Stylianos Kosmidis

Homeostatic Mechanisms for Iron Storage Revealed by Genetic Manipulations and Live Imaging of Drosophila Ferritin

Ferritin is a symmetric, 24-subunit iron-storage complex assembled of H and L chains. It is found in bacteria, plants, and animals and in two classes of mutations in the human L-chain gene, resulting in hereditary hyperferritinemia cataract syndrome or in neuroferritinopathy. Here, we examined systemic and cellular ferritin regulation and trafficking in the model organism Drosophila melanogaster. We showed that ferritin H and L transcripts are coexpressed during embryogenesis and that both subunits are essential for embryonic development. Ferritin overexpression impaired the survival of iron-deprived flies. In vivo expression of GFP-tagged holoferritin confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into hemolymph. A constant ratio of ferritin H and L subunits, secured via tight post-transcriptional regulation, is characteristic of the secreted ferritin in flies. Differential cellular expression, conserved post-transcriptional regulation via the iron regulatory element, and distinct subcellular localization of the ferritin subunits prior to the assembly of holoferritin are all important steps mediating iron homeostasis. Our study revealed both conserved features and insect-specific adaptations of ferritin nanocages and provides novel imaging possibilities for their in vivo characterization.

The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3

Consolidation of long-term memories depends on de novo protein synthesis. Several translational regulators have been identified, and their contribution to the formation of memory has been assessed in the mouse hippocampus. None of them, however, has been implicated in the persistence of memory. Although persistence is a key feature of long-term memory, how this occurs, despite the rapid turnover of its molecular substrates, is poorly understood. Here we find that both memory storage and its underlying synaptic plasticity are mediated by the increase in level and in the aggregation of the prion-like translational regulator CPEB3 (cytoplasmic polyadenylation element-binding protein). Genetic ablation of CPEB3 impairs the maintenance of both hippocampal long-term potentiation and hippocampus-dependent spatial memory. We propose a model whereby persistence of long-term memory results from the assembly of CPEB3 into aggregates. These aggregates serve as functional prions and regulate local protein synthesis necessary for the maintenance of long-term memory.

Molecular Mechanism for Age-Related Memory Loss: The Histone-Binding Protein RbAp48

A histone-binding protein that declines with age in the human dentate gyrus can reverse age-related memory deficits in mice. Try to Remember We do not know why, but we think we know where: Age-related memory loss begins in the dentate gyrus of the brain. (Alzheimer’s disease starts in nearby entorhinal cortex and other parts of the hippocampus.) To understand better why we forget more easily as we age, Pavlopoulos et al. gained access to eight human brains and looked carefully for proteins that rose and fell in the dentate gyrus with age. One was particularly notable, RbAp48, a histone-binding protein that regulates transcription and decreases in expression in older people. Exploring its function in mice led the authors to conclude that RbAp48 participates in the dentate gyrus dysfunction that becomes more prominent with aging and could potentially be a target for treatment of age-related memory decline. By first searching for proteins that are modulated in an age-related fashion in the human brain, the authors firmly rooted their investigation in the translational space. Their subsequent experiments in mice were designed to examine the protein’s function in ways that would have been impossible in humans. Key to validating RbAp48’s function was a transgenic mouse line that carried a dominant-negative inhibitor of RbAp48, but only in the forebrain. The authors could manipulate expression of RbAp48 at will by switching it on and off with artificial triggers. Prematurely inhibiting RbAp48 in young mice resulted in memory deficits just like those seen naturally in older mice. Increasing RbAp48 in older, more forgetful mice restored their memory to its youthful vigor. And not only behavior was affected. Functional magnetic resonance imaging showed that the dentate gyrus in the artificially impaired young mice did not work properly, and molecular assays showed abnormal histone acetylation. Further exploiting their transgenic mice, the authors also support the idea that RbAp48 acts through the protein kinase A (PKA)–cyclic adenosine monophosphate (cAMP) response element–binding protein 1 (CREB1)–CREB-binding protein (CBP), a well-understood signaling pathway. Agents that enhance signaling through PKA and CREB (cAMP signaling) are already known to improve age-related problems in hippocampal function in mice, so it is a logical next step to test these drugs for therapeutic use in treating age-related memory problems in people. To distinguish age-related memory loss more explicitly from Alzheimer’s disease (AD), we have explored its molecular underpinning in the dentate gyrus (DG), a subregion of the hippocampal formation thought to be targeted by aging. We carried out a gene expression study in human postmortem tissue harvested from both DG and entorhinal cortex (EC), a neighboring subregion unaffected by aging and known to be the site of onset of AD. Using expression in the EC for normalization, we identified 17 genes that manifested reliable age-related changes in the DG. The most significant change was an age-related decline in RbAp48, a histone-binding protein that modifies histone acetylation. To test whether the RbAp48 decline could be responsible for age-related memory loss, we turned to mice and found that, consistent with humans, RbAp48 was less abundant in the DG of old than in young mice. We next generated a transgenic mouse that expressed a dominant-negative inhibitor of RbAp48 in the adult forebrain. Inhibition of RbAp48 in young mice caused hippocampus-dependent memory deficits similar to those associated with aging, as measured by novel object recognition and Morris water maze tests. Functional magnetic resonance imaging studies showed that within the hippocampal formation, dysfunction was selectively observed in the DG, and this corresponded to a regionally selective decrease in histone acetylation. Up-regulation of RbAp48 in the DG of aged wild-type mice ameliorated age-related hippocampus-based memory loss and age-related abnormalities in histone acetylation. Together, these findings show that the DG is a hippocampal subregion targeted by aging, and identify molecular mechanisms of cognitive aging that could serve as valid targets for therapeutic intervention.

