Low intensity repetitive magnetic stimulation reduces expression of genes related to inflammation and calcium signalling in cultured mouse cortical astrocytes.

Repetitive transcranial magnetic stimulation (rTMS) is a form of non-invasive brain stimulation frequently used to induce neuroplasticity in the brain. Even at low intensities, rTMS has been shown to modulate aspects of neuronal plasticity such as motor learning and structural reorganisation of neural tissue. However, the impact of low intensity rTMS on glial cells such as astrocytes remains largely unknown. This study investigated changes in RNA (qPCR array: 125 selected genes) and protein levels (immunofluorescence) in cultured mouse astrocytes following a single session of low intensity repetitive magnetic stimulation (LI-rMS - 18 mT). Purified neonatal cortical astrocyte cultures were stimulated with either 1Hz (600 pulses), 10Hz (600 or 6000 pulses) or sham (0 pulses) LI-rMS, followed by RNA extraction at 5 h post-stimulation, or fixation at either 5 or 24-h post-stimulation. LI-rMS resulted in a two-to-four-fold downregulation of mRNA transcripts related to calcium signalling (Stim1 and Orai3), inflammatory molecules (Icam1) and neural plasticity (Ncam1). 10Hz reduced expression of Stim1, Orai3, Kcnmb4, and Ncam1 mRNA, whereas 1Hz reduced expression of Icam1 mRNA and signalling-related genes. Protein levels followed a similar pattern for 10Hz rMS, with a significant reduction of STIM1, ORAI3, KCNMB4, and NCAM1 protein compared to sham, but 1Hz increased STIM1 and ORAI3 protein levels relative to sham. These findings demonstrate the ability of 1Hz and 10Hz LI-rMS to modulate specific aspects of astrocytic phenotype, potentially contributing to the known effects of low intensity rTMS on excitability and neuroplasticity.

Subjective memory complaints predict baseline but not future cognitive function over three years: results from the Western Australia Memory Study.

ABSTRACTBackground:This study investigated the characteristics of subjective memory complaints (SMCs) and their association with current and future cognitive functions. METHODS: A cohort of 209 community-dwelling individuals without dementia aged 47-90 years old was recruited for this 3-year study. Participants underwent neuropsychological and clinical assessments annually. Participants were divided into SMCs and non-memory complainers (NMCs) using a single question at baseline and a memory complaints questionnaire following baseline, to evaluate differential patterns of complaints. In addition, comprehensive assessment of memory complaints was undertaken to evaluate whether severity and consistency of complaints differentially predicted cognitive function. RESULTS: SMC and NMC individuals were significantly different on various features of SMCs. Greater overall severity (but not consistency) of complaints was significantly associated with current and future cognitive functioning. CONCLUSIONS: SMC individuals present distinctive features of memory complaints as compared to NMCs. Further, the severity of complaints was a significant predictor of future cognition. However, SMC did not significantly predict change over time in this sample. These findings warrant further research into the specific features of SMCs that may portend subsequent neuropathological and cognitive changes when screening individuals at increased future risk of dementia.

Frequency-specific effects of repetitive magnetic stimulation on primary astrocyte cultures.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique that uses magnetic pulses over the cranium to induce electrical currents in underlying cortical tissue. Although rTMS has shown clinical utility for a number of neurological conditions, we have only limited understanding of how rTMS influences cellular function and cell-cell interactions. OBJECTIVE: In this study, we sought to investigate whether repeated magnetic stimulation (rMS) can influence astrocyte biology in vitro. METHOD: We tested four different rMS frequencies and measured the calcium response in primary neonatal astrocyte cultures. We also tested the effect of rMS on astrocyte migration and proliferation in vitro. We tested 3 to 4 culture replicates and 17 to 34 cells for each rMS frequency (sham, 1 Hz, cTBS, 10 Hz and biomemetic high frequency stimulation - BHFS). RESULTS: Of all frequencies tested, 1 Hz stimulation resulted in a statistically significant rise in intracellular calcium in the cytoplasmic and nuclear compartments of the cultured astrocytes. This calcium rise did not affect migration or proliferation in the scratch assay, though astrocyte hypertrophy was reduced in response to 1 Hz rMS, 24 hours post scratch injury. CONCLUSION: Our results provide preliminary evidence that rMS can influence astrocyte physiology, indicating the potential for a novel mechanism by which rTMS can influence brain activity.

