Amyloid precursor protein combinatorial phosphorylation code regulates AMPA receptor removal during distinct forms of synaptic plasticity.

Synaptic plasticity is essential for memory encoding and stabilization of neural network activity. Plasticity is impaired in neurodegenerative conditions including Alzheimer disease (AD). A central factor in AD is amyloid precursor protein (APP). Previous studies have suggested APP involvement in synaptic plasticity, but physiological roles of APP are not well understood. Here, we identified combinatorial phosphorylation sites within APP that regulate AMPA receptor trafficking during different forms of synaptic plasticity. Dual phosphorylation sites at threonine-668/serine-675 of APP promoted endocytosis of the GluA2 subunit of AMPA receptors during homeostatic synaptic plasticity. APP was also required for GluA2 internalization during NMDA receptor-dependent long-term depression, albeit via a distinct pair of phosphoresidues at serine-655/threonine-686. These data implicate APP as a central gate for AMPA receptor internalization during distinct forms of plasticity, unlocked by specific combinations of phosphoresidues, and suggest that APP may serve broad functions in learning and memory.

Plk2 promotes synaptic destabilization through disruption of N-cadherin adhesion complexes during homeostatic adaptation to hyperexcitation.

Synaptogenesis in the brain is highly organized and orchestrated by synaptic cellular adhesion molecules (CAMs) such as N-cadherin and amyloid precursor protein (APP) that contribute to the stabilization and structure of synapses. Although N-cadherin plays an integral role in synapse formation and synaptic plasticity, its function in synapse dismantling is not as well understood. Synapse weakening and loss are prominent features of neurodegenerative diseases, and can also be observed during homeostatic compensation to neuronal hyperexcitation. Previously, we have shown that during homeostatic synaptic plasticity, APP is a target for cleavage triggered by phosphorylation by Polo-like kinase 2 (Plk2). Here, we found that Plk2 directly phosphorylates N-cadherin, and during neuronal hyperexcitation Plk2 promotes N-cadherin proteolytic processing, degradation, and disruption of complexes with APP. We further examined the molecular mechanisms underlying N-cadherin degradation. Loss of N-cadherin adhesive function destabilizes excitatory synapses and promotes their structural dismantling as a prerequisite to eventual synapse elimination. This pathway, which may normally help to homeostatically restrain excitability, could also shed light on the dysregulated synapse loss that occurs in cognitive disorders.

ACh Transfers: Homeostatic Plasticity of Cholinergic Synapses.

The field of homeostatic plasticity continues to advance rapidly, highlighting the importance of stabilizing neuronal activity within functional limits in the context of numerous fundamental processes such as development, learning, and memory. Most homeostatic plasticity studies have been focused on glutamatergic synapses, while the rules that govern homeostatic regulation of other synapse types are less understood. While cholinergic synapses have emerged as a critical component in the etiology of mammalian neurodegenerative disease mechanisms, relatively few studies have been conducted on the homeostatic plasticity of such synapses, particularly in the mammalian nervous system. An exploration of homeostatic mechanisms at the cholinergic synapse may illuminate potential therapeutic targets for disease management and treatment. We will review cholinergic homeostatic plasticity in the mammalian neuromuscular junction, the autonomic nervous system, central synapses, and in relation to pathological conditions including Alzheimer disease and DYT1 dystonia. This work provides a historical context for the field of cholinergic homeostatic regulation by examining common themes, unique features, and outstanding questions associated with these distinct cholinergic synapse types and aims to inform future research in the field.

Inhibitory Parvalbumin Basket Cell Activity is Selectively Reduced during Hippocampal Sharp Wave Ripples in a Mouse Model of Familial Alzheimer's Disease.

