Cerebellar DNA Research Articles

Characterization of the Monoamine Oxidase-B (MAO-B) Expression in Postmortem Normal and Alzheimer’s Disease Brains (S39.006)

<h3>Objective:</h3> To characterize MAO-B expression in postmortem human control (CTRL) and Alzheimer’s disease (AD) brains. Specifically, we sought to: (1) Identify the cell type(s) expressing MAO-B; (2) Compare MAO-B immunoreactivity in CTRL vs. AD brains; (3) Correlate MAO-B levels with local measures of AD neuropathological changes, cortical thickness, and reactive glia; (4) Evaluate the impact of the <i>MAOB</i> rs1799836 SNP genotype on MAO-B immunoreactivity and all these other measures. <h3>Background:</h3> MAO-B has been proposed as a novel PET imaging biomarker of AD-associated reactive astrogliosis. However, a thorough characterization in postmortem CTRL and AD brains—including the impact of the <i>MAOB</i> rs1799836 SNP genotype, which may affect MAO-B enzymatic activity—is lacking. <h3>Design/Methods:</h3> We performed automated MAO-B immunohistochemistry on FFPE sections from multiple brain regions of CTRL and AD brain donors followed by whole-slide scan and quantification of cortical and white matter immunoreactivity using QuPath pixel classifier. <i>MAOB</i> rs1799836 SNP was genotyped in cerebellar DNA via Taqman assay. <h3>Results:</h3> We found that: (1) MAO-B is primarily expressed by cortical and white matter astrocytes; (2) MAO-B is elevated in AD vs. CTRL astrocytes uniformly across the temporal, frontal, and occipital cortex and white matter; (3) cortical MAO-B immunoreactivity correlates positively with the number of GFAP+ reactive astrocytes and CD68+ activated microglia, and negatively with cortical thickness, but not with Aβ plaque load or number of neurofibrillary tangles; (4) the <i>MAOB</i> rs1799836 SNP genotype does not affect MAO-B immunoreactivity, AD neuropathological changes, or reactive glia. <h3>Conclusions:</h3> Astrocytes are the main cellular source of MAO-B in the cerebral cortex. MAO-B levels are significantly increased in the AD cortex and parallel the number of GFAP+ reactive astrocytes. The <i>MAOB</i> rs1799836 SNP genotype does not impact any of these measures. MAO-B is a promising target for the development of PET imaging radiotracers of reactive astrogliosis. <b>Disclosure:</b> Dr. Jaisa-aad has nothing to disclose. Ms. Connors has nothing to disclose. An immediate family member of Dr. Hyman has received personal compensation for serving as an employee of novartis. Dr. Hyman has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for dewpoint. Dr. Hyman has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for abbvie. Dr. Hyman has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Arvinas. Dr. Hyman has received personal compensation in the range of $500-$4,999 for serving as a Consultant for avrobio. Dr. Hyman has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Biogen. Dr. Hyman has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Dept of Justice. Dr. Hyman has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Cell Signalling. Dr. Hyman has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sangamo. Dr. Hyman has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for sanofi. Dr. Hyman has received personal compensation in the range of $500-$4,999 for serving as a Consultant for vigil. Dr. Hyman has received personal compensation in the range of $500-$4,999 for serving as a Consultant for voyager. Dr. Hyman has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for biogen. Dr. Hyman has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for dept of justice. Dr. Hyman has stock in dewpoint. The institution of Dr. Hyman has received research support from nih. The institution of Dr. Hyman has received research support from JPB. The institution of Dr. Hyman has received research support from Cure Alz Fund. The institution of Dr. Hyman has received research support from Tau consortium. The institution of Dr. Hyman has received research support from abbvie. The institution of Dr. Hyman has received research support from BMS. Dr. Serrano-Pozo has received research support from NIH/NIA. Dr. Serrano-Pozo has received research support from Alzheimer’s Association. Dr. Serrano-Pozo has received research support from The Jack Satter Foundation. Dr. Serrano-Pozo has received research support from The Karen Toffler Charitable Trust. Dr. Serrano-Pozo has received research support from Ionis Pharmaceuticals, Inc. Dr. Serrano-Pozo has received research support from AbbVie, Inc..

