Murat S. Durakoglugil

ApoE4 reduces glutamate receptor function and synaptic plasticity by selectively impairing ApoE receptor recycling

Apolipoprotein E (ApoE) genotype is a powerful genetic modifier of Alzheimers disease (AD). The ApoE4 isoform significantly reduces the mean age-of-onset of dementia through unknown mechanisms. Here, we show that ApoE4 selectively impairs synaptic plasticity and NMDA receptor phosphorylation by Reelin, a regulator of brain development and modulator of synaptic strength. ApoE4 reduces neuronal surface expression of Apoer2, a dual function receptor for ApoE and for Reelin, as well as NMDA and AMPA receptors by sequestration in intracellular compartments, thereby critically reducing the ability of Reelin to enhance synaptic glutamate receptor activity. As a result, the ability of Reelin to prevent LTP suppression by extracts from AD-afflicted human brains in hippocampal slices from knockin mice expressing the human ApoE4 isoform is severely impaired. These findings show an isoform-specific role of ApoE in the localization and intracellular trafficking of lipoprotein and glutamate receptors and thereby reveal an alternative mechanism by which ApoE4 may accelerate onset of dementia and neuronal degeneration by differentially impairing the maintenance of synaptic stability.

Reelin signaling antagonizes β-amyloid at the synapse

Abnormal processing of the amyloid precursor protein (APP) and β-amyloid (Aβ) plaque accumulation are defining features of Alzheimer disease (AD), a genetically complex neurodegenerative disease that is characterized by progressive synapse loss and neuronal cell death. Aβ induces synaptic dysfunction in part by altering the endocytosis and trafficking of AMPA and NMDA receptors. Reelin is a neuromodulator that increases glutamatergic neurotransmission by signaling through the postsynaptic ApoE receptors Apoer2 and Vldlr and thereby potently enhances synaptic plasticity. Here we show that Reelin can prevent the suppression of long-term potentiation and NMDA receptors, which is induced by levels of Aβ comparable to those present in an AD-afflicted brain. This reversal is dependent upon the activation of Src family tyrosine kinases. At high concentrations of Aβ peptides, Reelin can no longer overcome the Aβ induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Aβ, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control.

Leptin induces a novel form of NMDA receptor-dependent long-term depression.

It is becoming apparent that the hormone leptin plays an important role in modulating hippocampal function. Indeed, leptin enhances NMDA receptor activation and promotes hippocampal long‐term potentiation (LTP). Furthermore, obese rodents with dysfunctional leptin receptors display impairments in hippocampal synaptic plasticity. Here we demonstrate that under conditions of enhanced excitability (evoked in Mg2+‐free medium or following blockade of GABAA receptors), leptin induces a novel form of long‐term depression (LTD) in area CA1 of the hippocampus. Leptin‐induced LTD was markedly attenuated in the presence of D‐(‐)‐2‐Amino‐5‐Phosphonopentanoic acid (D‐AP5), suggesting that it is dependent on the synaptic activation of NMDA receptors. In addition, low‐frequency stimulus‐evoked LTD occluded the effects of leptin. In contrast, metabotropic glutamate receptors (mGluRs) did not contribute to leptin‐induced LTD as mGluR antagonists failed to either prevent or reverse this process. The signalling mechanisms underlying leptin‐induced LTD were independent of the Ras‐Raf‐mitogen‐activated protein kinase signalling pathway, but were markedly enhanced following inhibition of either phosphoinositide 3‐kinase or protein phosphatases 1 and 2A. These data indicate that under conditions of enhanced excitability, leptin induces a novel form of homosynaptic LTD, which further underscores the proposed key role for this hormone in modulating NMDA receptor‐dependent hippocampal synaptic plasticity.

Developmental maturation of synaptic and extrasynaptic GABAA receptors in mouse thalamic ventrobasal neurones

Thalamic ventrobasal (VB) relay neurones express multiple GABAA receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABAA receptors of developing VB neurones and investigate the role of the α1 subunit during maturation of GABA‐ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and α10/0 mice and mice engineered to express diazepam‐insensitive receptors (α1H101R, α2H101R). In immature brain, rapid (P8/9–P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8–27) formed by the α4 and δ subunit occurred independently of the α1 subunit. Subsequently (≥ P15), synaptic α2 subunit/gephyrin clusters of WT VB neurones were replaced by those containing the α1 subunit. Surprisingly, in α10/0 VB neurones the frequency of mIPSCs decreased between P12 and P27, because the α2 subunit also disappeared from these cells. The loss of synaptic GABAA receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA‐ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABAA receptors in VB follows a developmental programme independent of the α1 subunit. Changes to synaptic GABAA receptor function and the increased expression of extrasynaptic GABAA receptors represent two distinct mechanisms for fine‐tuning GABA‐ergic control of thalamic relay neurone activity during development.

