Anat Boehm-Cagan

Reversal of apoE4-Driven Brain Pathology and Behavioral Deficits by Bexarotene

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimers disease (AD), is less lipidated than its corresponding AD-benign form, apoE3, and it has been suggested that the pathological effects of apoE4 are mediated by lipid-related mechanisms. ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1, respectively) are the most important apoE-lipidating proteins. The expression of these proteins, as well as that of apoE, is controlled by the transcription regulation retinoid X receptor (RXR)–liver X receptor (LXR) system. In the present study, we investigated the effects of the RXR agonist bexarotene on mRNA and protein levels of apoE, ABCA1, and ABCG1 in young, naive apoE3- and apoE4-targeted replacement mice and assessed the extent to which this reverses the apoE4-driven pathological phenotype. This investigation reveled that bexarotene increases the mRNA and protein levels of ABCA1 and ABCG1 in hippocampal neurons, but has no effect on the corresponding levels of apoE. These findings were associated with reversal of the lipidation deficiency of apoE4 and of the cognitive impairments of apoE4 mice in several tests. Furthermore, bexarotene reversed the apoE4-driven accumulation of Aβ42 and hyperphosphorylated tau in hippocampal neurons, as well as the apoE4-induced reduction in the levels of the presynaptic marker vesicular glutamatergic transporter 1 (VGluT1). In conclusion, the results show that treatment of apoE4 mice with the RXR agonist bexarotene reverses the apoE4-induced cognitive and neuronal impairments in vivo and suggest that this is due to reversal of the lipidation deficiency of apoE4. This puts forward the possibility that RXR activation and increased levels of ABCA1 and ABCG1 could be useful in the treatment of human apoE4 carriers.

ApoE4 induces Aβ42, tau, and neuronal pathology in the hippocampus of young targeted replacement apoE4 mice

BackgroundRecent findings suggest that the pathological effects of apoE4, the most prevalent genetic risk factor for Alzheimer’s disease (AD), start many years before the onset of the disease and are already detectable at a young age. In the present study we investigated the extent to which such pathological and cognitive impairments also occur in young apoE4 mice.ResultsThis study revealed that the levels of the presynaptic glutamatergic vesicular transporter, VGlut, in the CA3, CA1, and DG hippocampal subfields were lower in hippocampal neurons of young (4-month-old) apoE4-targeted replacement mice than in those of the apoE3 mice. In contrast, the corresponding inhibitory GABAergic nerve terminals and perikarya were not affected by apoE4.This synaptic effect was associated with hyperphosphorylation of tau in these neurons. In addition, apoE4 increased the accumulation of neuronal Aβ42 and induced mitochondrial changes, both of which were specifically pronounced in CA3 neurons. Spatial navigation behavioral studies revealed that these hippocampal pathological effects of apoE4 are associated with corresponding behavioral impairments. Time-course studies revealed that the effects of apoE4 on tau hyperphosphorylation and the mitochondria were already apparent at the age of 1 month and that the apoE4-driven accumulation of neuronal Aβ and reduced VGlut levels evolve later and are apparent at the age of 2–4 months. Furthermore, the levels of tau phosphorylation decrease in apoE3 mice and increase in apoE4 mice between 1 and 4 months, whereas the levels of Aβ42 decrease in apoE3 mice and are not affected in apoE4 mice over the same time period.ConclusionsThese findings show that apoE4 stimulates the accumulation of Aβ42 and hyperphosphorylated tau and reduces the levels of VGlut in hippocampal neurons of young apoE4-targeted replacement mice and that these neurochemical effects are associated with cognitive impairments. This model is not associated with hypothesis-driven mechanistic manipulations and is thus most suitable for unbiased studies of the mechanisms underlying the pathological effects of apoE4.

Hippocampus-Related Cognitive Impairments in Young apoE4 Targeted Replacement Mice

We presently investigated the effects of apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimers disease, on the cognitive performance of young targeted replacement apoE4 mice. We revealed that these mice were impaired in the object recognition and Morris water maze tests, both of which are associated with hippocampal learning and memory, relative to that of the apoE3 mice. These results are consistent with previous histological and biochemical findings that hippocampal neurons are specifically affected by apoE4. The suggestion that the behavioral impairments of the apoE4 mice are related to the hippocampal neuropathology of these mice is further supported by the fear conditioning test. This test revealed that the performance of the apoE4 mice in the contextual component, which is hippocampus related, was impaired, whereas their cued test response, which is amygdala driven, was not. The stress levels of the apoE4 and apoE3 mice, as unraveled by the light/dark anxiety test, were similar, suggesting that the observed cognitive impairments of the apoE4 mice are not related to differences in the basal anxiety levels of these mice. In conclusion, the present study shows that young apoE4 targeted replacement mice are impaired in numerous hippocampus-related learning and memory tasks.