Assessment of epigenetic changes and oxidative DNA damage in rat pups exposed to polychlorinated biphenyls and the protective effect of curcumin in the prenatal period.

Background Polychlorinated biphenyls (PCBs) are persistent organic chemicals that exert neurotoxic and endocrine disrupting effects. The aims of this study were to examine the effects of prenatal Aroclor 1254 (PCBs mixture) exposure on central nervous system tissues DNA and to evaluate the effects of curcumin. Methods Rat pups were assigned to three groups: [Group 1], Aroclor 1254 administrated group; [Group 2], Aroclor 1254 and curcumin administrated group; and [Group 3], control group. Plasma, cerebrum, cerebellum, pons and medulla oblongata tissue homogenates 8-hydroxy-2'-deoxyguanosine [8-(OH)DG] levels and plasma freeT4 levels were determined. Global DNA methylation and hydroxymethylation status were determined in cerebrum, cerebellum, pons and medulla oblongata. To this aim, DNA 5-hydroxymethylcytosine and 5-methylcytosine levels were measured, respectively. Results Mean cerebellum and cerebral cortex 5-hydroxymethylcytosine and 5-methylcytosine levels were higher in the control group than in the experimental groups. Mean plasma, cerebellum and cerebral cortex 8-(OH)DG concentrations were higher in Group 1 than the control group. No statistically significant difference was observed between Group 2 and the control group in terms of cerebellum and cerebral cortex 8-(OH)DG concentrations. Histopathological changes were also observed in the cerebral cortex and cerebellum of rat pups exposed to Aroclor 1254. PCBs exposure changes both DNA methylation and hypomethylation status and induces cerebellar and cerebral cortex DNA damage in the prenatal period. Exogenous curcumin may have protective effect on PCBs-induced DNA damage in cerebellum and cerebral cortex.

Ataxin-3, DNA damage repair, and SCA3 cerebellar degeneration: on the path to parsimony?

Ataxin-3, DNA damage repair, and SCA3 cerebellar degeneration: on the path to parsimony?

Cerebellar oxidative DNA damage and altered DNA methylation in the BTBR T+tf/J mouse model of autism and similarities with human post mortem cerebellum.

The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.

Litter and sex effects on maternal behavior and DNA methylation of the Nr3c1 exon 17 promoter gene in hippocampus and cerebellum

Early life events can alter gene expression through DNA methylation. The methylation status of the exon 17 promoter of the glucocorticoid receptor (Nr3c1 gene) in hippocampus associates with frequency of pup licking. Much of this work was conducted with male rats. Because dams more frequently lick male pups, this may contribute to sex differences in phenotypes through DNA methylation. Modifying litter gender composition (LGC), in which offspring of single-sex litters are compared to mixed-sex litters, alters maternal behavior. Previously, we demonstrated that LGC and sex affected pup licking times as well as anxiety and hippocampal DNA methylation of the Nr3c1 exon 17 promoter gene in adolescence. Now, we expand upon this work by examining effects in cerebellum and measuring mRNA levels. We also re-assessed DNA methylation in hippocampus using pyrosequencing and re-analyzed pup licking with the more commonly used frequency measure. Litters, culled to 8 pups on postnatal day 1 (PN1), were assigned to one of three conditions: all male (n=10), all female (n=12), or half of each sex (n=20). Licking was rated on PN4, 7, and 10. On PN35, hippocampal and cerebellar samples were obtained. Single-sex males were licked the least and mixed-sex males, the most. Hippocampal Nr3c1 mRNA levels were lowest in mixed females with no LGC or Sex effects in DNA methylation. Cerebellar DNA methylation levels were lowest in mixed males with no effect on mRNA levels. Maternal pup licking associated with DNA methylation of the Nr3c1 exon 17 promoter gene in cerebellum and with hippocampal mRNA.