Memory disruption in mild cognitive impairment (MCI) and Alzheimer's disease (AD) is poorly understood, particularly at early stages preceding neurodegeneration. In mouse models of AD, there are disruptions to sharp wave ripples (SWRs), hippocampal population events with a critical role in memory consolidation. However, the microcircuitry underlying these disruptions is under-explored. We tested whether a selective reduction in parvalbumin-expressing (PV) inhibitory interneuron activity underlies hyperactivity and SWR disruption. We employed the 5xFAD model of familial AD crossed with mouse lines labeling excitatory pyramidal cells (PCs) and inhibitory PV cells. We observed a 33% increase in frequency, 58% increase in amplitude, and 8% decrease in duration of SWRs in ex vivo slices from male and female three-month 5xFAD mice versus littermate controls. 5xFAD mice of the same age were impaired in a hippocampal-dependent memory task. Concurrent with SWR recordings, we performed calcium imaging, cell-attached, and whole-cell recordings of PC and PV cells within the CA1 region. PCs in 5xFAD mice participated in enlarged ensembles, with superficial PCs (sPCs) having a higher probability of spiking during SWRs. Both deep PCs (dPCs) and sPCs displayed an increased synaptic E/I ratio, suggesting a disinhibitory mechanism. In contrast, we observed a 46% spike rate reduction during SWRs in PV basket cells (PVBCs), while PV bistratified and axo-axonic cells were unimpaired. Excitatory synaptic drive to PVBCs was selectively reduced by 50%, resulting in decreased E/I ratio. Considering prior studies of intrinsic PV cell dysfunction in AD, these findings suggest alterations to the PC-PVBC microcircuit also contribute to impairment.SIGNIFICANCE STATEMENT We demonstrate that a specific subtype of inhibitory neuron, parvalbumin-expressing (PV) basket cells, have selectively reduced activity in a model of Alzheimer's disease (AD) during activity critical for the consolidation of memory. These results identify a potential cellular target for therapeutic intervention to restore aberrant network activity in early amyloid pathology. While PV cells have previously been identified as a potential therapeutic target, this study for the first time recognizes that other PV neuronal subtypes, including bistratified and axo-axonic cells, are spared. These experiments are the first to record synaptic and spiking activity during sharp wave ripple (SWR) events in early amyloid pathology and reveal that a selective decrease in excitatory synaptic drive to PV basket cells (PVBCs) likely underlies reduced function.

Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures.

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer's disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague-Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses.

Microtubule-associated protein 2 mediates induction of long-term potentiation in hippocampal neurons.

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Activation of nicotinic acetylcholine receptors induces potentiation and synchronization within in vitro hippocampal networks.

Nicotinic acetylcholine receptors (nAChRs) are known to play a role in cognitive functions of the hippocampus, such as memory consolidation. Given that they conduct Ca2+ and are capable of regulating the release of glutamate and γ-aminobutyric acid (GABA) within the hippocampus, thereby shifting the excitatory-inhibitory ratio, we hypothesized that the activation of nAChRs will result in the potentiation of hippocampal networks and alter synchronization. We used nicotine as a tool to investigate the impact of activation of nAChRs on neuronal network dynamics in primary embryonic rat hippocampal cultures prepared from timed-pregnant Sprague-Dawley rats. We perturbed cultured hippocampal networks with increasing concentrations of bath-applied nicotine and performed network extracellular recordings of action potentials using a microelectrode array. We found that nicotine modulated network dynamics in a concentration-dependent manner; it enhanced firing of action potentials as well as facilitated bursting activity. In addition, we used pharmacological agents to determine the contributions of discrete nAChR subtypes to the observed network dynamics. We found that ß4-containing nAChRs are necessary for the observed increases in spiking, bursting, and synchrony, while the activation of α7 nAChRs augments nicotine-mediated network potentiation but is not necessary for its manifestation. We also observed that antagonists of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs) partially blocked the effects of nicotine. Furthermore, nicotine exposure promoted autophosphorylation of Ca2+ /calmodulin-dependent kinase II (CaMKII) and serine 831 phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluA1. These results suggest that nicotinic receptors induce potentiation and synchronization of hippocampal networks and glutamatergic synaptic transmission. Findings from this work highlight the impact of cholinergic signaling in generating network-wide potentiation in the form of enhanced spiking and bursting dynamics that coincide with molecular correlates of memory such as increased phosphorylation of CaMKII and GluA1. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Kappa opioid receptors regulate hippocampal synaptic homeostasis and epileptogenesis.

