ANTECEDENTES GENERALES DE LOS PIP

The most obvious concern of any immunocytochemical detection of oxidative modi- fi cations in postmortem human tissues is whether the positive signal is simply due to postmortem artifact. To test for the effect of postmortem interval (PMI) on the accumu- lation of these modifi cations within tissue structures, we examined CEP in a rat model of PMI. Initial experiments found no CEP immunoreactivity in the brain in any of the samples up to 24 h postmortem delay using WKY rat strain. However, use of an addi- tional spontaneous hypertensive rat strain (SHR), proved to be very informative. Using

FIGURE 2.2 In autism, cellular processes contain choline acetyltransferase (A), in a pattern similar to that seen for CEP modifi cations (B).

this model, rat pups were kept at room temperature for different interval (0–8 h) prior to brain dissection to simulate increased PMI. All rat pups from all dams tested accumu- lated CEP in areas of the brain that overlapped signifi cantly with neurofi laments accu- mulation as seen using monoclonal antibody SMI-31 (Covance) (Figure 2.3). Image analysis was performed to measure both the percent area stained and the intensity of labeling for both CEP and neurofi laments. No correlation was found between either the amount or intensity of CEP accumulation and PMI. These data strongly argue for neu- rofi lament protein being a specifi c target for CEP modifi cation (Evans et al., 2008).

To further correlate the rat model data with human disease, the levels of CEP modifi cations were compared in autism and control brain sections, which had varying and disparate PMI. There was no signifi cant correlation between CEP and PMI, with times ranging from 13 to 39 h (Figure 2.4). Even in the control case with the longest PMI (36 h), there was no recognizable immunoreactivity to CEP (data not shown). Densitometric analysis of the staining in these autistic cases clearly did not show any signifi cant correlation with differences in PMI (Figure 2.4).

Taken together, the human and rat models provide strong evidence that CEP modifi cations do not accumulate after death, and therefore may be a direct result

FIGURE 2.3 In a rat model, CEP is localized to cellular processes within the brain strikingly similar to the CEP localization in autism. To test for any postmortem changes in CEP modifi cations, rat pup brains were fi xed following either 0 h (A), 4 h (B), or 8 h (C) postmortem delay. All rats displayed similar levels of CEP and following image analysis showed no signifi cant differences (not shown). Paralleling the fi ndings in human autism cases, CEP localization overlaps with neurofi lament protein accumulation on adjacent sec- tions (C, CEP and D, neurofi lament protein). Scale bar = 50 μm.

(A) (B)

of oxidative damage in the living brain. Although this excludes a direct correlation between postmortem time and staining, this does not account for any differences in the presence of these substances in the rat brain.

2.6 CONCLUSION

There is ample evidence suggesting systemic oxidative stress in autism patients and evidence for brain oxidative stress is now beginning to accumulate. Our immunocytochemical and biochemical studies provide the fi rst experimental evi- dence demonstrating lipid modifi cation in autistic brain and suggests CEP– and iso[4]LGE2–protein adducts, products of lipid peroxidation, as possible hallmark

oxidative stress markers for the autistic brain. Supplementary animal experiments show that CEP formation is unlikely due to postmortem artifact, supporting CEP as a specifi c oxidative stress marker. From a structural perspective, our fi ndings suggest that axons of cholinergic neurons in the white matter are the primary site of oxidative damage. At the molecular level, we have identifi ed NFH to be the major target for CEP modifi cation. Our fi ndings not only support the notion that brain oxidative stress plays an important role in autism but now warrant future in-depth mechanistic studies, which have the potential to provide new targets for therapeutic efforts.

FIGURE 2.4 In autism, CEP is localized specifi cally to processes within the brain. Morphologically, no differences are apparent between cases collected following a relatively short PMI of 13 h (A), or 24 h (B), or even after a long PMI of 39 h (C). Computer-assisted image analysis of the intensity of CEP accumulation in nine cases of autism found no signifi - cant correlation when compared with PMI (D). Scale bar = 50 μm.

(B) (A) (C) _(D) 0 5 10 15 20 25 30 35 0 10 20 30 40 50 PMI (h) Re la tive C EP in tensity

ACKNOWLEDGMENTS

This research was supported by Autism Research Institute. Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland under contracts N01-HD-4-3368 and N01-HD-4-3383.

