Fourth Assignment

Citation

Summarizing a Paper on NPF

Paper: Wen, T., Parrish, C., Wu, D., & Shen, P. (2005). Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity. PNAS. 102(6); 2141-2146. Available online, November 2010.

NPF’s action is mediated by G protein-coupled transmembrane receptors (NPFR1), and has been shown to regulate food intake, alcohol sensitivity, modulate aggression and more. The paper I have chosen to summarize is NPF and its effects on alcohol sensitivity.

The goal of this study was to determine if Neuropeptide F signalling with its receptor NPFR1 had an effect on alcohol sensitivity and ethanol sedation in Drosophila. In previous studies on mice it has been found that if they are “lacking NPY or Y1 [they] displayed increased ethanol consumption and resistance to alcohol sedation, whereas animals overexpressing NPY showed opposite behavioural phenotypes” (Wen, T. et al. 2005).

The hypothesis is that a deficiency in NPF signalling (test by knocking down NPF or its receptor NPFR1) will result in the flies being able to be exposed to higher than normal concentrations of ethanol vapours before having an effect on their behaviour (increased alcohol resistance).

Summary of results:

Test One: Disruption of NPF signalling:

To destroy the NPF cells, they crossed the npf-gal4 lines with UAS-DTI flies. As a results the NPF immunoreactivity was completely eliminated in the brain of the progeny. To determine the effectiveness of ethanol on these flies they took 20 y w (wild-type) female flies and sealed them in a container with 1 ml of ethanol solution (10%, 31%, 54%, or 100%) soaked in a piece of tissue. Initially they were all active and flew to the bottle top (negative geo-taxis). “The 10% ethanol solution had little sedative effect on the flies even after 1 h, whereas ethanol solutions of 31% or higher caused an increasingly rapid accumulation of sedated flies at the bottom of the container in a dose-dependent manner” They also tested transgenic flies with destroyed NPF neurons by using the same assay with 45% EtOH, and at 65 min all control flies were sedated, whereas the majority of the experimental flies, with their NPF cells ablated, (npf-gal4-1 X UAS-DTI flies and npf-gal4-2 X UAS-DTI flies) were still clinging to the bottle top. Therefore, this experiment in degrading the NPF cells had caused a delay in sedation, “indicating that NPF-neuron-deficient flies are significantly more resistant to ethanol sedation than are control flies.”

Test Two: Targeted disruption of NPFR1 signalling:

Then the npfr1-gal4 X UAS-DTI flies (these flies had their NPFR1 signalling disrupted) were tested for their resistance to ethanol vapour by using females in three separate trials (n=20 per trial), and they too showed higher ethanol resistance than the control flies (y w X UAS-DTI).  These receptor neuron deficient flies were even significantly more resistant than the npf-gal4 X UAS-DTI flies (which were missing the NPF neurons only). This therefore raises the question of; are the NPF receptors receiving signal from another neuronal circuit in addition to the NPF pathway?  Twenty sedated flies were kept sedated after exposure, and monitored over the period for recovery. They found that both NPF- and NPFR1- neuron deficient flies recovered significantly faster than the controls, “suggesting they were sedated to a lesser degree than controls under the same conditions.”

Test Three: Controlled disruption of NPF/NPFR1 neuronal activities:

Is alcohol resistance from disrupting NPF signalling in crosses directly due to the lack of NPF signalling? Or some other developmental defect from having knocked out NPF? They used a temperature sensitive allele (shi^ts1), that can block neurotransmitter release at a defined temperature. The flies with NPF ablated (npf-gal4 and npfr1-gal4 X UAS-shi^ts1) and without NPF ablated (w X UAS-shi^ts1) were tested at a temperature below the designated temp and they all acted similarly towards the alcohol vapours, but as soon as the temperature passed that of the critical point (29^oC) , the experimental flies were far more resistant to the ethanol vapours when compared to the control. Therefore, the alcohol resistance is not due to some downstream effects of not having NPF, but is due to missing NPF itself.

Test Four: Effect of NPF overexpression on acute alcohol sensitivity:

Can overexpressing NPF result in alcohol sensitivity? Transgenic flies with UAS-npf driven by 386Y-gal4 showed strong expression of NPF in the adult brain and these flies were tested for alcohol resistance against a 31% ethanol solution. They found that flies overexpressing NPF were significantly more sensitive to alcohol, even at the reduced concentration, than the controls.  Even further examination of the ethanol sensitivity of flies that overexpress NPF in the NPF neurons (npf-gal4 X UAS-npf flies), show they were significantly more sensitive to ethanol than the control flies.

