Pathological stress granules in Alzheimer's disease.

A feature of neurodegenerative disease is the accumulation of insoluble protein aggregates in the brain. In some conditions, including Amyotrophic Lateral Sclerosis and Frontotemporal lobar degeneration, the primary aggregating entities are RNA binding proteins. Through regulated prion-like assembly, RNA binding proteins serve many functions in RNA metabolism that are essential for the healthy maintenance of cells of the central nervous system. Those RNA binding proteins that are the core nucleating factors of stress granules (SGs), including TIA-1, TIAR, TTP and G3BP1, are also found in the pathological lesions of other neurological conditions, such as Alzheimer's disease, where the hallmark aggregating protein is not an RNA binding protein. This discovery suggests that the regulated cellular pathway, which utilizes assembly of RNA binding proteins to package and silence mRNAs during stress, may be integral in the aberrant pathological protein aggregation that occurs in numerous neurodegenerative conditions.

A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity.

Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients.

APOE modulates the effect of estrogen therapy on Aß accumulation EFAD-Tg mice.

The post-menopausal loss of estrogen is key in the increased incidence of Alzheimer's disease (AD) in women. However, estrogen therapy (ET) clinical trials have produced conflicting results. The APOE gene of apolipoprotein E (apoE) likely modulates the effects of ET in AD. APOE4 is the greatest genetic risk factor for AD, increasing risk up to 15-fold compared with APOE3, and the negative effect of APOE4 on AD risk and neuropathology is greater in women than men. The interactive effects of APOE and ET may converge on modulation of amyloid-beta (Aß) levels, as independently both the loss of estrogen and APOE4 increases Aß accumulation. Thus, in this study, 3-month old female EFAD mice (5XFAD mice crossed with apoE-targeted replacement mice), which express increased levels of Aß42 and human APOE were ovariectomized and treated for 3 months with either 17-ß estradiol (OVX(ET+), 0.25mg total) or vehicle control (OVX(ET-)) and the effects on Aß accumulation were determined. Compared to the OVX(ET-) cohort, in the OVX(ET+) cohort, extracellular amyloid and Aß deposition in the hippocampus and cortex were decreased with APOE2 and APOE3, but were increased with APOE4 by IHC. Biochemical analysis demonstrated increased total and insoluble Aß levels with APOE4, and decreased soluble Aß42 levels with both APOE3 and APOE4, after ET. These data suggest that ET administered at menopause may benefit APOE4 negative women by decreasing extracellular and soluble Aß42. However, for APOE4 carriers, the efficacy of ET will be dependent on the relative impact of extracellular and soluble Aß on AD-induced neurodegeneration.

Levels of soluble apolipoprotein E/amyloid-ß (Aß) complex are reduced and oligomeric Aß increased with APOE4 and Alzheimer disease in a transgenic mouse model and human samples.

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-ß (Aß) levels. Evidence suggests physical interactions between apoE and Aß are partially responsible for these functional effects. However, the apoE/Aß 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/Aß in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aß and an increase in soluble Aß, specifically oligomeric Aß (oAß), are associated with APOE4 and AD. Previously, soluble Aß42 and oAß 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/Aß levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aß levels isoform-specifically modulate soluble oAß clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aß levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aß levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aß42 levels decreased in AD patients compared with controls, oAß levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aß modulates oAß levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.

APOE4-specific changes in Aß accumulation in a new transgenic mouse model of Alzheimer disease.

