Structural Insights into the Human Mitochondrial Pyruvate Carrier Complexes.

Pyruvate metabolism requires the mitochondrial pyruvate carrier (MPC) proteins to transport pyruvate from the intermembrane space through the inner mitochondrial membrane to the mitochondrial matrix. The lack of the atomic structures of MPC hampers the understanding of the functional states of MPC and molecular interactions with the substrate or inhibitor. Here, we develop the de novo models of human MPC complexes and characterize the conformational dynamics of the MPC heterodimer formed by MPC1 and MPC2 (MPC1/2) by computational simulations. Our results reveal that functional MPC1/2 prefers to adopt an inward-open conformation, with the carrier open to the matrix side, whereas the outward-open states are less populated. The energy barrier for pyruvate transport in MPC1/2 is low enough, and the inhibitor UK5099 blocks the pyruvate transport by stably binding to MPC1/2. Notably, consistent with experimental results, the MPC1 L79H mutation significantly alters the conformations of MPC1/2 and thus fails for substrate transport. However, the MPC1 R97W mutation seems to retain the transport activity. The present de novo models of MPC complexes provide structural insights into the conformational states of MPC complexes and mechanistic understanding of interactions between the substrate/inhibitor and MPC proteins.

Extracellular gating of glucose transport through GLUT 1.

The ubiquitous glucose transporter 1 (GLUT1) is physiologically and pathologically relevant in energy metabolism of the CNS, skeletal muscles, cancer cells etc. Extensive experiments on GLUT1 produced thorough understandings of its expressions, functions, and structures which were recently resolved to atomic accuracy. However, theoretical understandings are still controversial about how GLUT1 facilitates glucose diffusion across the cell membrane. Molecular dynamics (MD) simulations of the current literature have GLUT1 embedded in a symmetric bilayer of a single lipid type. They provide atomistic illustrations of the alternating access theory (AAT), but the simulation results are inconsistent with the undisputed experimental data of kinetics showing rapid transport of glucose at near-physiological temperatures, high Arrhenius activation barrier in zero-trans uptake, and large trans-acceleration at sub-physiological temperatures. In this research, we embedded GLUT1 in an asymmetric bilayer of multiple lipids to better mimic the erythrocyte membrane. We ran unbiased MD simulations at 37 °C and at 5 °C and found a new mechanism of glucose transport via GLUT1: The extracellular (EC) gate opened wide for EC glucopyranose at 37 °C and, only in the presence of intracellular (IC) glucose, at 5 °C. In the absence of IC glucose at 5 °C, the EC gate opened narrowly for acyclic glucose, gating out glucopyranose. This EC-gating mechanism is simpler than AAT and yet it well explains for the rapid glucose transport at near-physiological temperatures and large trans-acceleration at sub-physiological temperatures. It also explains why zero-trans uptake (involving the pyranose-to-aldehyde transformation) has an Arrhenius barrier ∼20 kcal/mol higher than the equilibrium exchange transport.

The sterol regulatory element-binding protein 2 is dysregulated by tau alterations in Alzheimer disease.

Disturbed neuronal cholesterol homeostasis has been observed in Alzheimer disease (AD) and contributes to the pathogenesis of AD. As the master switch of cholesterol biosynthesis, the sterol regulatory element-binding protein 2 (SREBP-2) translocates to the nucleus after cleavage/activation, but its expression and activation have not been studied in AD which is the focus of the current study. We found both a significant decrease in the nuclear translocation of N-terminal SREBP-2 accompanied by a significant accumulation of C-terminal SREBP-2 in NFT-containing pyramidal neurons in AD. N-terminal- SREBP-2 is also found in dystrophic neurites around plaques in AD brain. Western blot confirmed a significantly reduced nuclear translocation of mature SREBP-2 (mSREBP-2) in AD brain. Interestingly, reduced nuclear mSREBP-2 was only found in animal models of tauopathies such as 3XTg AD mice and P301L Tau Tg mice but not in CRND8 APP transgenic mice, suggesting that tau alterations likely are involved in the changes of mSREBP-2 distribution and activation in AD. Altogether, our study demonstrated disturbed SREBP-2 signaling in AD and related models, and proved for the first time that tau alterations contribute to disturbed cholesterol homeostasis in AD likely through modulation of nuclear mSREBP-2 translocation.