Test Five: Acute Ether response of NPF-overexpressing flies:

So NPF overexpressing flies are more sensitive to ethanol solution, are they sensitive to other types of chemical sedatives (diethyl ether)? The tests showed that the experimental flies (npf-gal4 X UAS-npf ) and controls (e.g., y w X UAS-npf) showed no significant differences in their sensitivity to ether. Therefore, the NPF/NPFR1 pathway is specific to ethanol vapours and not other sedative agents.

Test Six: Ethanol absorption and metabolism:

Is the increase in alcohol resistance due to a decrease in ethanol absorption, or an increase in ethanol catabolism? The alcohol content in the bodies of the control and experimental flies that displayed either increased or decreased alcohol resistance was physically measured, and it was found that the alcohol levels in the extracts of NPF-overexpressing flies and controls were similar. Therefore, the changes in ethanol response are not due to these biochemical systems.

The authors found that:

1)      NPF expressed mostly in “subesophageal ganglion” region of the brain, this is important for maintenance of feeding and walking, and also in other sensory and motor neurons responsible for feeding, flight and locomotion.

2)     Flies with NPF and NPFR1 neurons degraded, show a higher resistance to alcohol sedation

3)     Flies with NPFR1 neurons degraded show a higher resistance to alcohol sedation when compared to flies that are NPF neuron deficient

4)     Alcohol resistance is due to flies missing NPF signalling pathway, and not a downstream effect of missing NPF during development

5)     Overexpressing NPF results in alcohol sensitivity

6)     The NPF-NPFR1 system is selective; it mediates acute sedation by ethanol vapour but not other sedatives like diethyl ether.

7)     Ethanol resistance is not due to changes in catabolism or metabolism, but in fact due to the altered NPF system

Critique:

The results seem to support the authors’ claims but there was not a lot of explanation as to why, I had to figure it out myself. They stated what they did and what it means, but not why.

I found this paper sometimes difficult to follow; the diagrams were difficult to understand at times. When your intended audience begins to read a paper, they begin with the results and figures. If they can’t understand them then they probably won’t finish the paper.

I was unsure what genotypes are over or under expressing NPF, I had to go back and search for it in the introduction and results sections. I did not know which graphs correspond to those genotypes, and some graphs did not even say what genotypes were being displayed! The axes of the graphs were not labelled well enough and the titles were exhaustive, to find what you’re looking for involved a lot of searching.

Some future experiments:

The neural circuits and pathways that are affected by alcohol are still somewhat uncertain, if these can be fully understood then further experimentation on this topic can be accomplished.

1. It was found that NPF expressed in “subesophageal ganglion” brain regions, which are important for regulating feeding and walking; climbing analyses would analyze if walking is negatively impacted when NPF is underexpressed. If so, than the flies would be unable to climb as well as the unaffected flies with normal NPF signalling.
2. Also, to address the question; are the NPF receptors receiving signal from another neuronal circuit in addition to the NPF pathway? They could set up an experiment to test other ethanol sensitive neuronal circuits to determine if there are others, and if they are involved with NPFR1. I believe they would discover that there is another pathway connected to NPFR1, because pathways are always complex. It’s just a matter of finding the pathway and identifying it.

Third Assignment

Function of Neuropeptide F

Functions:

Neuropeptide F is one of the many neuropeptides in the body; they are a group of quite diverse signal molecules that contribute to a wide range of different behaviours. NPF functions affect many different behaviours including:

• Feeding behaviour
• Stress response
• Alcohol sensitivity, and
• Modulate aggression

 Localization:

Drosophila NPF (dNPF) is localized in the neuronal network of the central nervous system, dNPF neurons are found on the “dorsal surface of the brain and in subesophageal ganglia” (Wu, et. al., 2003). NPF in flatworms is localized to the central and peripheral nervous systems, “mainly in the dorsal and ventral nerve plexi” (Dougan, et. al., 2002), a network of intersecting nerves.

Effects on different systems:

In Drosophila larvae that eat well have been shown to have high NPF gene expression, whereas older larvae, which are known to not eat as much, are shown to have decreased NPF gene expression. These larvae also show “increased mobility, food-dependent clumping and cooperative burrowing” (Wu, et. al., 2003). Transgenic larvae that are experimentally deficient in NPF have been shown to display the same behaviours as the older larvae (Wu, et. al., 2003).

Even when faced with stressful situations such as being placed in extremely cold temperatures that can lead to death, NPF is required for “cold-resistant feeding behaviour of fasted larvae” (Lingo, P.R. et. al., 2007). The overexpression of the NPF receptor (NPFR1) in fed larvae triggered this cold resistant feeding activity, which is normally seen in fasted larvae.  I believe that if the receptor expression is increased, NPF will be able to have a greater effect because it has more binding sites, therefore triggering the cold resistant feeding. It can be concluded that the NPF pathway is critical for responses to stressors of a thermal, gustatory or mechanical form (Lingo, P.R. et. al., 2007).

