A clinically relevant dose of cyclophosphamide chemotherapy impairs memory performance on the delayed spatial alternation task that is sustained over time as mice age.

BACKGROUND: Cyclophosphamide chemotherapy is a mainstay of adjuvant breast cancer treatment. Unfortunately, this drug is associated with cognitive impairments in cancer patients that may accelerate cognitive aging. Memory is particularly affected in many patients. In order to better understand the precise cognitive impairments caused by this chemotherapy agent, we investigated a clinically relevant dose and administration paradigm on delayed spatial memory abilities in C57BL/6 mice. We utilized a delayed alternation paradigm similar to a delayed match to sample paradigm reported to be sensitive in human neurotoxicology research. METHODS: A dose of 200mg/kg cyclophosphamide was administered intravenously (at weekly intervals) for 4 weeks to C57BL/6 mice starting at 6 ½ months of age. Memory was tested in mice using a reward-based delayed spatial alternation paradigm with delay values of 1.5, 3, 6.1, 12.4 and 25s presented randomly over 80 sessions (16 reinforcers per session), and testing began at the initiation of chemotherapy through 3 months. RESULTS: At the longest delay, i.e., that requiring the greatest memory, mice treated with chemotherapy exhibited a significant decline over time in percent correct which leveled off compared to controls that continued to improve slightly. CONCLUSIONS: Our clinically relevant model shows cyclophosphamide chemotherapy causes a slight decline in delayed spatial memories at the longest delay that is sustained over time as mice age.

Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

Characterization of binge-dosed methamphetamine-induced neurotoxicity and neuroinflammation.

Methamphetamine (MA) is a potent, highly addictive psychostimulant abused by millions of people worldwide. MA induces neurotoxicity, damaging striatal dopaminergic terminals, and neuroinflammation, with striatal glial activation leading to pro-inflammatory cytokine and reactive oxygen species production. It is unclear whether MA-induced neuroinflammation contributes to MA-induced neurotoxicity. In the current study, we examined the linkage between the time course and dose response of MA-induced neurotoxicity and neuroinflammation. Adult male mice underwent a binge dosing regimen of four injections given every 2h with doses of 2, 4, 6, or 8 mg/kg MA per injection, and were sacrificed after 1, 3, 7, or 14 days. Binge MA treatment dose-dependently caused hyperthermia and induced hypoactivity after one day, though activity returned to control levels within one week. Striatal dopamine (DA) was diminished one day after treatment with at least 4 mg/kg MA, while DA turnover rates peaked after seven days. Although striatal tyrosine hydroxylase and DA transporter levels were also decreased one day after treatment with at least 4 mg/kg MA, they trended toward recovery by day 14. All doses of MA activated striatal glia within one day. While astrocyte activation persisted, microglial activation was attenuated over the two weeks of the study. These findings help clarify the relationship between MA-induced neuroinflammation and neurotoxicity, particularly regarding their temporal and dose-specific dynamics.

The effects of postnatal exposure to low-dose bisphenol-A on activity-dependent plasticity in the mouse sensory cortex.

Bisphenol-A (BPA) is a monomer used in the production of polycarbonate plastics, epoxies and resins and is present in many common household objects ranging from water bottles, can linings, baby bottles, and dental resins. BPA exposure has been linked to numerous negative health effects throughout the body, although the mechanisms of BPA action on the developing brain are still poorly understood. In this study, we sought to investigate whether low dose BPA exposure during a developmental phase when brain connectivity is being organized can cause long-term deleterious effects on brain function and plasticity that outlast the BPA exposure. Lactating dams were orally exposed to 25 µg/kg/day of BPA (one half the U.S. Environmental Protection Agency's 50 µg/kg/day rodent dose reference) or vehicle alone from postnatal day (P)5 to P21. Pups exposed to BPA in their mother's milk exhibited deficits in activity-dependent plasticity in the visual cortex during the visual critical period (P28). To determine the possible mechanisms underlying BPA action, we used immunohistochemistry to examine histological markers known to impact cortical maturity and developmental plasticity and quantified cortical dendritic spine density, morphology, and dynamics. While we saw no changes in parvalbumin neuron density, myelin basic protein expression or microglial density in BPA-exposed animals, we observed increases in spine density on apical dendrites in cortical layer five neurons but no significant alterations in other morphological parameters. Taken together our results suggest that exposure to very low levels of BPA during a critical period of brain development can have profound consequences for the normal wiring of sensory circuits and their plasticity later in life.