Low intensity rTMS has sex-dependent effects on the local response of glia following a penetrating cortical stab injury.

Repetitive transcranial magnetic stimulation (rTMS), a non-invasive form of brain stimulation, has shown experimental and clinical efficacy in a range of neuromodulatory models, even when delivered at low intensity (i.e. subthreshold for action potential generation). After central nervous system (CNS) injury, studies suggest that reactive astrocytes and microglia can have detrimental but also beneficial effects; thus modulating glial activity, for example through application of rTMS, could potentially be a useful therapeutic tool following neurotrauma. Immunohistochemistry was used to measure the effect of low intensity rTMS (LI-rTMS) on GFAP (astrocyte), IBA1 (microglial), and CS56 (proteoglycan) expression in a unilateral penetrating cortical stab injury model of glial scarring in young adult and aged male and female C57BL6/J mice. Mice received contralateral low frequency, ipsilateral low frequency, ipsilateral high frequency or sham LI-rTMS (4-5mT intensity), for two weeks following injury. There was no significant difference in the overall volume of tissue containing GFAP positive (+) astrocytes, IBA1+ microglia, or proteoglycan expression, between sham and LI-rTMS-treated mice of all ages and sex. Importantly however, the density of GFAP+ astrocytes and IBA1+ microglia immediately adjacent to the injury was significantly reduced following ipsilateral low and high frequency stimulation in adult and aged females (p≤0.05), but was significantly increased in adult and aged males (p≤0.05). LI-rTMS effects were generally of greater magnitude in aged mice compared to young adult mice. These results suggest that sex differences need to be factored into therapeutic rTMS protocols. In particular, more work analyzing frequency and intensity specific effects, especially in relation to age and sex, is required to determine how rTMS can best be used to modify glial reactivity and phenotype following neurotrauma.

Serum high-density lipoprotein is associated with better cognitive function in a cross-sectional study of aging women.

Purpose/Aim of the study: Poor cardiovascular health, including obesity and altered lipid profiles at mid-life, are linked to increased risk of Alzheimer's disease (AD). The biological mechanisms linking cardiovascular health and cognitive function are unclear though are likely to be multifactorial. This study examined the association between various lipoproteins and cognitive functioning in ageing women. MATERIALS AND METHODS: We investigated the relationship between readily available biomarkers (i.e. serum lipoprotein) and cognitive decline in domains associated with increased risk of AD (e.g. episodic verbal memory performance and subjective memory complaint). We report cross-sectional data investigating the relationship between serum total cholesterol, triglycerides, high-density lipoprotein (HDL-C) and low-density lipoprotein with verbal memory and learning ability in 130 women with and without memory complaints (n = 71 and 59, respectively) drawn from a study investigating cognitively healthy Western Australians (average age 62.5 years old). RESULTS: After statistical modelling that controlled for the effects of age, depression and apolipoprotein E genotype, HDL-C was significantly associated with better verbal learning and memory performance, specifically short and long delay-free recalls (F = 3.062; p < .05 and F = 3.2670; p < .05, respectively). CONCLUSION: Our cross-sectional findings suggest that the positive effect of HDL-C on verbal memory may be present much earlier than previously reported and provide further support for the role of HDL-C in healthy brain ageing. Further exploration of the protective effect of HDL-C on cognitive function in ageing is warranted through follow-up, longitudinal studies.

Loss of the RNA-binding protein TACO1 causes late-onset mitochondrial dysfunction in mice.

The recognition and translation of mammalian mitochondrial mRNAs are poorly understood. To gain further insights into these processes in vivo, we characterized mice with a missense mutation that causes loss of the translational activator of cytochrome oxidase subunit I (TACO1). We report that TACO1 is not required for embryonic survival, although the mutant mice have substantially reduced COXI protein, causing an isolated complex IV deficiency. We show that TACO1 specifically binds the mt-Co1 mRNA and is required for translation of COXI through its association with the mitochondrial ribosome. We determined the atomic structure of TACO1, revealing three domains in the shape of a hook with a tunnel between domains 1 and 3. Mutations in the positively charged domain 1 reduce RNA binding by TACO1. The Taco1 mutant mice develop a late-onset visual impairment, motor dysfunction and cardiac hypertrophy and thus provide a useful model for future treatment trials for mitochondrial disease.