Gibbs Free-Energy Gradient along the Path of Glucose Transport through Human Glucose Transporter 3.

Fourteen glucose transporters (GLUTs) play essential roles in human physiology by facilitating glucose diffusion across the cell membrane. Due to its central role in the energy metabolism of the central nervous system, GLUT3 has been thoroughly investigated. However, the Gibbs free-energy gradient (what drives the facilitated diffusion of glucose) has not been mapped out along the transport path. Some fundamental questions remain. Here we present a molecular dynamics study of GLUT3 embedded in a lipid bilayer to quantify the free-energy profile along the entire transport path of attracting a ß-d-glucose from the interstitium to the inside of GLUT3 and, from there, releasing it to the cytoplasm by Arrhenius thermal activation. From the free-energy profile, we elucidate the unique Michaelis-Menten characteristics of GLUT3, low KM and high VMAX, specifically suitable for neurons' high and constant demand of energy from their low-glucose environments. We compute GLUT3's binding free energy for ß-d-glucose to be -4.6 kcal/mol in agreement with the experimental value of -4.4 kcal/mol ( KM = 1.4 mM). We also compute the hydration energy of ß-d-glucose, -18.0 kcal/mol vs the experimental data, -17.8 kcal/mol. In this, we establish a dynamics-based connection from GLUT3's crystal structure to its cellular thermodynamics with quantitative accuracy. We predict equal Arrhenius barriers for glucose uptake and efflux through GLUT3 to be tested in future experiments.

Macrophage TGF-ß1 and the Proapoptotic Extracellular Matrix Protein BIGH3 Induce Renal Cell Apoptosis in Prediabetic and Diabetic Conditions.

Metabolically stressed kidney is in part characterized by infiltrating macrophages and macrophage-derived TGF-ß1 that promote the synthesis of various ECM molecules. TGF-ß1 strongly enhances the expression of the gene TGFBI that encodes a cell-adhesion class, proapoptotic ECM protein called BIGH3. We hypothesized that in a diabetic environment a relationship between infiltrating macrophages, macrophage-derived TGF-ß1, and BIGH3 protein promotes renal cell death. To investigate this hypothesis, we used our mouse model of diabetic complications. Mice on a high-fat diet developed hypercholesterolemia, and exposure to streptozotocin rendered hypercholesterolemic mice diabetic. Immunohistochemical images show increased macrophage infiltration and BIGH3 protein in the kidney cortices of hypercholesterolemic and diabetic mice. Macrophages induced a two-fold increase in BIGH3 expression and an 86% increase in renal proximal tubule epithelial cell apoptosis. TGF-ß1 antibody and TGF-ß1 receptor chemical antagonist blocked macrophage-induced apoptosis. BIGH3 antibody completely blocked apoptosis that was induced by TGF-ß1, and blocked apoptosis induced by exogenous recombinant BIGH3. These results uncover a distinctive interplay of macrophage-derived TGF-ß1, BIGH3 protein, and apoptosis, and indicate that BIGH3 is central in a novel pathway that promotes diabetic nephropathy. Macrophage TGF-ß1 and BIGH3 are identified as prediabetic biomarkers, and potential therapeutic targets for intervention in prediabetic and diabetic individuals.

Modeling non-clinical and clinical drug tests in Gaucher disease.

