Millard Group - Molecular mechanisms for wiring the brain - School of Biomedical Sciences

*Clones of mushroom body neurons in the Drosophila central brain generated using mosaic analysis with a repressible cell marker (MARCM).*

The overall goal of the Millard lab is to understand how specificity is generated in the brain.

This problem is best exemplified by considering that 100 trillion synapses are generated and maintained in the human brain using a toolkit of only 20,000 genes. We have been approaching this problem using molecular genetics in the fruit fly, Drosophila melanogaster. Most projects in the lab revolve around how a broadly expressed cell surface protein, called Down syndrome cell adhesion molecule 2 (Dscam2), is able to perform specific functions in different neurons. We are also interested in mechanisms of neurological disease, particularly those that involve changes in synaptic function.

Projects in the lab

We use Drosophila molecular genetics to understand:

how brain wiring proteins work at the molecular level.
how brain structures and neural circuits form.
how the brain generates sufficient protein diversity to specify its numerous synaptic connections - focus on alternative splicing.
how cell surface proteins regulate synaptic physiology.

We also use flies to model human disease processes. ALS is our current focus.

Modelling neurodegeneration in the fly eye using candidate ALS risk factors.
Studying how ALS risk factors affect motor neuron morphology, synaptic makeup and physiology.

Lab work flow and techniques

We use molecular biology to manipulate genes or the genome, perform genetics to generate animals of the appropriate genotype and then score the resulting phenotypes using confocal microscopy, electrophysiology or external phenotypes in the fly.

CRISPR
RNAi
Cell-specific overexpression
Mosaic experiments (single cells, rather than the entire organism, manipulated)
High resolution imaging
High-throughput screening in a whole animal model

We are always interested in recruiting enthusiastic scientists and welcome new ideas and techniques. Please contact Sean to enquire about Honours, Masters, PhD or Postdoctoral positions in the lab. Below is a list of ongoing projects.

Dscam2-related projects

The role of Dscam2 in learning and memory

In collaboration with Krystyna Keleman at QBI.

We are using a courtship conditioning assay to assess short-term memory in flies. Dscam2 mutants have a memory defect and we aim to figure out the role that it plays in this proces

**Dscam2 is required for short-term memory.** (A) Schematic of the courtship conditioning assay used in the lab. Naïve males learn that the previously mated female is non-receptive during the training session and they court a different mated female less in the testing session. (B) Control flies demonstrate learning in the testing session. (C) Dscam2 mutant flies have a learning defect.

Molecular mechanisms of Dscam2 repulsion

We have found that Dscam2 homophilic binding signals through the PI3K pathway to induce repulsion. We have an in vivo assay set up to measure this and we are using it to uncover the molecular players involved and how homophilic binding leads to their recruitment.

**Dissecting Dscam2 repulsion.** A Dscam2 pull-down experiment performed by Sarah Kerwin identified several proteins that interact with the Dscam2 cytoplasmic domain. Sarah setup an in vivo Dscam2 repulsion assay for validating these hits using da neurons. These neurons normally express Dscam2B, but it does not induce repulsion between them as can be observed from the tight longitudinal nerve bundles that form within neuromeres of the ventral nerve cord. Overexpressing Dscam2B in these neurons causes them to repel each other and increases the width of these bundles (Ds2^M). Knocking down some Dscam2-interacting proteins like Chickadee, suppresses repulsion whereas knocking down others like shaggy, increases it. Future work will try to determine how Dscam2 homophilic binding leads to the activation or inhibition of these downstream regulatory pathways.

How does Dscam2 inhibit synaptic strength at the larval NMJ?

Dscam2 suppresses synaptic strength in motor neurons and we are investigating the cellular mechanisms involved. Synaptic vesicle recycling appears to be critical.

