Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2Ek/Ek) was lethal in mid-gestation (the equivalent of mouse E9.5–E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long–Evans strain Tsc2Ek/Ek embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: No accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR−/−] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB+/+)] with or without an apo(a) transgene [Tg(apoa+/−)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR−/−, (ii) LDLR−/−;Tg(apoa+/−), (iii) LDLR−/−;Tg(apoB+/+), and (iv)LDLR−/−;Tg(apoB+/+);Tg(apo+/−). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR−/− and LDLR−/−;Tg(apoa+/−) mice (1.0 ± 0.2% vs. 1.4 ± 0.3%). However, the LDLR−/−;Tg(apoB+/+) mice had ≈15-fold greater mean lesion area than the LDLR−/− mice. No significant difference was found in percent lesion area in the LDLR−/−;Tg(apoB+/+) mice whether or not they expressed apo(a) [18.5 ± 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 ± 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR−/−;Tg(apoB+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR−/−; Tg(apoB+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.

Dynamic characteristics and adaptability of mouse vestibulo-ocular and optokinetic response eye movements and the role of the flocculo-olivary system revealed by chemical lesions

The dynamic characteristics of reflex eye movements were measured in two strains of chronically prepared mice by using an infrared television camera system. The horizontal vestibulo-ocular reflex (HVOR) and horizontal optokinetic response (HOKR) were induced by sinusoidal oscillations of a turntable, in darkness, by 10° (peak to peak) at 0.11–0.50 Hz and of a checked-pattern screen, in light, by 5–20°at 0.11–0.17 Hz, respectively. The gains and phases of the HVOR and HOKR of the C57BL/6 mice were nearly equivalent to those of rabbits and rats, whereas the 129/Sv mice exhibited very low gains in the HVOR and moderate phase lags in the HOKR, suggesting an inherent sensory-motor anomaly. Adaptability of the HOKR was examined in C57BL/6 mice by sustained screen oscillation. When the screen was oscillated by 10° at 0.17 Hz, which induced sufficient retinal slips, the gain of the HOKR increased by 0.08 in 1 h on average, whereas the stimuli that induced relatively small or no retinal slips affected the gain very little. Lesions of the flocculi induced by local applications of 0.1% ibotenic acid and lesions of the inferior olivary nuclei induced by i.p. injection of 3-acetylpyridine in C57BL/6 mice little affected the dynamic characteristics of the HVOR and HOKR, but abolished the adaptation of the HOKR. These results indicate that the olivo-floccular system plays an essential role in the adaptive control of the ocular reflex in mice, as suggested in other animal species. The data presented provide the basis for analyzing the reflex eye movements of genetically engineered mice.

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1−/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: Toward a genetic model of meningioma progression

Nineteen benign [World Health Organization (WHO) grade I; MI], 21 atypical (WHO grade II; MII), and 19 anaplastic (WHO grade III; MIII) sporadic meningiomas were screened for chromosomal imbalances by comparative genomic hybridization (CGH). These data were supplemented by molecular genetic analyses of selected chromosomal regions and genes. With increasing malignancy grade, a marked accumulation of genomic aberrations was observed; i.e., the numbers (mean ± SEM) of total alterations detected per tumor were 2.9 ± 0.7 for MI, 9.2 ± 1.2 for MII, and 13.3 ± 1.9 for MIII. The most frequent alteration detected in MI was loss on 22q (58%). In MII, aberrations most commonly identified were losses on 1p (76%), 22q (71%), 14q (43%), 18q (43%), 10 (38%), and 6q (33%), as well as gains on 20q (48%), 12q (43%), 15q (43%), 1q (33%), 9q (33%), and 17q (33%). In MIII, most of these alterations were found at similar frequencies. However, an increase in losses on 6q (53%), 10 (68%), and 14q (63%) was observed. In addition, 32% of MIII demonstrated loss on 9p. Homozygous deletions in the CDKN2A gene at 9p21 were found in 4 of 16 MIII (25%). Highly amplified DNA sequences were mapped to 12q13–q15 by CGH in 1 MII. Southern blot analysis of this tumor revealed amplification of CDK4 and MDM2. By CGH, DNA sequences from 17q were found to be amplified in 1 MII and 8 MIII, involving 17q23 in all cases. Despite the high frequency of chromosomal aberrations in the MII and MIII investigated, none of these tumors showed mutations in exons 5–8 of the TP53 gene. On the basis of the most common aberrations identified in the various malignancy grades, a model for the genomic alterations associated with meningioma progression is proposed.