Furthermore, studies have shown the NPF and its receptor NPFR1 can have a mild affect on alcohol sensitivity. If Drosophila were deficient in NPF/NPFR1 signalling then there is a significant decrease in alcohol sensitivity. For example, when flies had their NPF/NPFR1 signalling pathways disrupted they showed a significant resistance in ethanol sedation (Wen, et. al., 2005). Coincidentally, flies that overexpress NPF pathways display the opposite phenotype. The neural circuits and pathways that are affected by alcohol are still somewhat uncertain, if these can be fully understood then further experimentation on this topic can be accomplished.

NPF is also important for sexual dimorphism in adult Drosophila (Lee, et. al., 2006). This study shows that male flies lacking NPF expression also lack male sexual behaviours.

Pathologies:

Neuropeptides act as neuroregulators along the central nervous system, if a problem arises within the regulatory system then diseases and consequences will be the outcome.

Neuropeptide Y (vertebrate homologue of NPF) has been linked to neurodegenerative diseases such as Alzheimer’s disease and Huntington’s disease. Studies have shown that concentrations on NPY are reduced in the cerebral cortex of Alzheimer’s patients, and this may be correlated to a loss of its’ corresponding (nonpyramidal) neurons (Dr. Flint Beal, M., & Dr. Martin, J.B., 2008 and Dr. Kowall, N.W., & Dr. Flint Beal, M. 1988). In Huntington’s disease, studies have shown an increase in the amount of NPY, which has been correlated to the preservation of the associated neurons (Flint Beal, M., et. al., 1986).

Future developments:

All these studies are on complex systems and neuronal pathways that are not yet completely understood. I believe if we are to reliably know the role of NPY in vertebrates and NPF in invertebrates then we have to understand these complicated systems first. After we posses that knowledge, we can further analyze these neuropeptides to gain a complete understanding of their effects and capabilities.

REFERENCES

1)  Dougan, P.M., Mair, G.R., Halton, D.W., Curry, W.J., Day, T.A., & Maule, A.G. (2002). Gene Organization and Expression of a Neuropeptide Y Homolog from the Land Planarian Arthurdendyus triangulates. The journal of comparative neurology. 454; 58-64.

2)  Dr. Flint Beal, M., & Dr. Martin, J.B. (2008). Neuropeptides in neurological disease. Annals of Neurology. 20(5); 547-565.

3)  Dr. Kowall, N.W., & Dr. Flint Beal, M. (1988). Cortical somatostatin, neuropeptides Y, and NADPH diaphorase neurons: Normal anatomy and alterations in Alzheimer’s disease. Annals of Neurology. 23(2); 105-114.

4)  Flint Beal, M., Kowall, N.W., Ellison, D.W., Mazurek, M.F., Swartz, J.K., & Martin, J.B. (1986). Replication of the neurochemical characteristics of Huntington’s disease by quinolinic acid. Nature. 321; 168-171.

5)  Lee, G., Bahn, J.H., & Park, J.H. (2006). Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila. PNAS. 103(33); 12580-12585.

6)  Lingo, P.R., Zhao, Z., & Shen, P. (2007). Co-regulation of Cold-Resistant Food Acquisition by insulin- and Neuropeptide Y-like Systems in Drosophila melanogaster. Neuroscience. 148(2): 371-374.

7)  Wen, T., Parrish, C., Wu, D., & Shen, P. (2005). Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity. PNAS. 102(6); 2141-2146.

8)  Wu, Q., Wen, T., Lee, G., Park, J.H., Cai, H.N., & Shen, P. (2003). Developmental Control of Foraging and Social Behaviour by the Drosophila Neuropeptide Y-like System. Neuron. 39; 147-161.

Second Assignment

Structure of my favorite hormone: Neuropeptide F


Mature NPF in invertebrates is 362 base pairs long, the precursor is comprised of a "signal peptide followed by the mature peptide of 36 amino acids in length" (Dougan, P.M., et. al., 2002). NPF is a homologue of NPY in vertebrates, NPY in Homo sapiens is 37 amino acids in length (Dougan, P.M., et. al., 2002). One important difference in the structure of NPF and NPY is that NPF has a phenylalanine (Phe) residue at the C-terminus, whereas NPY has a Tyrosine (Tyr) (Lee, K., et. al, 2004).
The typical receptor for these peptides is the G-protein receptor, which are transmembrane receptors, the peptide will bind to the receptor on the cell surface and send signals via signal transduction.
Structure more often than not equals function, meaning the protein will not function properly if the structure is altered. We can assume that any conserved areas of the protein alignment (figure 1) will be the important active areas of the protein. They may be crucial for binding to the receptor or any other activity of the peptide.

