Thus, RICK inhibition
in microglia represents a novel therapeutic strategy for the treatment of MS.
1283 BETA-SECRETASE GENE EXPRESSION AND ACTIVITY
IN MURINE GT1-7 HYPOTHALAMIC NEURONS
EXPOSED TO CHOLESTEROL SECOALDEHYDE.
S. Babu 1 , A. C. Raghavamenon 1 , R. J. Martin 2 , B. Prakhya 3 and R. M. Uppu 1 .
1
Environmental Toxicology, Southern University and A&M College, Baton Rouge, LA,
2
Neurobehavior Laboratory, Pennington Biomedical Research Center, Baton Rouge,
LA and 3 Genetic Toxicology, International Institute of Biotechnology and Toxicology,
Padappai, India.
Previous studies from our laboratory have demonstrated that 3β-hydroxy-5-oxo-
5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known
to be formed at inflammatory sites as a result of myeloperoxidase/H 2
O 2
/Cl − - or
singlet oxygen-mediated oxidation of cholesterol, promotes Aβ aggregation in
GT1-7 hypothalamic neurons. Since Aβ aggregation critically depends on the
cleavage of amyloid precursor protein (APP), and since increased activity and expression
of secretases are often seen in Alzheimer’s disease, we examined the activity
of β-secretase in neuronal cells exposed to low levels of ChSeco (1-5 μM) for 24 h.
The results showed that the β-secretase activity was not altered in response to the
exposure of ChSeco. Also, co-incubation of the ChSeco (5 μM)-exposed neuronal
cells with Trolox (0.2 mM)caused a little or no change in the activity of β-secretase.
To examine the significance of these results at gene expression level, we isolated the
total RNA from the control and the ChSeco-treated neuronal cells, prepared
cDNA, and performed RT-PCR analysis for β-secretase isoenzyme-1, APP, and β-
actin (housekeeing gene). It was found that the mRNA levels of β-secretase isoenzyme-1
and APP in the ChSeco (0-2.5 μM)-exposed neuronal cells are about the
same as those in the control cells that were never exposed to ChSeco. While the results
need further confirmation employing higher but sub-cytotoxic concentrations
of ChSeco and the measurement of other secretases and, possibly, the proteosomal
pathway, it appears that oxidative stress status but not increased activity or expression
of β-secretase plays a major role in the ChSeco-induced Aβ aggregation and
subsequent cytotoxicity. [Funding support from NIH (P20 RR16456) and US
Department of Education (PO31B040030) is acknowledged. Corresponding author’s
email: rao_uppu@subr.edu].
1284 STRUCTURALLY DIVERSE CATIONIC
NEUROTOXICANTS ATTENUATE ATP-DEPENDENT
CALCIUM SIGNALING IN ASTROCYTES.
B. Trout, K. M. Streifel and R. B. Tjalkens. Environmental Radiological and
Health Science, Colorado State University, Fort Collins, CO.
Calcium signaling throughout networks of astrocytes is initiated by synaptic activity
and in order to increase regional cerebral blood flow (rCBF) in response to the
local demand for oxygen and glucose. This increase in intracellular calcium
[Ca2+]i, in perivascular astrocytes causes a release of vasoactive factors that causes a
rapid, local dilation of arterioles. Deprecations in rCBF are well described in patients
with various neurodegenerative diseases but the mechanisms underlying these
decreases are unknown. To examine the possible contribution of astrocyte dysfunction
to this phenomenon, we postulated that several structurally diverse cationic
neurotoxicants of the basal midbrain would inhibit transmitter-induced calcium
signaling in cultured astrocytes: MPP+, the active metabolite of the model parkinsonian
neurotoxicant, 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP);
Paraquat (PQ); 6-Hydroxydopamine (6-OHDA); and Manganese (Mn2+). Using
calcium imaging in primary cultured cortical astrocytes, we investigated the effect
of acute treatment with each neurotoxicant on ATP-induced intracellular calcium
transients. We observed a dose dependent decrease in ATP-induced [Ca2+]i transients
with acute application of PQ, 6-OHDA and MPP+. In addition, mechanically-induced
intercellular [Ca2+]i waves were inhibited in the presence of MPP+,
an effect that was reversible following washout of the compound. Like MPP+, PQ,
6-OHDA, and Mn2+ similarly inhibit [Ca2+]i waves, however Mn2+ requires a
higher concentration to produce equivalent calcium wave inhibition. These findings
indicate that endogenous and exogenous chemicals that are structurally diverse
but that have cationic properties inhibit physiological calcium signaling in astrocytes.
