Project 1- Cellular and molecular mechanisms underlying axon morphogenesis of cortical neurons

The cerebral cortex is composed of billions of neurons connected through trillions of synapses. The proper function of the cortex requires appropriate generation of these two classes of neurons, their proper migration into specific layers and the establishment of a balanced number of excitatory and inhibitory synapses between these subpopulations of neurons.

Over the past three decades, the field has made important progress in the identification of the molecular mechanisms regulating neurogenesis and neuronal migration of these various classes of cortical neurons. However, our understanding of the cellular and molecular mechanisms patterning the connectivity of these classes of neurons is still limited. In particular, what are the molecular mechanisms underlying the ability of long-range projecting pyramidal neurons and locally-projecting interneurons to form an axon, for these axons to grow and branch and finally find their appropriate synaptic targets?

One major effort in our lab is focusing on the identification of the molecular mechanisms underlying axon development and patterning of cortical connectivity. We have identified the kinase LKB1 as a ‘master’ regulator of axogenesis (Barnes et al. Cell 2007), axon growth and branching (Courchet, Lewis et al. Cell 2013) in pyramidal long-range projecting neurons.

 We are currently focusing on understanding how this LKB1-dependent kinase pathway and some of its 14 potential downstream effector kinases control axon specification, axon growth, branching and presynaptic function.
Our current results point to the critical role play by this LKB1-dependent pathway in regulating mitochondria localization and function during axon branching (through presynaptic mitochondria anchoring) and and presynaptic function (through regulation of presynaptic Ca2+ homeostasis- See Kwon et al. PLoS Biology (2016)).
We are also exploring the potential cellular and molecular mechanisms anchoring mitochondria at presynaptic boutons.
 figure1_Lewis_Courchet

Figure summarizing the three major steps of axon development in the CNS: axon specification during neuronal polarization, axon growth and guidance, axon branching and presynaptic development (Modified from Lewis, Courchet and Polleux, The Journal of Cell Biology, 2013).

Publications related to Project 1

Articles

Courchet J.*, Lewis T. Jr*, Aizawa S. and Polleux F. (2013) Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture.
Cell 153:1510-1525.

*Contributed equally to this work.

Listen to a Cell Paperclip on this paper

Yi J.J., Barnes A.P., Hand R., Polleux F. and Ehlers M.D. (2010) TGFβ signaling specifies axons during brain development. Cell 142:144-57.

Barnes A.P., Lilley B., Pan, A. Plummer L, Powell A., Raines, A, Sanes J.R., Polleux F. (2007) LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons. Cell 129 :549-563.

Hand R*, Bortone D*, Mattar P, N’Guyen L, Heng JI-T, Guerrier S, Boutt E, Peters E, Barnes, AP, Parras C, Schuurmans C, Guillemot F and Polleux F. (2005)
Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex. Neuron 48:45-62.

Polleux F., Morrow T. and Ghosh A. (2000)
Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404:567-573. (Full Article +Cover +News & Views)

Polleux F., Giger R.J., Ginty, D.D. Kolodkin A.L. and Ghosh A. (1998) Patterning of cortical efferent projections by semaphorin-neuropilin interactions. Science 282: 1904-1906.

Reviews

Lewis T.L. Jr, Courchet J. and Polleux F. (2013)
Cell Biology of Neuroscience: Cellular and molecular mechanisms underlying axon formation, growth and branching.
The Journal of Cell Biology 202:837-42.

Barnes A.P., and Polleux F. (2009)
Establishment of Axon-Dendrite Polarity in Developing Neurons.
Annual Review of Neuroscience 32:347-81.

Project 2- Genetic mechanisms underlying human brain evolution: role of human-specific gene duplications during cortical development

What makes us human?  In particular, during recent evolution, what genetic mechanisms have allowed the emergence of specific traits characterizing the human brain? These questions represent some of the most challenging problems remaining in biology and have fascinated generations of philosophers, sociologists, anthropologists, geneticists, neuroscientists and evolutionary biologists. The identification of the genetic mechanisms underlying human-specific brain development during evolution will transform our ability to decipher the pathophysiological mechanisms underlying neurodevelopmental disorders affecting humans such as autism or schizophrenia. 

