Unsilencing Angelman Syndrome

Angelman syndrome is a severe disorder with symptoms that include speech impairment, intellectual disability and seizures. This lifelong disorder profoundly impacts patients and their families, yet no effective treatment currently exists. It is well established that this disorder is caused by genetic alterations in the maternally-inherited copy of a gene called Ube3a.

In collaboration with Drs. Ben Philpot and Bryan Roth, we found that topoisomerase inhibitors unsilence a dormant but functional copy of Ube3a in mice. We aim to advance our understanding of how these drugs work, with the ultimate goal of developing treatments for this debilitating, lifelong disorder.

For more information, please visit: http://www.cidd.unc.edu/Angelman-Syndrome/.

Molecules and Mechanisms for Pain

Chronic pain is a major medical issue, affecting more Americans than heart disease, diabetes and cancer combined (American Pain Foundation). In our laboratory, we are developing new approaches to treat chronic pain. In addition, we study the neural circuits that transmit pain-producing stimuli using molecular, genetic, electrophysiological and behavioral approaches.

Figure 1.

Ectonucleotidases in nociceptive circuits.

We recently found that Prostatic Acid Phosphatase (PAP, also know as ACPP) is expressed in nociceptive (pain-sensing) neurons and functions as an ectonucleotidase, converting adenosine monophosphate (AMP) to adenosine. The released adenosine potently suppresses inflammatory pain and neuropathic pain by acting through A1 adenosine receptors.

Our studies suggest it might be possible to treat chronic pain using recombinant PAP protein or small-molecules that mimic the effects of PAP.

Neural circuit-based approaches.

In mammals, pain signals are transmitted from the periphery to the CNS by two neural circuits; the so called peptidergic and non-peptidergic circuits (Fig. 1). Peptidergic neurons contain neuropeptides, like CGRP, while non-peptidergic neurons bind the lectin IB4.

Figure 2. Nerve endings in glabrous skin

We recently found a G protein-coupled receptor (GPCR) called Mrgprd which is expressed in a majority of all non-peptidergic neurons. Mrgprd is not expressed in CGRP⁺ neurons, nor is it expressed anywhere else in the brain or body.

To identify the tissues that Mrgprd-expressing neurons innervate, we engineered knock-in mice that express a membrane-tethered version of enhanced Green Fluorescent Protein (EGFPf) from the Mrgprd locus (MrgprdΔ^EGFPf). Surprisingly, we found that Mrgprd-expressing neurons only innervate the epidermis of the skin (Fig. 2). Joints and internal organs were not innervated, suggesting pain signals are transmitted from these tissues by molecularly-distinct circuits.

Figure 3. Dorsal spinal cord. CGRP (red) and Mrgprd (green) axons. PKCγ interneurons (blue)

In addition to molecular differences, Mrgprd-expressing axons and CGRP⁺ axons terminate within different zones of the epidermis (Fig. 2). Mrgprd-expressing axons (green) also terminate beneath the red-labeled CGRP lamina in the dorsal spinal cord (Fig. 3). Taken together, these findings suggest peptidergic and non-peptidergic neurons might have unique functions and connectivity.

Although studied for over 20 years, it is still not known why mammals have peptidergic and non-peptidergic circuits, both of which respond to noxious stimuli. Do these two molecularly different circuits have redundant or non-redundant functions in nociception? In our laboratory, we are trying to answer this fundamental question using a variety of approaches. As an example, we are making and studying circuit knockout mice. These mice are specifically missing either peptidergic or non-peptidergic neurons. We are studying the consequences of these ablations using molecular, electrophysiological and behavioral methodologies. We are also using Channelrhodopsin-2 (ChR2), a light-gated ion channel, to better understand how these circuits interface with pain-related regions of the central nervous system.

Techniques used in our lab:

Molecular biology and cell culture
In situ hybridization and immunofluorescence staining
Construction and characterization of knock-in and transgenic mice
Mouse behavioral experiments
Bioinformatics
FACS of neurons
Expression profiling with Affymetrix GeneChip arrays
Calcium imaging and electrophysiology

Recent Publications:

Hurt JK, Zylka MJ. PAPupuncture has localized and long-lasting antinociceptive effects in mouse models of acute and chronic pain. Mol Pain. 2012 Apr 23;8(1):28. [Epub ahead of print]

Huang HS, Allen JA, Mabb AM, King IF, Miriyala J, Taylor-Blake B, Sciaky N, Dutton JW Jr, Lee HM, Chen X, Jin J, Bridges AS, Zylka MJ, Roth BL, Philpot BD. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature. 2011 Dec 21;481(7380):185-9. doi: 10.1038/nature10726.

