Elizabeth M. Sajdel-Sulkowska

Increase in Cerebellar Neurotrophin-3 and Oxidative Stress Markers in Autism

Autism is a neurodevelopmental disorder characterized by social and language deficits, ritualistic–repetitive behaviors and disturbance in motor functions. Data of imaging, head circumference studies, and Purkinje cell analysis suggest impaired brain growth and development. Both genetic predisposition and environmental triggers have been implicated in the etiology of autism, but the underlying cause remains unknown. Recently, we have reported an increase in 3-nitrotyrosine (3-NT), a marker of oxidative stress damage to proteins in autistic cerebella. In the present study, we further explored oxidative damage in the autistic cerebellum by measuring 8-hydroxydeoxyguanosine (8-OH-dG), a marker of DNA modification, in a subset of cases analyzed for 3-NT. We also explored the hypothesis that oxidative damage in autism is associated with altered expression of brain neurotrophins critical for normal brain growth and differentiation. The content of 8-OH-dG in cerebellar DNA isolated by the proteinase K method was measured using an enzyme-linked immunosorbent assay (ELISA); neurotrophin-3 (NT-3) levels in cerebellar homogenates were measured using NT-3 ELISA. Cerebellar 8-OH-dG showed trend towards higher levels with the increase of 63.4% observed in autism. Analysis of cerebellar NT-3 showed a significant (p = 0.034) increase (40.3%) in autism. Furthermore, there was a significant positive correlation between cerebellar NT-3 and 3-NT (r = 0.83; p = 0.0408). These data provide the first quantitative measure of brain NT-3 and show its increase in the autistic brain. Altered levels of brain NT-3 are likely to contribute to autistic pathology not only by affecting brain axonal targeting and synapse formation but also by further exacerbating oxidative stress and possibly contributing to Purkinje cell abnormalities.

Perinatal exposure to polychlorinated biphenyls differentially affects cerebellar development and motor functions in male and female rat neonates

Perinatal exposure to polychlorinated biphenyls (PCBs) interacts with genetics and impacts the course of the central nervous system (CNS) development in both humans and animals. To test the hypothesis that the neurobehavioral impairments, and specifically motor dysfunctions following perinatal PCB exposure in rats are associated with changes in a specific brain region, the cerebellum, we compared neurodevelopment, motor behavior, cerebellar structure, and protein expression in rat neonates exposed to the PCB mixture Aroclor 1254 (A1254, 10.0 mg/kg/day) from gestational day 11 until postnatal day (P) 21 with that of controls. Body mass of PCB-exposed pups was not affected at birth, but was significantly lower than that of controls between birth and weaning; by P21 the difference was greater in females than in males. A1254 exposure delayed ear unfolding and impaired performance on the following behavioral tests: (1) righting response on P3-P6; (2) negative geotaxis on P5-P7; (3) startle response on P10-P12; and (4) a rotorod on P12, with PCB-male pups more severely affected than female. Changes in the behavior of PCB pups were associated with changes in cerebellar structure and protein expression. Cerebellar mass was more reduced in PCB-male than PCB-female pups. Analysis of selected cerebellar proteins revealed an increase in GFAP expression, greater in male than in female, and a decrease in L1 expression in both sexes. These results suggest that PCB exposure affects behavior and cerebellar development differently in male and female rat neonates, with greater effects in males. Further studies of neonatal PCB exposure will establish whether the environmental pollutants can contribute to the sex-related preponderance of certain neuropsychiatric disorders.

The beneficial effect of early excision on clinical response and thymic activity after burn injury.

Catabolic response and immunodepression were studied in guinea pigs with scald burns that were excised on day 1 postburn and those that were scald burned but unexcised. Weight gain returned to normal by day 6 in the excised group but remained depressed in burned but unexcised animals whose wounds were untreated, or in unexcised animals whose wounds were treated by topical silver sulfadiazine. Thymic DNA synthesis returned to normal by day 6 in the excised group but remained depressed in the unexcised group. Plasma and thymic-free cortisol returned to normal by day 6 in the excised group but remained markedly elevated in the burned, unexcised animals. These studies indicate early excision and early wound closure reduce the catabolic response and immunodepression of guinea pigs following burn injury.

Sexual dimorphism in cerebellar structure, function, and response to environmental perturbations.

Sexual dimorphism of CNS structure and function has been observed in humans and animals, but remains relatively unrecognized in the context of the cerebellum. Recent research in our laboratory has examined whether these gender differences extend to cerebellar structure and function, as well as the impact of environmental factors on the developing cerebellum. Perinatal exposure to both chemical and physical perturbations in the environment (in our experiments, PCBs or hypergravity) affects growth, neurodevelopment, and motor coordination differently in males and females. These neurodevelopmental and behavioral effects are accompanied by sex-related changes in cerebellar mass and cerebellar protein expression. Exposure to chemical toxins (PCBs) resulted in more dramatic neurodevelopmental and behavioral changes in male neonates. It is possible that gender-related differences in male and female cerebellar structure and function are related to sex-specific development of the cerebellum and sex-specific distribution of specific receptors, local synthesis of trophic factors, and maturation of the pituitary hypophesial axis. These sex-related differences may underlie the sex-specific preponderance of certain neuropsychiatric disorders, and must be incorporated in the design of future basic and clinical investigations.

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)-Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Abstract: Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)‐containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)‐cellulose‐purified poly(A)‐containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L‐[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9‐ to 20‐fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two‐dimensional polyacrylamide gel electrophoresis (PAGE): size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)‐cellulose chromatography yielded 1.8 μg/g and 2.0 μg/g, respectively, of poly(A)‐containing mRNA; this represents a two‐ to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two‐dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.

