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Activating Transcription Factor 4 (ATF4) has been postulated as a key regulator of learning and memory. We previously reported that specific hippocampal ATF4 down-regulation causes deficits in synaptic plasticity and memory and reduction of glutamatergic functionality. Here we extend our studies to address ATF49s role in neuronal excitability. We find that long-term ATF4 knockdown in cultured rat hippocampal neurons significantly increases the frequency of spontaneous action potentials. This effect is associated with decreased functionality of metabotropic GABA B receptors (GABA B Rs). Knocking down ATF4 results in significant reduction of GABA B R-induced GIRK-currents and increased mIPSCs frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABA B R 1a and 1b subunits, indicating that ATF4 regulates GABA B R trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABA B R2s, several GABA B R-coupled ion channels or β2 and Ɣ2 GABA A Rs. Pharmacologic manipulations confirmed the relationship between GABA B R functionality and action potential frequency in our cultures. Specifically, the effects of ATF4 down-regulation cited-above are fully rescued by transcriptionally active, but not by transcriptionally-inactive, shRNA-resistant, ATF4. We previously reported that ATF4 promotes stabilization of the actin-regulatory protein Cdc42 by a transcription-dependent mechanism. To test the hypothesis that this action underlies the mechanism by which ATF4 loss affects neuronal firing rates and GABA B R trafficking, we down-regulated Cdc42 and found that this phenocopies the effects of ATF4 knockdown on these properties. In conclusion, our data favor a model in which ATF4, by regulating Cdc42 expression, affects trafficking of GABA B Rs, which in turn modulates the excitability properties of neurons. Significance statement: GABA B receptors (GABA B Rs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABA B Rs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABA B Rs trafficking is influenced by Activating Transcription Factor 4 (ATF4), a protein that has a pivotal role in hippocampal memory processes. We found that ATF4 down-regulation in hippocampal neurons reduces membrane-bound GABA B R levels and thereby increases intrinsic excitability. These effects are mediated by loss of the small GTPase Cdc42 following ATF4 down-regulation. These findings reveal a critical role for ATF4 in regulating the modulation of neuronal excitability by GABA B Rs.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/− /PS1 +/−). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Qu et al. present the first evidence that mDia1-mediated stabilization of dynamic microtubules (MTs) and induction of tubulin posttranslational modifications drive tau hyperphosphorylation and synaptotoxicity, providing a novel target for therapy and the first mechanistic explanation as to why a dynamic MT cytoskeleton must be maintained to prevent neurodegenerative disease., Oligomeric Amyloid β1–42 (Aβ) plays a crucial synaptotoxic role in Alzheimer’s disease, and hyperphosphorylated tau facilitates Aβ toxicity. The link between Aβ and tau, however, remains controversial. In this study, we find that in hippocampal neurons, Aβ acutely induces tubulin posttranslational modifications (PTMs) and stabilizes dynamic microtubules (MTs) by reducing their catastrophe frequency. Silencing or acute inhibition of the formin mDia1 suppresses these activities and corrects the synaptotoxicity and deficits of axonal transport induced by Aβ. We explored the mechanism of rescue and found that stabilization of dynamic MTs promotes tau-dependent loss of dendritic spines and tau hyperphosphorylation. Collectively, these results uncover a novel role for mDia1 in Aβ-mediated synaptotoxicity and demonstrate that inhibition of MT dynamics and accumulation of PTMs are driving factors for the induction of tau-mediated neuronal damage.

Activating transcription factor 4 (ATF4/CREB2), in addition to its well-studied role in stress responses, is proposed to play important physiologic functions in regulating learning and memory. However, the nature of these functions has not been well defined and is subject to apparently disparate views. Here, we provide evidence that ATF4 is a regulator of excitability during synaptic plasticity. We evaluated ATF49s role in mature hippocampal cultures subjected to a brief chemical-LTP (cLTP) induction protocol that results in changes in mEPSC properties and synaptic AMPA receptor density one hour later, with return to baseline by 24 hours. We find that ATF4 protein, but not its mRNA, is rapidly depleted by about 50% in response to cLTP induction via NMDA receptor activation. Depletion is detectable in dendrites within 15 min and in cell bodies by 1 hour and returns to baseline by 8 hours. Such changes correlate with a parallel depletion of phospho-eIF2a, suggesting that ATF4 loss is driven by decreased translation. To probe the physiologic role of cLTP-induced ATF4 depletion, we constitutively overexpressed the protein. Reversing ATF4 depletion by over-expression blocked the recovery of synaptic activity and AMPA receptor density to baseline values that would otherwise occur 24 hours after cLTP induction. This reversal was not reproduced by a transcriptionally inactive ATF4 mutant. These findings support ATF49s role as a required element in resetting baseline synaptic responsiveness after cLTP. Significance The mechanism(s) by which synaptic responsiveness is reset after LTP are not well understood. Resetting avoids LTP "saturation" and uncontrolled feed-forward potentiation and may play a part in synaptic "scaling”. In the work reported here we have found that the transcription factor ATF4 is translationally down-regulated following cLTP induction and acts as a regulator of long-term synaptic plasticity, resetting the synapse to the de-potentiated state. Our findings may serve to begin to reconcile the conflicting views regarding the role of ATF4 in synaptic plasticity and further illuminate the role of ATF4 in learning and memory.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.