Mabb AM, Judson MC, Zylka MJ, Philpot BD. Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes. Trends Neurosci. 2011 Jun;34(6):293-303. [cover]

Zylka MJ, Sowa NA. NT5E mutations and arterial calcifications. N Engl J Med. 2011 Apr 21;364(16):1579; author reply 1579-80.

Zylka MJ. Pain-relieving prospects for adenosine receptors and ectonucleotidases. Trends Mol Med. 2011 Apr;17(4):188-96. [cover]

Street SE, Zylka MJ. (2011). Emerging roles for ectonucleotidases in pain-sensing neurons. Neuropsychopharmacology 36:358.

Sowa NA, Street SE, Vihko P, Zylka MJ. (2010). Prostatic acid phosphatase reduces thermal sensitivity and chronic pain sensitization by depleting phosphatidylinositol 4,5-bisphosphate. J. Neurosci. 30:10282-10293.

Sowa NA, Voss MK, Zylka MJ. (2010) Recombinant ecto-5’-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A₁ receptor activation. Molecular Pain. 6:20.

Sowa NA, Taylor-Blake B, Zylka MJ. (2010) Ecto-5’-nucleotidase (CD73) inhibits nociception by hydrolyzing AMP to adenosine in nociceptive circuits. J. Neurosci. 30:2235-2244.

Taylor-Blake B, Zylka MJ. (2010) Prostatic acid phosphatase is expressed in peptidergic and nonpeptidergic nociceptive neurons of mice and rats. PLOS One 5(1):e8674. doi:10.1371/journal.pone.0008674.

Wang H, Zylka MJ. (2009) Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons. J. Neurosci. 29:13202-13209.

Rau KK, McIlwrath SL, Wang H, Lawson JJ, Jankowski MP, Zylka MJ, Anderson DJ, Koerber HR. (2009) Mrgprd enhances excitability in specific populations of cutaneous murine polymodal nociceptors. J Neurosci. 29(26):8612-9.

Larsen RS, Zylka MJ, Scott JE. (2009) A high throughput assay to identify small molecule modulators of prostatic acid phosphatase. Current Chemical Genomics, 3:42-49.

Cavanaugh D, Lee H, Lo L, Shields S, Zylka MJ, Basbaum AI, Anderson DJ. (2009) Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli. Proc. Natl. Acad. Sci. USA, 106:9075-9080.

Sowa NA, Vadakkan K, Zylka MJ. (2009) Recombinant mouse PAP has pH-dependent ectonucleotidase activity and acts through A1-adenosine receptors to mediate antinociception. PLOS One, 4(1): e4248. doi:10.1371/journal.pone.0004248.

Zylka MJ, Sowa NA, Taylor-Blake B, Twomey MA, Herrala A, Voikar V, Vihko P. (2008) Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine. Neuron 60:111-22.

Dussor G, Zylka MJ, Anderson DJ, McCleskey EW. (2008) Cutaneous sensory neurons expressing the Mrgprd receptor sense extracellular ATP and are putative nociceptors. J. Neurophysiol. 99:1581-9.

Liu Y, Yang FC, Okuda T, Dong X, Zylka MJ, Chen CL, Anderson DJ, Kuner R, Ma Q. (2008) Mechanisms of compartmentalized expression of Mrg class G protein-coupled sensory receptors. J. Neurosci. 28: 125-32.

Campagnola L, Wang H, Zylka MJ. (2008) Fiber-coupled light-emitting diode for localized photostimulation of neurons expressing channelrhodopsin-2.  J Neurosci Methods. 169:27-33.

Liu Q, Vrontou S, Rice FL, Zylka MJ, Dong XD, Anderson DJ. (2007) Molecular genetic visualization of a rare subset of unmyelinated sensory neurons that may detect gentle touch. Nature Neurosci. 10:946-8

Zylka MJ. (2005) Nonpeptidergic circuits feel your pain. Neuron 47:771-772.

Zylka M, Rice FL, Anderson, DJ. (2005) Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 45:17-25.

Zylka MJ, Dong X, Southwell AL, Anderson DJ. (2003) Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family. Proc. Natl. Acad. Sci . USA 100:10043-10048.

Han S-K, Dong X, Hwang J-I, Zylka MJ, Anderson DJ, Simon MI. (2002) Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated by RF-amide-related peptides through the G a q/11 pathway. Proc. Natl. Acad. Sci . USA 99:14740-14745.

Dong X, Han S-K, Zylka MJ, Simon MI, Anderson DJ. (2001) A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 106:619-632.