Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease.

Initially identified in high-grade gliomas, mutations in the PTEN tumor-suppressor are also found in many sporadic cancers and a few related autosomal dominant hamartoma syndromes. PTEN is a 3'-specific phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) phosphatase and functions as a negative regulator of PI3K signaling. We generated a tissue-specific deletion of the mouse homolog Pten to address its role in brain function. Mice homozygous for this deletion (PtenloxP/loxP;Gfap-cre), developed seizures and ataxia by 9 wk and died by 29 wk. Histological analysis showed brain enlargement in PtenloxP/loxP;Gfap-cre mice as a consequence of primary granule-cell dysplasia in the cerebellum and dentate gyrus. Pten mutant cells showed a cell-autonomous increase in soma size and elevated phosphorylation of Akt. These data represent the first evidence for the role of Pten and Akt in cell size regulation in mammals and provide an animal model for a human phakomatosis condition, Lhermitte-Duclos disease (LDD).

Inhibitors of lysosomal enzymes: accumulation of lipofuscin-like dense bodies in the brain.

Injections of leupeptin (a thiol proteinase inhibitor) or chloroquine (a general lysosomal enzyme inhibitor) into the brains of young rats induced the formation of lysosome-associated granular aggregates (dense bodies) which closely resembled the ceroid-lipofuscin that accumulates in certain disease states and during aging. The dense material increased in a dose- and time-dependent fashion and was differentially distributed across brain regions and cell types. These observations provide clues to the origins of ceroid-lipofuscin and suggest means for studying the consequences of its accumulation.

Induction of a heat shock gene at the site of tissue injury in the rat brain.

Our objective was to investigate whether localized tissue injury induces expression of a gene encoding the major 70 kd heat shock protein (hsp70) in the mammalian nervous system. A small surgical cut was made in the rat cerebral cortex. By 2 hr postsurgery a dramatic and highly localized induction of hsp70 mRNA was detected at the lesion site using in situ hybridization with labeled riboprobe. By 12 hr the intensity of the signal had diminished, and by 24 hr only a few cells along the walls of the cut demonstrated a high level of hsp70 mRNA. Both neurons and glial cells at the site of the surgical cut responded to tissue injury by induction of hsp70 mRNA. Induction was not observed in other brain regions, nor was the pattern of constitutive expression affected by the surgical procedure.

Long-term treatment of male F344 rats with deprenyl: assessment of effects on longevity, behavior, and brain function.

L-Deprenyl (selegiline) was chronically administered to male Fischer 344 rats via their drinking water beginning at 54 weeks of age (estimated daily dose: 0.5 mg/kg/day). Beginning at 84 weeks of age, the rats were behaviorally evaluated using a sensorimotor battery, a motor-learning task, and the Morris water maze. At 118 weeks of age, cerebellar noradrenergic function was evaluated in the surviving rats using in vivo electrochemistry. The rats were then sacrificed to measure brain monoamine oxidase activity and perform quantitative autoradiography to evaluate the effect of chronic deprenyl treatment on beta-adrenergic receptors in the cerebellum, alpha 2-adrenergic receptors several brain regions, and D1 and D2 dopamine receptors in the striatum. Deprenyl treatment reduced brain monoamine oxidase B activity by 85%, but had no effect on brain monoamine oxidase A. A clear effect of chronic deprenyl treatment upon longevity was not observed. Several measures of CNS function were altered in the deprenyl-treated animals: 1) spatial learning in the Morris water maze was improved; 2) electrochemical signals recorded following local application of NE were reduced, and the responsiveness to the reuptake blocker nomifensine was enhanced, in the cerebellum; 3) beta-adrenergic receptor binding affinity was increased in the cerebellum; 4) alpha 2-adrenergic receptor density was increased in the inferior colliculus; and 5) striatal D1 dopamine receptor density was reduced but binding affinity was enhanced. In contrast, chronic deprenyl treatment did not cause changes in: 1) sensorimotor function, as evaluated by balance beam, inclined screen, or wire hang tasks; 2) motor learning; 3) alpha 2-adrenergic receptor density in any region examined except for the inferior colliculus, or binding affinity in any region examined; or 4) striatal D2 dopamine receptor number or affinity. Thus, long-term oral administration of deprenyl extended the functional life span of rats with respect to cognitive, but not motor, performance.