Deletion or activation of the aryl hydrocarbon receptor alters adult hippocampal neurogenesis and contextual fear memory.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxin and serves multiple developmental roles. In the adult brain, while we now localize AhR mRNA to nestin-expressing neural progenitor cells in the dentate gyrus (DG) of the hippocampus, its function is unknown. This study tested the hypothesis that AhR participates in hippocampal neurogenesis and associated functions. AhR deletion and activation by the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), adversely impacted neurogenesis and cognition. Adult AhR-deficient mice exhibited impaired hippocampal-dependent contextual fear memory while hippocampal-independent memory remained intact. AhR-deficient mice displayed reduced cell birth, decreased cell survival, and diminished neuronal differentiation in the DG. Following TCDD exposure, wild-type mice exhibited impaired hippocampal-dependent contextual memory, decreased cell birth, reduced neuronal differentiation, and fewer mature neurons in the DG. Glial differentiation and apoptosis were not altered in either TCDD-exposed or AhR-deficient mice. Finally, defects observed in TCDD-exposed mice were dependent on AhR, as TCDD had no negative effects in AhR-deficient mice. Our findings suggest that AhR should be further evaluated as a potential transcriptional regulator of hippocampal neurogenesis and function, although other sites of action may also warrant consideration. Moreover, TCDD exposure should be considered as an environmental risk factor that disrupts adult neurogenesis and potentially related memory processes.

An azobenzene photoswitch sheds light on turn nucleation in amyloid-ß self-assembly.

Amyloid-ß (Aß) self-assembly into cross-ß amyloid fibrils is implicated in a causative role in Alzheimer's disease pathology. Uncertainties persist regarding the mechanisms of amyloid self-assembly and the role of metastable prefibrillar aggregates. Aß fibrils feature a sheet-turn-sheet motif in the constituent ß-strands; as such, turn nucleation has been proposed as a rate-limiting step in the self-assembly pathway. Herein, we report the use of an azobenzene ß-hairpin mimetic to study the role turn nucleation plays on Aß self-assembly. [3-(3-Aminomethyl)phenylazo]phenylacetic acid (AMPP) was incorporated into the putative turn region of Aß42 to elicit temporal control over Aß42 turn nucleation; it was hypothesized that self-assembly would be favored in the cis-AMPP conformation if ß-hairpin formation occurs during Aß self-assembly and that the trans-AMPP conformer would display attenuated fibrillization propensity. It was unexpectedly observed that the trans-AMPP Aß42 conformer forms fibrillar constructs that are similar in almost all characteristics, including cytotoxicity, to wild-type Aß42. Conversely, the cis-AMPP Aß42 congeners formed nonfibrillar, amorphous aggregates that exhibited no cytotoxicity. Additionally, cis-trans photoisomerization resulted in rapid formation of native-like amyloid fibrils and trans-cis conversion in the fibril state reduced the population of native-like fibrils. Thus, temporal photocontrol over Aß turn conformation provides significant insight into Aß self-assembly. Specifically, Aß mutants that adopt stable ß-turns form aggregate structures that are unable to enter folding pathways leading to cross-ß fibrils and cytotoxic prefibrillar intermediates.

