Roman Vlkolinsky

The reduction of NADH ubiquinone oxidoreductase 24- and 75-kDa subunits in brains of patients with Down syndrome and Alzheimer's disease.

NADH: ubiquinone oxidoreductase (complex I), one of the most complicated multi-protein enzyme complexes, is important for energy metabolism because it is the initial enzyme of the mitochondrial respiratory chain. Deficiency of complex I is frequently found in various tissues of patients with neurodegenerative disease. Here we studied the protein levels of complex I 24- and 75-kDa subunits in several brain regions from patients with Down syndrome (DS) and Alzheimers disease (AD). We determined protein levels of complex I 24-, 75-kDa subunits and mitochondrial marker proteins mitochondrial matrix protein P1 (hsp60) and aconitate hydratase from seven brain regions of patients with DS, AD and controls. Proteins were separated by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Complex I 24-kDa subunit was significantly reduced in occipital cortex and thalamus in patients with DS and temporal and occipital cortices in patients with AD. Complex I 75-kDa subunit was significantly reduced in brain regions from patients with DS (temporal, occipital and caudate nucleus) and AD (parietal cortex). Reductions of two subunits of complex I may lead to the impairment of energy metabolism and result in neuronal cell death (apoptosis), a hallmark of both neurodegenerative disorders.

Decreased levels of complex III core protein 1 and complex V β chain in brains from patients with Alzheimer’s disease and Down syndrome

Abstract. Ubiquinol:cytochrome c oxidoreductase (complex III) and ATP synthase (complex V) are important enzymes in the mitochondrial electron transport chain. Defects in mitochondrial respiratory enzymes have been reported for several neurodegenerative diseases. In this study, we applied the proteomic approach to investigate protein levels of complex III core protein 1 and complex V β chain in brain regions of Alzheimer’s disease (AD) and Down syndrome (DS) patients. Complex III core protein 1 was significantly reduced in the temporal cortex of AD patients. Complex V β chain was significantly reduced in the frontal cortex of DS patients. We conclude that decreased mitochondrial respiratory enzymes could contribute to the impairment of energy metabolism observed in DS. These decreases could also cause the generation of reactive oxygen species and neuronal cell death (apoptosis) in DS as well as AD.

Traumatic Brain Injury in Young Rats Leads to Progressive Behavioral Deficits Coincident with Altered Tissue Properties in Adulthood

Traumatic brain injury (TBI) affects many infants and children, and results in enduring motor and cognitive impairments with accompanying changes in white matter tracts, yet few experimental studies in rodent juvenile models of TBI (jTBI) have examined the timeline and nature of these deficits, histologically and functionally. We used a single controlled cortical impact (CCI) injury to the parietal cortex of rats at post-natal day (P) 17 to evaluate behavioral alterations, injury volume, and morphological and molecular changes in gray and white matter, with accompanying measures of electrophysiological function. At 60 days post-injury (dpi), we found that jTBI animals displayed behavioral deficits in foot-fault and rotarod tests, along with a left turn bias throughout their early developmental stages and into adulthood. In addition, anxiety-like behaviors on the zero maze emerged in jTBI animals at 60 dpi. The final lesion constituted only ∼3% of brain volume, and morphological tissue changes were evaluated using MRI, as well as immunohistochemistry for neuronal nuclei (NeuN), myelin basic protein (MBP), neurofilament-200 (NF200), and oligodendrocytes (CNPase). White matter morphological changes were associated with a global increase in MBP immunostaining and reduced compound action potential amplitudes at 60 dpi. These results suggest that brain injury early in life can induce long-term white matter dysfunction, occurring in parallel with the delayed development and persistence of behavioral deficits, thus modeling clinical and longitudinal TBI observations.