OBJECTIVE: Homeostatic synaptic plasticity (HSP) serves as a gain control mechanism at central nervous system (CNS) synapses, including those between the dentate gyrus (DG) and CA3. Improper circuit control of DG-CA3 synapses is hypothesized to underlie epileptogenesis. Here, we sought to (1) identify compounds that preferentially modulate DG-CA3 synapses in primary neuronal culture and (2) determine if these compounds would delay or prevent epileptogenesis in vivo. METHODS: We previously developed and validated an in vitro assay to visualize the behavior of DG-CA3 synapses and predict functional changes. We used this "synapse-on-chip" assay (quantification of synapse size, number, and type using immunocytochemical markers) to dissect the mechanisms of HSP at DG-CA3 synapses. Using chemogenetic constructs and pharmacological agents we determined the signaling cascades necessary for gain control at DG-CA3 synapses. Finally, we tested the implicated cascades (using kappa opioid receptor (OR) agonists and antagonists) in two models of epileptogenesis: electrical amygdala kindling in the mouse and chemical (pentylenetetrazole) kindling in the rat. RESULTS: In vitro, synapses between DG mossy fibers (MFs) and CA3 neurons are the primary homeostatic responders during sustained periods of activity change. Kappa OR signaling is both necessary and sufficient for the homeostatic elaboration of DG-CA3 synapses, induced by presynaptic DG activity levels. Blocking kappa OR signaling in vivo attenuates the development of seizures in both mouse and rat models of epilepsy. SIGNIFICANCE: This study elucidates mechanisms by which synapses between DG granule cells and CA3 pyramidal neurons undergo activity-dependent homeostatic compensation, via OR signaling in vitro. Modulation of kappa OR signaling in vivo alters seizure progression, suggesting that breakdown of homeostatic closed-loop control at DG-CA3 synapses contributes to seizures, and that targeting endogenous homeostatic mechanisms at DG-CA3 synapses may prove useful in combating epileptogenesis.

Hexa (ethylene glycol) derivative of benzothiazole aniline promotes dendritic spine formation through the RasGRF1-Ras dependent pathway.

Our recent study demonstrated that an amyloid-ß binding molecule, BTA-EG4, increases dendritic spine number via Ras-mediated signaling. To potentially optimize the potency of the BTA compounds, we synthesized and evaluated an amyloid-ß binding analog of BTA-EG4 with increased solubility in aqueous solution, BTA-EG6. We initially examined the effects of BTA-EG6 on dendritic spine formation and found that BTA-EG6-treated primary hippocampal neurons had significantly increased dendritic spine number compared to control treatment. In addition, BTA-EG6 significantly increased the surface level of AMPA receptors. Upon investigation into the molecular mechanism by which BTA-EG6 promotes dendritic spine formation, we found that BTA-EG6 may exert its effects on spinogenesis via RasGRF1-ERK signaling, with potential involvement of other spinogenesis-related proteins such as Cdc42 and CDK5. Taken together, our data suggest that BTA-EG6 boosts spine and synapse number, which may have a beneficial effect of enhancing neuronal and synaptic function in the normal healthy brain.

A mobile APP for sharing contacts on your cell: An Editorial Highlight for 'The physiological role of the amyloid precursor protein as an adhesion molecule in the developing nervous system' on page 11.

The Review highlighted in this Editorial followed a CAEN Return Home Grant.

Evidence for glycinergic GluN1/GluN3 NMDA receptors in hippocampal metaplasticity.

Hebbian, or associative, forms of synaptic plasticity are considered the molecular basis of learning and memory. However, associative synaptic modifications, including long-term potentiation (LTP) and depression (LTD), can form positive feedback loops which must be constrained for neural networks to remain stable. One proposed constraint mechanism is metaplasticity, a process whereby synaptic changes shift the threshold for subsequent plasticity. Metaplasticity has been functionally observed but the molecular basis is not well understood. Here, we report that stimulation which induces LTP recruits GluN2B-lacking GluN1/GluN3 NMDA receptors (NMDARs) to excitatory synapses of hippocampal pyramidal neurons. These unconventional receptors may compete against conventional GluN1/GluN2 NMDARs to favor synaptic depotentiation in response to subsequent "LTP-inducing" stimulation. These results implicate glycinergic GluN1/GluN3 NMDAR as molecular brakes on excessive synaptic strengthening, suggesting a role for these receptors in the brain that has previously been elusive.

Motor skill training induces coordinated strengthening and weakening between neighboring synapses.

Motor skill training promotes the formation of parallel fiber multiple-synapse boutons (MSBs) contacting dendritic spine pairs of Purkinje cells in the rat cerebellum. However, the dendritic origin of such spine pairs is unknown. Here, we used three-dimensional electron microscopy reconstruction of synaptic connectivity to demonstrate that motor skill training selectively induced MSBs contacting two spines arising from the same dendrite, consistent with strengthening of local synaptic efficacy. However, excitatory synapses near MSBs were smaller in motor-trained animals, suggesting compensatory depression of MSB-neighbor synapses. Concerted strengthening and weakening of adjacent synapses may enhance synaptic weight differences for information encoding while maintaining stable overall activity levels within local dendritic segments.