ABCA1 Agonist Reverses the ApoE4-Driven Cognitive and Brain Pathologies

The allele ɛ4 of apolipoprotein E (apoE4) is the most prevalent genetic risk factor for Alzheimers disease (AD) and is therefore a promising therapeutic target. Human and animal model studies suggest that apoE4 is hypolipidated; accordingly, we have previously shown that the retinoid X receptor (RXR) agonist bexarotene upregulates ABCA1, the main apoE-lipidating protein, resulting in increased lipidation of apoE4, and the subsequent reversal of the pathological effects of apoE4, namely: accumulation of Aβ42 and hyperphosphorylated tau, as well as reduction in the levels of synaptic markers and cognitive deficits. Since the RXR system has numerous other targets, it is important to devise the means of activating ABCA1 selectively. We presently utilized CS-6253, a peptide shown to directly activate ABCA1 in vitro, and examined the extent to which it can affect the degree of lipidation of apoE4 in vivo and counteract the associated brain and behavioral pathologies. This revealed that treatment of young apoE4-targeted replacement mice with CS-6253 increases the lipidation of apoE4. This was associated with a reversal of the apoE4-driven Aβ42 accumulation and tau hyperphosphorylation in hippocampal neurons, as well as of the synaptic impairments and cognitive deficits. These findings suggest that the pathological effects of apoE4 in vivo are associated with decreased activation of ABCA1 and impaired lipidation of apoE4 and that the downstream brain-related pathology and cognitive deficits can be counteracted by treatment with the ABCA1 agonist CS-6253. These findings have important clinical ramifications and put forward ABCA1 as a promising target for apoE4-related treatment of AD.

Involvement of the Apoer2 and Lrp1 receptors in mediating the pathological effects of ApoE4 in vivo

This study investigated the possible role of the ApoE receptors Lrp1 and Apoer2 in mediating the pathological effects of ApoE4 in ApoE-targeted-replacement mice expressing either the human ApoE3 or ApoE4 allele. In this study we show that activation of the amyloid cascade by inhibition of the Aβ-degrading enzyme neprilysin results in upregulation of the ApoE receptor Lrp1 in the CA1 hippocampal neurons of 4-month-old ApoE4 mice, but not in the corresponding ApoE3 or ApoE-deficient (KO) mice. These results are in accordance with the previous findings that activation of the amyloid cascade induces Aβ accumulation in the CA1 neurons of ApoE4 mice, but not in ApoE3 or ApoE-KO mice. This suggests that the apoE4-driven elevation of Lrp1 is mediated via a gain of function mechanism and may play a role in mediating the effects of ApoE4 on Aβ. In contrast, no changes were observed in the levels of the corresponding Apoer2 receptor following the neprilysin inhibition. The ApoE receptors of naive ApoE4 mice were also affected differentially and isoform specifically by ApoE4. However, under these conditions, the effect was an ApoE4-driven reduction in the levels of Apoer2 in CA1 and CA3 pyramidal neurons, whereas the levels of Lrp1 were not affected. RT-PCR measurements revealed that the levels of Apoer2 and Lrp1 mRNA in the hippocampus of naïve and neprilysin-inhibited mice were not affected by ApoE4, suggesting that the observed effects of ApoE4 on the levels of these receptors is posttranscriptional. In conclusion, this study shows that the levels of hippocampal ApoE receptors Lrp1 and Apoer2 in vivo are affected isoform specifically by ApoE4 and that the type of receptor affected is context dependent.

Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimers disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.

Reversal of ApoE4-Driven Brain Pathology by Vascular Endothelial Growth Factor Treatment

Apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for Alzheimers disease (AD), is associated with increased neurodegeneration and vascular impairments. Vascular endothelial growth factor (VEGF), originally described as a key angiogenic factor, has recently been shown to play a crucial role in the nervous system. The objective of this research is to examine the role of VEGF in mediating the apoE4-driven pathologies. We show that hippocampal VEGF levels are lower in apoE4 targeted replacement mice compared to the corresponding apoE3 mice. This effect was accompanied by a specific decrease in both VEGF receptor-2 and HIF1-α. We next set to examine whether upregulation of VEGF can reverse apoE4-driven pathologies, namely the accumulation of hyperphosphorylated tau (AT8) and Aβ42, and reduced levels of the pre-synaptic marker, VGluT1, and of the ApoE receptor, ApoER2. This was first performed utilizing intra-hippocampal injection of VEGF-expressing-lentivirus (LV-VEGF). This revealed that LV-VEGF treatment reversed the apoE4-driven cognitive deficits and synaptic pathologies. The levels of Aβ42 and AT8, however, were increased in apoE3 mice, masking any potential effects of this treatment on the apoE4 mice. Follow-up experiments utilizing VEGF-expressing adeno-associated-virus (AAV-VEGF), which expresses VEGF specifically under the GFAP astrocytic promoter, prevented this effects on apoE3 mice, and reversed the apoE4-related increase in Aβ42 and AT8. Taken together, these results suggest that apoE4-driven pathologies are mediated by a VEGF-dependent pathway, resulting in cognitive impairments and brain pathology. These animal model findings suggest that the VEGF system is a promising target for the treatment of apoE4 carriers in AD.

