Diffusion tensor imaging analysis along the perivascular space in the UK biobank.

BACKGROUND: The recently discovered glymphatic system may support the removal of neurotoxic proteins, mainly during sleep, that are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's Disease. Diffusion tensor image analysis along the perivascular space (DTI-ALPS) has been suggested as a method to index the health of glymphatic system (with higher values indicating a more intact glymphatic system). Indeed, in small-scale studies the DTI-ALPS index has been shown to correlate with age, cognitive health, and sleep, and is higher in females than males. OBJECTIVE: To determine whether these relationships are stable we replicated previous findings associating the DTI-ALPS index with demographic, sleep-related, and cognitive markers in a large sample of participants from the UK Biobank. METHODS: We calculated the DTI-ALPS index in UK Biobank participants (n = 17723). Using Bayesian and Frequentist analysis approaches, we replicate previously reported relationships between the DTI-ALPS index. RESULTS: We found the predicted associations between the DTI-ALPS index and age, longest uninterrupted sleep window (LUSWT) on a typical night, cognitive performance, and sex. However, these effects were substantially smaller than those found in previous studies. Parameter estimates from this study may be used as priors in subsequent studies using a Bayesian approach. These results suggest that the DTI-ALPS index is consistently, and therefore predictably, associated with demographics, LUWST, and cognition. CONCLUSION: We propose that the metric, calculated for the first time in a large-scale, population-based cohort, is a stable measure, but one for which stronger links to glymphatic system function are needed before it can be used to understand the relationships between glymphatic system function and health outcomes reported in the UK Biobank.

ArcKR expression modifies synaptic plasticity following epileptic activity: Differential effects with in vitro and in vivo seizure-induction protocols.

OBJECTIVES: Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which is critically involved in protein-synthesis-dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor-mediated long-term depression (G1-mGluR-LTD) following seizures. METHODS: We used a knock-in mice line that express ArcKR and two hyperexcitation models: an in vitro model, where hippocampal slices were exposed to zero Mg2+, 6 mM K+; and an in vivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1-mGluR-LTD was induced chemically with the group 1 mGluR agonist DHPG. RESULTS: In the in vitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1-mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the in vivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1-mGluR-LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice. SIGNIFICANCE: We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1-mGluR-LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an in vitro model and a reduction in the kainate mouse model.

Impairments in sleep and brain molecular clearance in people with cognitive deterioration and biological evidence of AD: a report of four cases.

BACKGROUND: Recent evidence suggests that the failure of the glymphatic system - the brain's waste clearance system, which is active during sleep - plays a key role in the pathophysiology of Alzheimer's Disease (AD). Glymphatic function can be investigated using serial MRIs after intrathecal gadobutrol injection. This technique can reveal the health of the glymphatic system, but has not yet been used in participants with cognitive impairment due to AD. CASE REPORT: This report describes the sleep and gadobutrol tracer clearance patterns of four participants diagnosed with mild to moderate cognitive impairment with evidence of AD pathology (pathological levels of Ab and p-tau in cerebrospinal fluid). We performed polysomnography and MRI studies before tracer injection and MRI scans at 1.5-2 h, 5-6 h, and 48 h after injection. Despite participants reporting no sleep problems, polysomnography revealed that all participants had moderate to severe sleep disturbances, including reduced sleep efficiency during the study and obstructive sleep apnea. Severe side-effects related to tracer administration were observed, impeding the completion of the protocol in two participants. Participants who finished the protocol displayed delayed and persistent tracer enrichment in the cortex and white matter, even 48 h after injection. These outcomes have not been observed in previous studies in participants without AD. CONCLUSION: The findings suggest that brains with sleep impairment and AD pathology have poor glymphatic function, and therefore cannot clear the contrast tracer efficiently. This is likely to have caused the severe side effects in our participants, that have not been reported in healthy individuals. Our results may therefore represent the only available data acquired with this technique in participants with AD pathology.

Arc expression regulates long-term potentiation magnitude and metaplasticity in area CA1 of the hippocampus in ArcKR mice.