**Dscam2 is trafficked to different intracellular compartments.** We have been studying how Dscam2 affects synaptic vesicle recycling using electrophysiology and electron microscopy. Different intracellular compartments accumulate in the Dscam2 mutant and PhD student Tarosi Senapati has been trying to identify them. We use an APEX-GBP labelling system developed by Rob Parton’s group to label specific intracellular structures. Shown in B is a GFP-tagged version of Dscam2. Labelling can be seen on the plasma membrane as well as on vesicles within this motor neuron terminal. We are trying to figure out how removing Dscam2 leads to an increased number of synaptic vesicles at active zones, resulting in an increase in synaptic strength.

Do different Dscam2 cytoplasmic isoforms have different functions?

Dscam2 has 5 alternatively spliced cytoplasmic isoforms that have very different protein compositions. We are investigating whether they have different functions at the larval NMJ.

**Alternative Dscam2 cytoplasmic domains may contribute to different Dscam2 functions.** Homophilic repulsion and the suppression of synaptic strength are two known functions of Dscam2, but the signalling mechanisms that lead to these different outcomes are uncharacterised. Five different alternatively spliced cytoplasmic domains have been identified at the RNA level. Each have unique protein sequences. Two of the cytoplasmic domains contain a PDZ binding domain, a protein motif commonly used to scaffold proteins at the synapse. Three of isoforms contain large stretches of low complexity prion-like domains that could participate in liquid liquid phase separation, as has been shown for other synaptic proteins. We are investigating how these domains affect Dscam2 function through rescue experiments and single molecule imaging.

ALS project

How the ALS risk factor, GGNBP2, contributes to autophagy

In collaboration with Naomi Wray’s group at IMB.

We have been studying how the gene, GGNBP2, contributes to ALS pathology using the fly. In humans, increased expression of this gene is correlated with increased risk of ALS. We have shown that it is required for synaptic development in fly motor neurons and that it is involved in autophagy, a process commonly defective in ALS.

**GGNBP2 regulates autophagy.** (A) Autophagy is a process that removes damaged organelles and aggregated proteins from cells by capturing them in vesicles and fusing with lysosomes. (I-L) Using a fluorescent version of an autophagic protein that gets degraded during this process (Ref(2)P in flies p62 in vertebrates) we observe that both loss of GGNBP2 function (K) and overexpression of GGNBP2 (L) causes defects in autophagic flux. (M) Quantification of the density of Ref(2)P puncta in motor neurons of different GGNBP2 genotypes.

Mitophagy project

Mechanisms of Fbxl4-mediated mitophagy in Drosophila

In collaboration with Julia Pagan’s lab at SBMS.

Mitophagy is the selective degradation of damaged mitochondria using autophagic machinery. The Pagan lab has identified a ubiquitin ligase (FbxL4) that limits mitophagy by degrading receptors on mitochondria required for this process. We are trying to understand the mechanisms involved using Drosophila.

Group Head

Associate Professor Sean Millard

Associate Professor

School of Biomedical Sciences

Staff

Miss Anna Pilaar

Senior Research Assistant

School of Biomedical Sciences

Students

Miss Tarosi Senapati

PhD student

School of Biomedical Sciences
Mr Brendan Townsend

PhD student

School of Biomedical Sciences

Former students

Sarah Kerwin (PhD) – Postdoctoral Fellow at Rockefeller University (Bargmann lab)
Amber Kewin (MPhil) – Research Assistant at the Queensland Brain Institute (van Swinderen lab)
Lorenzo Odierna (PhD) – Postdoctoral Fellow at the University of Tasmania (Blizzard lab)
Joshua Li (PhD) – Postdoctoral Fellow at Harvard University (Perrimon lab)
Kevin Mutemi (MPhil) – PhD student at EMBL Heidelberg (Arendt lab)

For a full list of publications, visit eSpace

2020

Dscam2 suppresses synaptic strength through a PI3K-dependent endosomal pathway.
Odierna GL, Kerwin SK, Harris LE, Shin GJ, Lavidis NA, Noakes PG, Millard SS.
J Cell Biol. 2020 Jun 1;219(6):e201909143. doi: 10.1083/jcb.201909143.
PMID: 32259198