Cerebral Glucose Metabolism is Associated with Verbal but not Visual Memory Performance in Community-Dwelling Older Adults.

Increasing evidence suggests that Alzheimer's disease (AD) sufferers show region-specific reductions in cerebral glucose metabolism, as measured by [18F]-fluoro-2-deoxyglucose positron emission tomography (18F-FDG PET). We investigated preclinical disease stage by cross-sectionally examining the association between global cognition, verbal and visual memory, and 18F-FDG PET standardized uptake value ratio (SUVR) in 43 healthy control individuals, subsequently focusing on differences between subjective memory complainers and non-memory complainers. The 18F-FDG PET regions of interest investigated include the hippocampus, amygdala, posterior cingulate, superior parietal, entorhinal cortices, frontal cortex, temporal cortex, and inferior parietal region. In the cohort as a whole, verbal logical memory immediate recall was positively associated with 18F-FDG PET SUVR in both the left hippocampus and right amygdala. There were no associations observed between global cognition, delayed recall in logical memory, or visual reproduction and 18F-FDG PET SUVR. Following stratification of the cohort into subjective memory complainers and non-complainers, verbal logical memory immediate recall was positively associated with 18F-FDG PET SUVR in the right amygdala in those with subjective memory complaints. There were no significant associations observed in non-memory complainers between 18F-FDG PET SUVR in regions of interest and cognitive performance. We observed subjective memory complaint-specific associations between 18F-FDG PET SUVR and immediate verbal memory performance in our cohort, however found no associations between delayed recall of verbal memory performance or visual memory performance. It is here argued that the neural mechanisms underlying verbal and visual memory performance may in fact differ in their pathways, and the characteristic reduction of 18F-FDG PET SUVR observed in this and previous studies likely reflects the pathophysiological changes in specific brain regions that occur in preclinical AD.

Vascular insufficiency, not inflammation, contributes to chronic gliosis in a rat CNS transplantation model.

PURPOSE: There is considerable variability in the extent and nature of the glial response to injury and neurodegeneration. Transplantation of fetal cortical tissue onto the brain of neonatal host rats or mice results in region-specific changes dependent on where the fetal tissue is placed. These changes include chronic astrocytic and microglial gliosis, oxidative stress, and altered metabolism of a number of proteins associated with the pathogenesis of Alzheimer's disease. Such changes are only observed in heterotopic (cortex-to-midbrain) grafts and are not observed in homotopic cortex-to-cortex grafts. We investigated two possible triggers for the region-specific gliosis observed in our transplant model hypothesizing that either i) poor vascularization and lack of blood brain barrier integrity or ii) an inflammatory response initiated by the transplantation process, contributed to establishing chronic pathological changes. METHODS: We analyzed the time course of neovascularization, blood brain barrier permeability and inflammation using a combination of immunohistochemistry, enzyme-linked immunosorbant assay and Evan's blue dye extravasation techniques. RESULTS: Blood brain barrier permeability and altered neovascularization occurred prior to the onset of gliosis in heterotopic grafts. CONCLUSION: These data suggest that ischemic conditions and blood brain barrier damage can be a primary mechanism that initiates chronic gliosis and associated inflammatory changes in central nervous system tissue.

Bone mineral density, adiposity, and cognitive functions.

Cognitive decline and dementia due to Alzheimer's disease (AD) have been associated with genetic, lifestyle, and environmental factors. A number of potentially modifiable risk factors should be taken into account when preventive or ameliorative interventions targeting dementia and its preclinical stages are investigated. Bone mineral density (BMD) and body composition are two such potentially modifiable risk factors, and their association with cognitive decline was investigated in this study. 164 participants, aged 34-87 years old (62.78 ± 9.27), were recruited for this longitudinal study and underwent cognitive and clinical examinations at baseline and after 3 years. Blood samples were collected for apolipoprotein E (APOE) genotyping and dual energy x-ray absorptiometry (DXA) was conducted at the same day as cognitive assessment. Using hierarchical regression analysis, we found that BMD and lean body mass, as measured using DXA were significant predictors of episodic memory. Age, gender, APOE status, and premorbid IQ were controlled for. Specifically, the List A learning from California Verbal Learning Test was significantly associated with BMD and lean mass both at baseline and at follow up assessment. Our findings indicate that there is a significant association between BMD and lean body mass and episodic verbal learning. While the involvement of modifiable lifestyle factors in human cognitive function has been examined in different studies, there is a need for further research to understand the potential underlying mechanisms.

