The Q/R editing site of AMPA receptor GluA2 subunit acts as an epigenetic switch regulating dendritic spines, neurodegeneration and cognitive deficits in Alzheimer's disease.

BACKGROUND: RNA editing at the Q/R site of GluA2 occurs with ~99% efficiency in the healthy brain, so that the majority of AMPARs contain GluA2(R) instead of the exonically encoded GluA2(Q). Reduced Q/R site editing infcreases AMPA receptor calcium permeability and leads to dendritic spine loss, neurodegeneration, seizures and learning impairments. Furthermore, GluA2 Q/R site editing is impaired in Alzheimer's disease (AD), raising the possibility that unedited GluA2(Q)-containing AMPARs contribute to synapse loss and neurodegeneration in AD. If true, then inhibiting expression of unedited GluA2(Q), while maintaining expression of GluA2(R), may be a novel strategy of preventing synapse loss and neurodegeneration in AD. METHODS: We engineered mice with the 'edited' arginine codon (CGG) in place of the unedited glutamine codon (CAG) at position 607 of the Gria2 gene. We crossbred this line with the J20 mouse model of AD and conducted anatomical, electrophysiological and behavioural assays to determine the impact of eliminating unedited GluA2(Q) expression on AD-related phenotypes. RESULTS: Eliminating unedited GluA2(Q) expression in AD mice prevented dendritic spine loss and hippocampal CA1 neurodegeneration as well as improved working and reference memory in the radial arm maze. These phenotypes were improved independently of Aß pathology and ongoing seizure susceptibility. Surprisingly, our data also revealed increased spine density in non-AD mice with exonically encoded GluA2(R) as compared to their wild-type littermates, suggesting an unexpected and previously unknown role for unedited GluA2(Q) in regulating dendritic spines. CONCLUSION: The Q/R editing site of the AMPA receptor subunit GluA2 may act as an epigenetic switch that regulates dendritic spines, neurodegeneration and memory deficits in AD.

Fasting for 20 h does not affect exercise-induced increases in circulating BDNF in humans.

Intermittent fasting and exercise provide neuroprotection from age-related cognitive decline. A link between these two seemingly distinct stressors is their capability to steer the brain away from exclusively glucose metabolism. This cerebral substrate switch has been implicated in upregulating brain-derived neurotrophic factor (BDNF), a protein involved in neuroplasticity, learning and memory, and may underlie some of these neuroprotective effects. We examined the isolated and interactive effects of (1) 20-h fasting, (2) 90-min light exercise, and (3) high-intensity exercise on peripheral venous BDNF in 12 human volunteers. A follow-up study isolated the influence of cerebrovascular shear stress on circulating BDNF. Fasting for 20 h decreased glucose and increased ketones (P ≤ 0.0157) but had no effect on BDNF (P ≥ 0.4637). Light cycling at 25% of peak oxygen uptake ( V ̇ O 2 peak ${\dot V_{{{\rm{O}}_{\rm{2}}}{\rm{peak}}}}$ ) increased serum BDNF by 6 ± 8% (independent of being fed or fasted) and was mediated by a 7 ± 6% increase in platelets (P < 0.0001). Plasma BDNF was increased from 336 pg l-1 [46,626] to 390 pg l-1 [127,653] by 90-min of light cycling (P = 0.0128). Six 40-s intervals at 100% of V ̇ O 2 peak ${\dot V_{{{\rm{O}}_{\rm{2}}}{\rm{peak}}}}$ increased plasma and serum BDNF, as well as the BDNF-per-platelet ratio 4- to 5-fold more than light exercise did (P ≤ 0.0044). Plasma BDNF was correlated with circulating lactate during the high-intensity intervals (r = 0.47, P = 0.0057), but not during light exercise (P = 0.7407). Changes in cerebral shear stress - whether occurring naturally during exercise or induced experimentally with inspired CO2 - did not correspond with changes in BDNF (P ≥ 0.2730). BDNF responses to low-intensity exercise are mediated by increased circulating platelets, and increasing either exercise duration or particularly intensity is required to liberate free BDNF. KEY POINTS: Intermittent fasting and exercise both have potent neuroprotective effects and an acute upregulation of brain-derived neurotrophic factor (BDNF) appears to be a common mechanistic link. Switching the brain's fuel source from glucose to either ketone bodies or lactate, i.e. a cerebral substrate switch, has been shown to promote BDNF production in the rodent brain. Fasting for 20 h caused a 9-fold increase in ketone body delivery to the brain but had no effect on any metric of BDNF in peripheral circulation at rest. Prolonged (90 min) light cycling exercise increased plasma- and serum-derived BDNF irrespective of being fed or fasted and seemed to be independent of changes in cerebral shear stress. Six minutes of high-intensity cycling intervals increased every metric of circulating BDNF by 4 to 5 times more than prolonged low-intensity cycling; the increase in plasma-derived BDNF was correlated with a 6-fold increase in circulating lactate irrespective of feeding or fasting. Compared to 1 day of fasting with or without prolonged light exercise, high-intensity exercise is a much more efficient means to increase BDNF in circulation.