There is need for modeling biological systems to accelerate drug pipelines for treating metabolic diseases. The eliglustat treatment for Gaucher disease is approved by the FDA with a companion genomic test. The Transcriptome-To-Metabolome™ biosimulation technology was used to model, in silico, a standard non-clinical eliglustat test with an in vitro canine kidney cell system over-expressing a human gene; and a clinical test using human fibroblasts from control and Gaucher disease subjects. Protein homology modeling and docking studies were included to gather affinity parameters for the kinetic metabolic model. Pharmacodynamics and metabolomics analyses of the results replicated published findings and demonstrated that processing and transport of lysosomal proteins alone cannot explain the metabolic disorder. This technology shows promise for application to other diseases.

Epigenetic regulation by chromatin activation mark H3K4me3 in primate progenitor cells within adult neurogenic niche.

Histone 3 lysine 4 trimethylation (H3K4me3) is known to be associated with transcriptionally active or poised genes and required for postnatal neurogenesis within the subventricular zone (SVZ) in the rodent model. Previous comparisons have shown significant correlation between baboon (Papio anubis) and human brain. In this study, we demonstrate that chromatin activation mark H3K4me3 is present in undifferentiated progenitor cells within the SVZ of adult baboon brain. To identify the targets and regulatory role of H3K4me3 within the baboon SVZ, we developed a technique to purify undifferentiated SVZ cells while preserving the endogenous nature without introducing culture artifact to maintain the in vivo chromatin state for genome-wide studies (ChIP-Seq and RNA-Seq). Overall, H3K4me3 is significantly enriched for genes involved in cell cycle, metabolism, protein synthesis, signaling pathways, and cancer mechanisms. Additionally, we found elevated levels of H3K4me3 in the MRI-classified SVZ-associated Glioblastoma Multiforme (GBM), which has a transcriptional profile that reflects the H3K4me3 modifications in the undifferentiated progenitor cells of the baboon SVZ. Our findings highlight the importance of H3K4me3 in coordinating distinct networks and pathways for life-long neurogenesis, and suggest that subtypes of GBM could occur, at least in part, due to aberrant H3K4me3 epigenetic regulation.

Biomarkers from biosimulations: Transcriptome-To-Reactome™ Technology for individualized medicine.

We validated a model of the TGF-ß signaling pathway using reactions from Reactome. Using a patentpending technique, gene expression profiles from individual patients are used to determine model parameters. Gene expression profiles from 45 women, normal, or benign tumor and malignant breast cancer were used as training and validating sets for assessing clinical sensitivity and specificity. Biomarkers were identified from the biosimulation results using sensitivity analyses and derivative properties from the model. A membrane signaling marker had sensitivity of 80% and specificity of 60%; while a nuclear transcription factor marker had sensitivity of 80% and specificity of 90% to predict malignancy. Use of Fagan's nomogram increased probability from 7.5% for positive mammogram to 39% with positive results of the biosimulation for the nuclear marker. Our technology will allow researchers to identify and develop biomarkers and assist clinicians in diagnostic and treatment decision making.

Apolipoprotein E4 prevents growth of malaria at the intraerythrocyte stage: implications for differences in racial susceptibility to Alzheimer's disease.

Apolipoprotein E 4 (ApoE 4) has been linked to pathogenesis of Alzheimer's disease and has been suggested to be maintained through evolutionary pressure via a protective role in malaria infection. We evaluated Plasmodium falciparum viability at the intraerythrocyte stage by exposure to plasma from human subjects with ApoE 4/4 or ApoE 3/3. Plasma samples from ApoE 4/4 but not ApoE 3/3 donors inhibited growth and disrupted morphology of P. falciparum. Evolutionary history is characterized by war between pathogenic microorganisms and defense mechanisms countering their pathogenicities. ApoE 4 frequency is highest in sub-Saharan Africa and other isolated populations (e.g., Papua New Guinea) that exhibit endemic malaria. High ApoE frequency may offer selective advantage protecting against some infectious diseases (e.g., Plasmodium falciparum). These results implicate evolutionary pressure by malaria selecting humans with ApoE 4/4, even considering lower survival in late life. These selective advantages may be relevant in the exploration of possible disparities between Black and Whites in the incidence of Alzheimer's Disease.