Studying the cerebellar DNA damage response in the tissue culture dish

The cerebellum is exquisitely sensitive to deficiencies in the cellular response to specific DNA lesions. Genetic disorders caused by such deficiencies involve relentless, progressive cerebellar atrophy with striking loss of Purkinje and granule neurons. The reason for the extreme sensitivity of these cells to defective response to certain DNA lesions is unclear. This is particularly true for ataxia-telangiectasia (A-T) – a genomic instability syndrome whose major symptom is cerebellar atrophy. It is important to understand whether the DNA damage response in the cerebellum, particularly in Purkinje neurons, has special characteristics that stem from the unique features of these cells. Murine cerebellar organotypic cultures provide a valuable experimental system for this purpose since they retain the tissue organization for several weeks in culture and appear to provide the delicate Purkinje neurons with a physiological environment close to that in vivo. We have optimized this system and are using it to examine the Atm-mediated DNA damage response (DDR) in the cerebellum, with special emphasis on Purkinje cells. Our results to date, which indicate special chromatin organization in Purkinje cells that affects certain pathways of the DDR, demonstrate the usefulness of cerebellar organotypic cultures for addressing the above questions.

DNA methylation at the Igf2/H19 imprinting control region is associated with cerebellum mass in outbred mice

BackgroundInsulin-like growth factor 2 (Igf2) is a paternally expressed imprinted gene regulating fetal growth, playing an integral role in the development of many tissues including the brain. The parent-of-origin specific expression of Igf2 is largely controlled by allele-specific DNA methylation at CTCF-binding sites in the imprinting control region (ICR), located immediately upstream of the neighboring H19 gene. Previously we reported evidence of a negative correlation between DNA methylation in this region and cerebellum weight in humans.ResultsWe quantified cerebellar DNA methylation across all four CTCF binding sites spanning the murine Igf2/H19 ICR in an outbred population of Heterogeneous Stock (HS) mice (n = 48). We observe that DNA methylation at the second and third CTCF binding sites in the Igf2/H19 ICR shows a negative relationship with cerebellar mass, reflecting the association observed in human post-mortem cerebellum tissue.ConclusionsGiven the important role of the cerebellum in motor control and cognition, and the link between structural cerebellar abnormalities and neuropsychiatric phenotypes, the identification of epigenetic factors associated with cerebellum growth and development may provide important insights about the etiology of psychiatric disorders.

Mammalian DNA δ15N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level

We report the first high-precision characterization of molecular and intramolecular δ(15)N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid (15)N or (13)C values. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. The cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ(15)N and δ(13)C values characterized. Mean diet δ(15)N was 0.45 ± 0.33‰, compared with cerebellar whole tissue and DNA δ(15) N= +4.1 ± 0.7‰ and -4.5 ± 0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ(15)N were +1.4 ± 0.4, -2.1 ± 0.9, -7.2 ± 0.3, and -10.4 ± 0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ(15) N values due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ(15)N values originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ(15)N(ASP-PUR), δ(15)N(ASP-PYR), δ(15) N(GLN), and δ(15) N(GLY) +11.9 ± 2.3‰, +7.0 ± 2.0‰, -9.1 ± 2.4‰, and -31.8 ± 8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ(15) N values of 4.3 ± 3.2‰ and 13.8 ± 3.1‰, respectively, and δ(15)N values for gly and asp were 12.6 ± 2.2‰ and 15.2 ± 0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ(15)N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism from that in adult life. Our data show that cerebellar nucleoside intramolecular δ(15)N values vary over more than 40‰ and are not influenced by dietary protein level or age.