The therapeutic potential of the neuroactive peptides of soluble amyloid precursor protein-alpha in Alzheimer's disease and related neurological disorders.

Soluble amyloid precursor protein-alpha (sAPPα) is a multi-functional brain-derived protein that has neuroprotective, neurogenic and neurotropic properties. Moreover, it is known to facilitate synaptic function and promote neural repair. These properties suggest sAPPα may be useful as a therapeutic agent for the treatment of neurological diseases characterized by synaptic failure and neuronal loss, such as occurs in Alzheimer's disease, and for neural repair following traumatic brain injury and stroke. However, sAPPα's relatively large size and the difficulty of ongoing delivery of therapeutics to the brain mean this is not currently practicable. Importantly, however, sAPPα is composed of several neuroactive domains that each possess properties that collectively are remarkably similar to those of sAPPα itself. Here, we review the molecular structure of sAPPα and identify the domains that contribute to its overall functionality. Four peptide motifs present as possible targets for therapeutic development. We review their physiochemical and neuroactive properties, both within sAPPα and as isolated peptides, and discuss their potential for future development as multipurpose therapeutic agents for the treatment of Alzheimer's disease and other disorders of neuronal function. Further, we discuss the role of heparin binding sites, found within sAPPα's structure and overlapping with the neuroactive domains, as sites for interactions with effector proteins and synaptic receptors. The potential role of the neuroactive peptides known as Cationic Arginine-Rich Peptides (CARPs) as neuroprotective motifs is also reviewed. Mechanisms of peptide delivery to the brain are briefly discussed. Finally, we summarise the potential benefits and pitfalls of using the isolated peptides, either individually or in combination, for the treatment of neurological diseases.

Partial Endothelial Nitric Oxide Synthase Deficiency Exacerbates Cognitive Deficit and Amyloid Pathology in the APPswe/PS1ΔE9 Mouse Model of Alzheimer's Disease.

Increasing evidence implicates endothelial dysfunction in the pathogenesis of Alzheimer's disease (AD). Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is essential in maintaining cerebrovascular function and can modulate the production and clearance of amyloid beta (Aß). APPswe/PSdE1 (APP/PS1) mice display age-related Aß accumulation and memory deficits. In order to make the model more clinically relevant with an element of endothelial dysfunction, we generated APP/PS1/eNOS+/- mice by crossing complete eNOS deficient (eNOS-/-) mice and APP/PS1 mice. APP/PS1/eNOS+/- mice at 8 months of age displayed a more severe spatial working memory deficit relative to age-matched APP/PS1 mice. Moreover, immunohistochemistry and immunoblotting revealed significantly increased Aß plaque load in the brains of APP/PS1/eNOS+/- mice, concomitant with upregulated BACE-1 (hence increased Aß production), downregulated insulin-degrading enzyme (hence reduced Aß clearance) and increased immunoreactivity and expression of microglia. The present study, for the first time, demonstrated that partial eNOS deficiency exacerbated behavioral dysfunction, Aß brain deposition, and microglial pathology in APP/PS1 mice, further implicating endothelial dysfunction in the pathogenesis of AD. The present findings also provide the scientific basis for developing preventive and/or therapeutic strategies by targeting endothelial dysfunction.

