Tina Bilousova

Minocycline promotes dendritic spine maturation and improves behavioural performance in the fragile X mouse model

Background: Fragile X syndrome (FXS) is the most common single gene inherited form of mental retardation, with behaviours at the extreme of the autistic spectrum. Subjects with FXS and fragile X mental retardation gene knock out (Fmr1 KO) mice, an animal model for FXS, have been shown to exhibit defects in dendritic spine maturation that may underlie cognitive and behavioural abnormalities in FXS. Minocycline is a tetracycline analogue that has been used in clinical trials for stroke, multiple sclerosis and several neurodegenerative conditions. Methods: We evaluated the effects of minocycline on dendritic spine development in the hippocampus of young Fmr1 KO mice, and in primary cultures of hippocampal neurons isolated from those mice. Cognitive effects of minocycline in young WT and Fmr1 KO mice were also evaluated using established behavioural tests for general cognition, activity and anxiety. Results: Our studies demonstrate that minocycline promotes dendritic spine maturation both in cultures and in vivo. The beneficial effects of minocycline on dendritic spine morphology are also accompanied by changes in the behavioural performance of 3-week-old Fmr1 KO mice. Minocycline treated Fmr1 KO mice show less anxiety in the elevated plus maze and more strategic exploratory behaviour in the Y maze as compared to untreated Fmr1 KO mice. Our data suggest that these effects of minocycline may relate to its inhibitory action on MMP-9 expression and activity, which are higher in the hippocampus of Fmr1 KO mice. Conclusion: These findings establish minocycline as a promising therapeutic for the treatment of fragile X mental retardation.

A Rose by Any Other Name? The Potential Consequences of Microglial Heterogeneity During CNS Health and Disease

SummaryMicroglial activation and macrophage infiltration into the CNS are common features of CNS autoimmune disease and of chronic neurodegenerative diseases. Because these cells largely express an overlapping set of common macrophage markers, it has been difficult to separate their respective contributions to disease onset and progression. This problem is further confounded by the many types of macrophages that have been termed microglia. Several approaches, ranging from molecular profiling of isolated cells to the generation of irradiation chimeric rodent models, are now beginning to generate rudimentary definitions distinguishing the various types of microglia and macrophages found within the CNS and the potential roles that these cells may play in health and disease.

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples

Background: An ELISA was developed to determine the role of apoE/Aβ on soluble Aβ accumulation. Results: In AD transgenic mouse brain and human synaptosomes and CSF, levels of soluble apoE/Aβ are lower and oligomeric Aβ levels are higher with APOE4 and AD. Conclusion: Isoform-specific apoE/Aβ levels modulate soluble oligomeric Aβ levels. Significance: ApoE/Aβ and oligomeric Aβ represent a mechanistic approach to AD biomarkers. Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-β (Aβ) levels. Evidence suggests physical interactions between apoE and Aβ are partially responsible for these functional effects. However, the apoE/Aβ complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aβ in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aβ and an increase in soluble Aβ, specifically oligomeric Aβ (oAβ), are associated with APOE4 and AD. Previously, soluble Aβ42 and oAβ levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aβ levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aβ levels isoform-specifically modulate soluble oAβ clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aβ levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aβ levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aβ42 levels decreased in AD patients compared with controls, oAβ levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aβ modulates oAβ levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.

Pre-synaptic C-terminal truncated tau is released from cortical synapses in Alzheimer's disease

The microtubule‐associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimers disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal‐specific antibodies show that in many synaptosome samples tau lacks a C‐terminus. Flow cytometry experiments to quantify the extent of C‐terminal truncation reveal that only 15–25% of synaptosomes are positive for intact C‐terminal tau. Potassium‐induced depolarization demonstrates release of tau and tau fragments from pre‐synaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well‐positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the pre‐synaptic compartment in AD.

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases.

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.