A tetra(ethylene glycol) derivative of benzothiazole aniline enhances Ras-mediated spinogenesis.

The tetra(ethylene glycol) derivative of benzothiazole aniline, BTA-EG4, is a novel amyloid-binding small molecule that can penetrate the blood-brain barrier and protect cells from Aß-induced toxicity. However, the effects of Aß-targeting molecules on other cellular processes, including those that modulate synaptic plasticity, remain unknown. We report here that BTA-EG4 decreases Aß levels, alters cell surface expression of amyloid precursor protein (APP), and improves memory in wild-type mice. Interestingly, the BTA-EG4-mediated behavioral improvement is not correlated with LTP, but with increased spinogenesis. The higher dendritic spine density reflects an increase in the number of functional synapses as determined by increased miniature EPSC (mEPSC) frequency without changes in presynaptic parameters or postsynaptic mEPSC amplitude. Additionally, BTA-EG4 requires APP to regulate dendritic spine density through a Ras signaling-dependent mechanism. Thus, BTA-EG4 may provide broad therapeutic benefits for improving neuronal and cognitive function, and may have implications in neurodegenerative disease therapy.

Synapses need coordination to learn motor skills.

Experience-dependent remodeling of synaptic structure and function underlies information storage in the mammalian central nervous system. Although accumulating evidence suggests synergistic roles of long-term depression (LTD) and long-term potentiation (LTP) in cerebellar motor learning, their structural correlates and operational mechanisms have not been clearly addressed. A recent three-dimensional electron microscopic study provides insight for a potential complementary interplay between LTP and LTD in local dendritic segments of Purkinje cells of motor skill-trained animals. Complex motor skill training induced strengthening of a subset of parallel fiber synapses onto Purkinje cells by forming multiple-synapse boutons (MSBs) contacting spine pairs arising from the same dendrite, whereas MSB-neighboring synapses were weakened by reducing the size of the postsynaptic density. Here, we discuss these orchestrated structural modifications of neighboring synapses that may sharpen synaptic weight contrast in local dendritic segments, leading to enhanced signal-to-noise ratio for optimal motor skill retention.

Antihypertensive drug Valsartan promotes dendritic spine density by altering AMPA receptor trafficking.

Recent studies demonstrated that the antihypertensive drug Valsartan improved spatial and episodic memory in mouse models of Alzheimer's Disease (AD) and human subjects with hypertension. However, the molecular mechanism by which Valsartan can regulate cognitive function is still unknown. Here, we investigated the effect of Valsartan on dendritic spine formation in primary hippocampal neurons, which is correlated with learning and memory. Interestingly, we found that Valsartan promotes spinogenesis in developing and mature neurons. In addition, we found that Valsartan increases the puncta number of PSD-95 and trends toward an increase in the puncta number of synaptophysin. Moreover, Valsartan increased the cell surface levels of AMPA receptors and selectively altered the levels of spinogenesis-related proteins, including CaMKIIα and phospho-CDK5. These data suggest that Valsartan may promote spinogenesis by enhancing AMPA receptor trafficking and synaptic plasticity signaling.

Wherefore Art Tau? Functional importance of site-specific tau phosphorylation in diverse subcellular domains.

Tau has canonically been considered as an axonal protein, but studies have observed tau localization in other subcellular domains of neurons. This relocated tau has been identified in both physiological and pathological conditions, and it is often labeled mislocalized. Furthermore, these forms of tau are referred to as "hyperphosphorylated" without specifying the phosphosites involved. On the contrary, we speculate that tau may have multiple physiological functions in various locations regulated via specific phosphorylation sites, although this picture is obscured by a lack of comprehensive phosphosite analysis. Here, we examine findings in the literature on the subcellular location of tau and potential roles tau has in those regions. We intentionally focus on the site-specific phosphorylated patterns involved in governing these properties, which are not well elucidated. To facilitate understanding of these events, we have begun establishing a comprehensive map of tau phosphorylation signatures. Such efforts may clarify tau's diverse physiological functions beyond the axon as well as promote development of novel therapeutic strategies directed against distinct tau subpopulations.

Wherefore art thou, homeo(stasis)? Functional diversity in homeostatic synaptic plasticity.