Lentivirus-Mediated Expression of Human Secreted Amyloid Precursor Protein-Alpha Promotes Long-Term Induction of Neuroprotective Genes and Pathways in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Secreted amyloid precursor protein-alpha (sAPPα) can enhance memory and is neurotrophic and neuroprotective across a range of disease-associated insults, including amyloid-ß toxicity. In a significant step toward validating sAPPα as a therapeutic for Alzheimer's disease (AD), we demonstrated that long-term overexpression of human sAPPα (for 8 months) in a mouse model of amyloidosis (APP/PS1) could prevent the behavioral and electrophysiological deficits that develop in these mice. OBJECTIVE: To explore the underlying molecular mechanisms responsible for the significant physiological and behavioral improvements observed in sAPPα-treated APP/PS1 mice. METHODS: We assessed the long-term effects on the hippocampal transcriptome following continuous lentiviral delivery of sAPPα or empty-vector to male APP/PS1 mice and wild-type controls using Affymetrix Mouse Transcriptome Assays. Data analysis was carried out within the Affymetrix Transcriptome Analysis Console and an integrated analysis of the resulting transcriptomic data was performed with Ingenuity Pathway analysis (IPA). RESULTS: Mouse transcriptome assays revealed expected AD-associated gene expression changes in empty-vector APP/PS1 mice, providing validation of the assays used for the analysis. By contrast, there were specific sAPPα-associated gene expression profiles which included increases in key neuroprotective genes such as Decorin, betaine-GABA transporter and protocadherin beta-5, subsequently validated by qRT-PCR. An integrated biological pathways analysis highlighted regulation of GABA receptor signaling, cell survival and inflammatory responses. Furthermore, upstream gene regulatory analysis implicated sAPPα activation of Interleukin-4, which can counteract inflammatory changes in AD. CONCLUSION: This study identified key molecular processes that likely underpin the long-term neuroprotective and therapeutic effects of increasing sAPPα levels in vivo.

Altered brain arginine metabolism with age in the APPswe/PSEN1dE9 mouse model of Alzheimer's disease.

Amyloid-beta (Aß) cleaved from amyloid precursor protein (APP) has been proposed to play a central and causative role in the aetiology of Alzheimer's disease (AD). APPswe/PSEN1dE9 (APP/PS1) transgenic mice display chronic Aß accumulation and deposition in the brain. L-arginine is a semi-essential amino acid with a number of bioactive metabolites, and altered arginine metabolism has been implicated in the pathogenesis and/or the development of AD. This study systematically investigated how arginine metabolic profiles changed in the frontal cortex, hippocampus, parahippocampal region and cerebellum of male APP/PS1 mice at 4, 9 and 17 months of age relative to their sex- and age-matched wildtype controls. Immunohistochemistry demonstrated age-related Aß deposition in the brain. High-performance liquid chromatography and mass spectrometry revealed age-related increases in glutamine, spermidine and spermine in APP/PS1 mice in a region-specific manner. Notably, genotype-related increases in spermine were found in the frontal cortex at the 9-month age point and in the frontal cortex, hippocampus and parahippocampal region at 17 months of age. Given the existing literature indicating the role of polyamines (spermine in particular) in modulating the aggregation and toxicity of Aß oligomers, increased spermidine and spermine levels in APP/PS1 mice may be a neuroprotective mechanism to combat Aß toxicity. Future research is required to better understand the functional significance of these changes.

The Tripeptide RER Mimics Secreted Amyloid Precursor Protein-Alpha in Upregulating LTP.