Expression of the immediate early gene Arc/Arg3.1 (Arc), a key mediator of synaptic plasticity, is enhanced by neural activity and then reduced by proteasome-dependent degradation. We have previously shown that the disruption of Arc degradation, in an Arc knock-in mouse (ArcKR), where the predominant Arc ubiquitination sites were mutated, reduced the threshold to induce, and also enhanced, the strength of Group I metabotropic glutamate receptor-mediated long-term depression (DHPG-LTD). Here, we have investigated if ArcKR expression changes long-term potentiation (LTP) in CA1 area of the hippocampus. As previously reported, there was no change in basal synaptic transmission at Schaffer collateral/commissural-CA1 (SC-CA1) synapses in ArcKR versus wild-type (WT) mice. There was, however, a significant increase in the amplitude of synaptically induced (with low frequency paired-pulse stimulation) LTD in ArcKR mice. Theta burst stimulation (TBS)-evoked LTP at SC-CA1 synapses was significantly reduced in ArcKR versus WT mice (after 2 h). Group 1 mGluR priming of LTP was abolished in ArcKR mice, which could also potentially contribute to a depression of LTP. Although high frequency stimulation (HFS)-induced LTP was not significantly different in ArcKR compared with WT mice (after 1 h), there was a phenotype in environmentally enriched mice, with the ratio of LTP to short-term potentiation (STP) significantly reduced in ArcKR mice. These findings support the hypothesis that Arc ubiquitination supports the induction and expression of LTP, likely via limiting Arc-dependent removal of AMPA receptors at synapses.

The MK2 cascade mediates transient alteration in mGluR-LTD and spatial learning in a murine model of Alzheimer's disease.

A key aim of Alzheimer disease research is to develop efficient therapies to prevent and/or delay the irreversible progression of cognitive impairments. Early deficits in long-term potentiation (LTP) are associated with the accumulation of amyloid beta in rodent models of the disease; however, less is known about how mGluR-mediated long-term depression (mGluR-LTD) is affected. In this study, we have found that mGluR-LTD is enhanced in the APPswe /PS1dE9 mouse at 7 but returns to wild-type levels at 13 months of age. This transient over-activation of mGluR signalling is coupled with impaired LTP and shifts the dynamic range of synapses towards depression. These alterations in synaptic plasticity are associated with an inability to utilize cues in a spatial learning task. The transient dysregulation of plasticity can be prevented by genetic deletion of the MAP kinase-activated protein kinase 2 (MK2), a substrate of p38 MAPK, demonstrating that manipulating the mGluR-p38 MAPK-MK2 cascade at 7 months can prevent the shift in synapse dynamic range. Our work reveals the MK2 cascade as a potential pharmacological target to correct the over-activation of mGluR signalling.

Clathrin adaptor AP-1-mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments.

One of the hallmarks of Alzheimer's disease is the accumulation of toxic amyloid-ß (Aß) peptides in extracellular plaques. The direct precursor of Aß is the carboxyl-terminal fragment ß (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer's disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aß. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aß release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.

The p38MAPK-MK2 Signaling Axis as a Critical Link Between Inflammation and Synaptic Transmission.

p38 is a mitogen-activated protein kinase (MAPK), that responds primarily to stress stimuli. p38 has a number of targets for phosphorylation, including MAPK-activated protein kinase 2 (MK2). MK2 primarily functions as a master regulator of RNA-binding proteins, indirectly controlling gene expression at the level of translation. The role of MK2 in regulating the synthesis of pro-inflammatory cytokines downstream of inflammation and cellular stress is well-described. A significant amount of evidence, however, now points to a role for the p38MAPK-MK2 signaling axis in mediating synaptic plasticity through control of AMPA receptor trafficking and the morphology of dendritic spines. These processes are mediated through control of cytoskeletal dynamics via the activation of cofilin-1 and possibly control of the expression of Arc/Arg3.1. There is evidence that MK2 is necessary for group I metabotropic glutamate receptors long-term depression (mGluR-LTD). Disruption of this signaling may play an important role in mediating cognitive dysfunction in neurological disorders such as fragile X syndrome and Alzheimer's disease. To date, the role of neuronal MK2 mediating synaptic plasticity in response to inflammatory stimuli has not yet been investigated. In immune cells, it is clear that MK2 is phosphorylated following activation of a broad range of cell surface receptors for cytokines and other inflammatory mediators. We propose that neuronal MK2 may be an important player in the link between inflammatory states and dysregulation of synaptic plasticity underlying cognitive functions. Finally, we discuss the potential of the p38MAPK-MK2 signaling axis as target for therapeutic intervention in a number of neurological disorders.