REFERENCES

Ahlsen, G., Rosengren, L., Belfrage, M., Palm, A., Haglid, K., Hamberger, A., and Gillberg, C. (1993). Glial fi brillary acidic protein in the cerebrospinal fl uid of children with autism and other neuropsychiatric disorders. Biol. Psychiatry 33:734–743.

Alvarez, R. A., Aguirre, G. D., Acland, G. M., and Anderson, R. E. (1994). Docosapentaenoic acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected miniature poodle dogs. Invest. Ophthalmol. Vis. Sci. 35:402–408.

Bailey, A., Le Couteur, A., Gottesman, I., Bolton, P., Simonoff, E., Yuzda, E., and Rutter, M. (1995). Autism as a strongly genetic disorder: Evidence from a British twin study. Psychol. Med. 25:63–77.

Bailey, A., Luthert, P., Dean, A., Harding, B., Janota, I., Montgomery, M., Rutter, M., and Lantos, P. (1998). A clinicopathological study of autism. Brain 121 (Pt 5):889–905. Chauhan, A. and Chauhan, V. (2006). Oxidative stress in autism. Pathophysiology

13:171–181.

Chauhan, A., Chauhan, V., Brown, W. T., and Cohen, I. (2004). Oxidative stress in autism: Increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin— the antioxidant proteins. Life Sci. 75:2539–2549.

Chez, M. G., Buchanan, C. P., Aimonovitch, M. C., Becker, M., Schaefer, K., Black, C., and Komen, J. (2002). Double-blind, placebo-controlled study of l-carnosine supplemen- tation in children with autistic spectrum disorders. J. Child Neurol. 17:833–837. Chung, M. K., Dalton, K. M., Alexander, A. L., and Davidson, R. J. (2004). Less white matter

concentration in autism: 2D voxel-based morphometry. Neuroimage 23:242–251. Comi, A. M., Zimmerman, A. W., Frye, V. H., Law, P. A., and Peeden, J. N. (1999). Familial

clustering of autoimmune disorders and evaluation of medical risk factors in autism. J. Child Neurol. 14:388–394.

Connolly, A. M., Chez, M., Streif, E. M., Keeling, R. M., Golumbek, P. T., Kwon, J. M., Riviello, J. J., Robinson, R. G., Neuman, R. J., and Deuel, R. M. (2006). Brain-derived neurotrophic factor and autoantibodies to neural antigens in sera of children with autistic spectrum disorders, Landau–Kleffner syndrome, and epilepsy. Biol. Psychiatry 59:354–363.

Courchesne, E., Townsend, J., and Saitoh, O. (1994). The brain in infantile autism: Posterior fossa structures are abnormal. Neurology 44:214–223.

Dolske, M. C., Spollen, J., McKay, S., Lancashire, E., and Tolbert, L. (1993). A preliminary trial of ascorbic acid as supplemental therapy for autism. Prog. Neuropsychopharmacol. Biol. Psychiatry 17:765–774.

Evans, T., Siedlak, S. L., Lu, L., Fu, X., Wang, Z., McGinnis, W. R., Fakhoury, E., Castellani, R. J., Hazen, S. L., Walsh, W. L., Lewis, A. T., Salomon, R. G., Smith, M. A., Perry, G., and Zhu, X. (2008). The autistic phenotype exhibits a remarkably localized modifi cation of brain protein by products of free radical-induced lipid oxidation. Am. J. Biochem. Biotechnol. (Special Issue on Autism Spectrum Disorders) 4:61–72.

Filipek, P. A., Juranek, J., Smith, M., Mays, L. Z., Ramos, E. R., Bocian, M., Masser-Frye, D., Laulhere, T. M., Modahl, C., Spence, M. A., and Gargus, J. J. (2003). Mitochondrial dys- function in autistic patients with 15q inverted duplication. Ann. Neurol. 53:801–804. Golse, B., Debray-Ritzen, P., Durosay, P., Puget, K., and Michelson, A. M. (1978). Alterations

in two enzymes: Superoxide dismutase and glutathione peroxidase in developmental infantile psychosis (infantile autism). Rev. Neurol. 134:699–705.

Grice, D. E. and Buxbaum, J. D. (2006). The genetics of autism spectrum disorders. Neuromol. Med. 8:451–460.

