PKC activator therapeutic for mild traumatic brain injury in mice.

Traumatic brain injury (TBI) is a frequent consequence of vehicle, sport and war related injuries. More than 90% of TBI patients suffer mild injury (mTBI). However, the pathologies underlying the disease are poorly understood and treatment modalities are limited. We report here that in mice, the potent PKC activator bryostatin1 protects against mTBI induced learning and memory deficits and reduction in pre-synaptic synaptophysin and post-synaptic spinophylin immunostaining. An effective treatment has to start within the first 8h after injury, and includes 5 × i.p. injections over a period of 14 days. The treatment is dose dependent. Exploring the effects of the repeated bryostatin1 treatment on the processing of the amyloid precursor protein, we found that the treatment induced an increase in the putative α-secretase ADAM10 and a reduction in ß-secretase activities. Both these effects could contribute towards a reduction in ß-amyloid production. These results suggest that bryostatin1 protects against mTBI cognitive and synaptic sequela by rescuing synapses, which is possibly mediated by an increase in ADAM10 and a decrease in BACE1 activity. Since bryostatin1 has already been extensively used in clinical trials as an anti-cancer drug, its potential as a remedy for the short- and long-term TBI sequelae is quite promising.

Apoptotic changes in the cortex and hippocampus following minimal brain trauma in mice.

Interpretation of the cellular and molecular pathogenic basis of post-minimal traumatic brain injury is a significant clinical and scientific problem, especially due to the high prevalence of motor vehicle--and other accidents. Pathogenetic brain mechanisms following traumatic impact are usually investigated by using models of severe or moderate trauma. Apoptotic neuronal degeneration after notable brain trauma is a well-known phenomenon, but the source of its activation is not clear, especially after mild, subclinical brain trauma. In the present study, we used a closed head weight-drop model to induce minimal brain injury in mice. Pellets of 5, 10, 15, 20, 25 and 30 g were dropped on the right side of mice's head kept under light ether anesthesia. No abnormal behavioral or neurophysiological changes were seen following the head trauma. Morphological assessment was done 72 h after the traumatic impact using TUNEL assay and silver staining. We found gradual increase of TUNEL-positive and silver-impregnated cells number in different cortical and hippocampal regions of both injured and contralateral hemispheres. The threshold of traumatic impact that caused a significant activation was 10-15 g pellets (evident by silver staining), and 15-20 g for apoptosis. The most sensitive zones for trauma were anterior cingulate cortex and CA3 area of hippocampus. No bilateral hemispheric differences were found. Our results demonstrate that even closed head minimal traumatic brain injury can cause diffused neuronal damage and apoptosis. This results correlate well with cognitive and behavioral deficits described for mice suffering similar mTBI and can also explain the wide variety of mental disturbances described for post-concussion syndrome in patients who suffered mild head injury.

Morphine protects for head trauma induced cognitive deficits in mice.

Victims of minor traumatic brain injury (mTBI) can show long lasting cognitive, emotional and concentration difficulties, amnesia, depression, apathy and anxiety. The symptoms are generally known as a post-concussive syndrome without clear morphological brain defects. Endogenous opiates are released after impact to the brain, suggesting they may play a role in TBI pathophysiology. Furthermore, the administration of opiates to the brain of injured animals has been shown to affect the injury, induce cellular changes and also have protective qualities for neurological impairments. Here, we examined the protective properties of the opiate morphine on cognitive performances following minimal brain injury in mice. For this purpose, we have used our non-invasive closed-head weight drop model in mice, which closely mimics real life mTBI and examined mice performance in the Morris water maze. Our procedure did not cause visible structural or neurological damage to the mice. A single morphine injection administrated immediately after the induction of minimal TBI protected the injured mice from cognitive impairment, checked 30, 60 and 90 days post injury. However, mice injected with morphine that were examined 7 days after the injury did not show better performance than the saline injected mice. Our results indicate that morphine has long but not short-term effects on the cognitive ability of brain-injured mice. Although the exact nature of opioid neuroprotection is still unknown, its elucidation may lead to the much-needed treatment for traumatic brain injury.