A Drosophila ortholog of the human cylindromatosis tumor suppressor gene regulates triglyceride content and antibacterial defense

The cylindromatosis (CYLD) gene is mutated in human tumors of skin appendages. It encodes a deubiquitylating enzyme (CYLD) that is a negative regulator of the NF-κB and JNK signaling pathways, in vitro. However, the tissue-specific function and regulation of CYLD in vivo are poorly understood. We established a genetically tractable animal model to initiate a systematic investigation of these issues by characterizing an ortholog of CYLD in Drosophila. Drosophila CYLD is broadly expressed during development and, in adult animals, is localized in the fat body, ovaries, testes, digestive tract and specific areas of the nervous system. We demonstrate that the protein product of Drosophila CYLD (CYLD), like its mammalian counterpart, is a deubiquitylating enzyme. Impairment of CYLD expression is associated with altered fat body morphology in adult flies, increased triglyceride levels and increased survival under starvation conditions. Furthermore, flies with compromised CYLD expression exhibited reduced resistance to bacterial infections. All mutant phenotypes described were reversible upon conditional expression of CYLD transgenes. Our results implicate CYLD in a broad range of functions associated with fat homeostasis and host defence in Drosophila.

Differential Effects of Tau on the Integrity and Function of Neurons Essential for Learning in Drosophila

Tauopathies are a heterogeneous group of neurodegenerative dementias involving perturbations in the levels, phosphorylation, or mutations of the microtubule-binding protein Tau. The heterogeneous pathology in humans and model organisms suggests differential susceptibility of neuronal types to wild-type (WT) and mutant Tau. WT and mutant human Tau-encoding transgenes expressed pan-neuronally in the Drosophila CNS yielded specific and differential toxicity in the embryonic neuroblasts that generate the mushroom body (MB) neurons, suggesting cell type-specific effects of Tau in the CNS. Frontotemporal dementia with parkinsonism-17-linked mutant isoforms were significantly less toxic in MB development. Tau hyperphosphorylation was essential for these MB aberrations, and we identified two novel putative phosphorylation sites, Ser238 and Thr245, on WT hTau essential for its toxic effects on MB integrity. Significantly, blocking putative Ser238 and Thr245 phosphorylation yielded animals with apparently structurally normal but profoundly dysfunctional MBs, because animals accumulating this mutant protein exhibited strongly impaired associative learning. Interestingly, the mutant protein was hyperphosphorylated at epitopes typically associated with toxicity and neurodegeneration, such as AT8, AT100, and the Par-1 targets Ser262 and Ser356, suggesting that these sites in the context of adult intact MBs mediate dysfunction and occupation of these sites may precede the toxicity-associated Ser238 and Thr245 phosphorylation. The data support the notion that phosphorylation at particular sites rather than hyperphosphorylation per se mediates toxicity or dysfunction in a cell type-specific manner.

Cell type-specific processing of human Tau proteins in Drosophila.