The aryl hydrocarbon receptor contributes to the proliferation of human medulloblastoma cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic helix-loop-helix (bHLH)/PER-ARNT-SIM (PAS) transcription superfamily, is known to regulate the toxicity of polyaromatic halogenated hydrocarbon environmental chemicals, most notably dioxin. However, the AhR has also been implicated in multiple stages of tumorigenesis. Medulloblastoma (MB), a primary cerebellar brain tumor arising in infants and children, is thought to originate from abnormally proliferating cerebellar granule neuron precursors (GNPs). GNPs express high levels of the AhR in the external germinal layer of the developing cerebellum. Moreover, our laboratory has previously reported that either abnormal activation or deletion of the AhR leads to dysregulation of GNP cell cycle activity and maturation. These observations led to the hypothesis that the AhR promotes the growth of MB. Therefore, this study evaluated whether the AhR serves a pro-proliferative role in an immortalized MB tumor cell line (DAOY). We produced a stable AhR knockdown DAOY cell line [AhR short hairpin RNA (shRNA)], which exhibited a 70% reduction in AhR protein levels. Compared with wild-type DAOY cells, AhR shRNA DAOY cells displayed an impaired G(1)-to-S cell cycle transition, decreased DNA synthesis, and reduced proliferation. Furthermore, these cell cycle perturbations were correlated with decreased levels of the pro-proliferative gene Hes1 and increased levels of the cell cycle inhibitor p27(kip1). Supplementation experiments with human AhR restored the proliferative activity in AhR shRNA DAOY cells. Taken together, our data show that the AhR promotes proliferation of MB cells, suggesting that this pathway should be considered as a potential therapeutic target for MB treatment.

Turn nucleation perturbs amyloid ß self-assembly and cytotoxicity.

The accumulation of senile plaques composed of amyloid ß (Aß) fibrils is a hallmark of Alzheimer's disease, although prefibrillar oligomeric species are believed to be the primary neurotoxic congeners in the pathogenesis of Alzheimer's disease. Uncertainty regarding the mechanistic relationship between Aß oligomer and fibril formation and the cytotoxicity of these aggregate species persists. ß-Turn formation has been proposed to be a potential rate-limiting step during Aß fibrillogenesis. The effect of turn nucleation on Aß self-assembly was probed by systematically replacing amino acid pairs in the putative turn region of Aß (residues 24-27) with d-ProGly ((D)PG), an effective turn-nucleating motif. The kinetic, thermodynamic, and cytotoxic effects of these mutations were characterized. It was found that turn formation dramatically accelerated Aß fibril self-assembly dependent on the site of turn nucleation. The cytotoxicity of the three (D)PG-containing Aß variants was significantly lower than that of wild-type Aß40, presumably due to decreased oligomer populations as a function of a more rapid progression to mature fibrils; oligomer populations were not eliminated, however, suggesting that turn formation is also a feature of oligomer structures. These results indicate that turn nucleation is a critical step in Aß40 fibril formation.

Subchronic polychlorinated biphenyl (Aroclor 1254) exposure produces oxidative damage and neuronal death of ventral midbrain dopaminergic systems.

Recent epidemiologic studies have demonstrated a link between organochlorine and pesticide exposure to an enhanced risk for neurodegenerative disorders such as Parkinson's disease (PD). A common biological phenomenon underlying cell injury associated with both polychlorinated biphenyl (PCB) exposure and dopaminergic neurodegeneration during aging is oxidative stress (OS). In this study, we tested the hypothesis that oral PCB exposure, via food ingestion, impairs dopamine systems in the adult murine brain. We determined whether PCB exposure was associated with OS in dopaminergic neurons, a population of cells that selectively degenerate in PD. After 4 weeks of oral exposure to the PCB mixture Aroclor 1254, several congeners, mostly ortho substituted, accumulated throughout the brain. Significant increases in locomotor activity were observed within 2 weeks, which persisted after cessation of PCB exposure. Stereologic analyses revealed a significant loss of dopaminergic neurons within the substantia nigra and ventral tegmental area. However, striatal dopamine levels were elevated, suggesting that compensatory mechanisms exist to maintain dopamine homeostasis, which could contribute to the observed increases in locomotor activity following PCB exposure. Biochemical experiments revealed alterations in OS markers, including increases in SOD and HO-1 levels and the presence of oxidatively modified lipids and proteins. These findings were accompanied by elevated iron levels within the striatal and midbrain regions, perhaps due to the observed dysregulation of transferrin receptors and ferritin levels following PCB exposure. In this study, we suggest that both OS and the uncoupling of iron regulation contribute to dopamine neuron degeneration and hyperactivity following PCB exposure.