Differential expression of molecular chaperones in brain of patients with Down syndrome

Heat shock proteins (HSPs) in their molecular capacity as chaperones have been reported to regulate the apoptotic pathway and also play a critical role in protein conformational diseases such as Alzheimers disease (AD). As all Down syndrome (DS) brains display AD‐like neuropathology, neuronal loss in DS was shown to be mediated by apoptosis. We decided to investigate the expression patterns of HSPs in seven brain regions of adults with DS using two‐dimensional polyacrylamide gel electrophoresis (2‐DE). Following 2‐DE, approximately 120 protein spots were successfully identified by matrix‐assisted laser desorption/ionization – mass spectrometry (MALDI‐MS) followed by quantification of the identified proteins. We unambiguously identified and quantified nine different chaperone proteins. Accordingly, all but three chaperone proteins did exhibit a significant change in expression. HSP 70 RY, heat shock cognate (HSC) 71 and glucose‐regulated protein (GRP) 75 showed a significant decrease (P<0.05) in DS temporal cortex whereas HSP 70.1 and GRP 78 were significantly increased (P<0.05) in cerebellum. Whilst T‐complex 1 (TCP‐1) epsilon subunit showed a significant decrease (P<0.05) in parietal cortex, a similar extent of increase (P<0.05) as that observed in cerebellum was obtained in parietal levels of GRP 78. Alpha‐crystallin B, HSP 60 and GRP 94 did not show any detectable changes in expression patterns. This report presents the first approach to quantify nine different chaperones simultaneously at the protein level in different brain regions and provides evidence for aberrant chaperone expression patterns in DS. The relevance of this aberrant expression patterns are discussed in relation to the biochemical and neuropathological abnormalities in DS brain.

28Silicon Radiation-Induced Enhancement of Synaptic Plasticity in the Hippocampus of Naïve and Cognitively Tested Mice

The space radiation environment consists of multiple species of high-energy charge particles (HZE), including 56Fe and 28Si nuclei, that may impact neuronal cells, but their damaging effects on the central nervous system (CNS) have been poorly defined. Hippocampus-dependent memory functions have been shown to be highly sensitive to 56Fe HZE particles, which poses a significant risk to the cognitive performance of astronauts during space missions. While low doses of 56Fe radiation do not induce cell death of mature neurons, they affect synaptic plasticity in the CA1 region, the principal neuronal output of the hippocampal formation involved in memory formation. The effects of 28Si on the CNS have not been defined. Compared to behaviorally naïve mice, cognitive testing might affect synaptic plasticity and the effects of 28Si radiation on synaptic plasticity might be modulated by prior cognitive testing. Therefore, in the current study, we quantified the effects of whole-body 28Si radiation (600 MeV/n, 0.25 and 1 Gy) on hippocampus-dependent contextual freezing and synaptic plasticity in the CA1 region of animals not exposed (behaviorally naïve mice) and animals exposed to the contextual freezing test (cognitively tested mice). In behaviorally naïve mice exposed to 0.25 and 1 Gy of 28Si radiation, the magnitude of long-term potentiation (LTP) was enhanced. However, in mice irradiated with 0.25 Gy contextual fear conditioning was enhanced and was associated with a further enhancement of the LTP magnitude. Such increase in synaptic plasticity was not seen in cognitively tested mice irradiated with 1 Gy. Thus, low dose 28Si radiation has effects on synaptic plasticity in the CA1 region of the hippocampus and these effects are modulated by cognitive testing in a contextual fear-conditioning test.

Exposure to 56Fe-Particle Radiation Accelerates Electrophysiological Alterations in the Hippocampus of APP23 Transgenic Mice