Short-term low intensity PMF does not improve functional or histological outcomes in a rat model of transient focal cerebral ischemia.

Stimulation with pulsed magnetic fields (PMF) is a non-invasive technique that can modulate neural activity and has the potential to facilitate functional recovery and tissue preservation/repair following brain injury. The effect of low intensity (8 mT) PMF on functional recovery and infarct tissue volume was assessed in a middle cerebral artery occlusion model of transient focal ischemia in Spontaneously Hypertensive rats. Rats received a combination of PMF protocols, including high and low frequencies and recovery was monitored over eight days. PMF treatment had no effect on functional recovery or infarct volume. Infarcted tissue accounted for ≈8% of total brain volume, encompassing both cortical and subcortical structures. The microglial and astrocytic response to PMF treatment was monitored and there was no change in glial scarring, however there was increased macrophage infiltration in animals that received chronic high (6-9 Hz) and low (1 Hz) stimulation. There was no effect of PMF on the degree of cell death observed within the ischemic core, with no TUNEL positive cells observed in the non-infarcted tissue. No detrimental side-effects of PMF were observed, indicating that low-intensity PMF may have limited safety concerns for future human and animal studies.

Direct exposure of guinea pig CNS to human luteinizing hormone increases cerebrospinal fluid and cerebral beta amyloid levels.

BACKGROUND/AIMS: Luteinizing hormone (LH) has been shown to alter the metabolism of beta amyloid (Aß), a key protein in Alzheimer's disease (AD) pathogenesis. While LH and components required for LH receptor signalling are present in the brain, their role in the CNS remains unclear. In vitro, LH has been shown to facilitate neurosteroid production and alter Aß metabolism. However, whether LH can directly modulate cerebral Aß levels in vivo has not previously been studied. In this study, we investigated the effect of chronic administration of LH to the guinea pig CNS on cerebral Aß levels. METHODS: Gonadectomised male animals were administered, via cortical placement, either placebo or LH slow-release pellets. At 14 and 28 days after treatment, animals were sacrificed. Brain, plasma and CSF were collected and Aß levels measured via ELISA. Levels of the Aß precursor protein (APP) and the neurosteroidogenic enzyme cytochrome P450 side-chain cleavage enzyme (P450scc) were also assayed. RESULTS: An increase in CSF Aß40 levels was observed 28 days following treatment. These CSF data also reflected changes in Aß40 levels observed in brain homogenates. No change was observed in plasma Aß40 levels but APP and its C-terminal fragments (APP-CTF) were significantly increased in response to LH exposure. Protein expression of P450scc was increased after 28 days of LH exposure, suggesting activation of the LH receptor. CONCLUSION: These data indicate that direct exposure of guinea pig CNS to LH results in altered brain Aß levels, perhaps due to altered APP expression/metabolism.

Blocking the apolipoprotein E/amyloid-ß interaction reduces fibrillar vascular amyloid deposition and cerebral microhemorrhages in TgSwDI mice.