Neural precursor cell proliferation is disrupted through activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Neurogenesis involves the proliferation of multipotent neuroepithelial stem cells followed by differentiation into lineage-restricted neural precursor cells (NPCs) during the embryonic period. Interestingly, these progenitor cells express robust levels of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates expression of genes important for growth regulation, and xenobiotic metabolism. Upon binding 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pervasive environmental contaminant and potent AhR ligand, AhR, is activated and disrupts gene expression patterns to produce cellular toxicity. Because of its widespread distribution in the brain during critical proliferative phases of neurogenesis, it is conceivable that AhR participates in NPC expansion. Therefore, this study tested the hypothesis that AhR activation by TCDD disrupts signaling events that regulate NPC proliferation. The C17.2 NPC line served as a model system to (1) assess whether NPCs are targets for TCDD-induced neurotoxicity and (2) characterize the effects of TCDD on NPC proliferation. We demonstrated that C17.2 NPCs express an intact AhR signaling pathway that becomes transcriptionally active after TCDD exposure. (3)H-thymidine and alamar blue reduction assays indicated that TCDD suppresses NPC proliferation in a concentration-dependent manner without the loss of cell viability. Cell cycle distribution analysis by flow cytometry revealed that TCDD-induced growth arrest results from an impaired G1 to S cell cycle transition. Moreover, TCDD exposure altered p27( kip1) and cyclin D1 cell cycle regulatory protein expression levels consistent with a G1 phase arrest. Initial studies in primary NPCs isolated from the ventral forebrain of embryonic mice demonstrated that TCDD reduced cell proliferation through a G1 phase arrest, corroborating our findings in the C17.2 cell line. Together, these observations suggest that the inappropriate or sustained activation of AhR by TCDD during neurogenesis can interfere with signaling pathways that regulate neuroepithelial stem cell/NPC proliferation, which could adversely impact final cell number in the brain and lead to functional impairments.

IGF-1 partially restores chemotherapy-induced reductions in neural cell proliferation in adult C57BL/6 mice.

Chemotherapeutic agents produce persistent difficulties in memory through an unknown mechanism. We tested the hypothesis that chemotherapeutic agents readily able to cross the blood-brain barrier (cyclophosphamide and fluorouracil), as opposed to those not known to readily cross the barrier (paclitaxel and doxorubicin), reduce neural cell proliferation following chemotherapy. We found that 5-bromo-2-deoxyuridine labeling following chemotherapy given to C57BL/6 mice revealed a similar reduction in neural cell proliferation in the dentate gyrus for all four agents. Insulin-like growth factor 1, a molecule implicated in promoting neurogenesis, counteracted the effects of high doses of chemotherapy on neural cell proliferation.

The aryl hydrocarbon receptor has a normal function in the regulation of hematopoietic and other stem/progenitor cell populations.

The aryl hydrocarbon receptor (AhR) is known mainly as the mediator for the toxicity of certain xenobiotics. However, there is also much information to indicate that this transcription factor has important biological functions. Here we review the evidence that the AhR has a significant role in the regulation of hematopoietic stem cells (HSCs). Data to support this come from studies with xenobiotic AhR ligands, phenotypic analyses of mice lacking AhR, examining the presence and regulation of the AhR within HSCs, knowledge of genes and signaling pathways regulated by the AhR, and investigations of hematopoietic disorders. Based on this information, we hypothesize that AhR expression is necessary for the proper maintenance of quiescence in HSCs, and that AhR down-regulation is essential for "escape" from quiescence and subsequent proliferation of these cells. This implicates the AhR as a negative regulator of hematopoiesis with a function of curbing excessive or unnecessary proliferation. This provides an important advantage by preventing the premature exhaustion of HSCs and sensitivity to genetic alterations, thus preserving HSC function and long-term multi-lineage generation over the lifespan of the organism. This also implicates a role of the AhR in aging processes. AhR dysregulation may result in the altered ability of HSCs to sense appropriate signals in the bone marrow microenvironment leading to hematopoietic disease. It is also reasonable to hypothesize that this protein has an important function in the regulation of other tissue stem cell populations. Suggestive evidence is consistent with a role in skin and neural stem cells.

2,3,7,8-Tetracholorodibenzo-p-dioxin exposure disrupts granule neuron precursor maturation in the developing mouse cerebellum.

