John T. Weber

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

Altered calcium signaling following traumatic brain injury

Cell death and dysfunction after traumatic brain injury (TBI) is caused by a primary phase, related to direct mechanical disruption of the brain, and a secondary phase which consists of delayed events initiated at the time of the physical insult. Arguably, the calcium ion contributes greatly to the delayed cell damage and death after TBI. A large, sustained influx of calcium into cells can initiate cell death signaling cascades, through activation of several degradative enzymes, such as proteases and endonucleases. However, a sustained level of intracellular free calcium is not necessarily lethal, but the specific route of calcium entry may couple calcium directly to cell death pathways. Other sources of calcium, such as intracellular calcium stores, can also contribute to cell damage. In addition, calcium-mediated signal transduction pathways in neurons may be perturbed following injury. These latter types of alterations may contribute to abnormal physiology in neurons that do not necessarily die after a traumatic episode. This review provides an overview of experimental evidence that has led to our current understanding of the role of calcium signaling in death and dysfunction following TBI.

The role of calcium in synaptic plasticity and motor learning in the cerebellar cortex.

The cerebellum is important for motor coordination, as well as motor learning and memories. Learning is believed to occur in the cerebellar cortex, in the form of synaptic plasticity. Central to motor learning theory are Purkinje cells (PCs), which are the sole output neurons of the cerebellar cortex. Motor memories are postulated to be stored in the form of long-term depression (LTD) at parallel fiber synapses with PCs, once thought to be the only plastic synapse in the cerebellar cortex. However, in the past few decades many studies have demonstrated that several other synapses in the cerebellar cortex are indeed plastic, and that LTD or long-term potentiation at these various synapses could affect the overall output signal of PCs from the cerebellar cortex. Almost all of these forms of synaptic plasticity are dependent on calcium to some extent. In the current review we discuss various types of synaptic plasticity in the cerebellar cortex and the role of calcium in these forms of plasticity.

Chemical analysis and effect of blueberry and lingonberry fruits and leaves against glutamate-mediated excitotoxicity.

Phenolic compounds are a large class of phytochemicals that are widespread in the plant kingdom and known to have antioxidant capacities. This study aimed to determine the antioxidant capacities as well as the content of total soluble phenolics, anthocyanins, tannins, and flavonoids in the fruits and leaves of blueberries and lingonberries growing in Newfoundland. This study also determined the potential neuroprotective effect of extracts from fruits and leaves against glutamate-mediated excitotoxicity, which is believed to contribute to disorders such as stroke and neurodegenerative diseases. Lingonberry and blueberry plants were found to be rich sources of phenolic compounds. Total antioxidant capacities in terms of radical scavenging activity and reducing power were much higher in leaves of both plants as compared to their fruits. These results were in correlation with phenolic contents including total flavonoids, anthocyanins, and tannins. Brain-derived cell cultures from rats were prepared and grown for about 2 weeks. Cell cultures were treated with glutamate (100 μM) for 24 h, and the effect of extracts was determined on cells subjected to this excitotoxicity. Glutamate treatment caused approximately 23% cell loss when measured after 24 h of exposure. Whereas lingonberry fruit extract did not provide protection from glutamate toxicity, blueberry fruit extracts were extremely protective. Leaf extracts of both lingonberry and blueberry showed a significant neuroprotective effect. The greater protective effect of leaf extracts was in correlation with the levels of phenolics and antioxidant capacity. These findings suggest that berries or their components may contribute to protecting the brain from various pathologies.

Effects of intermittent binge alcohol exposure on long-term motor function in young rats.

Ethanol has well described acute effects on motor function, and chronic alcoholism can damage the cerebellum, which is associated with motor coordination, as well as motor learning. Binge drinking is common among preadolescents and adolescents, and this type of ethanol exposure may lead to long-term nervous system damage. In the current study, we analyzed the effects of periadolsecent/adolescent ethanol exposure on motor function in both male and female Sprague-Dawley rats. To simulate binge drinking, animals received an intraperitoneal injection of 25% (v/v) ethanol (3 g/kg) on postnatal days (PND) 25, 26, 29, 30, 33, 34, 37 and 38. On PND 42 and PND 61 animals were tested on their ability to traverse both square and round beams. There were no significant differences in the time to traverse the beams, or the amount of foot slips, between treated and untreated animals. On PND 48 and PND 62, animals were tested using a horizontal ladder walking apparatus. On PND 48 there were no differences in the ability of treated and untreated animals to traverse the ladder. On PND 62, there were no differences in the time to traverse the ladder, but ethanol treated animals had more foot slips than controls. On PND 43, we conducted footprint analysis of control and treated animals, which included measurements of stride length, paw overlap, and angle of foot placement. There was a significant difference in the angle of foot placement between treated and control animals, and this finding was significant for both male and female animals. There was also a significant overall difference in paw overlap between treatment groups. Although this effect was manifested in male animals there was no significant difference in females. These findings suggest that adolescent ethanol exposure can produce long-lasting effects on motor coordination, and that overall, effects are similar in males and females. In a second set of experiments, male rats received i.p. ethanol (3 g/kg) for 7 days (P31-37) or 4 days (P31,33,35,37). No significant differences were detected by footprint analysis when compared to control animals. However, ethanol treated animals had significantly less cerebellar Purkinje cells at 3 weeks after the last ethanol exposure. Altered motor function suggests a possible neurodegenerative effect in the cerebellum initiated by adolescent ethanol exposure, and may depend on the extent of exposure during the preadolescent and/or adolescent brain periods.

Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells

The waddles (wdl) mouse is characterized by a namesake side-to-side waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication.

Biochemical Properties and Neuroprotective Effects of Compounds in Various Species of Berries

Several species of berries, such as blueberries (Vaccinium angustifolium) and lingonberries (Vaccinium vitis-idaea L.), have attracted much scientific attention in recent years, especially due to their reported antioxidant and anti-inflammatory properties. Berries, as with other types of plants, have developed metabolic mechanisms to survive various environmental stresses, some of which involve reactive oxygen species. In addition, the fruits and leaves of berries have high amounts of polyphenols, such as flavonoids, which act as potent antioxidants. These compounds could potentially be beneficial for brain aging and neurodegenerative disorders. There are now several studies documenting the beneficial effects of various berries in cell models of neurotoxicity as well as in vivo models of neurodegenerative disease. In the current review, we discuss the metabolic strategies that plants and animals have developed in order to combat reactive oxygen species. We then discuss issues of bioavailability of various compounds in mammals and provide a synopsis of studies demonstrating the neuroprotective ability of berries and polyphenols. We also summarize findings from our own research group. For example, we have detected various polyphenols in samples of blueberries and lingonberries and have found that the leaves have a much higher antioxidant capacity than the fruits. Extracts from these species have also demonstrated neuroprotective effects in cellular models of toxicity and inflammation, which are being further pursued in animal models.

Animal Models of Traumatically-Induced Dementia

Dementia in humans following traumatic brain injury (TBI) has been well documented in clinical populations, either after a single TBI or repeated mild TBI (rMTBI). In most cases, trauma-induced dementia follows a slow, chronic time-course, and in many cases mild injuries accumulate over time. Both single TBI and rMTBI have been modeled experimentally in vivo, most often in rodents, and various treatment strategies have been studied in order to reduce brain damage after injury. Here, we review the recent literature with regard to currently used in vivo models of TBI in animals, and studies conducted with them to investigate the link between TBI and the development of dementia-like pathology, such as that associated with Alzheimer’s disease (AD). We also discuss the potential use of in vitro models of trauma for investigating a link between trauma and dementia.