Homeostatic plasticity has emerged as a fundamental regulatory principle that strives to maintain neuronal activity within optimal ranges by altering diverse aspects of neuronal function. Adaptation to network activity is often viewed as an essential negative feedback restraint that prevents runaway excitation or inhibition. However, the precise importance of these homeostatic functions is often theoretical rather than empirically derived. Moreover, a remarkable multiplicity of homeostatic adaptations has been observed. To clarify these issues, it may prove useful to ask: why do homeostatic mechanisms exist, what advantages do these adaptive responses confer on a given cell population, and why are there so many seemingly divergent effects? Here, we approach these questions by applying the principles of control theory to homeostatic synaptic plasticity of mammalian neurons and suggest that the varied responses observed may represent distinct functional classes of control mechanisms directed toward disparate physiological goals.

Reelin supplementation enhances cognitive ability, synaptic plasticity, and dendritic spine density.

Apolipoprotein receptors belong to an evolutionarily conserved surface receptor family that has intimate roles in the modulation of synaptic plasticity and is necessary for proper hippocampal-dependent memory formation. The known lipoprotein receptor ligand Reelin is important for normal synaptic plasticity, dendritic morphology, and cognitive function; however, the in vivo effect of enhanced Reelin signaling on cognitive function and synaptic plasticity in wild-type mice is unknown. The present studies test the hypothesis that in vivo enhancement of Reelin signaling can alter synaptic plasticity and ultimately influence processes of learning and memory. Purified recombinant Reelin was injected bilaterally into the ventricles of wild-type mice. We demonstrate that a single in vivo injection of Reelin increased activation of adaptor protein Disabled-1 and cAMP-response element binding protein after 15 min. These changes correlated with increased dendritic spine density, increased hippocampal CA1 long-term potentiation (LTP), and enhanced performance in associative and spatial learning and memory. The present study suggests that an acute elevation of in vivo Reelin can have long-term effects on synaptic function and cognitive ability in wild-type mice.

The cytoplasmic adaptor protein X11alpha and extracellular matrix protein Reelin regulate ApoE receptor 2 trafficking and cell movement.

The goal of this study was to determine the effect of X11alpha on ApoE receptor 2 (ApoEr2) trafficking and the functional significance of this interaction on cell movement in MCF 10A epithelial cells. We found that X11alpha increased surface levels of ApoEr2 by 64% compared to vector control, as determined by surface protein biotinylation. To examine the functional significance of this effect, we tested whether ApoEr2 played a novel role in cell movement in a wound-healing assay. We found that overexpression of ApoEr2 in MCF 10A cells increased cell migration velocity by 87% (P<0.01, n=4) compared to GFP control. Cotransfection of X11alpha had an additive effect on average velocity compared to ApoEr2 alone (13%; P<0.05, n=4). In addition, we tested whether ApoEr2 ligands altered the effect of ApoEr2 on cell movement. We found that treatment with concentrated medium containing the extracellular matrix protein Reelin, but not control medium, further increased the velocity of ApoEr2- but not APP-transfected cells (20%; P<0.001, n=4). Similarly, Reelin treatment increased cell velocity in the presence of ApoEr2 and X11alpha (10%; P<0.05, n=4). In the present study, we are the first to demonstrate that ApoEr2 regulates cell movement, and both X11alpha and Reelin enhance this effect.

Postsynaptic PDLIM5/Enigma Homolog binds SPAR and causes dendritic spine shrinkage.

Dendritic spine morphology is thought to play important roles in synaptic development and plasticity, and morphological derangements in spines are correlated with several neurological disorders. Here, we identified an interaction between Spine-Associated RapGAP (SPAR), a postsynaptic protein that reorganizes actin cytoskeleton and drives dendritic spine head growth, and PDLIM5/Enigma Homolog (ENH), a PDZ-LIM (postsynaptic density-95/Discs large/zona occludens 1-Lin11/Isl-1/Mec3) family member. PDLIM5 has been implicated in susceptibility to bipolar disorder, major depression, and schizophrenia, but its function in neurological disease is poorly understood. We show that PDLIM5 is present in the postsynaptic density, where it promotes decreased dendritic spine head size and longer, filopodia-like morphology. Conversely, RNA interference against PDLIM5 or loss of PDLIM5 interaction with SPAR caused increased spine head diameter. Furthermore, PKC activation promoted delivery of PDLIM5 into dendritic spines and increased its spine colocalization with SPAR. These data reveal new postsynaptic functions for PDLIM5 in shrinkage of dendritic spines that may be relevant to its association with psychiatric illness.