The ontogeny of the distribution of callosal projection neurons in the rat parietal cortex.

The ontogeny of callosal projection neurons in the rat parietal cortex was examined using the retrograde and anterograde transport of horseradish peroxidase (HRP), as well as Golgi and Nissl stains. From postnatal day 0 (PND 0) to early PND 4, the callosal projection neurons are distributed as two continuous horizontal bands of cells which extend throughout the subplate in layers Va and Vc-upper VIa. Neurons within the cortical plate (CP), however, do not transport HRP from a contralateral injection site until PND 3 to early PND 4, when a few cells at the lower CP border are generally labeled. However, by late on PND 4, and more consistently by PND 5, several changes in the distribution of callosal projection neurons take place. First, cells at all levels of the CP become labeled in a sequential fashion, from the lower border upward. Second, gaps, or areas devoid of HRP, become apparent in layer IV of the barrel field area. Third, in the cortical areas containing the gaps, as well as in other areas which are destined not to be callosally connected in the adult, there is a noticeable decrease in the number of cells labeled with HRP. This decrease continues through PND 15 and possibly into adulthood. The foregoing developmental events are compared to cortical maturation as seen in both Golgi- and Nissl-stained material. By PND 15, the basic adult pattern of callosal projection neurons is established. The neurons reside mainly in layers III and Va, with fewer in layers II and Vc-upper VIa, and fewer still in the other cortical layers. They are aligned in vertical arrays in discrete areas of the cortex.

Pharmacological modifications of endogenous antioxidant enzymes with special reference to the effects of deprenyl: a possible antioxidant strategy.

Limited information is available on the upregulation of endogenous antioxidant enzymes by means of administering various pharmaceuticals and/or chemicals. It has been reported that ursodeoxycholic acid (UDCA), a bile acid originally identified from black bear bile (a Chinese medicine, Yutan) increased glutathione S-transferase (GST) activities in mouse livers, resulting in a decrease in systemic lethal toxicity of orally challenged 1-2-dichloro-4-nitrobenzene (DCNB). Also, ursolic acid found in herbal medicines (e.g. leaves of loquat) was reported to increase catalase (CAT) activities in mouse liver. Interestingly, the chemical structures of these two compounds are surprisingly similar to each other, despite the difference in their original sources. These results suggest that in the future, more and more compounds will be found to have effects on increasing endogenous antioxidant enzyme activities. Deprenyl is a monoamine oxidase B inhibitor but also possesses many other different pharmacological activities. Among these various pharmacological effects of deprenyl, a possible causal relationship between two effects of deprenyl, namely the prolongation of the survival of animals and upregulation of antioxidant enzymes in selective brain regions, has been postulated by the authors. In at least four different animal species (rats, mice, hamsters and dogs), a significant prolongation of survival by chronic administration of the drug has been reported by different groups including that of the authors. This group has reported that repeated administration of the drug for 2-3 weeks can significantly increase activities of both types of superoxide dismutase (SODs) (Cu, Zn-, and Mn-SODs) as well as of CAT selectively in brain dopaminergic regions. Both effects are dose dependent but excessive dosages become less effective and even cause an adverse effect (i.e. a decrease in enzyme activities and shortening of life span). The parallelism of the dose-effect relationship between the two phenomena suggests that modification of SOD and CAT levels is one possible mechanism for deprenyl's ability to prolong the life span of animals.

Leupeptin causes an accumulation of lipofuscin-like substances in liver cells of young rats.