NMDA receptor-dependent signalling pathways regulate arginine vasopressin expression in the paraventricular nucleus of the rat.

The antidiuretic hormone arginine vasopressin (AVP) regulates water homeostasis, blood pressure and a range of stress responses. It is synthesized in the hypothalamus and released from the posterior pituitary into the general circulation upon a range of stimuli. While the mechanisms leading to AVP secretion have been widely investigated, the molecular mechanisms regulating AVP gene expression are mostly unclear. Here we investigated the neurotransmitters and signal transduction pathways that activate AVP gene expression in the paraventricular nucleus (PVN) of the rat using acute brain slices and quantitative real-time PCR. We show that stimulation with l-glutamate robustly induced AVP gene expression in acute hypothalamic brain slices containing the PVN. More specifically, we show that AVP transcription was stimulated by NMDA. Using pharmacological treatments, our data further reveal that the activation of ERK1/2 (PD184352), CaMKII (KN-62) and PI3K (LY294002; 740 Y-P) is involved in the NMDA-induced AVP gene expression in the PVN. Together, this study identifies NMDA-mediated cell signalling pathways that regulate AVP gene expression in the rat PVN.

The MK2 cascade regulates mGluR-dependent synaptic plasticity and reversal learning.

The ability to either erase or update the memories of a previously learned spatial task is an essential process that is required to modify behaviour in a changing environment. Current evidence suggests that the neural representation of such cognitive flexibility involves the balancing of synaptic potentiation (acquisition of memories) with synaptic depression (modulation and updating previously acquired memories). Here we demonstrate that the p38 MAPK/MAPK-activated protein kinase 2 (MK2) cascade is required to maintain the precise tuning of long-term potentiation and long-term depression at CA1 synapses of the hippocampus which is correlated with efficient reversal learning. Using the MK2 knockout (KO) mouse, we show that mGluR-LTD, but not NMDAR-LTD, is markedly impaired in mice aged between 4 and 5 weeks (juvenile) to 7 months (mature adult). Although the amplitude of LTP was the same as in wildtype mice, priming of LTP by the activation of group I metabotropic receptors was impaired in MK2 KO mice. Consistent with unaltered LTP amplitude and compromised mGluR-LTD, MK2 KO mice had intact spatial learning when performing the Barnes maze task, but showed specific deficits in selecting the most efficient combination of search strategies to perform the task reversal. Findings from this study suggest that the mGluR-p38-MK2 cascade is important for cognitive flexibility by regulating LTD amplitude and the priming of LTP.

The Temporal Dynamics of Arc Expression Regulate Cognitive Flexibility.

Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasome-dependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition.

The mechanistic link between Arc/Arg3.1 expression and AMPA receptor endocytosis.

The activity-regulated cytoskeleton associated protein (Arc/Arg3.1) plays a key role in determining synaptic strength through facilitation of AMPA receptor (AMPAR) endocytosis. Although there is considerable data on the mechanism by which Arc induction controls synaptic plasticity and learning behaviours, several key mechanistic questions remain. Here we review data on the link between Arc expression and the clathrin-mediated endocytic pathway which internalises AMPARs and discuss the significance of Arc binding to the clathrin adaptor protein 2 (AP-2) and to endophilin/dynamin. We consider which AMPAR subunits are selected for Arc-mediated internalisation, implications for synaptic function and consider Arc as a therapeutic target.

Does the MK2-dependent Production of TNFα Regulate mGluR-dependent Synaptic Plasticity?