Epileptic baboons have lower numbers of neurons in specific areas of cortex.

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.

Modeling cholesterol metabolism by gene expression profiling in the hippocampus.

An important part of the challenge of building models of biochemical reactions is determining reaction rate constants that transform substrates into products. We present a method to derive enzymatic kinetic values from mRNA expression levels for modeling biological networks without requiring further tuning. The core metabolic reactions of cholesterol in the brain, particularly in the hippocampus, were simulated. To build the model the baseline mRNA expression levels of genes involved in cholesterol metabolism were obtained from the Allen Mouse Brain Atlas. The model is capable of replicating the trends of relative cholesterol levels in Alzheimer's and Huntington's diseases; and reliably simulated SLOS, desmosterolosis, and Dhcr14/Lbr knockout studies. A sensitivity analysis correctly uncovers the Hmgcr, Idi2 and Fdft1 sites that regulate cholesterol homeostasis. Overall, our model and methodology can be used to pinpoint key reactions, which, upon manipulation, may predict altered cholesterol levels and reveal insights into potential drug therapy targets under diseased conditions.

Local pretreatment with the cannabinoid CB1 receptor antagonist AM251 attenuates methamphetamine intra-accumbens self-administration.

The endocannabinoid system is a potential target for therapeutic intervention of substance abuse. Cannabinoid CB1 receptor antagonist decreases intravenous methamphetamine self-administration in animal models. This study examined whether the nucleus accumbens (NAcc) is a site of interaction between methamphetamine and the CB1 receptor antagonist AM251. Male Sprague-Dawley rats were trained to lever press and then were surgically implanted with a guide cannula into the right NAcc. Rats were allowed one week to recover and then AM251 (0.1 or 1.0 µg/µL) was reverse dialyzed directly into the NAcc prior to methamphetamine (10 µg/µL) intra-accumbens self-administration. AM251 (1.0 µg/µL) reduced methamphetamine self-administration while AM251 (0.1 µg/µL) had an intermediary effect. The mechanism of self-administration attenuation is not known but could be mediated by AM251 affecting the negative feedback from the NAcc to the ventral tegmental area (VTA). This study provides evidence that the endocannabinoid system is involved with rewarding effects of methamphetamine and suggests a possible therapeutic intervention for methamphetamine abuse.

Cholesterol homeostasis markers are localized to mouse hippocampal pyramidal and granule layers.

Changes in brain cholesterol homeostasis are associated with multiple diseases, such as Alzheimer's and Huntington's; however, controversy persists as to whether adult neurons produce their own cholesterol, or if it is outsourced to astrocytes. To address this issue, we analyzed 25 genes most immediately involved in cholesterol homeostasis from in situ data provided by the Allen Brain Mouse Atlas. We compared the relative mRNA expression in the pyramidal and granule layers, populated with neurons, with the rest of the hippocampus which is populated with neuronal processes and glia. Comparing the expression of the individual genes to markers for neurons and astrocytes, we found that cholesterol homeostasis genes are preferentially targeted to neuronal layers. Therefore, changes in gene expression levels might affect neuronal populations directly.

Thiol oxidative stress induced by metabolic disorders amplifies macrophage chemotactic responses and accelerates atherogenesis and kidney injury in LDL receptor-deficient mice.