The accumulation of amyloid-ß (Aß) peptides as toxic oligomers, amyloid plaques, and cerebral amyloid angiopathy (CAA) is critical in the pathogenesis of Alzheimer's disease (AD). The binding of Aß peptides to apolipoprotein E (ApoE) plays an important role in modulation of amyloid deposition and clearance. We have shown that blocking the Aß/ApoE interaction with Aß(12-28P), a nontoxic blood-brain-barrier permeable and non-fibrillogenic synthetic peptide, constitutes a novel therapeutic approach for AD by reducing Aß parenchymal deposition. In the present study, we investigate this therapeutic effect on CAA in the transgenic (Tg) AD mice model (TgSwDI), which expresses Swedish (K670N/M671L), Dutch (E693Q)/Iowa (D694N) AßPP mutations. These mice develop abundant CAA beginning at the age of 6 months. Behavioral results show that Aß(12-28P) treated TgSwDI AD mice performed the same as wild-type mice, whereas vehicle treated TgSwDI were impaired in spatial memory. Furthermore, this treatment resulted in a significant reduction of total amyloid burden, especially the fibrillar vascular amyloid burden, which importantly was accompanied by a reduction in microhemorrhages and neuroinflammation. Measurement of Aß levels in the brain homogenate revealed a significant decrease in both the total amount of Aß and Aß oligomer levels in Aß(12-28P) treated TgSwDI mice. These findings suggest that blocking the Aß/ApoE interaction is a highly effective therapeutic approach for vascular amyloid deposition, in contrast to some other therapeutic approaches.

The relationship between memory complaints, perceived quality of life and mental health in apolipoprotein Eepsilon4 carriers and non-carriers.

Apolipoprotein E epsilon4 (APOE-epsilon4) is a major genetic risk factor for Alzheimer's disease. In this study, we addressed the question of whether possession of the APOE-epsilon4 allele results in adverse effects on perceived health-related quality of life (HRQL) and on symptoms of depression and anxiety in people with subjective memory complaints (SMC). 138 healthy, community-dwelling elderly volunteers, aged 52 to 85, were assessed for HRQL, depression, and anxiety. The participants were classified as i) APOE-epsilon4 carriers or ii) non-carriers with a) SMC or b) without memory complaints. The possible interactions of APOE genotype, gender, and SMC on HRQL, depression, and anxiety were investigated statistically. SMC was significantly associated with poorer outcomes on measures of depression, trait anxiety, and mental health. APOE-epsilon4 carriers did not significantly differ from non-carriers on HRQL, depression, and anxiety. However, significant interaction was found between APOE-epsilon4 genotype and SMC on depression. These findings are important from a health perspective and suggest that memory complaints are associated with markers of mental health and quality of life that are independent of possession of the APOE-epsilon4 allele, despite the importance of this polymorphism in the risk of AD and other health problems.

Olfactory dysfunction is associated with subjective memory complaints in community-dwelling elderly individuals.

Olfactory dysfunction has been reported in clinical and preclinical phases of Alzheimer's disease. Subjective memory complaints have been proposed as a potential early indicator for increased risk of Alzheimer's disease, but have also been associated with depression, personality characteristics, and health problems. In this study, we aimed to determine which of these putative markers can predict memory complaints in community-dwelling elderly individuals, focusing on olfactory symptoms. A cohort of 144 elderly volunteers (42 males and 102 females), aged 50 to 86, was recruited from an ongoing longitudinal study. Participants were assessed for olfactory capacities (threshold, discrimination, and identification), subjective memory complaints, depression, and cognitive functions. Subjective memory complaints were significantly associated with olfactory discrimination and identification but not with threshold. Olfactory functions and depressive symptoms were both significantly associated with subjective memory complaints. In addition, memory complainers were significantly worse than non-complainers with respect to olfactory discrimination, identification, and overall olfactory functioning. The findings suggest that olfactory capacity may be a potentially significant biomarker for identifying community-dwelling elderly with memory complaints who are at increased risk for age-related cognitive decline and Alzheimer's disease.

Association of cardiovascular factors and Alzheimer's disease plasma amyloid-beta protein in subjective memory complainers.

A strong link is indicated between cardiovascular disease (CVD) and risk for developing Alzheimer's disease (AD), which may be exacerbated by the major AD genetic risk factor apolipoprotein Eepsilon4 (APOEepsilon4). Since subjective memory complaint (SMC) may potentially be an early indicator for cognitive decline, we examined CVD risk factors in a cohort of SMC. As amyloid-beta (Abeta) is considered to play a central role in AD, we hypothesized that the CVD risk profile (increased LDL, reduced HDL, and increased body fat) would be associated with plasma Abeta levels. We explored this in 198 individuals with and without SMC (average age = 63 years). Correlations between Abeta40 and HDL were observed, which were stronger in non-APOEepsilon4 carriers (rho = -0.315, p < 0.001) and in SMC (rho = -0.322, p = 0.01). There was no relationship between percentage body fat and Abeta40 in this cohort. Age and HDL remained predictive for plasma Abeta40 using multivariate regression analysis. We report a novel negative association between HDL and Abeta, which if demonstrated to be causal has implications for the development of lifestyle interventions and/or novel therapeutics. The relationship between HDL and Abeta and the potential significance of such an association needs to be validated in a larger longitudinal study.

