Ana C. Calvo

Dynamic Encoding of Perception, Memory, and Movement in a C. elegans Chemotaxis Circuit

Brain circuits endow behavioral flexibility. Here, we study circuits encoding flexible chemotaxis in C. elegans, where the animal navigates up or down NaCl gradients (positive or negative chemotaxis) to reach the salt concentration of previous growth (the set point). The ASER sensory neuron mediates positive and negative chemotaxis by regulating the frequency and direction of reorientation movements in response to salt gradients. Both salt gradients and set point memory are encoded in ASER temporal activity patterns. Distinct temporal activity patterns in interneurons immediately downstream of ASER encode chemotactic movement decisions. Different interneuron combinations regulate positive versus negative chemotaxis. We conclude that sensorimotor pathways are segregated immediately after the primary sensory neuron in the chemotaxis circuit, and sensory representation is rapidly transformed to motor representation at the first interneuron layer. Our study reveals compact encoding of perception, memory, and locomotion in an experience-dependent navigational behavior in C. elegans.

Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l‐DOPA responsive and non‐responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)‐l‐erythro‐5,6,7,8‐tetrahydrobiopterin (BH4) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription–translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH4 supplementation to mice increased TH activity and protein levels in brain extracts, while the Th‐mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding‐aid effect of BH4 and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH4 crosses the blood–brain barrier and therapeutic regimes based on BH4 supplementation should thus consider the effect on TH. Furthermore, BH4 supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

Specific interaction of the diastereomers 7(R)- and 7(S)-tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo

Pterin‐4a‐carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)‐L‐erythro‐5,6,7,8‐tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)‐ and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki2.3–4.9 µM). Kinetic and binding studies, as well as characterization of the pterin‐enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin‐PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser‐251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser‐251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo. —Pey, A. L., Martinez, A., Charubala, R., Maitland, D. J., Teigen, K., Calvo, A., Pfleiderer, W., Wood, J. M., Schallreuter, K. U. Specific interaction of the diastereomers 7(R)‐ and 7(S)‐tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo FASEB J. 20, E1451–E1464 (2006)

Bidirectional thermotaxis in Caenorhabditis elegans is mediated by distinct sensorimotor strategies driven by the AFD thermosensory neurons

Significance The nematode Caenorhabditis elegans offers the opportunity to map complex behaviors to the specific roles of each neuron in a 302-neuron nervous system. Thermotaxis is a complex behavior where the worm inverts the behavioral mode—positive thermotaxis up gradients or negative thermotaxis down gradients—to move toward a remembered temperature. How are both long-term memory and multiple behavioral modes encoded? A long-standing model has been that separate circuits for positive and negative thermotaxis compete for control of body movement. In contrast, we find that different modes of thermotaxis are driven by one set of AFD thermosensory neurons. Circuits for different thermotactic behaviors diverge from the AFD neurons, probably by coupling sensory inputs to motor programs in different ways to create different thermotactic behaviors. The nematode Caenorhabditis elegans navigates toward a preferred temperature setpoint (Ts) determined by long-term temperature exposure. During thermotaxis, the worm migrates down temperature gradients at temperatures above Ts (negative thermotaxis) and performs isothermal tracking near Ts. Under some conditions, the worm migrates up temperature gradients below Ts (positive thermotaxis). Here, we analyze positive and negative thermotaxis toward Ts to study the role of specific neurons that have been proposed to be involved in thermotaxis using genetic ablation, behavioral tracking, and calcium imaging. We find differences in the strategies for positive and negative thermotaxis. Negative thermotaxis is achieved through biasing the frequency of reorientation maneuvers (turns and reversal turns) and biasing the direction of reorientation maneuvers toward colder temperatures. Positive thermotaxis, in contrast, biases only the direction of reorientation maneuvers toward warmer temperatures. We find that the AFD thermosensory neuron drives both positive and negative thermotaxis. The AIY interneuron, which is postsynaptic to AFD, may mediate the switch from negative to positive thermotaxis below Ts. We propose that multiple thermotactic behaviors, each defined by a distinct set of sensorimotor transformations, emanate from the AFD thermosensory neurons. AFD learns and stores the memory of preferred temperatures, detects temperature gradients, and drives the appropriate thermotactic behavior in each temperature regime by the flexible use of downstream circuits.

Rescuing Proteins of Low Kinetic Stability by Chaperones and Natural Ligands: Phenylketonuria, a Case Study

Publisher Summary Phenylketonuria (PKU) is a disease caused by deleterious mutations in phenylalanine hydroxylase (PAH) and constitutes a paradigm for misfolding diseases. Folding is the process by which a protein reaches a functional and stable native structure, while misfolding can be seen as the failure to attain this fully functional conformation. Natural substrates, cofactors, and inhibitors have effects on protein stability beyond their functional role in enzyme function by the same arguments as for other specific ligands and can be considered as natural chaperone ligands. To avoid pathogenic misfolding, the cell is equipped with protein quality control systems (QCS) mainly including chaperones, the ubiquitin proteasome pathway (UPP) and, in some instances, the aggresome. Binding of a ligand to a specific binding site on the native state of a protein will influence the unfolding equilibrium which will be shifted towards the natively folded state, resulting in an increase in protein stability.

Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans

In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah‐1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S0.5(L‐Phe)~150 μΜ; Κm for tetrahydrobiopterin (BH4)~35 μΜ and comparable Vmax], but cePAH is devoid of positive cooperativity for L‐Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L‐Phe. Pah‐1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah‐1 knockouts lack a yelloworange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah‐1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution.—Calvo, A. C., Pey, A. L., Ying, M., Loer, C. M., Martinez, A. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans. FASEB J. 22, 3046–3058 (2008)

Effect of pharmacological chaperones on brain tyrosine hydroxylase and tryptophan hydroxylase 2

J. Neurochem. (2010) 114, 853–863.

Cuticle Integrity and Biogenic Amine Synthesis in Caenorhabditis elegans Require the Cofactor Tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.

Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

Adsorption and Bioactivity of Tyrosine Hydroxylase on Gold Surfaces and Nanoparticles

Tyrosine hydroxylase is studied in terms of adsorption behaviour on gold surfaces and various passivating layers. Results reveal differences in layer formation, where mercaptoundecanoic acid-coated gold shows the best potential in terms of adsorbed mass. Nanoparticles with this coating are subsequently tested for enzymatic activity, which remains at attenuated levels.