Leupeptin, a thiol protease inhibitor, has previously been shown to cause a dense accumulation of substances resembling age pigment and called ceroid-lipofuscin, in brain cells of young rats. Thus far, however, attempts to produce age pigments in hepatocytes of normal young rats with protease inhibitor(s) have not been successful. The present study provides the first demonstration that leupeptin induces lipofuscin-like substances in normal young rat hepatocytes. Male Fischer-344 rats (age 4-6 weeks) were continuously infused with leupeptin or saline i.p. for 2 weeks by an osmotic minipump (dosage, 1-50 mg/100 g per day). Liver tissues were then examined by light, fluorescence and electron microscopy. Both hepatocytes and non-parenchymal cells of livers treated with leupeptin, but not saline, showed a dense accumulation of pigments which stained deeply with toluidine blue, were PAS-positive and were brightly autofluorescent. After UV excitation the pigments had an emission spectrum with a broad peak at 480-540 nm extending to 650 nm resembling the spectrum of age pigment from livers of normal aged rats. Electron microscopic examination revealed numerous lipofuscin-like deposits with heterogeneous morphology in the cytoplasm of both hepatocytes and non-parenchymal cells; lipid and myelin-like bodies were also present in hepatocytes. The results indicate that the perturbation of proteolytic activity in liver by leupeptin causes an accumulation of substances which by several criteria resemble lipofuscin. These results thus provide further support for the 'Protease Inhibitor Model of Lipofuscin Formation' as well as a potential experimental model for studying hepatocellular aging processes.

Sequential changes in activities of superoxide dismutase and catalase in brain regions and liver during (-)deprenyl infusion in male rats.

A continuous s.c. infusion of (-)deprenyl in young male rats at a dose of 2.0 mg/kg/day for 1 week significantly increased total superoxide dismutase (SOD) activities due to increases in both Cu Zn-SOD and Mn-SOD activities in certain brain regions such as the substantia nitra and striatum, but not in the hippocampus or cerebellum, or in the liver. With continuing infusion, enzyme activities of SOD were further increased in the following weeks, reaching a plateau at 3 weeks. In some cerebral cortices the increase became significant at 3 weeks. In contrast to SOD activities, an increase in catalase (CAT) activity became significant only after 2 weeks of infusion, and only in the brain regions where SOD activities were increased earlier. The delay in the increase in CAT activity following deprenyl infusion suggests that this increased CAT activity is an adaptive response to the earlier increase in deprenyl-induced SOD activities rather than a direct effect of deprenyl on CAT activity, although the latter possibility cannot be excluded.

The effect of a long term (6 months) treatment with (-)deprenyl on antioxidant enzyme activities in selective brain regions in old female Fischer 344 rats.

The effect of long term treatment with (-)deprenyl (s.c. injection three times a week for 6 months) on superoxide dismutase (SOD) and catalase (CAT) in selective brain regions was examined in old (22 months) female Fischer 344 rats. The three doses of deprenyl used (0.1, 0.25 and 0.5 mg/kg/day) increased the activities of both enzymes in substantia nigra, striatum and cerebral cortices essentially in a dose dependent manner. However, for CAT activities in cerebral cortices, the smallest dose of 0.1 mg/kg/day was most effective, while the highest dose (0.5 mg/kg/day) had no effect. In contrast to these brain regions, there were no significant differences in enzyme activities between control and deprenyl-treated groups in the hippocampus and cerebellum. If the effect of deprenyl on the life span of female F-344 rats is causally related to its effect on antioxidant enzyme activities in selective brain regions as shown in this study, then a dose of 0.25 or 0.5 mg/kg/day appears to be most appropriate. Since this dose is much lower than the dose suggested by our previous short term (3 week) experiments, an even longer term experiment is necessary to determine the optimal dose of deprenyl to increase free radical scavenging and thus possibly extend lifespan.

Protease inhibitors as a model for NCL disease, with special emphasis on the infantile and adult forms.

An animal model of NCL disease has been developed with the use of protease inhibitors. Young rats received a continuous infusion of various specific protease inhibitors or of physiological saline into the lateral ventricle of the brain using osmotic mini-pumps. Treatment lasted for 2 weeks, at which time animals were sacrificed and the brains were removed and processed for light or electron microscopic analysis. The thiol protease inhibitors leupeptin and E64C, but not saline or the serine protease inhibitor aprotinin, caused a massive accumulation of ceroid-lipofuscin (CL) in brain cells that bore a strong morphological resemblance to the CL in the infantile and adult forms of NCL disease, and bore similarity to the CL of the late infantile and juvenile forms. Leupeptin also caused the death of cerebellar Purkinje cells, as is seen in the infantile and adult forms of NCL. Further evidence is presented in support of the hypothesis (Ivy et al.: Science 226:985-987, 1984) that decreased or defective lysosomal thiol proteases or their substrates may underly the pathogenesis of at least the infantile and adult forms of NCL disease. Administration of protease inhibitors to the brains of young rats provides an important model for studying the cellular mechanisms of ceroid-lipofuscino-genesis.