SummaryPrior studies suggested that the transcription factor ATF4 negatively regulates synaptic plastic and memory. By contrast, we provide evidence from direct in vitro and in vivo knockdown of ATF4 in rodent hippocampal neurons and from ATF4-null mice that implicate ATF4 as essential for normal synaptic plasticity and memory. In particular, hippocampal ATF4 downregulation produces deficits in long-term spatial memory and behavioral flexibility without affecting associative memory or anxiety-like behavior. ATF4 knockdown or loss also causes profound impairment of both long-term potentiation (LTP) and long-term depression (LTD) as well as decreased glutamatergic function. We conclude that ATF4 is a key regulator of the physiological state necessary for neuronal plasticity and memory.

Tau is a highly abundant and multifunctional brain protein that accumulates in neurofibrillary tangles (NFTs), most commonly in Alzheimer's disease (AD) and primary age-related tauopathy. Recently, microRNAs (miRNAs) have been linked to neurodegeneration; however, it is not clear whether miRNA dysregulation contributes to tau neurotoxicity. Here, we determined that the highly conserved brain miRNA miR-219 is downregulated in brain tissue taken at autopsy from patients with AD and from those with severe primary age-related tauopathy. In a Drosophila model that produces human tau, reduction of miR-219 exacerbated tau toxicity, while overexpression of miR-219 partially abrogated toxic effects. Moreover, we observed a bidirectional modulation of tau levels in the Drosophila model that was dependent on miR-219 expression or neutralization, demonstrating that miR-219 regulates tau in vivo. In mammalian cellular models, we found that miR-219 binds directly to the 3'-UTR of the tau mRNA and represses tau synthesis at the post-transcriptional level. Together, our data indicate that silencing of tau by miR-219 is an ancient regulatory mechanism that may become perturbed during neurofibrillary degeneration and suggest that this regulatory pathway may be useful for developing therapeutics for tauopathies.

Activating Transcription Factor 4 (ATF4) has been postulated as a key regulator of learning and memory. We previously reported that specific hippocampal ATF4 downregulation causes deficits in synaptic plasticity and memory and reduction of glutamatergic functionality. Here we extend our studies to address ATF4's role in neuronal excitability. We find that long-term ATF4 knockdown in cultured rat hippocampal neurons significantly increases the frequency of spontaneous action potentials. This effect is associated with decreased functionality of metabotropic GABA(B) receptors (GABA(B)Rs). Knocking down ATF4 results in significant reduction of GABA(B)R-induced GIRK currents and increased mIPSC frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABA(B)R 1a and 1b subunits, indicating that ATF4 regulates GABA(B)R trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABA(B)R2s, several GABA(B)R-coupled ion channels or β2 and γ2 GABA(A)Rs. Pharmacologic manipulations confirmed the relationship between GABA(B)R functionality and action potential frequency in our cultures. Specifically, the effects of ATF4 downregulation cited above are fully rescued by transcriptionally active, but not by transcriptionally inactive, shRNA-resistant, ATF4. We previously reported that ATF4 promotes stabilization of the actin-regulatory protein Cdc42 by a transcription-dependent mechanism. To test the hypothesis that this action underlies the mechanism by which ATF4 loss affects neuronal firing rates and GABA(B)R trafficking, we downregulated Cdc42 and found that this phenocopies the effects of ATF4 knockdown on these properties. In conclusion, our data favor a model in which ATF4, by regulating Cdc42 expression, affects trafficking of GABA(B)Rs, which in turn modulates the excitability properties of neurons. SIGNIFICANCE STATEMENT GABA(B) receptors (GABA(B)Rs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABA(B)Rs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABA(B)Rs trafficking is influenced by Activating Transcription Factor 4 (ATF4), a protein that has a pivotal role in hippocampal memory processes. We found that ATF4 downregulation in hippocampal neurons reduces membrane-bound GABA(B)R levels and thereby increases intrinsic excitability. These effects are mediated by loss of the small GTPase Cdc42 following ATF4 downregulation. These findings reveal a critical role for ATF4 in regulating the modulation of neuronal excitability by GABA(B)Rs.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.