Secreted amyloid precursor protein-alpha (sAPPα), generated by enzymatic processing of the APP, possesses a range of neurotrophic and neuroprotective properties and plays a critical role in the molecular mechanisms of memory and learning. One of the key active regions of sAPPα is the central APP domain (E2) that contains within it the tripeptide sequence, RER. This sequence is exposed on the surface of a coiled coil substructure of E2. RER has by itself displayed memory-enhancing properties, and can protect newly formed engrams from interference in a manner similar to that displayed by sAPPα itself. In order to determine whether RER mimics other properties of sAPPα, we investigated the electrophysiological effects of the N-terminal protected acetylated RER (Ac-RER) and an isoform containing a chiral switch in the first amino acid from an l- to a d-orientation (Ac-rER), on synaptic plasticity. We found that, like sAPPα, exogenous perfusion with nanomolar concentrations of Ac-RER or Ac-rER enhanced the induction and stability of long-term potentiation (LTP) in area CA1 of rat and mouse hippocampal slices, in a protein synthesis- and trafficking-dependent manner. This effect did not occur with a control Ac-AAA or Ac-IFR tripeptide, nor with a full-length sAPPα protein where RER was substituted with AAA. Ac-rER also protected LTP against amyloid-beta (Aß25 - 35)-induced LTP impairment. Our findings provide further evidence that the RER-containing region of sAPPα is functionally significant and by itself can produce effects similar to those displayed by full length sAPPα, suggesting that this tripeptide, like sAPPα, may have therapeutic potential.

Tumor Necrosis Factor-α-Mediated Metaplastic Inhibition of LTP Is Constitutively Engaged in an Alzheimer's Disease Model.

LTP, a fundamental mechanism of learning and memory, is a highly regulated process. One form of regulation is metaplasticity (i.e., the activity-dependent and long-lasting changes in neuronal state that orchestrate the direction, magnitude, and persistence of future synaptic plasticity). We have previously described a heterodendritic metaplasticity effect, whereby strong high-frequency priming stimulation in stratum oriens inhibits subsequent LTP in the stratum radiatum of hippocampal area CA1, potentially by engagement of the enmeshed astrocytic network. This effect may occur due to neuron-glia interactions in response to priming stimulation that leads to the release of gliotransmitters. Here we found in male rats that TNFα and associated signal transduction enzymes, but not interleukin-1ß (IL-1ß), were responsible for mediating the metaplasticity effect. Replacing priming stimulation with TNFα incubation reproduced these effects. As TNFα levels are elevated in Alzheimer's disease, we examined whether heterodendritic metaplasticity is dysregulated in a transgenic mouse model of the disease, either before or after amyloid plaque formation. We showed that TNFα and IL-1ß levels were significantly increased in aged but not young transgenic mice. Although control LTP was impaired in the young transgenic mice, it was not TNFα-dependent. In the older transgenic mice, however, LTP was impaired in a way that occluded further reduction by heterosynaptic metaplasticity, whereas LTP was entirely rescued by incubation with a TNFα antibody, but not an IL-1ß antibody. Thus, TNFα mediates a heterodendritic metaplasticity in healthy rodents that becomes constitutively and selectively engaged in a mouse model of Alzheimer's disease.SIGNIFICANCE STATEMENT The proinflammatory cytokine TNFα is known to be capable of inhibiting LTP and is upregulated several-fold in brain tissue, serum, and CSF of Alzheimer's disease (AD) patients. However, the mechanistic roles played by TNFα in plasticity and AD remain poorly understood. Here we show that TNFα and its downstream signaling molecules p38 MAPK, ERK, and JNK contribute fundamentally to a long-range metaplastic inhibition of LTP in rats. Moreover, the impaired LTP in aged APP/PS1 mice is rescued by incubation with a TNFα antibody. Thus, there is an endogenous engagement of the metaplasticity mechanism in this mouse model of AD, supporting the idea that blocking TNFα might be of therapeutic benefit in the disease.

Safety and neurochemical profiles of acute and sub-chronic oral treatment with agmatine sulfate.