Abstract An unavoidable complication of space travel is exposure to high-charge, high-energy (HZE) particles. In animal studies, exposure of the CNS to HZE-particle radiation leads to neurological alterations similar to those seen in aging or Alzheimers disease. In this study we examined whether HZE-particle radiation accelerated the age-related neuronal dysfunction that was previously described in transgenic mice overexpressing human amyloid precursor protein (APP). These APP23 transgenic mice exhibit age-related behavioral abnormalities and deficits in synaptic transmission. We exposed 7-week-old APP23 transgenic males to brain-only 56Fe-particle radiation (600 MeV/nucleon; 1, 2, 4 Gy) and recorded synaptic transmission in hippocampal slices at 2, 6, 9, 14 and 18–24 months. We stimulated Schaeffer collaterals and recorded field excitatory postsynaptic potentials (fEPSP) and population spikes (PS) in CA1 neurons. Radiation accelerated the onset of age-related fEPSP decrements recorded at the PS threshold from 14 months of age to 9 months and reduced synaptic efficacy. At 9 months, radiation also reduced PS amplitudes. At 6 months, we observed a temporary deficit in paired-pulse inhibition of the PS at 2 Gy. Radiation did not significantly affect survival of APP23 transgenic mice. We conclude that irradiation of the brain with HZE particles accelerates Alzheimers disease-related neurological deficits.

Radiation-Induced Alterations in Synaptic Neurotransmission of Dentate Granule Cells Depend on the Dose and Species of Charged Particles

The evaluation of potential health risks associated with neuronal exposure to space radiation is critical for future long duration space travel. The purpose of this study was to evaluate and compare the effects of low-dose proton and high-energy charged particle (HZE) radiation on electrophysiological parameters of the granule cells in the dentate gyrus (DG) of the hippocampus and its associated functional consequences. We examined excitatory and inhibitory neurotransmission in DG granule cells (DGCs) in dorsal hippocampal slices from male C57BL/6 mice at 3 months after whole body irradiation with accelerated proton, silicon or iron particles. Multielectrode arrays were used to investigate evoked field synaptic potentials, an extracellular measurement of synaptic excitability in the perforant path to DG synaptic pathway. Whole-cell patch clamp recordings were used to measure miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in DGCs. Exposure to proton radiation increased synaptic excitability and produced dose-dependent decreases in amplitude and charge transfer of mIPSCs, without affecting the expression of γ-aminobutyric acid type A receptor α2, β3 and γ2 subunits determined by Western blotting. Exposure to silicon radiation had no significant effects on synaptic excitability, mEPSCs or mIPSCs of DGCs. Exposure to iron radiation had no effect on synaptic excitability and mIPSCs, but significantly increased mEPSC frequency at 1 Gy, without changes in mEPSC kinetics, suggesting a presynaptic mechanism. Overall, the data suggest that proton and HZE exposure results in radiation dose- and species-dependent long-lasting alterations in synaptic neurotransmission, which could cause radiation-induced impairment of hippocampal-dependent cognitive functions.

Proton Radiation Alters Intrinsic and Synaptic Properties of CA1 Pyramidal Neurons of the Mouse Hippocampus

High-energy protons constitute at least 85% of the fluence of energetic ions in interplanetary space. Although protons are only sparsely ionizing compared to higher atomic mass ions, they nevertheless significantly contribute to the delivered dose received by astronauts that can potentially affect central nervous system function at high fluence, especially during prolonged deep space missions such as to Mars. Here we report on the long-term effects of 1 Gy proton irradiation on electrophysiological properties of CA1 pyramidal neurons in the mouse hippocampus. The hippocampus is a key structure for the formation of long-term episodic memory, for spatial orientation and for information processing in a number of other cognitive tasks. CA1 pyramidal neurons form the last and critical relay point in the trisynaptic circuit of the hippocampal principal neurons through which information is processed before being transferred to other brain areas. Proper functioning of CA1 pyramidal neurons is crucial for hippocampus-dependent tasks. Using the patch-clamp technique to evaluate chronic effects of 1 Gy proton irradiation on CA1 pyramidal neurons, we found that the intrinsic membrane properties of CA1 pyramidal neurons were chronically altered at 3 months postirradiation, resulting in a hyperpolarization of the resting membrane potential (VRMP) and a decrease in input resistance (Rin). These small but significant alterations in intrinsic properties decreased the excitability of CA1 pyramidal neurons, and had a dramatic impact on network function in a computational model of the CA1 microcircuit. We also found that proton-radiation exposure upregulated the persistent Na+ current (INaP) and increased the rate of miniature excitatory postsynaptic currents (mEPSCs). Both the INaP and the heightened rate of mEPSCs contribute to neuronal depolarization and excitation, and at least in part, could compensate for the reduced excitability resulting from the radiation effects on the VRMP and the Rin. These results show long-term alterations in the intrinsic properties of CA1 pyramidal cells after realistic, low-dose proton irradiation.