In recent years, many potential genetic mechanisms have been proposed to participate in human brain evolution. These include molecular evolution of transcription factors or changes in transcriptional regulation (Enard et al., 2002; Konopka et al., 2009), accelerated evolution of small non-coding RNAs (Pollard et al., 2006), changes in the tissue-specificity of enhancer elements (McLean et al., 2011; Prabhakar et al., 2008), or changes in patterns of alternative splicing of specific genes (Calarco et al., 2007). So far, few studies have assessed the functional consequences of these genomic changes.

Gene duplication is one of the major forces driving evolution and speciation (Ohno, 1970). Recent breakthroughs in evolutionary genomics show that a burst of gene duplications occurred in the human lineage during its separation from non-human primates approximately 6 million years ago (Bailey et al., 2002; Fortna et al., 2004; Marques-Bonet et al., 2009). This has led to the hypothesis that these evolutionarily recent gene duplications might have participated in the emergence of human-specific traits of brain development and function (Bailey and Eichler, 2006; Stankiewicz and Lupski, 2010). However, so far, this portion of the human genome has largely remained unexplored largely because, for technical reasons, it is still poorly assembled (Bailey et al., 2002). The long-term scientific goal of this project is aimed at determining the role of hominoid- and human-specific gene duplications during brain development and evolution. This is a unique scientific paradigm: the first publication determining the role of human-specific gene duplications during brain development came out of our laboratory recently (Charrier et al., 2012) and represents a milestone in our understanding of the genetic and neurobiological mechanisms underlying the emergence of human-specific traits of brain development, for example neoteny during synaptic maturation (Benavides-Piccione et al., 2002; Petanjek et al., 2011).

We first focused on SRGAP2 and its human-specific paralogs because we previously reported that this gene plays important roles during neocortical development, in particular regulating neuronal migration and dendritic branching (Guerrier et al., 2009). SRGAP2 has undergone two main human-specific duplications ((Fortna et al., 2004; Sudmant et al., 2010); see Fig. 1A). We found that the two main human-specific gene duplications (SRGAP2B and SRGAP2C) are partial and encode a truncated F-BAR domain involved in membrane deformation. SRGAP2C (but not SRGAP2B) is detected both at the mRNA and protein levels in the fetal and adult human brain and dimerizes through its truncated F-BAR domain with ancestral SRGAP2 but strongly inhibits its function (Charrier et al. 2012). In the mouse neocortex, we discovered that SRGAP2 is required for proper spine maturation and limits spine density in vivo (Figure 1C). Expression of SRGAP2C in cortical neurons in vivo phenocopies SRGAP2 genetic loss-of-function (Charrier et al., 2012). Remarkably, its expression in vivo, in mouse cortical pyramidal neurons, leads to the emergence of human-specific features, including neoteny of spine maturation resulting in increased density of spines with long necks. The group of Dr Evan Eichler has dated the emergence of these human-specific gene duplications to approximately 3.4 and 2.4 million years ago respectively (Dennis et al., 2012) (Figure 1A-B). Of particular interest, the second duplication that gave rise to SRGAP2C arose 2.4 mya which corresponds approximately to the time during evolution where the Australopithecus and Homo lineages diverged and in the fossil record corresponds to the beginning of brain expansion characterizing the Homo lineage (Dennis et al., 2012). These results suggest that inhibition of SRGAP2 function by its human-specific paralogs has contributed to the evolution of the human neocortex by slowing the rate of excitatory synapse maturation and allowing the emergence of more spines per pyramidal neuron, which is a critical feature of human pyramidal neurons (Benavides-Piccione et al., 2002).

We are currently using this new paradigm to define the expression and function of other hominoid- and human-specific gene duplications during brain development and evolution.