The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

Alzheimers disease (AD) and synucleinopathies share common pathological mechanisms. Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for AD, also increases the risk for dementia in pure synucleinopathies. We presently examined the effects of α‐synuclein deficiency (α‐syn−/−) and sex on apoE4‐driven pathologies.

THE NOVEL APOE4 THERAPEUTIC CS6253 PENETRATES THE BLOOD BRAIN BARRIER, UPREGULATES THE ABCA1 TRANSPORTER AND PREVENTS ALZHEIMER’S DISEASE PATHOGENESIS

neuronal cells (lipid accumulations). Lipid metabolism is fundamental for brain development and function, but its roles in normal and pathological neural stem cell (NSC) regulation remain largely unexplored. Moreover, while genetic, biochemical and biomarker studies have made correlative links between abnormal lipid metabolism and AD, further advances have been hindered by technical limitations in the ability to localize, identify, and decipher the biological impacts of dysregulated brain lipids. Methods: We used a multidisciplinary approach including mass spectrometry, microarray, in vitro and in vivo stem cell assays, and both mouse and human samples in this study. Results:We localized prominent lipid accumulations to the ependymal cells that form the brain-cerebrospinal fluid (CSF) interface in both AD patients and the 3xTg-AD mouse model. We identified the accumulating lipids as 12 specific triglycerides that are particularly enriched with oleic acid sidechains, and show that infusion of oleic acid into the lateral ventricle of wild-type mice is sufficient to recapitulate the AD-associated triglyceride phenotype. Screening of the plasma and CSF of 3xTg-AD versus control mice showed no differences in circulating levels of AD-associated triglycerides or their component fatty acids, suggesting brain-specific alterations in lipid metabolism. Interestingly, microarray analyses of the subventricular zone proper revealed extensive alterations in lipid-related and neurogenesis/neural stem cell gene expression. In wild-type mice, locally increasing oleic acid was sufficient to recapitulate the AD-associated ependymal triglyceride phenotype and inhibit neural stem cell proliferation. Moreover, inhibiting the rate-limiting enzyme of oleic acid synthesis completely rescued proliferative defects in both the lateral ventricle and hippocampal adult neurogenic niches of 3xTg-AD mice. Conclusions: These studies support a novel pathogenic mechanism in which AD-induced perturbation of fatty acid metabolism within niche ependymal cells suppresses the homeostatic and regenerative functions of adult NSCs.

COGPEP™, AN APOE-DERIVED ABCA1 AGONIST PEPTIDE, REVERSES ALZHEIMER'S DISEASE PHENOTYPE IN HUMAN APOE4 REPLACEMENT MICE

human APOE3 or APOE4) were treated with Bex, LG268 (a more selective RXR agonist), or vehicle control in 3 treatment paradigms: T1) 7-day oral gavage (5.75-6M); T2) 7-day hydrogel (5.75-6M); and T3) 30-day hydrogel (5-6M). Hydrogel provides a steady dosage of drug throughout the awake period of the mice. Brains were harvested, dissected, and homogenized by 3-step serial extraction.Results: In brain regions with lowAb levels at treatment, RXR agonists did not change soluble levels of Ab 42 and oAb in E3FAD or E4FAD mice. In brain regions with intermediate Ab levels, RXR agonist treatment induced an increase in soluble Ab 42 and oAb levels in E3FAD and E4FADmice. However, in the hippocampus of E4FADmice, with high Ab levels at treatment, RXR agonists induced a decrease in soluble Ab 42 and oAb levels and an increase in synaptic proteins. Importantly, total apoE levels were unaffected for all treatment groups, suggesting an alternate mechanism of action for RXR agonists. Our data further demonstrate that the beneficial effects of RXR agonists in E4FAD mice are mediated via: increased ABCA1 and ABCG1 expression, increased apoE4 association with lipoproteins, increased apoE/Ab complex levels, reduced oAb levels and enhanced synaptic viability. Conclusions: Collectively, our data demonstrate that RXR agonist efficacy is determined by the levels of Ab pathology at time of treatment, exhibiting no effect, or even an increase the levels of neurotoxic Ab in prevention paradigms where Ab levels are likely sub-pathological. However, in later stages of AD, RXR agonists may address the loss of function associated with APOE4 by increasing apoE4 lipidation and apoE4/Ab complex formation. Future studies are necessary to determine whether this pathway is relevant for APOE3 carriers with high Ab pathology, or if RXR agonists are an APOE4specific AD therapeutic.