Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics.

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases, including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of an active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics show that these new compounds, such as LJ2 or its specific isomers LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. In agreement with the established role of Casp2 in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~5,500,000 M-1 s-1), and the most selective inhibitor, LJ3a, has close to a 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that the spatial configuration of Cα at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit Casp2-mediated S1P cleavage and thus SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with ß-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

Rapid ATF4 Depletion Resets Synaptic Responsiveness after cLTP.

Activating transcription factor 4 [ATF4 (also called 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 the role of ATF4 in mature hippocampal cultures subjected to a brief chemically induced LTP (cLTP) protocol that results in changes in mEPSC properties and synaptic AMPA receptor density 1 h later, with return to baseline by 24 h. We find that ATF4 protein, but not its mRNA, is rapidly depleted by ∼50% in response to cLTP induction via NMDA receptor activation. Depletion is detectable in dendrites within 15 min and in cell bodies by 1 h, and returns to baseline by 8 h. 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 overexpression blocked the recovery of synaptic activity and AMPA receptor density to baseline values that would otherwise occur 24 h after cLTP induction. This reversal was not reproduced by a transcriptionally inactive ATF4 mutant. These findings support the role of ATF4 as a required element in resetting baseline synaptic responsiveness after cLTP.

Activating Transcription Factor 4 (ATF4) Regulates Neuronal Activity by Controlling GABABR Trafficking.

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 GABAB receptors (GABABRs). Knocking down ATF4 results in significant reduction of GABABR-induced GIRK currents and increased mIPSC frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABABR 1a and 1b subunits, indicating that ATF4 regulates GABABR trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABABR2s, several GABABR-coupled ion channels or ß2 and γ2 GABAARs. Pharmacologic manipulations confirmed the relationship between GABABR 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 GABABR 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 GABABRs, which in turn modulates the excitability properties of neurons.SIGNIFICANCE STATEMENT GABAB receptors (GABABRs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABABRs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABABRs 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 GABABR 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 GABABRs.

Brain-Derived Neurotrophic Factor Elevates Activating Transcription Factor 4 (ATF4) in Neurons and Promotes ATF4-Dependent Induction of Sesn2.

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.

Stabilization of dynamic microtubules by mDia1 drives Tau-dependent Aß1-42 synaptotoxicity.

Oligomeric Amyloid ß1-42 (Aß) plays a crucial synaptotoxic role in Alzheimer's disease, and hyperphosphorylated tau facilitates Aß toxicity. The link between Aß and tau, however, remains controversial. In this study, we find that in hippocampal neurons, Aß 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 Aß. 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 Aß-mediated synaptotoxicity and demonstrate that inhibition of MT dynamics and accumulation of PTMs are driving factors for the induction of tau-mediated neuronal damage.

Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.

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 Aß 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.

Activating Transcription Factor 4 (ATF4) modulates Rho GTPase levels and function via regulation of RhoGDIα.

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.

Gene-wise association of variants in four lysosomal storage disorder genes in neuropathologically confirmed Lewy body disease.

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<0.001). A significant increase and accumulation of several species for the lipid classes, ceramides and sphingolipids, was observed in LBD brains carrying GBA mutations compared to controls (p range: p<0.05-p<0.01). INTERPRETATION: Our study indicates that variants in GBA, SMPD1 and MCOLN1 are associated with LB pathology. Biochemical data comparing GBA mutation carrier to non-carriers support these findings, which have important implications for biomarker development and therapeutic strategies.

Specific downregulation of hippocampal ATF4 reveals a necessary role in synaptic plasticity and memory.

Prior 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.

Assembly and interrogation of Alzheimer's disease genetic networks reveal novel regulators of progression.

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.

Dysregulation of microRNA-219 promotes neurodegeneration through post-transcriptional regulation of tau.

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.

Functional role of RNA polymerase II and P70 S6 kinase in KCl withdrawal-induced cerebellar granule neuron apoptosis.

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-ß-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.

A systems approach to drug discovery in Alzheimer's disease.

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.

Primary age-related tauopathy (PART): a common pathology associated with human aging.