Accumulation of hyperphosphorylated Tau is associated with a number of neurodegenerative diseases collectively known as tauopathies. Differences in clinical and cognitive profiles among them suggest differential sensitivity of neuronal populations to Tau levels, phosphorylation and mutations. We used tissue specific expression of wild type and mutant human tau transgenes to demonstrate differential phosphorylation and stability in a cell type‐specific manner, which includes different neuronal types and does not correlate with the level of accumulated protein. Rather, they likely reflect the spatial distribution or regulation of Tau‐targeting kinases and phosphatases.

MicroRNA-22 Gates Long-Term Heterosynaptic Plasticity in Aplysia through Presynaptic Regulation of CPEB and Downstream Targets

The maintenance phase of memory-related long-term facilitation (LTF) of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT)-triggered presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22) that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC), another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.

Phosphorylation differentiates tau-dependent neuronal toxicity and dysfunction

The heterogeneous pathology of tauopathies and the differential susceptibility of different neuronal types to WT (wild-type) and mutant tau suggest that phosphorylation at particular sites rather than hyperphosphorylation mediates toxicity or dysfunction in a cell-type-specific manner. Pan-neuronal accumulation of tau in the Drosophila CNS (central nervous system) specifically affected the MBs (mushroom body neurons), consistent with neuronal type-specific effects. The MB aberrations depended, at least in part, on occupation of two novel phosphorylation sites: Ser(238) and Thr(245). The degree of isoform-specific MB aberrations was paralleled by defects in associative learning, as blocking putative Ser(238) and Thr(245) phosphorylation yielded structurally normal, but profoundly dysfunctional, MBs, as animals accumulating the mutant protein exhibited strongly impaired associative learning. Similarly dysfunctional MBs were obtained by temporally restricting tau accumulation to the adult CNS, which also altered the tau phosphorylation pattern. Our data clearly distinguish tau-dependent neuronal degeneration and dysfunction and suggest that temporal differences in occupation of the same phosphorylation sites are likely to mediate these distinct effects of tau.

Gpr158 mediates osteocalcin’s regulation of cognition

That osteocalcin (OCN) is necessary for hippocampal-dependent memory and to prevent anxiety-like behaviors raises novel questions. One question is to determine whether OCN is also sufficient to improve these behaviors in wild-type mice, when circulating levels of OCN decline as they do with age. Here we show that the presence of OCN is necessary for the beneficial influence of plasma from young mice when injected into older mice on memory and that peripheral delivery of OCN is sufficient to improve memory and decrease anxiety-like behaviors in 16-mo-old mice. A second question is to identify a receptor transducing OCN signal in neurons. Genetic, electrophysiological, molecular, and behavioral assays identify Gpr158, an orphan G protein–coupled receptor expressed in neurons of the CA3 region of the hippocampus, as transducing OCN’s regulation of hippocampal-dependent memory in part through inositol 1,4,5-trisphosphate and brain-derived neurotrophic factor. These results indicate that exogenous OCN can improve hippocampal-dependent memory in mice and identify molecular tools to harness this pathway for therapeutic purposes.

Behavioral decline and premature lethality upon pan-neuronal ferritin overexpression in Drosophila infected with a virulent form of Wolbachia

Iron is required for organismal growth. Therefore, limiting iron availability may be a key part of the host’s innate immune response to various pathogens, for example, in Drosophila infected with Zygomycetes. One way the host can transiently reduce iron bioavailability is by ferritin overexpression. To study the effects of neuronal-specific ferritin overexpression on survival and neurodegeneration we generated flies simultaneously over-expressing transgenes for both ferritin subunits in all neurons. We used two independent recombinant chromosomes bearing UAS-Fer1HCH, UAS-Fer2LCH transgenes and obtained qualitatively different levels of late-onset behavioral and lifespan declines. We subsequently discovered that one parental strain had been infected with a virulent form of the bacterial endosymbiont Wolbachia, causing widespread neuronal apoptosis and premature death. This phenotype was exacerbated by ferritin overexpression and was curable by antibiotic treatment. Neuronal ferritin overexpression in uninfected flies did not cause evident neurodegeneration but resulted in a late-onset behavioral decline, as previously reported for ferritin overexpression in glia. The results suggest that ferritin overexpression in the central nervous system of flies is tolerated well in young individuals with adverse manifestations appearing only late in life or under unrelated pathophysiological conditions.