Quantification and distribution of beta-secretase alternative splice variants in the rat and human brain.

Beta-amyloid (Abeta) is formed by sequential cleaving of the amyloid precursor protein by two proteolytic enzymes, beta- and gamma-secretases. Beta-secretase (BACE) is a type I transmembrane aspartic proteinase that is highly expressed in the mammalian brain. Four alternative splice variants of BACE are currently known and each encodes for a protein isoform with a different enzymatic activity. In Alzheimer's disease (AD) patients, the enzymatic activity and protein levels of BACE are increased in the neocortex, suggesting their differential expression may have a role in Abeta plaque formation. We have determined the differential expression of BACE mRNA and its splice variants in eight regions of the rat and two of the human brain. In humans, the frontal cortex which shows Abeta deposition in AD, expressed three-fold more BACE than the cerebellum and four fold more than the rats' frontal cortex both of which do not form Abeta plaques. The highest BACE levels of rats were found in the frontal cortex and less in other areas. Although most human and rat brain regions expressed all four BACE variants, the human cerebellum did not express the I-457 BACE variant. Human and rat frontal cortex expressed high levels of the I-501 and I-457 variants, but I-432 was highly expressed only in the rat. Species-specific differences were evident between human and rat brain areas, suggesting that BACE transcript variants may have different evolutionary conservation. Differential expression of BACE variants may explain the broad spectrum of phenotypic abnormalities and possible pathogenetic mechanisms underlying Alzheimer's disease.

Distribution of parkin in the adult rat brain.

A mutation in the parkin gene has been identified as the cause for an autosomal recessively inherited form of Parkinson's disease (PD). The authors have recently isolated the mRNA coding for the rat homolog of parkin and showed its widespread expression in the central nervous system (CNS) by in situ hybridization. In the present study, we investigated the distribution of parkin in the rat CNS with a polyclonal antibody that reacts with a approximately 52-kDa protein, mainly localized in the cytoplasm and corresponding to the predicted molecular mass of parkin. Immunohistochemistry on adult rat brain sections showed a widespread distribution of parkin. This included labeling of cell bodies, nuclei as well as processes in the hippocampus, cerebral cortex, cerebellum, and several nuclei in the brainstem. The regional expression of parkin-immunoreactivity (IR) correlated well with the parkin-mRNA levels assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). This study provides the detailed analysis of the regional and cellular distribution of parkin in the rat brain and may be useful in elucidating its pathophysiological role.

Behavioral consequences of minimal traumatic brain injury in mice.

Victims of minor traumatic brain injury (mTBI), who show no clear morphological brain defects, frequently manifest cognitive, behavioral and emotional difficulties that can be long-lasting. In this paper we present a modified weight drop model used to deliver a closed head minimal traumatic brain injury to mice, which closely mimics real-life injuries and the symptoms observed in mTBI patients. Our choice of impact force does not produce structural damage to the brain and its surrounding tissue (as examined by MRI), any skull fracture, no edema and no evident damage to the blood-brain barrier (BBB). Moreover, our mTBI mice show no abnormal behavior on recovering from the weight drop, or any change in other brain functions such as reflexes, balance, exploration, strength, locomotor activity and swim speed. Since our mTBI model does not produce neurological, motor or sensory damage to the mice, it allows the direct evaluation of mTBI sequelae on the mice behavior and cognitive abilities. Using a variety of cognitive and behavioral tests (Morris water maze, staircase test, passive avoidance test, water T-maze, hot palate, elevated plus maze and forced swimming test) we assessed the short- and long-term sequelae induced by our model. Our results indicate that our closed head mTBI cause profound and long-lasting, irreversible learning and memory impairments, accompanied by a depressive-like behavior in mice that are evident even 90 days post injury. Our results indicate that the closed head mTBI model presented here may be useful in the development of novel therapeutic approaches, such as neuroprotective agents, for mTBI.

A mouse model of blast-induced mild traumatic brain injury.

