Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

The effect of mobile phone on the number of Purkinje cells: a stereological study.

PURPOSE: The World Health Organisation proposed an investigation concerning the exposure of animals to radiofrequency fields because of the possible risk factor for health. At power frequencies there is evidence to associate both childhood leukaemia and brain tumours with magnetic field exposures. There is also evidence of the effect of mobile phone exposure on both cognitive functions and the cerebellum. Purkinje cells of the cerebellum are also sensitive to high dose microwave exposure in rats. The present study investigated the effect of exposure to mobile phone on the number of Purkinje and granule neurons in the developing cerebellum. MATERIAL AND METHODS: Male and female Swiss albino mice were housed as control and mobile phone-exposed groups. Pregnant animals in the experimental group were exposed to Global System for Mobile Communication (GSM) mobile phone radiation at 890-915 MHz at 0.95 W/Kg specific absorption rate (SAR). The cerebella were processed by frozen microtome. The sections obtained were stained with Haematoxylin-eosin and cresyl violet. For cell counting by the optical fractionator method, a pilot study was firstly performed. Cerebellar areas were analysed by using Axiovision software running on a personal computer. The optical dissectors were systematically spaced at random, and focused to the widest profile of the neuron cell nucleus. RESULTS: A significant decrease in the number of Purkinje cells and a tendency for granule cells to increase in cerebellum was found. CONCLUSION: Further studies in this area are needed due to the popular use of mobile telephones and relatively high exposure on developing brain.

Radiation damage increases Purkinje neuron heterokaryons in neonatal cerebellum.

OBJECTIVE: Recent studies have shown that in radiated and bone marrow transplanted mice, bone marrow-derived cells (BMDCs) fuse with Purkinje neurons resulting in the formation of binucleated heterokaryons. Here we investigated whether radiation plays a role in the formation of Purkinje neuron heterokaryons. METHODS: Fused cells were identified by reporter gene expression in mice, carrying floxed LacZ (R26R-LacZ) in all cells and Cre in hematopoietic-derived cells. Cell fusion was confirmed by the presence of two nuclei. The number of fused Purkinje neurons was studied in: 1) whole-body radiated newborn and adult R26R-LacZ mice, transplanted with bone marrow cells expressing Cre; 2) in newborn and adult mice that received different doses of radiation to the head; and 3) in radiated and non-radiated newborns treated with a myeloablative drug before bone marrow transplantation. RESULTS: In neonatal, but not in adult cerebelleum, radiation-in a dose-dependent manner-induces a dramatic increase in the number of fused Purkinje neurons. INTERPRETATION: Increase recruitment of BMDCs into the cerebellum, radiation damage to cerebellar cells, or both, increase the formation of fused Purkinje cells. BMDC-Purkinje heterokaryons formation may reflect an endogeneous neuronal repair mechanism, or it could be a by-product of radiation-induced inflammation. In either case, fused Purkinje neurons increase following radiation damage in the developing cerebellum. The above observations reveal a novel consequence of head radiation in neonatal rodents. It will be interesting to determine if similar increase in the number of binucleated Purkinje neurons, occurs in children that receive radiation during early development. Ann Neurol 2009;66:100-109.

Cerebellar heterokaryon formation increases with age and after irradiation.

Hematopoietic cells have been demonstrated to survive in many nonhematopoietic tissues after transplantation. Apparent "bone marrow-derived" cerebellar Purkinje cells in fact result from fusion events and it has been suggested that fusion may be a natural physiological phenomenon to rescue dysfunctioning cells. Here, we show that fusion of transplanted bone marrow cells with resident Purkinje cells is age-dependent and is strongly enhanced when Purkinje cells are damaged by high-dose irradiation. In addition, Purkinje heterokaryons occur in increased frequencies in the cerebellum of normal, unperturbed, aged mice compared to young animals. Our data suggest that age- and/or irradiation-induced dysfunctioning of Purkinje cells in the cerebellum is required for cell fusion.

Kv3 K+ channels enable burst output in rat cerebellar Purkinje cells.