The widespread environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity associated with abnormal cerebellar maturation in both humans and rodents. TCDD mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of xenobiotic metabolizing enzymes and growth regulatory molecules. Our previous studies demonstrated that cerebellar granule neuron precursor cells (GNPs) express transcriptionally active AhR during critical developmental periods. TCDD exposure also impaired GNP proliferation and survival in vitro. Therefore, this study tested the hypothesis that TCDD exposure disrupts cerebellar development by interfering with GNP differentiation. In vivo experiments indicated that TCDD exposure on postnatal day (PND) 6 resulted in increased expression of a mitotic marker and increased thickness of the external granule layer (EGL) on PND10. Expression of the early differentiation marker TAG-1 was also more pronounced in postmitotic, premigratory granule neurons of the EGL, and increased apoptosis of GNPs was observed. On PND21, expression of the late GNP differentiation marker GABA(A alpha 6) receptor (GABAR(A alpha 6)) and total estimated cell numbers were both reduced following exposure on PND6. Studies in unexposed adult AhR(-/-) mice revealed lower GABAR(A alpha 6) levels and DNA content. In vitro studies showed elevated expression of the early differentiation marker p27/Kip1 and the GABAR(A alpha 6) in GNPs following TCDD exposure, and the expression patterns of proteins related to granule cell neurite outgrowth, beta III-tubulin and polysialic acid neural cell adhesion molecule, were consistent with enhanced neuroblast differentiation. Together, our data suggest that TCDD disrupts a normal physiological role of AhR, resulting in compromised GNP maturation and neuroblast survival, which impacts final cell number in the cerebellum.

Heme-oxygenase-1 promotes polychlorinated biphenyl mixture aroclor 1254-induced oxidative stress and dopaminergic cell injury.

Dopaminergic (DAergic) systems have been identified as putative targets for polycholorinated biphenyl (PCB) actions. However, the precise mechanisms leading to neurotoxicity are unresolved. Reactive oxygen species (ROS) were recently shown to mediate injury in DAergic MN9D cells following exposure to Aroclor 1254 (A1254), a commercial PCB mixture. The oxidative stress response in DAergic cells included a persistent expression of heme oxygenase-1 (HO-1). This study tested the hypothesis that a sustained PCB-induced HO-1 response leads to abnormally high Fe levels, which generates ROS production and mediates death in the MN9D DAergic cell model. Accordingly, results indicated that A1254 augmented intracellular Fe levels in MN9D cells after 24 h. Fe chelation by desferoxamine or pharmacologic inhibition of HO activity with tin-protoporphyrin reduced Fe accumulation, ROS production, and cytotoxicity following A1254 exposure. HO-1 over-expression predisposed MN9D DAergic cells to enhanced ROS production and cell death in response to PCBs. Conversely, antisense inhibition of HO-1 expression prevented PCB-induced ROS production and cell death. These observations suggest that enhanced HO-1 catalytic activity and subsequent liberation of Fe participate in neurotoxic DAergic cell injury caused by A1254 exposure in vitro.

Aryl hydrocarbon receptor expression and activity in cerebellar granule neuroblasts: implications for development and dioxin neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent teratogen that produces neurobehavioral abnormalities associated with both cognitive and locomotor systems, yet the precise regional and cellular targets of developmental neurotoxicity remain largely unknown. Most, if not all, TCDD-induced pathology is mediated via binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) superfamily. Upon ligand binding, AhR translocates to the nucleus, dimerizes with the AhR nuclear translocator protein (Arnt), and regulates transcription by interaction with dioxin-response elements (DREs) in target genes, most notably specific cytochrome P450 (CYP) family members. To assess whether developing cerebellar granule neuroblasts are potential direct targets for TCDD toxicity, AhR expression and transcriptional activity were examined. AhR and Arnt proteins were present in mouse cerebellum from birth throughout postnatal development. AhR protein levels peaked between postnatal day (PND) 3-10, a critical period for granule neuroblast growth and maturation. Transcriptionally active AhR was detected in immature cerebellar granule cells in a transgenic dioxin-responsive lacZ mouse model after acute TCDD exposure. AhR and Arnt were also expressed in cerebellar granule neuroblast cultures. AhR localized to the nucleus in granule cells 15 min after TCDD treatment. TCCD elicited time-dependent and concentration-dependent increases in CYP1A1 and 1B1 mRNA and protein levels. Moreover, TCDD treatment reduced both thymidine incorporation and granule neuroblast survival in a concentration-dependent manner. These data suggest that (1) granule neuroblasts are direct targets for developmental AhR-mediated TCDD neurotoxicity and (2) TCDD exposure may disrupt granule cell neurogenesis.