Expression of the gene encoding the beta-subunit of S-100 protein in the developing rat brain analyzed by in situ hybridization.

To investigate patterns of expression of the gene encoding the beta-subunit of S-100 protein during development of the rat brain we have used Northern blotting and in situ hybridization histochemistry. During late prenatal development beta-S-100 mRNA was observed first in the germinal zone lining the 4th ventricle. In the postnatal cerebellum this mRNA accumulated primarily in Bergmann glia and astrocytes of the deep white matter. In the hindbrain, expression of S-100 mRNA increased steadily in specific regions during the first postnatal week while levels remained low in more anterior brain regions. By the end of the second postnatal week, a dense punctate signal was distributed throughout the midbrain and hindbrain. Expression in forebrain, first observed at E18, was confined to cells lining the ventricle until the second postnatal week when accumulation of mRNA was observed in specific regions of the hippocampus, neocortex and olfactory bulb. The adult brain pattern of beta-S-100 mRNA distribution is attained during the third postnatal week. These results demonstrate a caudal-rostral gradient in expression of the beta-S-100 gene during rat brain development, as well as pronounced regional differences which may reflect the differentiation of subpopulations of astrocytes.

Plasticity in the maternal circuit: effects of experience and partum condition on brain astrocyte number in female rats.

Female rats that have received a maternal experience undergo enhanced c-fos expression in a number of brain sites when reexposed to pups. The present 2 studies examined changes in the expression of another brain protein, glial fibrillary acidic protein (GFAP), which is a major unit of the astrocytic cytoskeleton. In both experiments, primiparous and multiparous female rats were given varying amounts of postpartum contact with pups and overdosed after varying intervals, with no pups. Brains were prepared for GFAP immunohistochemical analysis. In both studies, Day 5 postpartum multiparous subjects given additional postpartum contact with pups, when compared with pup-exposed primiparous subjects, were found to have significantly higher numbers of GFAP positive cells in the medial preoptic area of the hypothalamus, an area critical for the expression of maternal behavior, but not in control sites. In Experiment 2, an opposite effect of parity was found in the medial amygdala and habenula.

(-)Deprenyl increases the life span as well as activities of superoxide dismutase and catalase but not of glutathione peroxidase in selective brain regions in Fischer rats.

(-)Deprenyl, a MAO-B inhibitor that is also known to be effective for symptoms of Parkinson's disease, when injected subcutaneously (sc) in male Fischer-344 rats at a dose of 0.5 mg/kg per day (3 times a week) from 18 months of age, significantly increased the remaining life expectancy. The average life span after 24 months was 34% greater in treated rats than in saline-treated control animals. Furthermore, a short-term (3 wk) continuous sc infusion of deprenyl significantly increased activities of superoxide dismutase and catalase but not of glutathione peroxidase in selective brain regions such as s. nigra, striatum, and cerebral cortex, but not in hippocampus or cerebellum, or the liver. The optimal dose for increasing these activities, however, differed greatly depending on the sex and age of animals, with a 10-fold lower value for young female than male rats. Interestingly, aging caused an increase and a decrease in the optimal dose in female and male rats, respectively. In addition, treatment for a longer term tended to reduce the optimal dosage in the same animal group. The results clearly demonstrate that deprenyl increases antioxidant enzyme activities in selective brain regions. If this effect of deprenyl is causally related to its life-prolonging effect, the dosage to be used for any life span study would be a critical factor, with the dosage differing widely depending on sex, age of animal, and mode and duration of drug administration.

Assessing the effects of deprenyl on longevity and antioxidant defenses in different animal models.