Improvised explosive devices (IEDs) are one of the main causes for casualties among civilians and military personnel in the present war against terror. Mild traumatic brain injury from IEDs induces various degrees of cognitive, emotional and behavioral disturbances but knowledge of the exact brain pathophysiology following exposure to blast is poorly understood. The study was aimed at establishing a murine model for a mild BI-TBI that isolates low-level blast pressure effects to the brain without systemic injuries. An open-field explosives detonation was used to replicate, as closely as possible, low-level blast trauma in the battlefield or at a terror-attack site. No alterations in basic neurological assessment or brain gross pathology were found acutely in the blast-exposed mice. At 7 days post blast, cognitive and behavioral tests revealed significantly decreased performance at both 4 and 7 m distance from the blast (5.5 and 2.5 PSI, respectively). At 30 days post-blast, clear differences were found in animals at both distances in the object recognition test, and in the 7 m group in the Y maze test. Using MRI, T1 weighted images showed an increased BBB permeability 1 month post-blast. DTI analysis showed an increase in fractional anisotropy (FA) and a decrease in radial diffusivity. These changes correlated with sites of up-regulation of manganese superoxide dismutase 2 in neurons and CXC-motif chemokine receptor 3 around blood vessels in fiber tracts. These results may represent brain axonal and myelin abnormalities. Cellular and biochemical studies are underway in order to further correlate the blast-induced cognitive and behavioral changes and to identify possible underlying mechanisms that may help develop treatment- and neuroprotective modalities.

Cutting edge: MHC class I-Ly49 interaction regulates neuronal function.

MHC class I molecules (MHC-I) have been implicated in nervous system development in the mouse. In this study we present evidence for the interaction of MHC-I with the NK cell receptor Ly49 in primary cortical neuronal cultures. We show that MHC-I and Ly49 are expressed on neuronal soma and axon surfaces, with Ly49 also present on dendrites. Anti-MHC-I Abs reduce synapsin-I expression and enhance neurite outgrowth and neuronal death. Conversely, anti-Ly49 mAbs increase synapsin-I expression, reduce neurite outgrowth, and promote neuron viability. Because we show that Ly49 genes are selectively expressed in the adult brain, these findings suggest an unsuspected role for the MHC-I-Ly49 interaction in the development and function of the brain.

MAP kinase signaling cascade dysfunction specific to Alzheimer's disease in fibroblasts.

Mitogen-activated protein kinases (such as Erk1/2) regulate phosphorylation of the microtubule-associated protein tau and processing of the amyloid protein beta, both events critical to the pathophysiology of Alzheimer's disease (AD). Here we report that enhanced and prolonged Erk1/2 phosphorylation in response to bradykinin (BK) was detected in fibroblasts of both familial and sporadic AD, but not age-matched controls (AC). The AD-associated abnormality in Erk1/2 phosphorylation was not seen in fibroblasts from Huntington's disease patients with dementia. The elevation of Erk1/2 phosphorylation occurred immediately after BK stimulation and required an IP3-sensitive Ca(2+) release as well as activation of PKC and c-src as upstream events. Treatment of cells with the PI-3 kinase blocker LY924002 partially inhibited the BK-stimulated Erk1/2 phosphorylation in AC, but had no effect in AD cells, suggesting that the BK-induced Erk1/2 phosphorylation in AD cells is independent of PI-3 kinase. Activation of the cAMP-responsive element binding protein (CREB) monitored as an increase in phosphorylation at Ser-133 was also observed after BK stimulation. Unlike the AD-specific differences for Erk1/2, however, the BK-stimulated CREB phosphorylation was not different between AC and AD cells. Abnormal Erk1/2 activities may alter downstream cellular processes such as gene transcription, amyloid precursor protein processing, and tau protein phosphorylation, which contribute to the pathogenesis of AD. Moreover, detection of AD-specific differences in MAP kinase in peripheral tissues may provide an efficient means for early diagnosis of AD as well as help us to identify therapeutic targets for drug discovery.