Agmatine (decarboxylated arginine) exerts numerous central nervous system (CNS) dependent pharmacological effects and may potentially modulate altered neurochemistry seen in neurological disorders. In preclinical studies, injection has been the predominant route of systemic administration. However, a significant translational step would be the use of oral agmatine treatment at therapeutic doses and better understanding of L-arginine metabolic profiles in the CNS post-treatment. The present study systematically investigated the tolerability, safety and brain-plasma neurochemistry following daily oral agmatine sulfate treatment (via gavage) to wild-type (WT) mice up to 900 mg/kg for one week (Experiment 1) or WT and APPswe/PS1ΔE9 transgenic (Tg) mice at 300 mg/kg for fifteen weeks (Experiment 2). Agmatine treatment in both experiments was well tolerated with no marked behavioural impairments, and gross necropsy and organ histology revealed no pathological alterations after 15-week dosing. Moreover, oral treatment increased agmatine levels in the hippocampus and plasma of WT mice (Experiment 1), and in 6 brain regions examined (but not plasma) of WT and Tg mice (Experiment 2), at 30 minutes or 24 hours post-treatment respectively. This study provides fundamental pre-clinical evidence that daily oral delivery of agmatine sulfate to both WT and Tg mice is safe and well tolerated. Exogenous agmatine passes through the blood brain barrier and accumulates in the brain to a greater extent in Tg mice. Furthermore exogenous agmatine has differential actions in the brain and periphery, and its effect on brain putrescine appears to be dependent on the time post-treatment.

A C-terminal peptide from secreted amyloid precursor protein-α enhances long-term potentiation in rats and a transgenic mouse model of Alzheimer's disease.

Processing of the amyloid precursor protein by alternative secretases results in ectodomain shedding of either secreted amyloid precursor protein-α (sAPPα) or its counterpart secreted amyloid precursor protein-ß (sAPPß). Although sAPPα contains only 16 additional amino acids at its C-terminus compared to sAPPß, it displays significantly greater potency in neuroprotection, neurotrophism and enhancement of long-term potentiation (LTP). In the current study, this 16 amino acid peptide sequence (CTα16) was characterised for its ability to replicate the synaptic plasticity-enhancing properties of sAPPα. An N-acetylated version of CTα16 produced concentration-dependent increases in the induction and persistence of LTP at Schaffer collateral/commissural synapses in area CA1 of young adult rat hippocampal slices. A scrambled peptide had no effect. CTα16 significantly enhanced de novo protein synthesis, and correspondingly its enhancement of LTP was blocked by the protein synthesis inhibitor cycloheximide, as well as by the α7-nicotinic receptor blocker α-bungarotoxin. The impaired LTP of 14-16 month old APPswe/PS1dE9 transgenic mice, a mouse model of Alzheimer's disease, was completely restored to the wild-type level by CTα16. These results indicate that the CTα16 peptide fragment of sAPPα mimics the larger protein's functionality with respect to LTP, stimulation of protein synthesis and activation of α7-nAChRs, and thus like sAPPα may have potential as a therapeutic agent against the plasticity and cognitive deficits observed in AD and other neurological disorders.

Glutamate Receptor Trafficking and Protein Synthesis Mediate the Facilitation of LTP by Secreted Amyloid Precursor Protein-Alpha.