Cloning of a rat α1,3-fucosyltransferase gene: a member of the fucosyltransferase IV family

We report the cloning of a rat α1,3-fucosyltransferase gene (rFuc-T), isolated from a rat genomic library by a PCR-cross-hybridization based cloning approach using primers derived from the conserved region of human α1,3-Fuc-T sequences. Comparison of the rFuc-T predicted amino acid sequence with those of previously cloned human and murine fucosyltransferases showed highest degree of homology to murine Fuc-TIV (87% identity) and human Fuc-TIV (78% identity), with lower homology (41–49% identity) to Fuc-TIII, V, VI, and VII. COS-1 cells transfected with the rFuc-T gene expressed a fucosyltransferase activity with type 2 (Galβ1→4GlcNAc)-containing oligosaccharides and the glycolipid acceptor neolactotetraosyl-ceramide but only low activity with sialylated substrates; the SSEA-1/Lex antigen was detected in transfected cells by immunocytochemistry. Based on these results, we surmise that rFuc-T is a member of the fucosyltransferase IV family. Northern blot analysis with a rFuc-T specific probe indicated a major transcript of 4.2 kb most abundantly expressed in rat spleen; minor transcripts of different sizes were detected in several tissues, including rat brain. Abbreviations: Fuc-T, fucosyltransferase; SSEA-1, stage specific embryonic antigen or Lewisx (Lex); ELFT, ELAM-1 ligand fucosyltransferase; ELAM, endothelial-leukocyte adhesion molecule; nLc4Cer, neolactotetraosylceramide (Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer); NEM, N-ethylmaleimide

Chapter 19 Transcriptional and translational regulatory mechanisms during normal aging of the mammalian brain and in Alzheimer's disease

Publisher Summary Irrespective of the etiology of Alzheimers disease (AD), increasing evidence supports the hypothesis that basic defects at the level of transcription or translation, or both, may be involved in the pathogenesis of this illness. Decreased levels of protein synthesis can be demonstrated in living patients with AD and the AD post-mortem brain messenger RNA has diminished capacity to stimulate high levels of protein synthesis in vitro. Although numerous factors may contribute to the observed decline in in vivo functional activity, the concentration of the brain RNA appears to be particularly important for regulating the rate of translation. During normal aging, the level of the brain RNA decreases. In AD, there appears to be an accelerated general decline in neuronal RNA and a further decrease within neurons containing neurofibrillary tangles. RNA extracted from affected regions supports the limited synthesis of proteins. Although the overall levels of in vitro synthesis tend to be low, the specific activity of certain proteins, such as glial fibrillary acidic protein, remains high. These observations may be related, in part, to increased degradation of neuronal RNA with sparing of glial RNA. The level of AD brain alkaline ribonuclease activity is increased due to the loss of inhibitor activity.

The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA

Although the precise role of astrocytes in the pathogenesis of Alzheimers disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimers disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.

Purkinje cell loss accompanies motor impairment in rats developing at altered gravity.

We have previously reported that the developmental exposure of rats to altered gravity (1.65 g) from gestational day 8 to postnatal day 21 impacts motor functions and cerebellar structure. The present study examined whether the decrease in cerebellar mass accompanied by impaired performance on a rotorod in hypergravity-exposed rats was related to a decrease in Purkinje cell number. The total number of Purkinje cells was determined on postnatal day 21 using a stereological analysis applied to paraformaldehyde-fixed cerebellar samples subsequently embedded in celloidin. Total Purkinje cell number was decreased by 17.7–25.3%. These results imply that exposure to altered gravity during Purkinje cell birth may affect their proliferation, resulting in a decrease in Purkinje cell number, which, in turn, leads to motor impairment.

Effects of Hypergravity Exposure on the Developing Central Nervous System: Possible Involvement of Thyroid Hormone

The present study examined the effects of hypergravity exposure on the developing brain and specifically explored the possibility that these effects are mediated by altered thyroid status. Thirty-four timed-pregnant Sprague-Dawley rats were exposed to continuous centrifugation at 1.5 G (HG) from gestational Day 11 until one of three key developmental points: postnatal Day (P) 6, P15, or P21 (10 pups/dam: 5 males/5 females). During the 32-day centrifugation, stationary controls (SC, n = 25 dams) were housed in the same room as HG animals. Neonatal body, forebrain, and cerebellum mass and neonatal and maternal thyroid status were assessed at each time point. The body mass of centrifuged neonates was comparatively lower at each time point. The mass of the forebrain and the mass of the cerebellum were maximally reduced in hypergravity-exposed neonates at P6 by 15.9% and 25.6%, respectively. Analysis of neonatal plasma suggested a transient hypothyroid status, as indicated by increased thyroid stimulating hormone (TSH) level (38.6%) at P6, while maternal plasma TSH levels were maximally elevated at P15 (38.9%). Neither neonatal nor maternal plasma TH levels were altered, suggesting a moderate hypothyroid condition. Thus, continuous exposure of the developing rats to hypergravity during the embryonic and neonatal periods has a highly significant effect on the developing forebrain and cerebellum and neonatal thyroid status (P < 0.05, Bonferroni corrected). These data are consistent with the hypothesized role of the thyroid hormone in mediating the effect of hypergravity in the developing central nervous system and begin to define the role of TH in the overall response of the developing organism to altered gravity.