Among many pharmaceuticals that have been tested for their effects on longevities of different animal rodents, deprenyl is unique in that its effects on longevity has been tested in at least four different animal species by independent research groups and that the effect has been postulated to be due to its effect of raising such antioxidant enzyme activities as superoxide dismutase (SOD) and catalase (CAT) in selective brain regions. Thus far, in all four species of animals examined (rats, mice, hamsters, and dogs), a positive effect was demonstrated, although the extent of its effect is quite variable. Our group has examined the effect on longevities in rats and mice and on antioxidant enzymes in rats, mice, and dogs. Although in rats of both sexes, we have obtained positive effects on longevity, two studies with different doses in mice did not reveal a significantly positive effect. We have observed, however, significantly positive effects on SOD (in Cu, Zn-, and Mn-) as well as CAT (but not glutathione peroxidase) activities in the brain dopaminergic system such as in the S. nigra and striatum (but not in hippocampus) in all rats, mice, and dogs, although the effects were quite variable, depending on the doses used. In mice, however, a long-term administration (3x/w, 3 months) caused a remarkable decrease in the magnitude of activity as well as a narrowing of the effective dose range, which may explain a relatively weak effect of the drug on mouse longevity. Further, a recent study on aging beagle dogs by Ruehl et al. showed a remarkable effect on longevity, which agrees with our SOD study in dogs. Although deprenyl has been claimed to have several other effects, such as a radical scavenging effect and a neuroprotective effect, past reports on its effects on longevities and antioxidant defenses are compatible with the notion that the drug prolongs the life span of animals by reducing the oxidative damage to the brain dopaminergic system during aging. Further, our studies on F-344 rats as well as a dog study by Ruehl et al. suggest that the drug may at least partially prolong the life span of animals by enhancing immune system function and preventing tumor development in animals.

Leupeptin causes an accumulation of lipofuscin-like substances and other signs of aging in kidneys of young rats: further evidence for the protease inhibitor model of aging.

The "protease inhibitor model of aging" has been proposed on the basis of observations that young rat brains, livers, and retinas exposed to a protease inhibitor, leupeptin, accumulate lipofuscin-like substances (LLS) that are similar to age pigment, and also display a variety of other manifestations of aging. In order to validate this hypothesis in more general terms, the present study reports attempts to induce age-like changes in kidney cells of young rats. Male F-344 rats (4-5 weeks of age) were continuously infused intraperitoneally (i.p.) with various doses of leupeptin (1-50 mg/100 g/day) for 2 weeks. Control animals received saline solution and normal aged rats (27-30 months-old) received no treatment. Animals were sacrificed and subjected to histological examination. In kidneys of leupeptin-, but not saline-treated rats, generally round-shaped PAS-positive particles were clearly observed, which were predominantly distributed in proximal convoluted tubules, and which resembled particles in normal aged kidneys. With increasing drug doses, particles tended to become bigger and more numerous. The dominant accumulation of LLS in cells of the proximal convoluted tubules had a fine morphologic configuration that resembled age pigments in old rats. Also, there was a concomitant thickening of the basement membrane that was present in leupeptin-treated and aged kidneys, but not in controls. The results, therefore, support the protease inhibitor model of aging and provide an experimental tool for probing the cellular mechanisms of aging.

Upregulation of antioxidant enzyme activities by deprenyl. Implications for life span extension.

In order to elucidate the exact role of antioxidant enzyme activities such as superoxide dismutase (SOD) in the aging process of animals, we compared various enzyme activities in different brain regions and in the liver of young (6-8 mo) and old (28-30 mo) Fischer-344 (F-344) rats. While Mn-SOD activities were elevated 3-5-fold in specific brain regions such as hippocampus, striatum and substantia nigra in brains of old male rats compared with the young, in females both forms of SOD (Cu, Zn- and Mn-) enzyme activities remained essentially unchanged with aging. Continued subcutaneous infusion of deprenyl for 3 weeks caused a 2-3-fold increase in activities of both Cu Zn- and Mn-SOD and a 50-60% increase in CAT activities in striatum and substantia nigra but not in hippocampus, cerebellum or the liver. Further, long-term treatment of old male rats with deprenyl caused a significant increase in the remaining life expectancy from 24 months of age by 34%. In conclusion, activities of antioxidant enzymes in these regions examined do not show any uniform age-associated change, suggesting that changes in these enzyme activities do not have any specific role in the life span of rodents in general terms. In contrast, the results of our deprenyl study suggests the possibility that the protection of catecholaminergic neurons by an upregulation of SOD and CAT activities plays a significant role in the life span of animals.