APOE4 is the greatest risk factor for Alzheimer disease (AD) and synergistic effects with amyloid-ß peptide (Aß) suggest interactions among apoE isoforms and different forms of Aß accumulation. However, it remains unclear how the APOE genotype affects plaque morphology, intraneuronal Aß, soluble Aß42, and oligomeric Aß (oAß), particularly in vivo. As the introduction of human APOE significantly delays amyloid deposition in transgenic mice expressing familial AD (FAD) mutations (FAD-Tg), 5xFAD-Tg mice, which exhibit amyloid deposition by age 2 months, were crossed with apoE-targeted replacement mice to produce the new EFAD-Tg mice. Compared with 5xFAD mice, Aß deposition was delayed by ∼4 months in the EFAD mice, allowing detection of early changes in Aß accumulation from 2-6 months. Although plaque deposition is generally greater in E4FAD mice, E2/E3FAD mice have significantly more diffuse and E4FAD more compact plaques. As a first report in FAD-Tg mice, the APOE genotypes had no effect on intraneuronal Aß accumulation in EFAD mice. In E4FAD mice, total apoE levels were lower and total Aß levels higher than in E2FAD and E3FAD mice. Profiles from sequential three-step extractions (TBS, detergent, and formic acid) demonstrated that the lower level of total apoE4 is reflected only in the detergent-soluble fraction, indicating that less apoE4 is lipoprotein-associated, and perhaps less lipidated, compared with apoE2 and apoE3. Soluble Aß42 and oAß levels were highest in E4FAD mice, although soluble apoE2, apoE3, and apoE4 levels were comparable, suggesting that the differences in soluble Aß42 and oAß result from functional differences among the apoE isoforms. Thus, APOE differentially regulates multiple aspects of Aß accumulation.

Intraneuronal Aß detection in 5xFAD mice by a new Aß-specific antibody.

BACKGROUND: The form(s) of amyloid-ß peptide (Aß) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aß accumulation is an issue of considerable controversy; even the existence of Aß deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aß. To further address this issue, an anti-Aß antibody was developed (MOAB-2) that specifically detects Aß, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aß in transgenic mice with increased levels of human Aß in 5xFAD and 3xTg mice. RESULTS: MOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aß residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aß residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aß40 and Aß42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunoreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aß, distinct from Aß associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues. CONCLUSIONS: Both intraneuronal Aß accumulation and extracellular Aß deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aß from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aß with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.

Introducing Human APOE into Aß Transgenic Mouse Models.

Apolipoprotein E (apoE) and apoE/amyloid-ß (Aß) transgenic (Tg) mouse models are critical to understanding apoE-isoform effects on Alzheimer's disease risk. Compared to wild type, apoE(-/-) mice exhibit neuronal deficits, similar to apoE4-Tg compared to apoE3-Tg mice, providing a model for Aß-independent apoE effects on neurodegeneration. To determine the effects of apoE on Aß-induced neuropathology, apoE(-/-) mice were crossed with Aß-Tg mice, resulting in a significant delay in plaque deposition. Surprisingly, crossing human-apoE-Tg mice with apoE(-/-)/Aß-Tg mice further delayed plaque deposition, which eventually developed in apoE4/Aß-Tg mice prior to apoE3/Aß-Tg. One approach to address hAPOE-induced temporal delay in Aß pathology is an additional insult, like head injury. Another is crossing human-apoE-Tg mice with Aß-Tg mice that have rapid-onset Aß pathology. For example, because 5xFAD mice develop plaques by 2 months, the prediction is that human-apoE/5xFAD-Tg mice develop plaques around 6 months and 12 months before other human-apoE/Aß-Tg mice. Thus, tractable models for human-apoE/Aß-Tg mice continue to evolve.

Proposed mechanism for lipoprotein remodelling in the brain.

Lipoprotein remodelling in the periphery has been extensively studied. For example, the processing of nascent apoAI particles to cholesterol-loaded HDL lipoproteins during reverse cholesterol transport involves a series of enzymes, transporters in peripheral tissue, as well as other apolipoproteins and lipoproteins. These extensive modifications and interconversions are well defined. Here, we present the hypothesis that a similar process occurs within the blood brain barrier (BBB) via glia-secreted lipid-poor apoE particles undergoing remodelling to become mature central nervous system (CNS) lipoproteins. We further pose several pressing issues and future directions for the study of lipoproteins in the brain.