BACKGROUND: Strengthening the macrophage glutathione redox buffer reduces macrophage content and decreases the severity of atherosclerotic lesions in LDL receptor-deficient (LDLR(-/-)) mice, but the underlying mechanisms were not clear. This study examined the effect of metabolic stress on the thiol redox state, chemotactic activity in vivo, and the recruitment of macrophages into atherosclerotic lesions and kidneys of LDL-R(-/-) mice in response to mild, moderate, and severe metabolic stress. METHODS AND RESULTS: Reduced glutathione (GSH) and glutathione disulfide (GSSG) levels in peritoneal macrophages isolated from mildly, moderately, and severe metabolically-stressed LDL-R(-/-) mice were measured by HPLC, and the glutathione reduction potential (E(h)) was calculated. Macrophage E(h) correlated with the macrophage content in both atherosclerotic (r(2)=0.346, P=0.004) and renal lesions (r(2)=0.480, P=0.001) in these mice as well as the extent of both atherosclerosis (r(2)=0.414, P=0.001) and kidney injury (r(2)=0.480, P=0.001). Compared to LDL-R(-/-) mice exposed to mild metabolic stress, macrophage recruitment into MCP-1-loaded Matrigel plugs injected into LDL-R(-/-) mice increased 2.6-fold in moderately metabolically-stressed mice and 9.8-fold in severely metabolically-stressed mice. The macrophage E(h) was a strong predictor of macrophage chemotaxis (r(2)=0.554, P<0.001). CONCLUSIONS: Thiol oxidative stress enhances macrophage recruitment into vascular and renal lesions by increasing the responsiveness of macrophages to chemoattractants. This novel mechanism contributes at least in part to accelerated atherosclerosis and kidney injury associated with dyslipidemia and diabetes in mice.

C-terminal fragment of transforming growth factor beta-induced protein (TGFBIp) is required for apoptosis in human osteosarcoma cells.

Transforming growth factor beta-induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-beta1 treatment increased expression of TGFBIp over 72 h (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37-150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-beta1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-beta1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins.

Prevalence of diabetes mellitus and correlation of urinary transforming growth factor-beta1 with blood hemoglobin A1C in the Atascosa Diabetes Study.

INTRODUCTION: This study was conducted to determine the prevalence of type 2 diabetes and prediabetes in the Atascosa Diabetes Study sample and to ascertain the relationship between urinary transforming growth factor-beta1 (TGF-beta1) and blood hemoglobin (Hgb) A1C. METHODS: Subjects (N = 526) classified as adjusted normal, at risk, prediabetes, and diabetes mellitus were given a one-hour and two-hour postprandial glucose (PPG) test. Morning urine samples were collected to test for a correlation of TGF-beta1 with blood HgbA1C. RESULTS: Of the subjects, 14.3% had diabetes, 31.6% had prediabetes, 7.9% were at risk, and 46.2% were adjusted normal. Sensitivity and specificity for one-hour PPG for prediabetes and diabetes were significant, with an efficiency of 80.2%-90.9% and a likelihood ratio of 4.7-10.2. Receiver operating characteristic analysis resulted in an area under the curve of .880 +/- .016 for one hour to prediabetes and diabetes and .960 +/- .016 for one hour to diabetes. Prediabetes was 1.07 times more prevalent in Hispanics, but diabetes was 1.65 times greater in Whites. Urinary TGF-beta1 was more than fivefold higher in poorly controlled versus controlled diabetic or normal subjects and had a significant positive correlation with HgbA1C. CONCLUSIONS: The percentage of subjects with type 2 diabetes was 1.64 times higher than the national average. Prevalence of prediabetes was equivalent in Hispanics and Whites, and the reversal for diabetes might reflect higher mortality rate from diabetes in Hispanics in Atascosa County. Use of one-hour PPG and urine markers for early kidney involvement could improve this disparity in such high-risk populations.

Differences in performance between Sprague-Dawley and Fischer 344 rats in positive reinforcement tasks.

This experimental investigation tested two different strains of rat, Sprague-Dawley (SD) and Fischer 344 (F344), in their ability to learn lever pressing for food (autoshaping) or intracranial self-administration (ICSA) of dextroamphetamine (AMPH) into the nucleus accumbens (NAcc). Additionally, a unique method of intracranial drug delivery was utilized, via reverse dialysis, by the use of a microdiaylsis probe. The experiments revealed definite behavioral differences between SD and F344 animals. The autoshaping data indicated that SD rats, on average, acquired lever pressing for food in fewer training days than F344 rats. Also, the ICSA experiment revealed that SD rats self-administered AMPH at a 30 mug/mul concentration. Lever pressing was significantly greater in those SD rats receiving AMPH than in the F344 drug group. Furthermore, the F344 rats never acquired lever pressing for intra-NAcc delivery of AMPH under our testing regime. These data reveal differences in performance of positively reinforced operant tasks between the inbred F344 rats as compared to the outbred SD strain.