Do antioxidant strategies work against aging and age-associated disorders? Propargylamines: a possible antioxidant strategy.

The free radical theory of aging was initially proposed by Harman half a century ago primarily to explain biological aging processes. Although administration of so-called antioxidant chemicals, which have been tested in the past for several decades, turned out to be mostly ineffective in prolonging the life spans of animals, the same theory of age-associated diseases appears to be increasingly supported in the last two decades. Despite these difficulties, the success in extending life span of 4 different animal species (mice, rats, hamsters, and dogs) with (-)deprenyl (including a study of our group) indicates that there might exist another type of antioxidant strategy in addition to a simple administration of antioxidant chemicals. (-)Deprenyl has also been shown to increase superoxide dismutase (SOD) and catalase (CAT) activities selectively in brain dopaminergic tissues. Interestingly, we have recently shown that another propargylamine, rasagiline not only increases antioxidant enzyme activities (CAT and SOD) in brain dopaminergic regions as (-)deprenyl does, but also increases CAT and SOD activities in extrabrain catecholaminergic systems such as the heart and kidneys as well. These recent observations coupled with previous observations on the life span of animals with (-)deprenyl suggest that pharmacological modulation of endogenous antioxidant enzyme activities could be one potential antioxidant strategy against aging and age-associated disorders. If the causal relationship between the two effects of (-)deprenyl exists as we hypothesized, we might be able to advance the elucidation of mechanism(s) of aging based on the free radical theory of aging.

Short intertrial intervals impair water maze performance in old Fischer 344 rats.

Male and female Fischer 344 rats (N = 55) aged approximately 18, 21, and 24 months were tested for spatial learning in the water maze with intertrial intervals of 1-4 min (Massed) or 23-33 min (Spaced). Animals tested in the Massed condition showed an age-related impairment on trials to criterion; rats aged 24 months performed more poorly than younger subjects. Spaced animals did not differ at any age nor did they differ from 18- or 21-month-old Massed subjects. The youngest rats in both groups were comparable to animals aged 7-8 months tested under Massed conditions. Tests on swim distance, swim speed, and escape latency produced similar results. Our data suggest that acquisition deficits in 24-month-old rats tested with long intertrial intervals are due at least in part to increased susceptibility to fatigue and/or thermal stress. Caution should be used, therefore, when interpreting age-related impairments in water maze performance.

Ephemeral cellular segmentation in the thalamus of the neonatal rat.

The distribution of thalamocortical relay cells in the rat ventrobasal complex was studied during the early postnatal period using the retrograde transport of horseradish peroxidase from the parietal cortex. It was found that the relay cells undergo marked changes in their distribution during the first two postnatal weeks. On postnatal days (PNDs) 0 and 1, the cells are rather homogeneously distributed throughout the ventrobasal complex. However, by PND 2, and more clearly by PND 3, the cells form a distinctly segmented pattern. This pattern consists of discrete curvilinear arrays of cells extending throughout most of the rostrocaudal extent of the nucleus. This distinct cellular pattern is present from PND 2 to about PND 8. In animals sacrificed on PND 15 or as adults, the pattern is no longer obvious. The cellular pattern seen at PND 8 was examined in the 3 standard planes of section and compared to both the somatotopic organization of the nucleus and to the organization of its major ascending and descending inputs. The developmental time course of the cellular segmentation was related to that of the lemniscal and corticothalamic afferents, which also show ephemeral segmentation patterns during the first two postnatal weeks.

Anomalous accumulation of tau and ubiquitin immunoreactivities in rat brain caused by protease inhibition and by normal aging: a clue to PHF pathogenesis?

Rats received intraventricular infusion of leupeptin or saline and brain sections were immunostained with antibodies to tau (anti-HFoPHF) or ubiquitin. Results were compared with immunostaining on normal aged rat brains and Alzheimer's disease (AD) brains. Both antibodies stained Purkinje cell perikarya and dendrites of leupeptin (but not saline)-treated and aged rat brains, as well as senile plaque neurites and neurofibrillary tangles in AD brains. The results are consistent with the hypothesis that paired helical filament (PHF) formation involves defective protein degradation.