Efthimios M. C. Skoulakis

Interaction of Akt-Phosphorylated Ataxin-1 with 14-3-3 Mediates Neurodegeneration in Spinocerebellar Ataxia Type 1

Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but the mechanism underlying this accumulation has been unclear. We have discovered that the 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing its normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. Our finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention.

Preferential expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory

Involvement of the cAMP cascade in Drosophila learning and memory is suggested by the aberrant behavioral phenotypes of the mutants dunce (cAMP phosphodiesterase) and rutabaga (adenylyl cyclase). Line DCO581, isolated via an enhancer detector screen for genes preferentially expressed in the mushroom bodies, contains a transposon in the first exon of the catalytic subunit gene (DCO) of protein kinase A (PKA). RNA in situ hybridization and immunohistochemistry show that DCO is preferentially expressed in the mushroom bodies. The DCO581 insertion and an independently isolated hypomorphic allele (DCOB10) each produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing heteroallelic yet viable DCO581/DCOB10 animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes.

Olfactory Learning Deficits in Mutants for leonardo, a Drosophila Gene Encoding a 14-3-3 Protein

Studies of Drosophila and other insects have indicated an essential role for the mushroom bodies in learning and memory. The leonardo gene encodes a Drosophila protein highly homologous to the vertebrate 14-3-3zeta isoform, a protein well studied for biochemical roles but without a well established biological function. The gene is expressed abundantly and preferentially in mushroom body neurons. Mutant alleles that reduce LEONARDO protein levels in the mushroom bodies significantly decrease the capacity for olfactory learning, but do not affect sensory modalities or brain neuroanatomy that are requisite for conditioning. These results establish a biological role for 14-3-3 proteins in mushroom body-mediated learning and memory processes, and suggest that proteins known to interact with them, such as RAF-1 or other protein kinases, may also have this biological function.

Leonardo, a Drosophila 14-3-3 Protein Involved in Learning, Regulates Presynaptic Function

The leonardo gene encodes a conserved member of the 14-3-3 protein family, which plays a role in Drosophila learning. Immunological localization of the protein shows that it is expressed at synaptic connections and enriched in presynaptic boutons of the neuromuscular junction (NMJ). Null leonardo mutants die as mature embryos. Electrophysiological assays of the mutant NMJ demonstrate that basal synaptic transmission is reduced by 30% and that transmission amplitude, fidelity, and fatigue resistance properties are reduced at elevated stimulation frequencies and in low external [Ca2+]. Moreover, transmission augmentation and post-tetanic potentiation (PTP) are disrupted in the mutant. These results suggest that Leonardo plays a role in the regulation of synaptic vesicle dynamics, a function which may underlie synaptic modulation properties enabling learning.

14-3-3 proteins in neuronal development and function

The 14-3-3 proteins are small, cytosolic, evolutionaritly conserved proteins expressed abundantly in the nervous system. Although they were discovered more than 30 yr ago, their function in the nervous system has remained enigmatic. Several recent studies have helped to clarify their biological function. Crystallographic investigations have revealed that 14-3-3 proteins exist as dimers and that they contain a specific region for binding to other proteins. The interacting proteins, in turn, contain a 14-3-3 binding motif; proteins that interact with 14-3-3 dimers include PKC and Raf, protein kinases with critical roles in neuronal signaling. These proteins are capable of activating Raf in vitro, and this role has been verified by in vivo studies inDrosophila. Most interestingly, mutations in theDrosophila 14-3-3 genes disrupt neuronal differentiation, synaptic plasticity, and behavioral plasticity, establishing a role for these proteins in the development and function of the nervous system.

The cyclic AMP system and Drosophila learning

The cyclic AMP (cAMP) system plays a critical role in olfactory learning in the fruit fly,Drosophila melanogaster, as evidenced by the following: [1] The dunce gene encodes a form of cAMP phosphodiesterase (PDE). Flies carrying mutations at this gene show reduced PDE activity, high cAMP levels, and deficits in olfactory learning and memory [2]. The rutabaga gene encodes one type of adenylyl cyclase (AC) similar in properties to the Type I AC characterized from vertebrate brain. This enzyme is activated by G-protein and Ca++ and has been postulated to be a molecular coincidence detector, capable of integrating information from two independent sources such as the conditioned stimulus (CS) and the unconditioned stimulus (US) delivered to animals during Pavlovian conditioning. Rutabaga mutant flies are deficient in AC activity and show behavioral defects similar to those exhibited by dunce mutants [3]. Flies carrying mutations in the gene (DC0) that encodes the catalytic subunit of protein kinase A (PKA), the major mediator of cAMP actions, show alterations in learning performance and a loss in PKA activity.All three genes are expressed preferentially in mushroom bodies, neuroanatomical sites that mediate olfactory learning. Interestingly, the PDE and the catalytic subunit of PKA are found primarily in axonal and dendritic compartments of the mushroom body cells, whereas the AC is found primarily in the axonal compartment. The reason for this differential compartmentalization is unclear, although the hypothetical role of AC as coincidence detector would predict that CS and US stimuli are integrated in the axonal compartment.These observations suggest that cAMP is a dominant second messenger utilized by mushroom body cells to modulate their physiology while the animal is learning and consolidating memory. However, many other types of molecules are likely involved in the physiological alterations that occur in these cells during learning, including cell surface proteins, transcription factors, and synaptic proteins.

Molecular vibration-sensing component in Drosophila melanogaster olfaction

A common explanation of molecular recognition by the olfactory system posits that receptors recognize the structure or shape of the odorant molecule. We performed a rigorous test of shape recognition by replacing hydrogen with deuterium in odorants and asking whether Drosophila melanogaster can distinguish these identically shaped isotopes. We report that flies not only differentiate between isotopic odorants, but can be conditioned to selectively avoid the common or the deuterated isotope. Furthermore, flies trained to discriminate against the normal or deuterated isotopes of a compound, selectively avoid the corresponding isotope of a different odorant. Finally, flies trained to avoid a deuterated compound exhibit selective aversion to an unrelated molecule with a vibrational mode in the energy range of the carbon–deuterium stretch. These findings are inconsistent with a shape-only model for smell, and instead support the existence of a molecular vibration-sensing component to olfactory reception.

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.

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.