Modulation of antioxidant defense systems by the environmental pesticide maneb in dopaminergic cells.

A lack of evidence supporting a role of heritability in the development of idiopathic Parkinson's disease (PD) has implicated exposures to environmental contaminants in the disease etiology. Epidemiological and clinical studies, as well as animal models of the PD phenotype, have consistently linked agrichemical exposure with dopaminergic (DAergic) damage, particularly through oxidative stress mechanisms. Maneb (MB) is a dithiocarbamate (DTC) fungicide that has specifically been implicated to have adverse effects on dopamine (DA) systems, but the role MB plays in modulating the oxidative state of DAergic cells has not previously been described. Since glutathione (GSH) is a major cellular antioxidant, it was hypothesized that exposure to MB would disrupt this system. The current study primarily utilized the PC12 cell line, which displays a catecholaminergic phenotype. Low concentrations of MB (50-1000 ng/ml) had little effect on cell viability, as measured by LDH release. These same concentrations, however, led to increases in GSH and its oxidized form, GSSG. Effects on viability and GSH were correlated to a primary mesencephalic culture system. Furthermore, these effects were markedly different from those observed with the classical oxidative stressor and pesticide, paraquat (PQ). To determine how MB would affect cells in which antioxidant systems were compromised, PC12 cells were treated with L-buthionine-(S,R)-sulfoximine (BSO) to deplete cellular GSH, followed by treatment with MB. Results suggest that following an insult to the GSH antioxidant system, MB can act as an additional insult to the system and prevent the normal recovery of those defenses. Altered protein levels of heme oxygenase-1 (HO-1) further indicated an oxidative stress response elicited by MB in PC12 cells. DAergic neurons, as a population, are inherently vulnerable to oxidative stress, and the disruption of antioxidant systems by the fungicide MB may contribute to the neurodegeneration of these cells, especially with concurrent exposures to other environmentally relevant oxidative stressors, such as PQ.

2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions.

Quantitative morphometric analyses have demonstrated that axon atrophy is the primary neuropathic feature in the CNS and PNS of rats intoxicated with 2,5-hexanedione (HD). Axon caliber is maintained by the exchange of mobile neurofilament (NF) subunits with the stationary polymer and, therefore, HD might produce atrophy by disrupting cytoskeletal turnover. To evaluate this possibility, groups of rats were exposed to HD at dosing schedules (175 mg/kg x 101 days or 400 mg/kg x 26 days) that produced moderate levels of neurological deficits and prevalent axon atrophy in spinal cord white matter tracts. Lumbar spinal cord regions from HD-intoxicated rats and their age-matched controls were Triton-extracted and separated by differential fractionation into a low-speed, insoluble pellet (P1) of NF polymer and a high-speed supernatant fraction (S2), which presumably contained mobile monomer. Cytoskeletal protein contents (NF-L, -M, -H, and beta-tubulin) in each fraction were determined by immunoblot analysis. Results show that regardless of HD dose-rate, the NF polymer in P1 remained unaffected, although soluble monomer in the S2 fraction was depleted significantly (60-80% reduction). Fractional beta-tubulin contents were inconsistently affected and abnormal higher-molecular-weight NF proteins were detected in the P1 fraction only. Studies with antibodies directed against phosphorylated (RT97) and nonphosphorylated (SMI32) epitopes on NF-H and measurements of corresponding isoelectric range suggested that alterations in phosphorylation were not involved. The selective depletion of Triton-soluble protein suggested that HD adduction of NFs interfered with the dynamic interactions of the polymeric and mobile monomeric pools.