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Natural polyamines (putrescine, spermidine and spermine) are ubiquitous molecules known to regulate a number of physiological processes and suspected to play a role also in various pathological conditions. Changes in polyamine levels and in their biosynthetic enzymes have been described for some neurodegenerative diseases but the available data are incomplete and somewhat contradictory. We report here alterations of the key enzyme of the polyamine pathway, ornithine decarboxylase (ODC) catalytic activity and polyamine levels in different CNS areas from SOD1 G39A transgenic mice, an animal model for amyotrophic lateral sclerosis (ALS). ODC catalytic activity, was found significantly increased both in the cervical and lumbar spinal cord and, to a lesser extent in the brain stem of transgenic mice at a symptomatic stage of the disease (125-day-old mice), while no differences were present at a pre-symptomatic stage (55-day-old mice). In parallel with the increase of ODC activity putrescine levels were several times increased in both cervical and lumbar spinal cord and in the brain stem of 125-day-old SOD1 G39A mice. Higher order polyamines were not increased except for a significant increase of spermidine in the cervical spinal cord. The present data demonstrate considerable alterations of the ODC/polyamine system in a reliable animal model of ASL, consistent with their role in neurodegeneration and in particular in motor neuron diseases.

We have investigated changes in ornithine decarboxylase (ODC) activity and in polyamine levels in the central nervous system of aged rats. We measured a significant increase of ODC catalytic activity in the spinal cord from 30 month-old rats (+105%) as compared to 4 month-old rats. No changes were noticed in the cerebellum, cortex and hippocampus from the same animals. A related putrescine increase was measured in the spinal cord of 30 month-old rats (+168%), together with a smaller increase of spermidine (+33%). A parallel increase (+78%) of the Ca2+-dependent transglutaminase activity was detected in the spinal cord of 30 month-old rats, while no changes were apparent in the cortex and cerebellum. Our observations indicate a possible role of the ODC/polyamine system during the normal process of ageing in rats and point to the spinal cord as the most sensitive area for this kind of modification. A possible role of protein polyamination by transglutaminase is discussed.

Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 mM lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.

Young adult rats were chronically treated with lithium (2.5 mmol/kg/day) for 16 days. The day after the last lithium administration, rats were injected s.c. with the excitotoxic convulsant kainic acid (10 mg/kg). As compared to saline controls, lithium-treated rats had no apparent attenuation of convulsions. Furthermore, the induction of brain ornithine decarboxylase and the consequent increase of putrescine levels, an index related to the convulsant effects of kainic acid, were similar in saline- and lithium-treated rats. Other rats were unilaterally injected with ibotenic acid into the nucleus basalis magnocellularis: no differences were measured in cortical choline acetyltransferase (ChAT) decrease among saline- and lithium-treated rats. In both the above experiments, apoptotic cell death was monitored in relevant brain regions of saline- or lithium-treated rats through a specific in situ labeling method for fragmented DNA. Whilst morphological evidence for a reduced damage in the olfactory cortex and hippocampus of kainic acid-injected rats was not obtained, lithium-treated rats showed a lower decrease of specific neurochemical markers: [3H]D-aspartate uptake and glutamate decarboxylase. This result suggests that mechanisms of recovery, absent in saline-treated animals, are elicited by the excitotoxic insult in lithium-treated rats.

Pregnant rats were treated for five consecutive days during gestation with s.c. injections of the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO). Treatment beginning at gestational days 13 or 14 was effective in inhibiting ODC and altering polyamine levels, and resulted in relatively small decreases in body and forebrain weight, but not in significant differences in adult neurochemistry. Neonatal rats were treated with DFMO from postnatal day 0 (PD 0) to PD 24. In addition to some somatic effects (decreased body weight, delayed eyelid opening and delayed fur growth) the postnatal treatment resulted in a permanent decrease in brain weight, which was mainly due to a dramatic decrease in cerebellar size. During treatment, and 3 days after the end of it, the levels of putrescine and spermidine, but not those of spermine, were consistently lower in the cerebellum and forebrain of DFMO-treated rats than in controls. On the other hand, ODC appeared strongly inhibited only during the first phase of the treatment and showed recovery, and also rebound of the activity, during the second part of the treatment. A screening of neurochemical markers related to cholinergic, GABAergic and glutamatergic neurons, as well to astrocytes and oligodendrocytes was performed in several brain regions (cerebellum, olfactory bulbs, cortex, striatum, hippocampus) of some of these rats once they became adults. Significant alterations for all the parameters tested, with the exception of the marker for the glutamatergic transmission, were measured in the undersized cerebellum of the neonatally DFMO-treated rats. A shorter neonatal treatment with DFMO (from PD 1 to 6) resulted, in the adult, in decreased cerebellar size and in neurochemical alterations, both very similar to those occurring after the prolonged treatment. In the other brain regions a few minor differences were noticed. The present results show that: (1) the brain polyamine system is differently regulated in foetuses with respect to newborns; (2) the effects of chronic ODC blockade are different on prenatally or postnatally proliferating neurons, due either to a lower sensitivity of gestationally proliferating neurons or to a subsequent recovery; and (3) chronic postnatal ODC inhibition has a strong effect on proliferating neurons, but little effect on further maturation of postmitotic neurons.

Neonatal rats were daily treated with increasing doses of the competitive NMDA antagonist CGP 39551 from postnatal day 1 to 22. At 75-80 days of age the rats were given an excitotoxic dose of kainic acid s.c. Histological examination suggested that the olfactory cortex of the neonatally CGP 39551-treated rats was less damaged than that of controls. This was quantitatively confirmed by assaying the decrease of appropriate neurochemical markers (D-[3H]aspartate uptake and glutamate decarboxylase) as an index of the extent of neuronal degeneration. By contrast, the decrease of the same markers was not different in the hippocampus. These results suggest a selective effect on some brain circuits of adult rats consequent to the neonatal blockade of NMDA receptors and, therefore, add new evidence to a developmental role of this receptor.

Neonatal rats were subjected to chronic blockade of the N-methyl-D-aspartate (NMDA) receptor through daily systemic administration of increasing doses of the competitive antagonist CGP 39551 from postnatal days 1-22. Treatment did not result in any significant alteration of the levels of putrescine, spermidine and spermine or in the constitutively expressed activity of the key enzyme for polyamine biosynthesis, ornithine decarboxylase (ODC), as evaluated at 10 and 20 days of age. However, in 30-day-old rats significant differences were observed in the process of excitotoxic ODC induction in the olfactory cortex and the hippocampus of chronically-treated rats: the increase of ODC activity caused by systemic administration of kainic acid took place more rapidly but it was shorter and apparently reached a smaller peak in treated animals as compared to controls. This result, in conjunction with previous data on neurochemistry and locomotor activity of similarly treated rats, strengthens the suggestion that functional alterations of some brain circuits may be the consequence of the blockade of NMDA receptor during the critical neonatal period of brain maturation.

The activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, dramatically increases after different types of brain injuries. The role of this induction is still unclear. We report here data on the temporal pattern of ODC induction caused by the excitotoxin kainic acid. After systemic administration, ODC activity increases severalfold peaking at 8 h in the prefrontal cortex and at 16 h in the olfactory cortex and hippocampus. After intrastriatal injection, the peak of induction is reached at 32 h, while a smaller and more transient increase is also observed in the contralateral, saline-injected striatum. We suggest that ODC induction is initially linked to overactivation of neural circuits and, later on, to the development of widespread neural damage.