In earlier studies, we showed that ATF4 down-regulation affects post-synaptic development and dendritic spine morphology in neurons through increased turnover of the Rho GTPase Cell Division Cycle 42 (Cdc42) protein. Here, we find that ATF4 down-regulation in both hippocampal and cortical neuron cultures reduces protein and message levels of RhoGDIα, a stabilizer of the Rho GTPases including Cdc42. This effect is rescued by an shATF4-resistant active form of ATF4, but not by a mutant that lacks transcriptional activity. This is, at least in part, due to the fact that Arhgdia, the gene encoding RhoGDIα, is a direct transcriptional target of ATF4 as is shown in ChIP assays. This pathway is not restricted to neurons. This is seen in an impairment of cell migration on ATF4 reduction in non-neuronal cells. In conclusion, we have identified a new cellular pathway in which ATF4 regulates the expression of RhoGDIα that in turn affects Rho GTPase protein levels, and thereby, controls cellular functions as diverse as memory and cell motility.

Alzheimer’s disease researchers are a pretty dispirited lot these days. In the first half of the 20th century research on this malady and an understanding of its impact on society were limited in scope and intensity. The dementia described by Alois Alzheimer was felt to refer to a relatively rare disease of middle age and different from senile dementia of the elderly, which was often attributed to cerebral atherosclerosis. It was not until the application of electron microscopy to the brain at the beginning of the second half of the 20th century that it became clear that the neurofibrillary degeneration seen in senile dementia was composed of the same, unique, paired helical filaments that were seen in Alzheimer’s disease and that there was an identical deposition of congophilic senile plaques and of synaptic changes in both entities. This entity in the elderly was first called “Senile Dementia of the Alzheimer’s type (SDAT)” and eventually both were grouped as Alzheimer’s disease (AD). There was a lively debate at that time as to whether AD was or was not an amyloidosis that was eventually resolved in a rapid series of discoveries that followed on the purification and identification of the amyloid peptide (Aβ or beta amyloid) from the brain by the late George Glenner. The findings that mutations in the Amyloid Precursor Protein (APP), from which Aβ is processed, and the proteins presenilin 1 (PS1) and presenilin 2 (PS2) were associated with early onset AD helped established the “Amyloid Hypothesis” that, reduced to its simplest form, overproduction of Aβ (or reductions in its clearance) leads to the histopathological and behavioral changes that characterize AD. In this model, the widespread intracellular neurofibrillary tangles composed of tau protein are believed to be downstream of the generation of Aβ. These discoveries led to the generation of transgenic mice that over-expressed mutant human APP, developed amyloid plaques quite similar to those seen in human AD and showed age-related alterations in memory. None of these mice showed significant neuronal loss nor development of neurofibrillary tangles, though increased tau phosphorylation was seen. These models have been the primary objects of biological study on AD over the past few years, and a large number of agents have shown the ability to reverse the electrophysiological and behavioral changes seen in them. These include a number of the classes of agents that are discussed in this volume. The development of clinical drugs has focused on the removal of Aβ and on blocking its production. These include active and passive immunization against Aβ and inhibition of the γ-secretase and β-secretases that cleave APP to produce Aβ. These approaches have been extremely successful in the rodent but clinical trials using them have all failed to improve established AD. This has led to the view that the mouse models reflect only the early stages of the disease and that latter stages of the disease—the stages with neurofibrillary tangles and neuronal loss—are irreversible and too late to treat. This question is being addressed by using these agents to treat patients who have dominant mutations in PS1 that will lead to AD in 100% of persons carrying the mutation. Therefore, treatment can be started before symptoms appear and alterations in the normally rapid progression of the disease assessed. The introduction of PET scanning ligands for Aβ and for tau also will allow for treating patients who are developing Aβ pathology prior to the clinical manifestations of the disease. Even if these approaches are successful, it is likely that more than one drug will be needed to treat the disease and arrest its progression. The articles in this volume focus on a number of systems that are affected in AD. They explore key topics such as energetics and mitochondria, the role of heavy metals, molecules to increase synaptic plasticity and approaches to the defects in protein sorting and in protein degradation. Given the limitations mentioned in current animal models, articles in this volume describe advances in the use of human stem cells and stem cell-derived neurons for the study of AD, as well as the application of the new areas of computational and systems biology to analyze data derived from post-mortem human brain to predict master regulators of AD progression. The sum total of the articles provides a snapshot of where we are at this place and time, the model systems that might be appropriate and some glimpse of future research directions. We apologize to authors and areas that have been omitted from this volume because of limits of space and time. We hope that the papers presented here will assist those in the field, those contemplating entering AD research and to the professionals involved in the care of AD patients.