Induction of toll-like receptor 9 signaling as a method for ameliorating Alzheimer's disease-related pathology.

The pathogenesis of Alzheimer's disease (AD) is thought to be related to the accumulation of amyloid beta (Abeta) in amyloid deposits and toxic oligomeric species. Immunomodulation is emerging as an effective means of shifting the equilibrium from Abeta accumulation to clearance; however, excessive cell mediated inflammation and cerebral microhemorrhages are two forms of toxicity which can occur with this approach. Vaccination studies have so far mainly targeted the adaptive immune system. In the present study, we have stimulated the innate immune system via the Toll-like receptor 9 (TLR9) with cytosine-guanosine-containing DNA oligodeoxynucleotides in Tg2576 AD model transgenic mice. This treatment produced a 66% and 80% reduction in the cortical (p = 0.0001) and vascular (p = 0.0039) amyloid burden, respectively, compared with nontreated AD mice. This was in association with significant reductions in Abeta42, Abeta40, and Abeta oligomer levels. We also show that treated Tg mice performed similarly to wild-type mice on a radial arm maze. Our data suggest that stimulation of innate immunity via TLR9 is highly effective at reducing the parenchymal and vascular amyloid burden, along with Abeta oligomers, without apparent toxicity.

Fluoro-2-deoxy-D-glucose (FDG)-PET in APOEepsilon4 carriers in the Australian population.

Apolipoprotein E-epsilon4 (APOEepsilon4) has been associated with increased risk of developing Alzheimer's disease (AD) and regional cerebral glucose hypometabolism, as measured by fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET). We report here preliminary data from studies that aim to determine whether cerebral glucose hypometabolism is observed in APOEepsilon4 positive, cognitively intact individuals between the ages of 50 and 80, and whether there is an additional impact of subjective memory complainer (SMC) status on glucose metabolism determined by NeuroStat analysis. FDG-PET was conducted in 30 community dwelling, APOE-epsilon4 carriers without clinical evidence of dementia and objective cognitive impairment as assessed using a neuropsychological battery. Neurological soft-signs (NSS) were also assessed. Glucose hypometabolism was demonstrated in the anterior and posterior cingulate cortex and in the temporal association cortices in APOEepsilon4 carriers compared to the normative NeuroStat database. This pattern was particularly evident in APOEepsilon4 heterozygous individuals. SMC showed hypometabolism in the aforementioned brain regions, whereas non-SMC showed no significant pattern of glucose hypometabolism. FDG-PET with NeuroStat analysis showed that APOEepsilon4 carriers have mild glucose hypometabolism in areas associated with AD. SMC may be associated with AD-related differences in regional cerebral glucose metabolism. These findings are currently being investigated in a larger group of APOEepsilon4 carriers.

Oxidative stress in a rat model of chronic gliosis.

Alzheimer's disease is a progressive neurodegenerative disorder characterised by abnormal extracellular deposition of a 4 kDa peptide termed beta-amyloid, neuronal loss, oxidative stress and chronic astrocytosis and microgliosis, but how the latter two features contribute to the progression of the disease is poorly understood. We have previously demonstrated in a novel in vivo transplantation model that chronic astro- and microgliosis resulted in molecular pathology similar to that observed in the Alzheimer's disease brain. We now report that these heterotopic, gliotic transplants exhibit prolonged oxidative stress, characterised by lipid peroxidation and protein carbonyl formation. Furthermore, we demonstrate that dietary additives can elevate endogenous anti-oxidant defences and reduce oxidative stress without attenuating astro- and microgliosis. We also show that administration of ibuprofen through the drinking water results in a similar reduction in oxidative stress but with no observable effect on glial reactivity. The present study lends support to the notion that dietary anti-oxidants and non-steroidal anti-inflammatory drugs may be potential preventative agents against some of the pathological processes associated with neurodegenerative disease.