Analysis of MDMA and its metabolites in urine and plasma following a neurotoxic dose of MDMA.

3,4-Methylenedioxymethamphetamine (MDMA), a commonly encountered drug of abuse, has been shown in a variety of studies to cause neurotoxic effects. Because MDMA itself is not neurotoxic, identifying the potential neurotoxic metabolite(s) was of significant importance. Evaluation of urine and plasma concentrations of MDMA and three of its main metabolites, 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA), following administration of a neurotoxic dose (20 mg/kg) to male Dark Agouti rats was accomplished. Currently there are no data available describing urine and plasma concentrations of MDMA and these metabolites over a period of 7 days. The rats received a single 20 mg/kg i.p. dose of MDMA. Blood and urine samples were collected prior to administration and at 2, 4, 8, 12, 16, 20, 24, 48, 96, and 168 h following drug administration. Plasma and urine samples were extracted using solid-phase extraction, derivatized with N-methyl-bis(trifluoroacetamide), then analyzed using gas chromatography-mass spectrometry. Urine samples showed peak concentrations of MDMA at 4 h, MDA at 8 h, HMMA at 12 h, and HMA at 16 h post dose. MDMA and its metabolites were detectable (limit of detection 25 ng/mL) in the urine for up to 168 h post dose. Plasma samples showed mean peak concentrations of MDMA and MDA at 2 h post dose and HMMA at 4 h. Although the highest mean concentration of HMA was seen at 24 h post dose, variability between sample results for this time point was significant. No detectable levels of MDMA, MDA, HMA, and HMMA (LOD 10 ng/mL) were found in plasma at 96 and 168 h post dose.

Acute effects of pulsed microwaves and 3-nitropropionic acid on neuronal ultrastructure in the rat caudate-putamen.

Ultrastructure of the medium sized "spiny" neuron in rat dorsal-lateral caudate-putamen was assessed after administration of 3-nitropropionic acid (3-NP) and exposure to pulsed microwaves. Sprague-Dawley male rats were given two daily intraperitoneal doses of 0 or 10 mg/kg 3-NP and 1.5 h after each dose were exposed to microwave radiation at a whole body averaged specific absorption rate (SAR) of 0 (sham exposure), 0.6, or 6 W/kg for 30 min. Microwave exposure consisted of 1.25 GHz radiation delivered as 5.9 micros pulses with repetition frequency 10 Hz. Tissue samples taken 2-3 h after the second sham or microwave exposure showed no injury with light microscope methods. Blinded qualitative assessment of ultrastructure of randomly selected neurons from the same samples did reveal differences. Subsequent detailed, quantitative measurements showed that, when followed by sham exposure, administration of 3-NP significantly increased endoplasmic reticulum (ER) intracisternal width, ER area density, and nuclear envelope thickness. Microwave exposure at 6 W/kg alone also significantly increased these measures. Exposure of 3-NP treated animals at 6 W/kg significantly increased effects of 3-NP on ultrastructure. Although exposure at 0.6 W/kg alone did not affect ultrastructure measures, exposure of 3-NP treated animals at 0.6 W/kg reduced the effects of 3-NP. We concluded that 3-NP changed neuronal ultrastructure and that the microwave exposures used here changed neuronal ultrastructure in ways that depended on microwave SAR and neuron metabolic status. The apparent cancellation of 3-NP induced changes by exposure to pulsed microwaves at 0.6 W/kg indicated the possibility that such exposure can protect against the effects of mitochondrial toxins on the nervous system.