Gu, X., Meer, S. G., Miyagi, M., Rayborn, M. E., Hollyfi eld, J. G., Crabb, J. W., and Salomon, R. G. (2003). Carboxyethylpyrrole protein adducts and autoantibodies, biomarkers for age-related macular degeneration. J. Biol. Chem. 278:42027–42035.

Halliwell, B., Zhao, K., and Whiteman, M. (1999). Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good: A personal view of recent controversies. Free Radic. Res. 31:651–669.

Horvath, K. and Perman, J. A. (2002). Autism and gastrointestinal symptoms. Curr. Gastroenterol. Rep. 4:251–258.

James, S. J., Cutler, P., Melnyk, S., Jernigan, S., Janak, L., Gaylor, D. W., and Neubrander, J. A. (2004). Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. Am. J. Clin. Nutr. 80:1611–1617.

James, S. J., Melnyk, S., Jernigan, S., Cleves, M. A., Halsted, C. H., Wong, D. H., Cutler, P., Bock, K., Boris, M., Bradstreet, J. J., Baker, S. M., and Gaylor, D. W. (2006). Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism. Am. J. Med. Genet. B Neuropsychiatr. Genet. 141B:947–956.

Joosten, E. A. and Houweling, D. A. (2004). Local acute application of BDNF in the lesioned spinal cord anti-infl ammatory and anti-oxidant effects. Neuroreport 15:1163–1166. Jyonouchi, H., Sun, S., and Le, H. (2001). Proinfl ammatory and regulatory cytokine produc-

tion associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression. J. Neuroimmunol. 120:170–179. Keller, F. and Persico, A. M. (2003). The neurobiological context of autism. Mol. Neurobiol.

28:1–22.

Kleijnen, J. and Knipschild, P. (1991). Niacin and vitamin B6 in mental functioning: A review of controlled trials in humans. Biol. Psychiatry 29:931–941.

Laurence, J. A. and Fatemi, S. H. (2005). Glial fi brillary acidic protein is elevated in superior frontal, parietal and cerebellar cortices of autistic subjects. Cerebellum 4:206–210.

Lee, H. G., Perry, G., Moreira, P. I., Garrett, M. R., Liu, Q., Zhu, X., Takeda, A., Nunomura, A., and Smith, M. A. (2005). Tau phosphorylation in Alzheimer’s disease: Pathogen or protector? Trends Mol. Med. 11:164–169.

McGinnis, W. R. (2004). Oxidative stress in autism. Altern. Ther. Health Med. 10:22–36. Meagher, E. A. and FitzGerald, G. A. (2000). Indices of lipid peroxidation in vivo: Strengths

and limitations. Free Radic. Biol. Med. 28:1745–1750.

Ming, X., Stein, T. P., Brimacombe, M., Johnson, W. G., Lambert, G. H., and Wagner, G. C. (2005). Increased excretion of a lipid peroxidation biomarker in autism. Prostaglandins Leukot. Essent. Fatty Acids 73:379–384.

Miyagi, M., Sakaguchi, H., Darrow, R. M., Yan, L., West, K. A., Aulak, K. S., Stuehr, D. J., Hollyfi eld, J. G., Organisciak, D. T., and Crabb, J. W. (2002). Evidence that light modulates protein nitration in rat retina. Mol. Cell Proteomics 1:293–303.

Miyazaki, K., Narita, N., Sakuta, R., Miyahara, T., Naruse, H., Okado, N., and Narita, M. (2004). Serum neurotrophin concentrations in autism and mental retardation: A pilot study. Brain Dev. 26:292–295.

Money, J., Bobrow, N. A., and Clarke, F. C. (1971). Autism and autoimmune disease: A family study. J. Autism Child Schizophr. 1:146–160.

Nunomura, A., Perry, G., Aliev, G., Hirai, K., Takeda, A., Balraj, E. K., Jones, P. K., Ghanbari, H., Wataya, T., Shimohama, S., Chiba, S., Atwood, C. S., Petersen, R. B., and Smith, M. A. (2001). Oxidative damage is the earliest event in Alzheimer disease. J. Neuropathol. Exp. Neurol. 60:759–767.