We recommend a new term, "primary age-related tauopathy" (PART), to describe a pathology that is commonly observed in the brains of aged individuals. Many autopsy studies have reported brains with neurofibrillary tangles (NFTs) that are indistinguishable from those of Alzheimer's disease (AD), in the absence of amyloid (Aß) plaques. For these "NFT+/Aß-" brains, for which formal criteria for AD neuropathologic changes are not met, the NFTs are mostly restricted to structures in the medial temporal lobe, basal forebrain, brainstem, and olfactory areas (bulb and cortex). Symptoms in persons with PART usually range from normal to amnestic cognitive changes, with only a minority exhibiting profound impairment. Because cognitive impairment is often mild, existing clinicopathologic designations, such as "tangle-only dementia" and "tangle-predominant senile dementia", are imprecise and not appropriate for most subjects. PART is almost universally detectable at autopsy among elderly individuals, yet this pathological process cannot be specifically identified pre-mortem at the present time. Improved biomarkers and tau imaging may enable diagnosis of PART in clinical settings in the future. Indeed, recent studies have identified a common biomarker profile consisting of temporal lobe atrophy and tauopathy without evidence of Aß accumulation. For both researchers and clinicians, a revised nomenclature will raise awareness of this extremely common pathologic change while providing a conceptual foundation for future studies. Prior reports that have elucidated features of the pathologic entity we refer to as PART are discussed, and working neuropathological diagnostic criteria are proposed.

Activating transcription factor 4 (ATF4) modulates post-synaptic development and dendritic spine morphology.

The ubiquitously expressed activating transcription factor 4 (ATF4) has been variably reported to either promote or inhibit neuronal plasticity and memory. However, the potential cellular bases for these and other actions of ATF4 in brain are not well-defined. In this report, we focus on ATF4's role in post-synaptic synapse development and dendritic spine morphology. shRNA-mediated silencing of ATF4 significantly reduces the densities of PSD-95 and GluR1 puncta (presumed markers of excitatory synapses) in long-term cultures of cortical and hippocampal neurons. ATF4 knockdown also decreases the density of mushroom spines and increases formation of abnormally-long dendritic filopodia in such cultures. In vivo knockdown of ATF4 in adult mouse hippocampal neurons also reduces mushroom spine density. In contrast, ATF4 over-expression does not affect the densities of PSD-95 puncta or mushrooom spines. Regulation of synaptic puncta and spine densities by ATF4 requires its transcriptional activity and is mediated at least in part by indirectly controlling the stability and expression of the total and active forms of the actin regulatory protein Cdc42. In support of such a mechanism, ATF4 silencing decreases the half-life of Cdc42 in cultured cortical neurons from 31.5 to 18.5 h while knockdown of Cdc42, like ATF4 knockdown, reduces the densities of mushroom spines and PSD-95 puncta. Thus, ATF4 appears to participate in neuronal development and plasticity by regulating the post-synaptic development of synapses and dendritic mushroom spines via a mechanism that includes regulation of Cdc42 levels.

Generation of iPSC lines from archived non-cryoprotected biobanked dura mater.

BACKGROUND: Induced pluripotent stem cells (iPSCs) derived from patients with neurodegenerative disease generally lack neuropathological confirmation, the gold standard for disease classification and grading of severity. The use of tissue with a definitive neuropathological diagnosis would be an ideal source for iPSCs. The challenge to this approach is that the majority of biobanked brain tissue was not meant for growing live cells, and thus was not frozen in the presence of cryoprotectants such as DMSO. RESULTS: We report the generation of iPSCs from frozen non-cryoprotected dural tissue stored at -80°C for up to 11 years. This autopsy cohort included subjects with Alzheimer's disease and four other neurodegenerative diseases. CONCLUSIONS: Disease-specific iPSCs can be generated from readily available, archival biobanked tissue. This allows for rapid expansion of generating iPSCs with confirmed pathology as well as allowing access to rare patient variants that have been banked.

Caspase-2 is required for dendritic spine and behavioural alterations in J20 APP transgenic mice.

Caspases have critical roles in Alzheimer's disease pathogenesis. Here we show that caspase-2 is required for the cognitive decline seen in human amyloid precursor protein transgenic mice (J20). The age-related changes in behaviour and dendritic spine density observed in these mice are absent when they lack caspase-2, in spite of similar levels of amyloid beta (Aß) deposition and inflammation. A similar degree of protection is observed in cultured hippocampal neurons lacking caspase-2, which are immune to the synaptotoxic effects of Aß. Our studies suggest that caspase-2 is a critical mediator in the activation of the RhoA/ROCK-II signalling pathway, leading to the collapse of dendritic spines. We propose that this is controlled by an inactive caspase-2/RhoA/ROCK-II complex localized in dendrites, which dissociates in the presence of Aß, allowing for their activation and entry in the spine. These findings directly implicate caspase-2 as key driver of synaptic dysfunction in Alzheimer's disease and offer novel therapeutic targets.

ß-Amyloid (Aß) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1.

We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aß oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aß-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aß may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.