Because these astrocytic signals are critical to regulation of rCBF, these data
suggest a new target for neurotoxicants that may provide insight into mechanisms
of decreased cerebral blood flow that may contribute to the onset of neurological
disease.
1285 BLOCKADE OF NUCLEAR FACTOR KAPPA B
PREVENTS MPTP-INDUCED EXPRESSION OF
INDUCIBLE NITRIC OXIDE SYNTHASE IN PRIMARY
ASTROCYTES.
K. A. Popichak 1 , D. L. Carbone 1 , J. A. Miller 1 , S. Safe 2 and R. B. Tjalkens 1 .
1
ERHS, Colorado State University, Fort Collins, CO and 2 Vet Physiol and
Pharmacology Texas A&M University, College Station, TX.
Neuroinflammation is associated with loss of dopaminergic neurons in Parkinson’s
disease(PD)but there are no approved therapeutics that block this
phenotype.Activation of microglia and astrocytes leads to overproduction of inflammatory
mediators such as tumor necrosis factor and nitric oxide that damage
dopaminergic neurons. Thus,increased neuronal protein nitration is detected postmortem
in PD patients.Expression of nitric oxide synthase(NOS2) and other inflammatory
genes in astrocytes is mediated largely by the transcription factor,NFkB,thought
to be a key pathway regulating inflammatory signaling in glial cells.We
therefore postulated that inhibitors of NF-kB signaling may have potential therapeutic
utility in preventing inflammatory expression of NOS in astrocytes.To address
this hypothesis,we examined the anti-inflammatory capacity of a representative
diindolylmethane-derived compound(cDIM)in primary astrocytes,as well as its
neuroprotective efficacy in vitro. Low doses of cDIM globally suppressed MPTPinduced
expression of NF-kB regulated genes in a qPCR array study and also exhibited
dose-dependent inhibition of Nos2 mRNA expression.cDIM treatment
prevented MPTP-induced protein nitration in astrocytes that mirrored the effect of
the NOS2 inhibitor,AMT,indicating the functional effect of inhibiting NOS2 activity.Using
live-cell fluorescence imaging in transgenic astrocytes expressing an
NF-kB reporter construct,it was discovered that low concentrations of cDIM also
directly prevented NF-kB-dependent gene expression.In co-culture studies with
primary astrocytes and striatal neurons,cDIM pretreatment prevented astrocyte-dependent
neuronal apoptosis,indicated by decreased activation of caspases and annexin
V staining.Collectively,these data suggest that use of pharmacologic inhibitors
of NF-kB could be effective in mitigating the effects of neuroinflammation
by activated glial cells.Work supported by:Michael J.Fox FDN for Parkinson’s
Disease Research(RBT)
1286 OXIDATIVE STRESS MAY LEAD TO
NEURODEGENERATION IN CAENORHABDITIS
ELEGANS FOLLOWING CHRONIC EXPOSURE TO
MANCOZEB.
R. Negga, O. Mirallas and V. A. Fitsanakis. Biology, King College, Bristol, TN.
High incidence of Parkinson’s disease (PD) is correlated with an increase in pesticide
usage in agricultural areas. Mitochondrial inhibition, which also can cause oxidative
stress, may lead to dopaminergic (DAergic) neuronal death and subsequent
neuromuscular dysfunction associated with PD. In order to test the hypothesis that
mancozeb (MZ: manganese/zinc ethylene-bis-dithiocarbamate) may contribute to
the etiology of PD, we chronically exposed various strains of the nematode C. elegans
to multiple concentrations of MZ. Image analysis of fluorescent photomicrographs
of NW1229 (all neurons tagged with GFP) indicate a statistically significant
decrease (p0.05; 0.1, 1.0, 1.5, 1.6 or 1.7%). Furthermore,
pixel number was also decreased (p