Reelin protects against amyloid β toxicity in vivo

Reelin prevents the deleterious effects of amyloid β on synaptic transmission, learning, and memory. Protecting neurons from amyloid β In the developing nervous system, the secreted protein Reelin helps to guide migrating neurons to their correct destination. In the adult nervous system, Reelin enhances synaptic plasticity and protects isolated neurons from the toxicity of amyloid β, the accumulation of which causes the neurodegeneration characteristic of Alzheimer’s disease. To avoid the developmental defects associated with Reelin deficiency, Lane-Donovan et al. generated mice with an inducible knockout of Reelin that accumulated amyloid β. Mice that lacked Reelin as adults showed greater defects in synaptic plasticity, learning, and memory in response to amyloid β accumulation, indicating that Reelin protects against the neurotoxicity of amyloid β in vivo. Alzheimer’s disease (AD) is a currently incurable neurodegenerative disorder and is the most common form of dementia in people over the age of 65 years. The predominant genetic risk factor for AD is the ε4 allele encoding apolipoprotein E (ApoE4). The secreted glycoprotein Reelin enhances synaptic plasticity by binding to the multifunctional ApoE receptors apolipoprotein E receptor 2 (Apoer2) and very low density lipoprotein receptor (Vldlr). We have previously shown that the presence of ApoE4 renders neurons unresponsive to Reelin by impairing the recycling of the receptors, thereby decreasing its protective effects against amyloid β (Aβ) oligomer–induced synaptic toxicity in vitro. We showed that when Reelin was knocked out in adult mice, these mice behaved normally without overt learning or memory deficits. However, they were strikingly sensitive to amyloid-induced synaptic suppression and had profound memory and learning disabilities with very low amounts of amyloid deposition. Our findings highlight the physiological importance of Reelin in protecting the brain against Aβ-induced synaptic dysfunction and memory impairment.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning

Glycosylation of the apolipoprotein E receptor Apoer2 is important for regulating synaptic function and cognition. Sugar for Normal Brain Function Alzheimer’s disease is a neurodegenerative disorder that results in dementia. Decreased signaling through the receptor Apoer2 exacerbates some of the molecular changes that occur in Alzheimer’s disease. Wasser et al. generated mice with a form of Apoer2 lacking the domain that is heavily glycosylated with O-linked sugars. The abundance of this mutant receptor in these mice was higher than that of Apoer2 in wild-type mice. Lack of this domain resulted in changes in synaptic morphology and composition, decreased synaptic efficacy, and defects in learning and memory. These neurological effects appeared to depend on the increased amount of the mutant receptor because they were absent in mice with lower amounts of the mutant receptor. Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase–dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory.

Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice

Alzheimers disease (AD) is the most common form of dementia in individuals over the age of 65 years. The most prevalent genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4), and novel AD treatments that target ApoE are being considered. One unresolved question in ApoE biology is whether ApoE is necessary for healthy brain function. ApoE knock-out (KO) mice have synaptic loss and cognitive dysfunction; however, these findings are complicated by the fact that ApoE knock-out mice have highly elevated plasma lipid levels, which may independently affect brain function. To bypass the effect of ApoE loss on plasma lipids, we generated a novel mouse model that expresses ApoE normally in peripheral tissues, but has severely reduced ApoE in the brain, allowing us to study brain ApoE loss in the context of a normal plasma lipid profile. We found that these brain ApoE knock-out (bEKO) mice had synaptic loss and dysfunction similar to that of ApoE KO mice; however, the bEKO mice did not have the learning and memory impairment observed in ApoE KO mice. Moreover, we found that the memory deficit in the ApoE KO mice was specific to female mice and was fully rescued in female bEKO mice. Furthermore, while the AMPA/NMDA ratio was reduced in ApoE KO mice, it was unchanged in bEKO mice compared with controls. These findings suggest that plasma lipid levels can influence cognition and synaptic function independent of ApoE expression in the brain. SIGNIFICANCE STATEMENT One proposed treatment strategy for Alzheimers disease (AD) is the reduction of ApoE, whose ε4 isoform is the most common genetic risk factor for the disease. A major concern of this strategy is that an animal model of ApoE deficiency, the ApoE knock-out (KO) mouse, has reduced synapses and cognitive impairment; however, these mice also develop dyslipidemia and severe atherosclerosis. Here, we have shown that genetic restoration of plasma ApoE to wild-type levels normalizes plasma lipids in ApoE KO mice. While this does not rescue synaptic loss, it does completely restore learning and memory in the mice, suggesting that both CNS and plasma ApoE are independent parameters that affect brain health.

Lrp4 Domains Differentially Regulate Limb/Brain Development and Synaptic Plasticity

Apolipoprotein E (ApoE) genotype is the strongest predictor of Alzheimer’s Disease (AD) risk. ApoE is a cholesterol transport protein that binds to members of the Low-Density Lipoprotein (LDL) Receptor family, which includes LDL Receptor Related Protein 4 (Lrp4). Lrp4, together with one of its ligands Agrin and its co-receptors Muscle Specific Kinase (MuSK) and Amyloid Precursor Protein (APP), regulates neuromuscular junction (NMJ) formation. All four proteins are also expressed in the adult brain, and APP, MuSK, and Agrin are required for normal synapse function in the CNS. Here, we show that Lrp4 is also required for normal hippocampal plasticity. In contrast to the closely related Lrp8/Apoer2, the intracellular domain of Lrp4 does not appear to be necessary for normal expression and maintenance of long-term potentiation at central synapses or for the formation and maintenance of peripheral NMJs. However, it does play a role in limb development.

Transient Intrauterine Hypotension Causes Apoptosis in Fetal Rat Brain and Affects Learning

Hypotensive episodes are frequent during pregnancy, and their functional effect on fetal brain has not been studied. We produced systemic hypotension for 30 min during mid-gestation in pregnant rats and examined their offspring on postnatal days 1 and 28. When compared with sham controls, the brain of the hypotensive group contained more TUNEL-positive cells in the hippocampal and periventricular regions on both time points. Spatial learning assessed by water milk maze test was impaired in 28-day-old pups of the hypotensive mothers. According to these results, transient maternal hypotension can induce apoptotic cell death in fetal brain and affect learning. Similar mechanisms may be considered and investigated in the pathogenesis of human learning disorders.