The molecular mechanisms and signalling cascades that trigger the induction of group I metabotropic glutamate receptor (GI-mGluR)-dependent long-term depression (LTD) have been the subject of intensive investigation for nearly two decades. The generation of genetically modified animals has played a crucial role in elucidating the involvement of key molecules regulating the induction and maintenance of mGluR-LTD. In this review we will discuss the requirement of the newly discovered MAPKAPK-2 (MK2) and MAPKAPK-3 (MK3) signalling cascade in regulating GI-mGluR-LTD. Recently, it has been shown that the absence of MK2 impaired the induction of GI-mGluR-dependent LTD, an effect that is caused by reduced internalization of AMPA receptors (AMPAR). As the MK2 cascade directly regulates tumour necrosis factor alpha (TNFα) production, this review will examine the evidence that the release of TNFα acts to regulate glutamate receptor expression and therefore may play a functional role in the impairment of GI-mGluRdependent LTD and the cognitive deficits observed in MK2/3 double knockout animals. The strong links of increased TNFα production in both aging and neurodegenerative disease could implicate the action of MK2 in these processes.

Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2.

The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc-AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2.

Negative feedback regulation of the ERK1/2 MAPK pathway.

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.

Hippocampal metabotropic glutamate receptor long-term depression in health and disease: focus on mitogen-activated protein kinase pathways.

Group I metabotropic glutamate receptor (mGluR) dependent long-term depression (LTD) is a major form of synaptic plasticity underlying learning and memory. The molecular mechanisms involved in mGluR-LTD have been investigated intensively for the last two decades. In this 60th anniversary special issue article, we review the recent advances in determining the mechanisms that regulate the induction, transduction and expression of mGluR-LTD in the hippocampus, with a focus on the mitogen-activated protein kinase (MAPK) pathways. In particular we discuss the requirement of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. The recent advances in understanding the signaling cascades regulating mGluR-LTD are then related to the cognitive impairments observed in neurological disorders, such as fragile X syndrome and Alzheimer's disease. mGluR-LTD is a form of synaptic plasticity that impacts on memory formation. In the hippocampus mitogen-activated protein kinases (MAPKs) have been found to be important in mGluR-LTD. In this 60th anniversary special issue article, we review the independent and complementary roles of two classes of MAPK, p38 and ERK1/2 and link this to the aberrant mGluR-LTD that has an important role in diseases. This article is part of the 60th Anniversary special issue.

Preferential assembly of heteromeric small conductance calcium-activated potassium channels.

The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits.

The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility.

The interplay between long-term potentiation and long-term depression (LTD) is thought to be involved in learning and memory formation. One form of LTD expressed in the hippocampus is initiated by the activation of the group 1 metabotropic glutamate receptors (mGluRs). Importantly, mGluRs have been shown to be critical for acquisition of new memories and for reversal learning, processes that are thought to be crucial for cognitive flexibility. Here we provide evidence that MAPK-activated protein kinases 2 and 3 (MK2/3) regulate neuronal spine morphology, synaptic transmission and plasticity. Furthermore, mGluR-LTD is impaired in the hippocampus of MK2/3 double knockout (DKO) mice, an observation that is mirrored by deficits in endocytosis of GluA1 subunits. Consistent with compromised mGluR-LTD, MK2/3 DKO mice have distinctive deficits in hippocampal-dependent spatial reversal learning. These novel findings demonstrate that the MK2/3 cascade plays a strategic role in controlling synaptic plasticity and cognition.

Triad3A regulates synaptic strength by ubiquitination of Arc.

Activity-dependent gene transcription and protein synthesis underlie many forms of learning-related synaptic plasticity. At excitatory glutamatergic synapses, the immediate early gene product Arc/Arg3.1 couples synaptic activity to postsynaptic endocytosis of AMPA-type glutamate receptors. Although the mechanisms for Arc induction have been described, little is known regarding the molecular machinery that terminates Arc function. Here, we demonstrate that the RING domain ubiquitin ligase Triad3A/RNF216 ubiquitinates Arc, resulting in its rapid proteasomal degradation. Triad3A associates with Arc, localizes to clathrin-coated pits, and is associated with endocytic sites in dendrites and spines. In the absence of Triad3A, Arc accumulates, leading to the loss of surface AMPA receptors. Furthermore, loss of Triad3A mimics and occludes Arc-dependent forms of synaptic plasticity. Thus, degradation of Arc by clathrin-localized Triad3A regulates the availability of synaptic AMPA receptors and temporally tunes Arc-mediated plasticity at glutamatergic synapses.

MSK1 regulates homeostatic and experience-dependent synaptic plasticity.

The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.