KCl withdrawal-induced apoptosis in cerebellar granule neurons is associated with aberrant cell cycle activation, and treatment with cyclin-dependent kinase (Cdk) inhibitors protects cells from undergoing apoptosis. Because the Cdk inhibitor flavopiridol is known to inhibit RNA polymerase II (Pol II)-dependent transcription elongation by inhibiting the positive transcription elongation factor b (P-TEFb, a complex of CDK9 and cyclin T), we examined whether inhibition of RNA Pol II protects neurons from apoptosis. Treatment of neurons with 5, 6-dichloro-1-β-D-ribobenzimidazole (DRB), an RNA Pol II-dependent transcription elongation inhibitor, and flavopiridol inhibited phosphorylation and activation of Pol II and protected neurons from undergoing apoptosis. In addition to Pol II, neurons subjected to KCl withdrawal showed increased phosphorylation and activation of p70 S6 kinase, which was inhibited by both DRB and flavopiridol. Immunostaining analysis of the neurons deprived of KCl showed increased nuclear levels of phospho-p70 S6 kinase, and neurons protected with DRB and flavopiridol showed accumulation of the kinase into large spliceosome assembly factor-positive speckle domains within the nuclei. The formation of these foci corresponded with cell survival, and removal of the inhibitors resulted in dispersal of the speckles into smaller foci with subsequent apoptosis induction. Because p70 S6 kinase is known to induce translation of mRNAs containing a 5'-terminal oligopyrimidine tract, our data suggest that transcription and translation of this subset of mRNAs may contribute to KCl withdrawal-induced apoptosis in neurons.

Objective Variants in GBA are associated with Lewy Body (LB) pathology. We investigated whether variants in other lysosomal storage disorder (LSD) genes also contribute to disease pathogenesis. Methods We performed a genetic analysis of four LSD genes including GBA, HEXA, SMPD1, and MCOLN1 in 231 brain autopsies. Brain autopsies included neuropathologically defined LBD without Alzheimer Disease (AD) changes (n = 59), AD without significant LB pathology (n = 71), Alzheimer disease and lewy body variant (ADLBV) (n = 68), and control brains without LB or AD neuropathology (n = 33). Sequencing of HEXA, SMPD1, MCOLN1 and GBA followed by ‘gene wise’ genetic association analysis was performed. To determine the functional effect, a biochemical analysis of GBA in a subset of brains was also performed. GCase activity was measured in a subset of brain samples (n = 64) that included LBD brains, with or without GBA mutations, and control brains. A lipidomic analysis was also performed in brain autopsies (n = 67) which included LBD (n = 34), ADLBV (n = 3), AD (n = 4), PD (n = 9) and control brains (n = 17), comparing GBA mutation carriers to non-carriers. Results In a ‘gene-wise’ analysis, variants in GBA, SMPD1 and MCOLN1 were significantly associated with LB pathology (p range: 0.03–4.14 x10-5). Overall, the mean levels of GCase activity were significantly lower in GBA mutation carriers compared to non-carriers (p

In the articles included in this volume, one feels a strong frustration among the writers with the slow course of therapeutics development for Alzheimer's disease and with the clinical failure of targeted therapeutic agents despite substantial progress in our understanding of the biology and biochemistry of the disease.

In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

Alzheimer's disease (AD) is a complex multifactorial disorder with poorly characterized pathogenesis. Our understanding of this disease would thus benefit from an approach that addresses this complexity by elucidating the regulatory networks that are dysregulated in the neural compartment of AD patients, across distinct brain regions. Here, we use a Systems Biology (SB) approach, which has been highly successful in the dissection of cancer related phenotypes, to reverse engineer the transcriptional regulation layer of human neuronal cells and interrogate it to infer candidate Master Regulators (MRs) responsible for disease progression. Analysis of gene expression profiles from laser-captured neurons from AD and controls subjects, using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe), yielded an interactome consisting of 488,353 transcription-factor/target interactions. Interrogation of this interactome, using the Master Regulator INference algorithm (MARINa), identified an unbiased set of candidate MRs causally responsible for regulating the transcriptional signature of AD progression. Experimental assays in autopsy-derived human brain tissue showed that three of the top candidate MRs (YY1, p300 and ZMYM3) are indeed biochemically and histopathologically dysregulated in AD brains compared to controls. Our results additionally implicate p53 and loss of acetylation homeostasis in the neurodegenerative process. This study suggests that an integrative, SB approach can be applied to AD and other neurodegenerative diseases, and provide significant novel insight on the disease progression.