Copy number variation of the SELENBP1 gene in schizophrenia

BackgroundSchizophrenia is associated with rare copy-number (CN) mutations. Screening for such alleles genome-wide, though comprehensive, cannot study in-depth the causality of particular loci, therefore cannot provide the functional interpretation for the disease etiology. We hypothesized that CN mutations in the SELENBP1 locus could associate with the disorder and that these mutations could alter the gene product's activity in patients.MethodsWe analyzed SELENBP1 CN variation (CNV) in blood DNA from 49 schizophrenia patients and 49 controls (cohort A). Since CN of genes may vary among tissues, we investigated SELENBP1 CN in age- sex- and postmortem interval-matched cerebellar DNA samples from 14 patients and 14 controls (cohort B). Since CNV may either be de-novo or inherited we analyzed CNV of the SELENBP1 locus in blood DNA from 26 trios of schizophrenia probands and their healthy parents (cohort C). SELENBP1 mRNA levels were measured by real-time PCR.ResultsIn cohort A reduced CN of the SELENBP1 locus was found in four patients but in none of the controls. In cohort B we found reduced CN of the SELENBP1 locus in two patients but in none of the controls. In cohort C three patients exhibited drastic CN reduction, not present in their parents, indicating de-novo mutation. A reduction in SELENBP1 mRNA levels in the postmortem cerebellar samples of schizophrenia patients was found.ConclusionsWe report a focused study of CN mutations in the selenium binding-protein1 (SELENBP1) locus previously linked with schizophrenia. We provide evidence for recurrence of decreased CN of the SELENBP1 locus in three unrelated patients' cohorts but not in controls, raising the possibility of functional involvement of these mutations in the etiology of the disease.

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.

Effects of neonatal thyroidectomy on thymidine metabolism and deoxyribonucleic acid synthesis in the developing rat cerebellum.

Thyroid deficiency at birth caused a temporal decrease of the DNA content in rat cerebellum during the periods from 7 to 21 days of life as compared with the normal. At 2 h following the subcutaneous injection of [3H]-thymidine, the specific radioactivity of cellular dTTP in thyroid deficient rats was apparently increased on the 14th and 21st days, although the endogenous pool sizes of dTTP did not significantly differ between the normal and thyroidectomized rats throughout the entire ages studied. The ratio of incorporation of thymidine into cerebellar DNA was initially determined by the specific radioactivity and then expressed as the ratio of [3H]-DNA to [3H]-dTTP, the relative specific radioactivity of DNA. The age peak of incorporation occurred at 7 days in the normal; on the other hand, the specific activity of DNA in thyroid deficiency attained a maximum at 14 days and the relative specific activity appeared to be prolonged up to 14 days. The corresponding changes were observed in the formation of dTTP, suggesting that a transient retardation of cerebellar DNA synthesis taking place by neonatal thyroidectomy may be in part attributable to the variance of phosphorylation of thymidine. Indeed, thymidine kinase activity that regulated salvage pathway for DNA synthesis displayed a parallel variation in thyroid deficiency to characteristic age dependence of DNA synthesis.

Pitfalls of PCR-Based Strategy for Genotyping Cre-Loxp Mice

Pitfalls of PCR-Based Strategy for Genotyping <i>Cre-Loxp</i> Mice

Guinea DNA nitrogen and carbon isotope ratios are depleted relative to tissue and mildly responsive to physiological state

Background. Natural, endogenous, δ13C and δ15N isotope ratios are routinely used to probe environmental systems but have not been much exploited for nutritional studies. Cerebellar neuronal DNA maintains a long-lived record of isotope ratio because it does not turnover. We investigated the endogenous δ13C and δ15N of DNA and bulk tissue of cerebellum to determine baseline isotope ratios and whether in utero physiology influences isotope ratios at this level. Methods. Pregnant guinea pigs were randomized to five different groups: Control (C), low protein (LP), high protein (HP), feed restricted (FR), and diabetic (D). Pups were fed the C diet from birth. Pup cerebellum δ13C and δ15N was analyzed for DNA and bulk by elemental analysis-isotope ratio mass spectrometry at 2 da and 120 da of age. All isotope ratios are expressed relative to that of the feed. Results. Cerebellar DNA averaged about δ15N = −5‰ at both 2 and 120 da of age. Bulk cerebellum was δ15N = +4‰. Cerebellar DNA δ13C was +3.4‰ at 2 da and about +5.4% at 120 da. Bulk cerebellar δ13C at 120 days followed the in utero protein level: −0.85, +1.3, +4.9‰ for LP, C and HP, respectively. Conclusions. We show for the first time that δ15N of DNA is depleted relative to feed and that it is largely unresponsive to physiological state. δ13C of whole tissue increased with in utero dietary protein level. Funding: NIH GM071534.