Secreted amyloid precursor protein-alpha (sAPPα) has growth factor-like properties and can modulate long-term potentiation (LTP) and memory. Here, we demonstrate that exposure to sAPPα converts short-lasting LTP into protein-synthesis-dependent late LTP in hippocampal slices from male rats. sAPPß had no discernable effect. We hypothesized that sAPPα facilitated LTP via regulated glutamate receptor trafficking and de novo protein synthesis. We found using a linear mixed model that sAPPα stimulated trafficking of GluA2-lacking AMPARs, as well as NMDARs to the extrasynaptic cell surface, in a calcium/calmodulin-dependent kinase II and protein kinase G-dependent manner. Both cell surface receptor accumulation and LTP facilitation were present even after sAPPα washout and inhibition of receptor trafficking or protein synthesis prevented all these effects. Direct visualization of newly synthesized proteins (FUNCAT-PLA) confirmed the ability of sAPPα to stimulate de novo protein synthesis and revealed GluA1 as one of the upregulated proteins. Therefore, sAPPα generates a coordinated synthesis and trafficking of glutamate receptors to the cell surface that facilitate LTP.SIGNIFICANCE STATEMENT Secreted amyloid precursor protein-alpha (sAPPα) is a neurotrophic and neuroprotective protein that can promote synaptic plasticity and memory, yet the molecular mechanisms underlying these effects are still not well understood. Here, we show that sAPPα facilitates long-term potentiation (LTP) in a concentration-dependent fashion through cellular processes involving de novo protein synthesis and trafficking of both GluA2-lacking AMPARs and NMDARs to the extrasynaptic cell surface. sAPPα also enhances GluA1, but not GluA2, synthesis. The trafficking effects, along with the LTP facilitation, persist after sAPPα washout, revealing a metaplastic capability of exogenous sAPPα administration. sAPPα thus facilitates LTP through coordinated activation of protein synthesis and trafficking of glutamate receptors to the cell surface, where they are positioned for priming LTP.

Circulating Plasma microRNAs are Altered with Amyloidosis in a Mouse Model of Alzheimer's Disease.

Pathological changes underlying Alzheimer's disease (AD) begin decades before the classical symptoms of memory loss become evident. As microRNAs are released from neurons and enter the bloodstream, circulating microRNAs may be reflective of AD progression and are ideal candidates as biomarkers for early-stage disease detection. Here, we provide a novel, in-depth analysis of how plasma microRNAs alter with aging, the most prominent risk factor for AD, and with development of amyloid-ß (Aß) plaque deposition. We assessed the circulating microRNAs in APPswe/PSEN1dE9 transgenic mice and wild-type controls at 4, 8 and 15 m (n = 8-10) using custom designed Taqman arrays representing 185 neuropathology-related microRNAs. We performed a linear mixed-effects model to investigate the effects of age and genotype on plasma microRNAs expression. Following this analysis, we found 8 microRNAs were significantly affected by age alone in wild-type animals and 12 microRNAs altered in APPswe/PSEN1dE9 mice, either prior to Aß plaque deposition (4 m) or during the development of AD-like pathogenesis (8 m or 15 m). Importantly, we found that differing sets of microRNAs were identified at each time point. Functional analysis of these data revealed that while common biological pathways, such as Inflammatory Response, were enriched throughout the disease process, Free Radical Scavenging, Immunological Disease, and Apoptosis Signaling were specifically enriched later in the disease process. Overall, this study reinforces that distinct biological processes underpin the early versus late stages of AD-like pathogenesis and highlights potential pre-symptomatic microRNAs biomarkers of neurodegeneration.

Lentivirus-mediated expression of human secreted amyloid precursor protein-alpha prevents development of memory and plasticity deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease driven in large part by accumulated deposits in the brain of the amyloid precursor protein (APP) cleavage product amyloid-ß peptide (Aß). However, AD is also characterised by reductions in secreted amyloid precursor protein-alpha (sAPPα), an alternative cleavage product of APP. In contrast to the neurotoxicity of accumulated Αß, sAPPα has many neuroprotective and neurotrophic properties. Increasing sAPPα levels has the potential to serve as a therapeutic treatment that mitigates the effects of Aß and rescue cognitive function. Here we tested the hypothesis that lentivirus-mediated expression of a human sAPPα construct in a mouse model of AD (APPswe/PS1dE9), begun before the onset of plaque pathology, could prevent later behavioural and electrophysiological deficits. Male mice were given bilateral intra-hippocampal injections at 4 months of age and tested 8-10 months later. Transgenic mice expressing sAPPα performed significantly better than untreated littermates in all aspects of the spatial water maze task. Expression of sAPPα also resulted in partial rescue of long-term potentiation (LTP), tested in vitro. These improvements occurred in the absence of changes in amyloid pathology. Supporting these findings on LTP, lentiviral-mediated expression of sAPPα for 3 months from 10 months of age, or acute sAPPα treatment in hippocampal slices from 18 to 20 months old transgenic mice, completely reversed the deficits in LTP. Together these findings suggest that sAPPα has wide potential to act as either a preventative or restorative therapeutic treatment in AD by mitigating the effects of Aß toxicity and enhancing cognitive reserve.