The ability of cells to generate an appropriate spike output depends on a balance between membrane depolarizations and the repolarizing actions of K(+) currents. The high-voltage-activated Kv3 class of K(+) channels repolarizes Na(+) spikes to maintain high frequencies of discharge. However, little is known of the ability for these K(+) channels to shape Ca(2+) spike discharge or their ability to regulate Ca(2+) spike-dependent burst output. Here we identify the role of Kv3 K(+) channels in the regulation of Na(+) and Ca(2+) spike discharge, as well as burst output, using somatic and dendritic recordings in rat cerebellar Purkinje cells. Kv3 currents pharmacologically isolated in outside-out somatic membrane patches accounted for approximately 40% of the total K(+) current, were very fast and high voltage activating, and required more than 1 s to fully inactivate. Kv3 currents were differentiated from other tetraethylammonium-sensitive currents to establish their role in Purkinje cells under physiological conditions with current-clamp recordings. Dual somatic-dendritic recordings indicated that Kv3 channels repolarize Na(+) and Ca(2+) spikes, enabling high-frequency discharge for both types of cell output. We further show that during burst output Kv3 channels act together with large-conductance Ca(2+)-activated K(+) channels to ensure an effective coupling between Ca(2+) and Na(+) spike discharge by preventing Na(+) spike inactivation. By contributing significantly to the repolarization of Na(+) and especially Ca(2+) spikes, our data reveal a novel function for Kv3 K(+) channels in the maintenance of high-frequency burst output for cerebellar Purkinje cells.

Effects of prenatal exposure to low dose ionizing radiation on the development of the cerebellar cortex in the rat.

The effects of maternal exposure to a single dose of whole body irradiation (0.5 Gy) on gestational days (GD) 17, 18, 19, or 20 on the development of the cerebellar cortex was examined in the offspring of Sprague Dawley rats at 21 and 28 days postnatally. No gross cerebellar anomalies were observed in the irradiated animals. However, compared to control animals, rat irradiated on each of GD-17, 18, 19 and 20 showed a significantly higher incidence (p < 0.05) of circumscribed cerebellar lesions (CL) distributed in the inner granular layer of the anterior and posterior lobes. These lesions were characterized by a loss of granule cells and atrophied and/or reduced number of Purkinje cells. In 21 days old rats, irradiation on GD-17 resulted in more CL anteriorly (75%) and in the vermis whereas on GD-20, the CL predominated posteriorly (100%) and in the lateral hemispheres. In 28 day old rats, following irradiation on each of GD-17 and GD-20, there was an equal distribution of CL in both the anterior and posterior lobes. However, with irradiation on both GD-17 and GD-20, these CL occurred more frequently in the lateral hemispheres of the anterior lobe, whereas in the posterior lobe they predominated in the vermis. These results suggest that a direct relationship exists between the proliferation, migration, development, and maturation of granule cells and their induction by Purkinje cells. The findings also support the view that both cell death and the regulation of granule cells by Purkinje cells maximize the effective development and organization of the cerebellum.

Purkinje and granule cells distribution in the cerebellum of the rat following prenatal exposure to low dose ionizing radiation.

The effects of maternal exposure to a single dose of whole body irradiation (0.5 Gy) on gestational days (GD) 17, 18, 19, or 20 on the number of Purkinje and granule cells in the pyramis of the cerebellar cortex was examined in the offspring of Sprague Dawley rats at 21 and 28 days postnatally. The laterolateral distribution of both granule and Purkinje cells in the pyramis were significantly reduced (p < 0.001) from controls in rats irradiated on each of GD-17, 18, 19 and 20. There was a greater deficit in granule cell number with irradiation on GD-20 than on GD17 (p < 0.05). Purkinje cells were reduced in number with irradiation on GD-17 and GD-20; however, the decrease did not correspond to the degree of reduction in the number of granule cells. There was a greater reduction of both granule and Purkinje cells in the vermis with irradiation on GD-17, whereas on GD-20, both granule (p < 0.05) and Purkinje cells (p < 0.001) were more reduced in the lateral hemispheres. The GC/PC ratio was smaller in rats irradiated on GD-20 than on GD-17. The GC/PC ratio between the irradiated animals and the controls were relatively similar. The findings show that irradiation does not affect the population of granule cells directly, but rather indirectly. The reduction in the number of granule cells could be an indirect consequence of reduced critical interaction with Purkinje cells. These results suggest that a direct relationship exists between the proliferation, migration, development, and maturation of granule cells and their induction by Purkinje cells.