Chronic gestational exposure to ethanol causes insulin and insulin‐like growth factor resistance in the developing brain: link to impaired acetylcholine biosynthesis

In an experimental model of fetal alcohol syndrome (FAS), cerebellar hypoplasia was found to be associated with increased apoptosis and impaired insulin-stimulated survival signaling. However, little is known about the effects produced following lower levels of ethanol exposure. This study characterizes the dose-effects of chronic gestational ethanol exposure on cerebellar development and the expression of genes required for insulin and insulin-like growth factor signaling, and also examines upstream mechanisms and downstream consequences of ethanol-impaired insulin and IGF signaling in relation to acetylcholine biosynthesis. Pregnant Long-Evans rats were fed isocaloric liquid diets in which ethanol comprised 0%, 8%, 18%, 26%, or 35.4% of the caloric content beginning on gestation day 6 and continuing throughout pregnancy. After examination of the rat pup cerebella (P0), the results demonstrated ethanol dose-dependent increases in the degree of cerebellar hypoplasia, DNA damage, and neuronal cell loss. In addition, ethanol dose-dependent reductions in brain insulin and choline acetyltransferase (ChAT) expression, and increased expression of acetylcholinesterase were observed. Despite normal receptor expression levels, ligand binding to insulin and IGF receptors was significantly reduced. Further studies linked the ethanol-impaired receptor binding and stimulation of ChAT to reduced membrane cholesterol content. In conclusion, FAS associated cerebellar hypoplasia is correlated with impaired neuronal cell survival and decreased levels of ChAT expression, mediated by pathological alterations in membrane lipid composition that result in impaired ligand-receptor binding.

A single peripheral injection of basic fibroblast growth factor (bFGF) stimulates granule cell production and increases cerebellar growth in newborn rats.

The control of neuronal number is critical for coordinating innervation and target organ requirements. Although basic fibroblast growth factor (bFGF) is known to regulate neuron number in the developing embryonic cortex, its potential role during postnatal brain development remains undefined. To address this issue, the cerebellum, a site of postnatal neurogenesis, was used. Previously, we found that a single peripheral injection of bFGF in newborn rats elicited mitosis of neuronal precursors in the external germinal layer (EGL) 8 h after administration. We now define the sustained effects of bFGF treatment on postnatal granule cell production and cerebellar growth. Seventy-two h after a single injection of bFGF (20 ng/g) in newborn rats, the fraction of BrdU-labeled cells in the EGL increased by 46% without altering apoptotic cell number, consistent with enhanced precursor proliferation. Moreover, bFGF increased mitotically labeled cells by 100% and total cell density by 33% in the internal granular layer (IGL), the final destination of the EGL precursors. Because cerebellar volume also increased by 22%, bFGF-induced proliferation enhanced generation of total IGL neurons and increased cerebellar growth. These morphometric measures were corroborated independently by using DNA quantitation: cerebellar DNA content increased 16% after bFGF injection, consistent with increased neuron number. Furthermore, using DNA quantitation as an index, increased total cerebellar cell number elicited by bFGF injection persisted beyond the neurogenetic period, until P35. We conclude that a single postnatal injection of bFGF increases granule neuron number and enhances cerebellar growth following mitotic stimulation.