The Drosophila melanogaster Homologue of the Xeroderma Pigmentosum D Gene Product Is Located in Euchromatic Regions and Has a Dynamic Response to UV Light-induced Lesions in Polytene Chromosomes

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne’s syndrome, and trichothiodystrophy. XPD has a 5′- to 3′-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Progressive entorhinal cortex lesions accelerate hippocampal sprouting and spare spatial memory in rats

Accelerating hippocampal sprouting by making unilateral progressive lesions of the entorhinal cortex spared the spatial memory of rats tested for retention of a learned alternation task. Subsequent transection of the sprouted crossed temporodentate pathway (CTD), as well as a simultaneous CTD transection and progressive entorhinal lesion, produced a persistent deficit on the memory task. These results suggest that CTD sprouting, which is homologous to the original perforant path input to the dentate gyrus of the hippocampus, is behaviorally significant and can ameliorate at least some of the memory deficits associated with hippocampal deafferentation.

Verb generation in patients with focal frontal lesions: A neuropsychological test of neuroimaging findings

What are the neural bases of semantic memory? Traditional beliefs that the temporal lobes subserve the retrieval of semantic knowledge, arising from lesion studies, have been recently called into question by functional neuroimaging studies finding correlations between semantic retrieval and activity in left prefrontal cortex. Has neuroimaging taught us something new about the neural bases of cognition that older methods could not reveal or has it merely identified brain activity that is correlated with but not causally related to the process of semantic retrieval? We examined the ability of patients with focal frontal lesions to perform a task commonly used in neuroimaging experiments, the generation of semantically appropriate action words for concrete nouns, and found evidence of the necessity of the left inferior frontal gyrus for certain components of the verb generation task. Notably, these components did not include semantic retrieval per se.

Severe deep white matter lesions and outcome in elderly patients with major depressive disorder: follow up study

Objective To determine the difference in outcome among elderly people with major depression who do and do not have severe white matter lesions on magnetic resonance imaging.

Response of human DNA polymerase ι to DNA lesions

Lesion bypass is an important mechanism to overcome replication blockage by DNA damage. Translesion synthesis requires a DNA polymerase (Pol). Human Pol ι encoded by the RAD30B gene is a recently identified DNA polymerase that shares sequence similarity to Pol η. To investigate whether human Pol ι plays a role in lesion bypass we examined the response of this polymerase to several types of DNA damage in vitro. Surprisingly, 8-oxoguanine significantly blocked human Pol ι. Nevertheless, translesion DNA synthesis opposite 8-oxoguanine was observed with increasing concentrations of purified human Pol ι, resulting in predominant C and less frequent A incorporation opposite the lesion. Opposite a template abasic site human Pol ι efficiently incorporated a G, less frequently a T and even less frequently an A. Opposite an AAF-adducted guanine, human Pol ι was able to incorporate predominantly a C. In both cases, however, further DNA synthesis was not observed. Purified human Pol ι responded to a template TT (6–4) photoproduct by inserting predominantly an A opposite the 3′ T of the lesion before aborting DNA synthesis. In contrast, human Pol ι was largely unresponsive to a template TT cis-syn cyclobutane dimer. These results suggest a role for human Pol ι in DNA lesion bypass.

Visual habit formation in monkeys with neurotoxic lesions of the ventrocaudal neostriatum

Visual habit formation in monkeys, assessed by concurrent visual discrimination learning with 24-h intertrial intervals (ITI), was found earlier to be impaired by removal of the inferior temporal visual area (TE) but not by removal of either the medial temporal lobe or inferior prefrontal convexity, two of TE's major projection targets. To assess the role in this form of learning of another pair of structures to which TE projects, namely the rostral portion of the tail of the caudate nucleus and the overlying ventrocaudal putamen, we injected a neurotoxin into this neostriatal region of several monkeys and tested them on the 24-h ITI task as well as on a test of visual recognition memory. Compared with unoperated monkeys, the experimental animals were unaffected on the recognition test but showed an impairment on the 24-h ITI task that was highly correlated with the extent of their neostriatal damage. The findings suggest that TE and its projection areas in the ventrocaudal neostriatum form part of a circuit that selectively mediates visual habit formation.

Abortive base-excision repair of radiation-induced clustered DNA lesions in Escherichia coli

It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called “clustered damages” or “multiply damaged sites”. Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted base-excision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco1 : High Amounts of Sorbitol Lead to Necrotic Lesions

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 μmol g−1 fresh weight were found. Plants that accumulated up to 2 to 3 μmol g−1 fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 μmol g−1 fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 μmol g−1 fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.