Diffusion Tensor Imaging Detected Optic Nerve Injury Correlates with Decreased Compound Action Potentials after Murine Retinal Ischemia

PURPOSE This study evaluated the function of mouse optic nerves after transient retinal ischemia using in vitro electrophysiologic recordings of compound action potentials (CAPs) correlated with diffusion tensor imaging (DTI) injury markers with confirmation by immunohistochemistry-determined pathology. METHODS Retinal ischemia was induced in 7- to 8-week-old female C57BL/6 mice by elevating intraocular pressure to 110 mm Hg for 60 minutes. At 3 and 7 days after retinal ischemia, optic nerves were removed for CAP measurements. The CAP amplitude was recorded using suction electrodes in isolated control and injured optic nerves followed by ex vivo DTI evaluation. After DTI, optic nerves were embedded in paraffin and cut for immunohistochemical analyses. RESULTS Consistent with previous in vivo DTI measurements, a 25% decrease in axial diffusivity with normal radial diffusivity was seen at 3 days after retinal ischemia, suggesting axonal injury without myelin damage. At 7 days, there was no additional change in axial diffusivity compared with that at 3 days, but radial diffusivity significantly increased by 50%, suggestive of significant myelin damage due to sustained axonal injury. The relative anisotropy (RA) progressively decreased after retinal ischemia when compared with that of the controls. The CAP amplitude in injured nerves also progressively decreased after retinal ischemia, which correlated with the reduced RA (r = 0.80). CONCLUSIONS This study suggests that CAP amplitude reflects both axonal and myelin integrity and RA is an optimal parameter for functional assessment compared with axial or radial diffusivity alone in murine optic nerves after retinal ischemia.

Brain irradiation improves focal cerebral ischemia recovery in aged rats

BACKGROUND Studies have shown that aging is a significant factor in worsening stroke outcomes. While many mechanisms may aggravate brain injury in the elderly, one such potential system may involve increased glial proliferation in the aged stroke patient that could result in increased scar formation. We hypothesized that in aged rats a single brain-only exposure to a low radiation dose prior to focal brain ischemia would reduce glial proliferation and confer a long-term neuroprotective effect. METHODS Brain-only proton irradiation (8 Gy) was performed ten days prior to middle cerebral artery occlusion (MCAO) in aged male rats. Magnetic resonance imaging (MRI) was undertaken in naive, radiation-only (Rad), MCAO, and MCAO+Rad groups at 2, 14 and 28 days post-stroke followed by immunohistochemistry (day 28). RESULTS Ischemic lesion volume in MCAO+Rad group was decreased by 50.7% with an accelerated temporal reduction in peri-lesional brain edema and increased water mobility within the ischemic core (39.8%) compared to MCAO-only rats. In the peri-lesional brain region of MCAO+Rad rats there was a decreased scar formation (49%, glial fibrillary acidic protein), brain tissue sclerosis (30%, aquaporin-4) and necrosis/apoptosis (58%, TUNEL positive cells) compared to those in MCAO animals. CONCLUSION In aged animals a single exposure to brain-only radiation prior to focal cerebral ischemia is neuroprotective as it prevents glial hyperproliferation, progressive brain tissue sclerosis and reduces the apoptosis/necrosis in the peri-lesional region. Decreased lesion volume is in agreement with accelerated reduction of brain edema in these animals.