Effects of neonatal naltrexone on neurological and somatic development in rats of both genders.

The effects of a daily injection of the opioid antagonist naltrexone (NALTX, 1 mg/kg, s.c.) from birth to weaning on various parameters were investigated in male and female rats during postnatal development until adulthood. NALTX increased cerebellar DNA and protein content as well as cerebellar weight at 7 days. Eye opening was not affected by NALTX but it appeared advanced by 1 day in all groups compared to other studies, possibly due to a handling effect caused by the daily injection. Water and food consumption were augmented by NALTX during days 23-32 and days 55-70. Treated preweaning animals showed lower body growth rates than controls. However, NALTX caused a moderate increase in body weight measured during postweaning until adulthood. The effects of NALTX on the parameters evaluated (excepting the cerebellar measurements on day 7), although clearly statistically significant, were small in absolute terms. The preweaning opioid blockade caused by our NALTX treatment seems to affect more markedly the neural and behavioural development than the somatic growth. This work also provides potentially useful baseline data for the study of male and female rats during postnatal development.

Calpain Activation and Not Oxidative Damage Mediatesl-2-Chloropropionic Acid-Induced Cerebellar Granule Cell Necrosis

Possible biochemical events involved inl-2-chloropropionic acid (L-CPA)-induced delayed cerebellar granule cell necrosis followingN-methyl-d-aspartate activation were studiedin vivo.We examined whether the calcium-sensitive proteolytic enzymes, the calpains, may be activated by L-CPA or whether the generation of excess quantities of cytotoxic free radicals may play a role in the neurotoxicity produced by oral administration of L-CPA (750 mg/kg, pH 7.0). Evidence for free radical-induced cellular damage was examined using biochemical approaches such as examining brains from L-CPA-treated rats for increased lipid peroxidation, DNA damage, or protein oxidation. Second, the ability of antioxidants to provide neuroprotective activity against L-CPA-induced neurotoxicity was examinedin vivo.Western blotting using antibodies against spectrin (α-fodrin) demonstrated evidence for calpain (EC 3.4.22.17) activation in the cerebellum, but not in the cerebral cortex of L-CPA-treated rats at 36 and 48 hr after L-CPA dosing. In contrast, there was no evidence for oxidative damage to cerebellar proteins or lipids in L-CPA-treated rat brains compared to controls. We also could not find evidence for DNA damage using the TUNEL method for the detection of single- and/or double-strand breakagein situin L-CPA-treated brains. We examined whether a number of reported antioxidants may be effective against L-CPA-induced neurotoxicity. The aminosteroids U74389G and U83836E, the free radical scavengers 3-methyl-1-phenylpyrazolin-5-one andN-tert-butylphenylnitrone, and the iron chelatorN-ethoxy-2-ethyl-3-hydroxypyridin-4-one were all ineffective in attenuating L-CPA neurotoxicity. We suggest that L-CPA-induced cerebellar necrosis is the result of calpain activation which results in the degradation of cytoskeletal proteins and other proteins necessary for cellular biochemistry. We could find no evidence of oxidative damage to cerebellar proteins, lipids, or DNA as a result of excess amounts of free radicals, and selective antioxidants were unable to provide neuroprotection against L-CPA neurotoxicity, suggesting that oxidative stress does not play a role in the granule cell necrosis.

In vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development in transgenic mice: evidence that IGF-I increases proliferation of granule cell progenitors