Publications related to Project 2

Fossati M, Pizzarelli R, Schmidt ER, Kupferman JV, Stroebel D, Polleux F*, Charrier C.* (2016) SRGAP2 and Its Human-Specific Paralog Co-Regulate the Development of Excitatory and Inhibitory Synapses. Neuron. 91(2):356-69. Link

*Co-corresponding authors

See Preview by Subramanian and Nedivi in the same Issue Link

Charrier C*, Joshi K*, Coutinho-Budd J, Kim JE, Lambert N, de Marchena J, Jin WL, Vanderhaeghen P, Ghosh A, Sassa T, Polleux F (2012) Inhibition of SRGAP2 function by its human-specific paralogs induces neoteny during spine maturation. Cell 149:923-35.

* Contributed equally to this work.

Related publications from the lab

Coutinho-Budd J, Ghukasyan V, Zylka MJ, Polleux F. (2012) The F-BAR domains from SRGAP1, SRGAP2 and SRGAP3 differentially regulate membrane deformation. Journal of Cell Science 125:3390-401. Epub 2012 Mar 30.

Guerrier S, Coutinho-Budd J, Sassa T, Vincent-Jordan N, Chen K, Jin WL, Frost A, and Polleux F (2009) The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis. Cell 138:990-1004.

Project 3- Signaling pathways underlying the synaptotoxic effects of Amyloid-beta during early stages of alzheimer’s disease progression

Alzheimer’s disease (AD) is the most prevalent form of dementia affecting more than 5 million people in the USA and over 25 million people worldwide. This neurodegenerative disease mainly affects people over 65 years old in its sporadic, late-onset form but can affect younger individuals in its genetically inherited, early-onset form. AD is thought to be caused by the abnormal accumulation of a 40-42 amino-acid long amyloid-β (Aβ) peptide derived from cleavage of the transmembrane protein amyloid-precursor protein (APP). Amyloid-β 42 (Aβ42) has a strong ability to oligomerize to form diffusible oligomers as well as larger polymers called fibrils that form insoluble amyloid plaques, a major hallmark of AD. Oligomeric forms of Amyloid-β 1-42 (Aβ42) are synaptotoxic for excitatory cortical and hippocampal neurons and causes drastic changes in excitatory/inhibitory balance that appears to play a crucial role in Alzheimer’s Disease (AD). Recent evidence suggested that Aβ42 can trigger activation of AMP-activated Kinase (AMPK) and that AMPK activation is increased in the brain of AD patients. Our results (Mairet-Coello et al. Neuron 2013) show that increase in intracellular calcium [Ca2+]i induced by NMDA receptor activation or membrane depolarization strongly activates AMPK through activatoin of Calcium/Calmodulin-Activated Kinase Kinase 2 (CAMKK2). Importantly, we found that the inhibition of the catalytic activity of CAMKK2 or AMPK protects hippocampal neurons against the synaptotoxic effects induced by acute exposure to Aβ42 oligomers in vitro and against the loss of dendritic spines observed in the hAPPSWE,IND-expressing transgenic mouse model (J20) in vivo. Furthermore, AMPK phosphorylates Tau on the KxGS-motif (S262) and expression of hTauS262A partially but significantly blocks the synaptotoxic effects of Aβ42 oligomers.

We are currently identifying the downstream effectors of this CAMKK2-AMPK kinase pathway including Tau and some key effectors of autophagy.

Publications related to Project 3

Mairet-Coello G., Courchet J., Pierault S., Courchet, V., Maximov A., Polleux F. (2013) The CAMKK2-AMPK kinase pathway mediates the synaptotoxic effects of Aβ oligomers in vivo through Tau phosphorylation. Neuron 78:94-108.

Williams T. *, Courchet J. *, Viollet B.  Brenman J.E. and Polleux F. (2011) AMPK kinase activity is not required for neuronal development but regulates axogenesis during metabolic stress. PNAS 108:5849-54. * Co-first authors.

Mairet-Coello G. and Polleux F. (2014) Involvement of ‘stress-response’ kinase pathways in Alzheimer’s disease progression. Curr. Opin. Neurobiol. 27:110-117.