AD synapses contain abundant Aβ monomer and multiple soluble oligomers, including a 56 kDa assembly

Much evidence indicates that soluble amyloid beta (Aβ) oligomers are key mediators of early cognitive loss, but the localization and key peptide species remain unclear. We have used flow cytometry analysis to demonstrate that surviving Alzheimers disease (AD) synapses accumulate both Aβ and phosphorylated tau (p-tau). The present experiments use peptide-specific X-map assays and Western blot analyses to identify the Aβ peptide species in synaptosome-enriched samples from normal human subjects, neurologic controls, and AD cases. Aβ40 peptide levels did not vary, but both Aβ42 and Aβ oligomers were increased in soluble AD extracts, with oligomer levels 20-fold higher in aqueous compared with detergent extracts. In Western blot analysis, a ladder of sodium dodecyl sulfate (SDS)-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kDa and larger. Multiple oligomers, including monomer, small oligomers, a 56-kDa assembly, and amyloid precursor protein (APP) were correlated with the Aβ level measured in flow cytometry-purified synaptosomes. These results suggest that multiple amyloid precursor protein processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction.

Apolipoprotein E level and cholesterol are associated with reduced synaptic amyloid beta in Alzheimer's disease and apoE TR mouse cortex.

The apolipoprotein E4 allele (APOE4) contributes to Alzheimer’s disease (AD) risk and APOE2 is protective, but the relevant cellular mechanisms are unknown. We have used flow cytometry analysis to measure apolipoprotein E (apoE) and amyloid beta peptide (Aβ) levels in large populations of synaptic terminals from AD and aged cognitively normal controls, and demonstrate that modest but significant increases in soluble apoE levels accompany elevated Aβ in AD cortical synapses and in an APP/PS1 rat model of AD. Dual labeling experiments document co-localization of apoE and Aβ in individual synapses with concentration of Aβ in a small population of apoE-positive synapses in both AD and controls. Consistent with a clearance role, the apoE level was higher in Aβ-positive synapses in control cases. In aged targeted replacement mice expressing human apoE, apoE2/4 synaptic terminals demonstrated the highest level of apoE and the lowest level of Aβ compared to apoE3/3 and apoE4/4 lines. In apoE2/4 terminals, the pattern of immunolabeling for apoE and Aβ closely resembled the pattern in human control cases, and elevated apoE was accompanied by elevated free cholesterol in apoE2/4 synaptic terminals. These results are consistent with a role for APOE in Aβ clearance in AD synapses, and suggest that optimal lipidation of apoE2 compared to E3 and E4 makes an important contribution to Aβ clearance and synaptic function.

Major histocompatibility complex class I molecules modulate embryonic neuritogenesis and neuronal polarization

We studied cultured hippocampal neurons from embryonic wildtype, major histocompatibility complex class I (MHCI) heavy chain-deficient (K(b)D(b)-/-) and NSE-D(b) (which have elevated neuronal MHCI expression) C57BL/6 mice. K(b)D(b)-/- neurons displayed slower neuritogenesis and establishment of polarity, while NSE-D(b) neurons had faster neurite outgrowth, more primary neurites, and tended to have accelerated polarization. Additional studies with ß2M-/- neurons, exogenous ß2M, and a self-MHCI monomer suggest that free heavy chain cis interactions with other surface molecules can promote neuritogenesis while tripartite MHCI interactions with classical MHCI receptors can inhibit axon outgrowth. Together with the results of others, MHCI appears to differentially modulate neuritogenesis and synaptogenesis.

Dietary DHA supplementation in an APP/PS1 transgenic rat model of AD reduces behavioral and Aβ pathology and modulates Aβ oligomerization.

Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimers disease (AD). These effects have been postulated to arise from DHAs pleiotropic effects on AD pathophysiology, including its effects on β-amyloid (Aβ) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aβ oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aβ indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aβ plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aβ oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aβ oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aβ oligomers and decreased prefibrillar (i.e. putatively more toxic) Aβ oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aβ aggregation by stabilizing soluble fibrillar Aβ oligomers and thus reduce the formation of both Aβ plaques and prefibrillar Aβ oligomers. However, since fibrillar Aβ oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aβ oligomer levels for more effective prevention of AD in clinical settings.

Enhanced neuronal expression of major histocompatibility complex class I leads to aberrations in neurodevelopment and neurorepair.

Mice deficient in classical major histocompatibility complex class I (MHCI) have aberrations in neurodevelopment. The consequences of upregulated neuronal MHCI expression have not been examined. We found that transgenic C57Bl/6 mice that are engineered to express higher levels of self-D(b) on their CNS neurons have alterations in their hippocampal morphology and retinogeniculate projections, as well as impaired neurorepair responses. Thus, enhanced neuronal classical MHCI expression can lead to aberrations in neural circuitry and neurorepair. These findings complement a growing body of knowledge concerning the neurobiological activities of MHCI and may have potential clinical relevance.