Nunomura, A., Chiba, S., Lippa, C. F., Cras, P., Kalaria, R. N., Takeda, A., Honda, K., Smith, M. A., and Perry, G. (2004). Neuronal RNA oxidation is a prominent feature of familial Alzheimer’s disease. Neurobiol. Dis. 17:108–113.

Poliakov, E., Brennan, M. L., Macpherson, J., Zhang, R., Sha, W., Narine, L., Salomon, R. G., and Hazen, S. L. (2003). Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo. FASEB J. 17:2209–2220.

Sajdel-Sulkowska, E. M., Lipinski, B., Windom, H., Audhya, T., and McGinnis, W. (2008). Oxidative stress in autism: Cerebellar 3-nitrotyrosine levels. Am. J. Biochem. Biotechnol. (Special Issue on Autism Spectrum Disorders) 4:73–84.

Sajdel-Sulkowska, E. M., Ming, X., and Koibuchi, N. (2009). Increase in brain neurotrophin-3 and oxidative stress in autism. Cerebellum [Epub PMID 19357934].

Schumann, C. M. and Amaral, D. G. (2005). Stereological estimation of the number of neurons in the human amygdaloid complex. J. Comp. Neurol. 491:320–329.

Skinner, E. R., Watt, C., Besson, J. A., and Best, P. V. (1993). Differences in the fatty acid composition of the grey and white matter of different regions of the brains of patients with Alzheimer’s disease and control subjects. Brain 116 (Pt 3):717–725.

Smith, M. A., Kutty, R. K., Richey, P. L., Yan, S. D., Stern, D., Chader, G. J., Wiggert, B., Petersen, R. B., and Perry, G. (1994). Heme oxygenase-1 is associated with the neurofi - brillary pathology of Alzheimer’s disease. Am. J. Pathol. 145:42–47.

Sogut, S., Zoroglu, S. S., Ozyurt, H., Yilmaz, H. R., Ozugurlu, F., Sivasli, E., Yetkin, O., Yanik, M., Tutkun, H., Savas, H. A., Tarakcioglu, M., and Akyol, O. (2003). Changes in nitric oxide levels and antioxidant enzyme activities may have a role in the pathophysiological mechanisms involved in autism. Clin. Chim. Acta 331:111–117.

Sweeten, T. L., Bowyer, S. L., Posey, D. J., Halberstadt, G. M., and McDougle, C. J. (2003). Increased prevalence of familial autoimmunity in probands with pervasive develop- mental disorders. Pediatrics 112:420–424.

Sweeten, T. L., Posey, D. J., Shankar, S., and McDougle, C. J. (2004). High nitric oxide production in autistic disorder: A possible role for interferon-gamma. Biol. Psychiatry 55:434–437.

Takeda, A., Smith, M. A., Avila, J., Nunomura, A., Siedlak, S. L., Zhu, X., Perry, G., and Sayre, L. M. (2000). In Alzheimer’s disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modifi cation. J. Neurochem. 75:1234–1241.

Vojdani, A., Campbell, A. W., Anyanwu, E., Kashanian, A., Bock, K., and Vojdani, E. (2002). Antibodies to neuron-specifi c antigens in children with autism: Possible cross-reaction with encephalitogenic proteins from milk, Chlamydia pneumoniae and Streptococcus group A. J. Neuroimmunol. 129:168–177.

Yao, Y., Walsh, W. J., McGinnis, W. R., and Pratico, D. (2006). Altered vascular phenotype in autism: Correlation with oxidative stress. Arch. Neurol. 63:1161–1164.

Yorbik, O., Sayal, A., Akay, C., Akbiyik, D. I., and Sohmen, T. (2002). Investigation of antioxidant enzymes in children with autistic disorder. Prostaglandins Leukot. Essent. Fatty Acids 67:341–343.

Zhu, X., Lee, H. G., Casadesus, G., Avila, J., Drew, K., Perry, G., and Smith, M. A. (2005). Oxidative imbalance in Alzheimer’s disease. Mol. Neurobiol. 31:205–217.

Zoroglu, S. S., Armutcu, F., Ozen, S., Gurel, A., Sivasli, E., Yetkin, O., and Meram, I. (2004). Increased oxidative stress and altered activities of erythrocyte free radical scavenging enzymes in autism. Eur. Arch. Psychiatry Clin. Neurosci. 254:143–147.

47

3

Oxidative Stress