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.

Mutations in the neuronal-specific microtubule-binding protein TAU are associated with several dementias and neurodegenerative diseases. However, the effects of elevated TAU accumulation on behavioral plasticity are unknown. We report that directed expression of wild-type vertebrate and Drosophila TAU in adult mushroom body neurons, centers for olfactory learning and memory in Drosophila, strongly compromised associative olfactory learning and memory, but olfactory conditioning-relevant osmotactic and mechanosensory responses remained intact. In addition, TAU accumulation in mushroom body neurons did not result in detectable neurodegeneration or premature death. Therefore, TAU-mediated structural or functional perturbation of the microtubular cytoskeleton in mushroom body neurons is likely causal of the behavioral deficit. These results indicate that behavioral plasticity decrements may be the earliest detectable manifestations of tauopathies.

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1. Neurofibromatosis-1 (NF1) syndrome is a common (1/3,000 births) genetic disorder affecting multiple organ systems, including the nervous system. Its clinical features include short stature, learning disabilities, and several types of benign and malignant tumors. NF1 is caused by mutations that inactivate the NF1 gene, a crucial negative regulator of Ras signaling. Although unregulated Ras signaling is a hallmark of NF1, the specific Ras signaling pathways responsible for disease development remain largely unknown. The Drosophila and human Nf1 genes are highly conserved; and, as in patients, mutant flies are smaller than usual and present deficient learning. Here, we identified the Drosophila Receptor Tyrosine Kinase dAlk as a negative regulator of organismal growth and olfactory learning. We show that excessive dAlk activation results in growth and learning defects similar to those of Nf1 mutants. Genetic suppression studies and pharmacological inhibition indicate dAlk as a critical upstream activator of Nf1-regulated neuronal Ras/ERK signals that contribute to size determination and learning. Importantly, our results strongly suggest that Alk represents a novel, highly specific, and promising therapeutic target in human NF1.

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 theDrosophilaCNS 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, Ser²³⁸and Thr²⁴⁵, on WT hTau essential for its toxic effects on MB integrity. Significantly, blocking putative Ser²³⁸and Thr²⁴⁵phosphorylation 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 Ser²⁶²and Ser³⁵⁶, suggesting that these sites in the context of adult intact MBs mediate dysfunction and occupation of these sites may precede the toxicity-associated Ser²³⁸and Thr²⁴⁵phosphorylation. 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.

Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation inDrosophilato uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the flyfmr1gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

Members of the conserved 14-3-3 protein family spontaneously self-assemble as homo- and heterodimers via conserved sequences in the first four (alphaA-alphaD) of the nine helices that comprise them. Dimeric 14-3-3s bind conserved motifs in diverse protein targets involved in multiple essential cellular processes including signaling, intracellular trafficking, cell cycle regulation, and modulation of enzymatic activities. However, recent mostly in vitro evidence has emerged, suggesting functional and regulatory roles for monomeric 14-3-3s. We capitalized on the simplicity of the 14-3-3 family in Drosophila to investigate in vivo 14-3-3zeta monomer properties and functionality. We report that dimerization is essential for the stability and function of 14-3-3zeta in neurons. Moreover, we reveal the contribution of conserved amino acids in helices A and D to homo- and heterodimerization and their functional consequences on the viability of animals devoid of endogenous 14-3-3zeta. Finally, we present evidence suggesting endogenous homeostatic adjustment of the levels of the second family member in Drosophila, D14-3-3epsilon, to transgenic monomeric and dimerization-competent 14-3-3zeta.