Therapeutic Potential of Secreted Amyloid Precursor Protein APPsα.

Cleavage of the amyloid precursor protein (APP) by α-secretase generates an extracellularly released fragment termed secreted APP-alpha (APPsα). Not only is this process of interest due to the cleavage of APP within the amyloid-beta sequence, but APPsα itself has many physiological properties that suggest its great potential as a therapeutic target. For example, APPsα is neurotrophic, neuroprotective, neurogenic, a stimulator of protein synthesis and gene expression, and enhances long-term potentiation (LTP) and memory. While most early studies have been conducted in vitro, effectiveness in animal models is now being confirmed. These studies have revealed that either upregulating α-secretase activity, acutely administering APPsα or chronic delivery of APPsα via a gene therapy approach can effectively treat mouse models of Alzheimer's disease (AD) and other disorders such as traumatic head injury. Together these findings suggest the need for intensifying research efforts to harness the therapeutic potential of this multifunctional protein.

Time-dependent changes in gene expression induced by secreted amyloid precursor protein-alpha in the rat hippocampus.

BACKGROUND: Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer's disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15). RESULTS: Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer's disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα. CONCLUSIONS: Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.

Calcium/calmodulin-dependent protein kinase II mediates group I metabotropic glutamate receptor-dependent protein synthesis and long-term depression in rat hippocampus.

Activation of Group I metabotropic glutamate receptors (mGluRs) in rat hippocampus induces a form of long-term depression (LTD) that is dependent on protein synthesis. However, the intracellular mechanisms leading to the initiation of protein synthesis and expression of LTD after mGluR activation are only partially understood. We investigated the role of several pathways linked to mGluR activation, translation initiation, and induction of LTD. We found that Group I mGluR-dependent protein synthesis and associated LTD, as induced by the agonist (RS)-3,5-dihydrophenylglycine (DHPG) or paired-pulse synaptic stimulation, was dependent on activation of calcium/calmodulin-dependent protein kinase IIα (CaMKII). DHPG induced a transient increase in the level of phospho-CaMKII (phospho-CaMKII(T286)) in synaptoneurosomes prepared from whole hippocampus and in CA1 minislices. In synaptoneurosomes, DHPG also induced an increase in phosphorylation of eIF4E, and an increase in protein synthesis that was abolished by translation inhibitors and the CaMKII inhibitors 1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chloro-cinnamyl)-N-methylbenzylamine (KN93). In field recordings from CA1, both the translation inhibitor cycloheximide and KN62 significantly reduced DHPG-induced LTD. Combined application did not further reduce the LTD, suggesting a common mechanism. In whole-cell recordings, a third CaMKII inhibitor, AIP (autocamtide-2-related inhibitory peptide), significantly reduced the DHPG-induced LTD of synaptic currents. Inhibition of the classical pathway mediating many Group I mGluR effects by blocking PKC (protein kinase C) or PLC (phospholipase C) did not impair DHPG-induced protein synthesis or LTD. Collectively, these findings demonstrate an important role for CaMKII in mediating the initiation of protein synthesis that then supports the postsynaptic expression of DHPG-induced LTD.

Metaplasticity: new insights through electrophysiological investigations.

The term synaptic plasticity describes the ability of excitatory synapses to undergo activity-driven long-lasting changes in the efficacy of basal synaptic transmission. This change may be expressed as a long-term potentiation (LTP) or as a long-term depression (LTD). Metaplasticity is a higher-order form of synaptic plasticity that regulates the expression of both LTP and LTD through processes that are initiated by cellular activity that precedes a later bout of plasticity-inducing synaptic activity. Activation by prior synaptic activity and later expression as a facilitation or inhibition of activity-dependent synaptic plasticity are fundamental properties of metaplasticity. The intracellular mechanisms which support metaplasticity appear to be closely linked to those of synaptic plasticity, hence there are significant technical challenges to overcome in order to elucidate those mechanisms specific to metaplasticity. This review will examine the progress in the characterization of metaplasticity over the last decade or so with a focus on findings gained using electrophysiological techniques. It will look at the techniques applied, the brain regions investigated and the knowledge gained from the application of a wide range of protocols designed to examine the influence of varied forms of prior synaptic activity on later, activity-induced, synaptic plasticity.