Noradrenergic innervation of ectopic granule cells following low-level X-irradiation.

The distribution pattern of the noradrenergic system within the cerebellar cortex following low-level X-irradiation was studied using immunocytochemistry. Following one X-irradiation exposure on postnatal day 1, the laminar distribution of tyrosine hydroxylase-like immunoreactive (THIR) fibers was similar to controls at postnatal day 30, but the orientation of the fibers in the molecular layer (ML) was slightly changed. Successive daily doses, however, produced alterations in both the pattern and concentration of THIR fibers within the ML of the cerebellar cortex. In addition, THIR fibers were found among ectopic cell clusters within the ML. This relationship suggests a potential role for the noradrenergic system in the development and/or maintenance of the ectopic cell clusters.

[Morphofunctional state of Purkinje cells in the cerebellum of newborn rats after exposure to laser and ionizing radiation]. / Morfofunktsionalnoe sostoianie kletok Purkiné mozzhechka krysiat pri deistvii lazernogo i ioniziruiushchego izluchenii.

Following of local laser (632.8 nm, 6.3 J/cm2) and whole-body 6.37 Gy gamma-ray exposures of new-born rats the contrast changes of morphometrical indices, RNA amount, and chromatophilia of Purkinje cells in the cerebellum were seen. The preliminary laser exposure of new-born rat cerebellum artially increased activity of karyogene structures of the cerebellum cells which were inhibited by 6.37 Gy gamma-rays.

Effect of nonionizing radiation on the Purkinje cells of the uvula in squirrel monkey cerebellum.

Pregnant squirrel monkeys were exposed to 2450-MHz (CW) microwaves at an equivalent power density of 10 mW/cm2 (SAR 3.4 mW/g) for three hours daily in a cavity-cage module. The exposure began when pregnancy was determined by a hormonal method, and continued through of offspring's first 9.5 months. After irradiation, the brain of the offspring were fixed with formaldehyde, and the inferior vermis of each cerebella was removed and processed for histologic observations. Purkinje cell density in the uvula was determined in sagittal serial section. There was no significant difference between control and experimental animals in the number of Purkinje cells per mm of Purkinje cell line (linear density), as well as in the density of Purkinje cells in the Purkinje cell layer.

Effect of nonionizing radiation on the Purkinje cells of the rat cerebellum.

In one experiment, Sprague Dawley rats (16-21 days of gestation) and their offspring were exposed to 100-MHz (CW) electromagnetic radiation at 46 mW/cm2 (SAR 2.77 mW/g) for 4 h/day for 97 days. In another experiment, the pregnant rats were irradiated daily from 17 to 21 days of gestation with 2450-MHz (CW) microwaves at 10 mW/cm2 (SAR 2 m W/G) for 21 h/day. In a third experiment, 6-day old rat pups were irradiated 7 h/day for five days with 2450-MHz radiation at 10 mW/cm2. Equal numbers of animals were sham irradiated in each group. Quantitative studies of Purkinje cells showed a significant and irreversible decrease in rats irradiated during fetal or fetal and early postnatal life. In animals exposed postnatally, and euthanized immediately after irradiation, significant decrease in the relative number of Purkinje cells was apparent. However, restoration apparently occurred after forty days of recovery.

Induction of mitosis in the differentiating Purkinje cells of the cerebellum.

The neonate rats of 5 days postnatal age were exposed to low-level X-ray irradiation or were administered microquantities of N-ethyl-N-nitrosourea directly into the cerebellum. Following the initial phase of cell degeneration and phagocytosis of the debris, the surviving Purkinje cells showed recovery of their differentiation. During this stage some Purkinje cells were observed to undergo mitosis, and they were identified in light- and electron-microscopic preparations. The relationship between tetraploidy in the Purkinje cells during their postnatal differentiation and induction of mitosis in them by experimental means is discussed.