The in vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development have been investigated in transgenic (Tg) mice (IGF-II/I Tg mice) in whom an IGF-II promoter-driven IGF-I transgene is highly expressed in cerebellum. Compared to normal littermates, the brains of IGF-II/I Tg mice exhibited overgrowth beginning from the second week of postnatal life. Among the brain regions examined, cerebellum exhibited the greatest increase in size, such that by 50 days of age cerebellar weight and DNA content were increased by 90% and 143%, respectively, compared to littermate controls. Morphological studies of adult IGF-II/I Tg mice showed that the total number of granule and Purkinje cells was increased by 82% and 20%, respectively, findings consistent with the increased cerebellar DNA content and indicating that the increased cerebellar weight was due in part to an increase in cell number. The thickness of the molecular layer also was increased in IGF-II/I Tg mice. During early postnatal development the number of external granular layer cells, as well as the number of BrdU labeled external granular cells, was increased. These data strongly indicate that IGF-I increases granule cell number by a mechanism that involves the stimulation of granule cell progenitor proliferation. Our findings also indicate that IGF-I influences the growth of Purkinje cells and possibly of other cell types in the cerebellum.

Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats.

This report deals with the mechanism involved in the cerebellar degeneration following experimental methylmercury poisoning of male Wistar rats. The cerebellar granule cells of animals that exhibited typical hind leg paresis were shrunken and displayed marked nuclear pyknosis. At the ultrastructural level, the nuclei of these cells were condensed and fragmented, features which are characteristic of apoptosis. In situ staining for DNA strand breaks revealed that the pyknotic nuclei were positively labeled. DNA fragmentation was confirmed by agarose gel electrophoresis; a ladder pattern of multiples of approximately 200-base pair fragments, typical of apoptosis, was observed with the cerebellar DNA of the methylmercury-treated animals. These observations suggest that the degeneration of cerebellar granule cells by alkyl mercury compounds involves an apoptotic process.

Nutritional insult and recovery in the neonatal rat cerebellum: insulin-like growth factors (IGFs) and their binding proteins (IGFBPs).

Alterations in growth caused by neonatal malnutrition may be mediated in part by changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression. Since the neonatal rat cerebellum undergoes a transient, proliferative growth phase in the first two weeks of life, this structure was used to determine whether alterations in circulating and tissue IGFs and IGFBPs may mediate effects of impaired nutrition on the developing central nervous system. Gravid rats were placed on a 4% (protein-calorie deprived, D) or 20% (control, C) protein diets one day prior to delivery and allowed to nurse their pups postpartum. Pups nursing from D mothers received a limited volume of milk and were calorically deprived. Some litters of D pups were foster fed by C mothers from day 8 to day 13 to constitute a recovery group (R). Cerebellar weight, protein, and DNA content in D pups were less than C, p < 0.001. In R pups, DNA and protein returned to C levels by day 13. Between days 6 and 13, serum IGF-I levels rose from 158 +/- 18 to 210 +/- 18 ng/ml in C but remained low in D (47 +/- 6 ng/ml and 25 +/- 3 ng/ml), respectively. In R pups, serum IGF-I partially recovered during this time, and increased from 49 +/- 5 to 110 +/- 7 ng/ml. In cerebellar extracts, IGF-I levels in both C and D were lower at 13 days than at 6 days, p < 0.05 and p < 0.005, respectively. IGF-I levels in C were similar at day 9 and day 11 and were consistently higher than D (11.84 +/- 0.83 vs 8.56 +/- 0.92 ng/g, p < 0.02 C vs D). In R, IGF-I was reduced on day 11, but was similar to C on day 13. Serum IGF-II in D was lower than C, p < 0.01, and did not increase in the R group. Cerebellar IGF-II was virtually undetectable in either group. Immunoprecipitation and ligand blotting studies of serum demonstrated that circulating levels of 32-34 K IGFBPs were increased 3-4 fold in D vs C, reflecting high levels of IGFBP-1 and/or -2, while levels of 24 K IGFBP-4 were lower in D vs C. By contrast, immunoprecipitation and ligand blotting of cerebellar extracts detected IGFBP-2 and -4, but did not detect IGFBP-1. Further, tissue levels of IGFBP-2 were not increased in D vs C, and levels of IGFBP-4 also were not markedly affected by nutritional deprivation. These results suggest that alterations in tissue content and the availability of IGF-I only modestly contributed to the effects of impaired nutrition in the developing central nervous system.