Dopamine D1/D5 receptor activation reverses NMDA receptor-dependent long-term depression in rat hippocampus.

Activation of dopamine D1/D5 receptors (D1/D5Rs) in area CA1 of the rat hippocampus modulates the expression of synaptic plasticity in a manner that is dependent on the timing of the D1/D5R activation. Here, we measured field EPSPs in rat hippocampal slices to examine the modulation of long-term depression (LTD) in CA1 by D1/D5Rs when activated immediately after the induction of LTD by low-frequency stimulation (LFS) or bath application of NMDA or the metabotropic glutamate receptor agonist DHPG [(RS)-3,5-dihydroxyphenylglycine]. Activation of D1/D5Rs by SKF 38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide] completely reversed a moderate LFS-induced LTD in a time-dependent manner, presumably through an adenylate cyclase/cAMP cascade. In support of this, general adenylate cyclase activation by forskolin ([3R-(3 alpha,4a beta,5 beta,6 beta,6a alpha,10 alpha,10a beta,10b alpha)]-5-(acetyloxy)-3-ethenyldodecahydro-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamenthyl-1H-naphtho[2,1-b]pyran-1-one) immediately, but not 60 min, after LFS also reversed the LTD. Beta-adrenergic receptor activation by isoproterenol failed to reverse the LTD, indicating that reversal is specific to D1/D5R-mediated increased cAMP production. SKF 38393 only partially reversed a more robust LFS-induced LTD, indicating that some components of consolidated LTD are resistant to reversal. LTD induced by bath application of NMDA, but not DHPG, was also reversed by SKF 38393. Western blot analysis of postsynaptic density fractions after NMDA-induced LTD revealed that the LTD was attributable to dephosphorylation of the AMPA receptor subunit glutamate receptor 1 (GluR1) at serine 845, without a change in total GluR content. Reversal of the LTD by SKF 38393 was associated with rephosphorylation of this same residue. Together, these findings demonstrate a new role for dopamine in the neuromodulation of hippocampal LTD.

Dopamine D1/D5 receptor activation fails to initiate an activity-independent late-phase LTP in rat hippocampus.

The role of dopamine in the hippocampus remains poorly defined. Numerous studies have suggested that it acts as a neuromodulator of late-phase long-term potentiation (L-LTP) in CA1, while other reports controversially indicate that D1/D5 receptor (D1/D5R) activation may directly initiate activity-independent LTP. We have further investigated this putative role of dopamine in area CA1 in rat hippocampal slices using field potential recording techniques. Application of the dopamine D1/D5 receptor agonists SKF 38393 and 6-bromo-APB at 100 microM for 20 min did not induce an activity-independent L-LTP. Varying the incubation conditions still did not permit either SKF 38393 or an alternative D1/D5R agonist, 6-chloro-PB, to induce L-LTP. To further determine if intracellular mechanisms, which may act to limit the expression of LTP, were preventing D1/D5R-induced L-LTP expression, we inhibited protein phosphatase 1 activity by reducing cyclin-dependent kinase 5 (cdk5) inhibition of inhibitor 1. Inhibition of cdk5 by roscovitine (10 microM, 40 min) did not facilitate the ability of SKF 38393 to induce L-LTP in CA1. Biochemical experiments confirmed that the concentration of agonist used significantly elevated intracellular cAMP levels, suggesting that effective D1/D5R activation was achieved. Furthermore, coactivation with NMDA receptors (NMDAR) resulted in a synergistic increase in cAMP. These findings demonstrate that D1/D5R activation in CA1 initiates intracellular second messenger accumulation, but that this is insufficient to induce an activity-independent L-LTP.