Elevated levels of Drosophila Wdr62 promote glial cell growth and proliferation through AURKA signalling to AKT and MYC.
Shohayeb B, Mitchell N, Millard SS, Quinn LM, Ng DCH.
Biochim Biophys Acta Mol Cell Res. 2020 Jul;1867(7):118713. doi: 10.1016/j.bbamcr.2020.118713. Epub 2020 Apr 1.
PMID: 32246948

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.
Shohayeb B, Ho U, Yeap YY, Parton RG, Millard SS, Xu Z, Piper M, Ng DCH.
Hum Mol Genet. 2020 Jan 15;29(2):248-263. doi: 10.1093/hmg/ddz281.
PMID: 31816041

2019

Li, JS and Millard SS (2019). Deterministic splicing of Dscam2 is regulated by Muscleblind. Sci Adv 5(1): eaav1678.

2018

Millard SS, Pecot MY (2018). Strategies for assembling columns and layers in the Drosophila visual system. Neural Development 13(1):11.

Kerwin SK, Li JS, Noakes PG, Shin GJ, Millard SS (2018). Regulated alternative splicing of Drosophila Dscam2 is necessary for attaining the appropriate number of photoreceptor synapses. Genetics 208 (2): 717-28.

2017

Lim NR, Shohayeb B, Zaytseva O, Mitchell N, Millard SS, Ng DCH, and Quinn LM (2017). Glial-specific functions of microcephaly protein WDR62 and interaction with the mitotic kinase AURKA are essential for Drosophila brain growth. Stem Cell Reports 9: 1-10.

2016

Tadros W, Xu S, Akin O, Yi CH, Shin GJ, Millard SS and Zipursky SL (2016). Dscam proteins direct dendritic targeting through adhesion. Neuron 89 (3):480-93.

2015

Li JS, Shin GJ, Millard SS (2015). Neuronal cell-type-specific alternative splicing: A mechanism for specifying connections in the brain? Neurogenesis 2:1, e1122699: 1-5.

Bosch DS, van Swinderen B and Millard SS (2015). Dscam2 affects visual perception in Drosophila melanogaster. Front. Behav. Neurosci. 9:149. doi: 10.3389/fnbeh.2015.00149.

2014

Lah GJ, Li JS, Millard SS (2014). Cell-specific alternative splicing of Drosophila Dscam2 is crucial for proper neuronal wiring. Neuron 17;83(6):1376-88.

2013

Li Q, Ha TS, Okuwa S, Wang Y, Wang Q Millard SS, Smith DP and Volkan-Cayirlioglu P (2013). Combinatorial rules of precursor specification underlying olfactory neuron diversity. Current Biol. 23, 2481-2490.

Paulk A, Millard SS, van Swinderen B. (2013). Vision in Drosophila: Seeing the World Through a Model's Eyes. Ann. Rev. Entomol. 58:313-32.

2010

Millard SS, Lu Z, Zipursky SL, Meinertzhagen IA. (2010) Drosophila Dscam proteins regulate postsynaptic specificity at multiple-contact synapses. Neuron 9;67(5):761-8.

2008

Millard SS, Zipursky SL. (2008) Dscam-mediated Repulsion Controls Tiling and Self-avoidance. Current Opinion in Neurobiology 18(1):84-9.

Hattori D, Millard SS, Wojtowicz W, Zipursky SL. (2008) Dscam-mediated Cell Recognition Regulates Neural Circuit Formation. Annual Reviews in Cell and Developmental Biology 24:597-620.

2007

Millard SS, Flanagan JJ, Pappu KS, Wu W, Zipursky SL. (2007) Dscam2 mediates axonal tiling in the Drosophila visual system. Nature 447(7145):720-4.

Millard Group - Molecular mechanisms for wiring the brain