Fig.1: Alignment of proteins for Fruit fly NPF (Genebank accession number NM_080493), silkworm NPF (accession number NM_001130883), Human NPY (accession number H15789) and goldfish NPY (accession number M87297). Click on the image for a larger image. Alignments were made using ClustalW software 
KEY: 

"*" means that the residues or nucleotides in that column are identical in all sequences in the alignment. 

":" means that conserved substitutions have been observed, according to the COLOUR table above. 

"." means that semi-conserved substitutions are observed.

SeqA Name           Len(aa)  SeqB Name           Len(aa)  Score

===============================================================

1    fly.npf        101      2    silkworm.npf   82       28  

1    fly.npf        101      3    human.npy      97       14  

1    fly.npf        101      4    goldfish.npy   96       15  

2    silkworm.npf   82       3    human.npy      97       20  

2    silkworm.npf   82       4    goldfish.npy   96       18  

3    human.npy      97       4    goldfish.npy   96       62  

===============================================================

Table 1.0: Percent similarity based on protein alignment, between NPY and NPF of the species: Drosophila melanogaster, Bombyx mori, Homo sapien, & Carassius auratus.


You can see in Table 1.0 that Human NPY and Goldfish NPY are the highest conserved peptides.


References:

(1) Dougan, P.M., Mair, G.R., Halton, D.W., Curry, W.J., Day, T.A., & Maule, A.G. (2002). Gene Organization and Expression of a Neuropeptide Y Homolog from the Land Planarian Arthurdendyus triangulates. The journal of comparative neurology. 454;58-64.

(2) Lee, K., You, K., Choo, J., Han, Y., & Yu, K. (2004). Drosophila Short Neuropeptide F regulates food intake and body size. The Journal of Biological Chemistry. 279(49); 50781-50789.

L

First Assignment

My Favorite Hormone: Neuropeptide F (NPF)


NPF is a 36 amino acid peptide in Drosophila Melanogaster, or fruit flies, that was found to be a homologue of the mammalian Neuropeptide Y hormone (Lee, K., et. al, 2004). NPY is synthesized and secreted by neurons into the peripheral nervous system and central nervous system. It can also be synthesized and secreted from islet cells of the pancreas, and via its Y1 receptor, it inhibits insulin secretion due to glucose. NPY has also been shown to be a potent stimulator in rodents in terms of food intake (Kronenberg, H.M., et. al, 2008). One important difference in the structure of NPF and NPY is that NPF has a Phenylalanine (Phe) residue at the C-terminus, whereas NPY has a Tyrosine (Tyr).
NPF has many diverse functions and affects many important processes in Drosophila, such as: behaviour, alcohol sensitivity, energy homeostasis, circadian rhythm, food intake, body size, reproduction, anxiety, seizure, learning and memory (Lee, G., et. al, 2006). 
NPF has a receptor known as NPFR1, which is linked to ethanol sedation. If there is a deficiency in NPF or this receptor, it has been shown that there is also a decrease in alcohol sensitivity. The opposite phenotype can be observed when there is overexpression of NPF or NPFR1 (Wen, T., et. al, 2005).
This same receptor, NPFR1, is linked to food intake as well. The activity of this receptor is mandatory for fasting larvae to participate in what is known as "cold-resistant feeding behaviour" (Lingo, P., et. al, 2007). In other words, these fasted larvae would not eat cold food if their lives depended on it, unless this receptor was active. From this study, it can be suggested that the mammalian NPF-like system (NPY) can play a central role in gustation.

References:

1)      Kronenberg, H.M., Melmed, S., Polonsky, K.S., & Larsen, P.R. (2008). Williams Textbook of Endocrinology. 1955. Reprint. Philadelphia, PA: Saunders Elsevier.

2)      Lee, G., Bahn, J.H., & Park, J.H. (2006). Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila. PNAS. 103(33); 12580-12585.

3)      Lee, K., You, K., Choo, J., Han, Y., & Yu, K. (2004). Drosophila Short Neuropeptide F regulates food intake and body size. The Journal of Biological Chemistry. 279(49); 50781-50789.

4)      Lingo, P., Zhao, Z., & Shen, P. (2007). Co-regulation of Cold-Resistant Food Acquisition by Insulin- and Neuropeptide Y-like Systems in Drosophila melanogaster. Neuroscience. 148(2); 371-374.

5)      Wen, T., Parrish, C., Wu, D., & Shen, P. (2005). Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity. PNAS. 102(6); 2141-2146.