The Molecular Mechanisms of Dendritic Growth:
We employ quantitative analysis of dendritic architecture including estimates for synapse and ion channel localization by 3-dimensional geometric reconstruction of dendritic shape from high resolution confocal images. These tools now allow us to quantitatively analyze the three dimensional architecture of neurons in great detail, both, following different experimental manipulations in the healthy CNS and in disease states.
Central neurons are amazingly beautiful and complex structures. For example, geometric reconstruction of the identified flight motoneuron, MN5, in adult flies reveals more than 6 mm total dendritic length, comprising more than 4000 dendritic branches (FIGURE1). We currently study the molecular mechanisms that regulate dendritic structure, dendritic territory coverage, dendritic self-avoidance and tiling as well as synaptogenesis.
Figure 1. Morphological variability of the identified motoneuron, MN5. A-C: Projection views of confocal image stacks from intracellular stainings of MN5 from three different animals with identical genetic background are superimposed with 3D reconstructions. D: Overlay of geometric dendrite reconstructions shown in A-C. Although similarities exist in the overall branching structure, and the dendritic territories are nearly identical in all three cells, marked differences exist in the fine branching structure. Asterisks mark neuropil areas devoid of dendrites and small arrows mark typical distal dendrites.
Example 1: (Vonhoff et al., 2013, Development. 2013 Feb 1;140(3):606-16): By combining live imaging, genetic manipulation, newly generated nuclear reporters for transcriptional events and quantitative neuroanatomy we have identified a critical period during pupal life, during which cholinergic synaptic activity and calcium influx through voltage gated channels is translated by CaMKII into AP-1 dependent transcription to promote dendritic growth. We now aim to identify targets of activity regulated transcription factors that regulate dendrite growth. We now aim to dissect apart the relative contributions of global calcium influx and subsequent transcriptional events as opposed to local calcium signals at sites of newly formed synapses.
Example 2: By combining targeted RNAi knock down and genetic mosaic analysis we have recently identified a new role for Down Syndrome Cell Adhesion Molecule (Dscam) in dendrite growth (Hutchinson et al., 2014, Jan 29;34(5):1924-31) and now study the molecular underpinnings (FIGURE 2).
The Function of Neuronal Dendrites:
Although it is clear that neurons have widely ramified 3-dimensional dendritic trees to provide sufficient space for large numbers of synaptic inputs and that dendritic structure strongly affects the computation of synaptic input, it remains unclear why dendrites are so staggeringly complex. Moreover, many neurogenerative and neurodevelopmental conditions correlate with structural dendritic defects, but, in most cases, it remains unclear whether these are the cause or the consequence of the disease. Therefore, understanding the function of dendritic structure is fundamental to basic research and to a better understanding of diseases.
We probe the functions dendritic architecture for by selectively manipulating neuronal dendrites, to then monitor the resulting consequences for neuronal input-output computations, firing patterns and ultimately for behavior. Our developmental studies on dendritic growth have identified molecules which selectively affect only dendritic growth and not any other neuronal properties. This now allows for producing identified neurons with different degrees of dendritic defects in an otherwise control CNS. See Ryglewski et al., (PNAS, 2014 Dec 16;111(50):18049-54) for an example for Drosophila wing depressor motoneuron dendrites.
Ion Channel Function in Motoneurons during Rhythmic Motor Patterns:
The generation of rhythmic motor patterns, such as breathing, flying, walking, and crawling, relies upon activity in central pattern generating (CPG) networks, which can produce rhythmic motor output in the absence of sensory feedback. Although CPGs play critical roles in generating the timing of complex patterned spiking output executed by motoneurons (MNs), the MNs are not merely passive interpreters of synaptic input from the CPG. Instead, MNs are equipped with intrinsic and conditional membrane properties that sculpt the final motor output. Therefore, knowing how MN ionic currents shape CPG output is fundamental to understanding the control of movement in all animals.
We patch onto Drosophila larval motoneurons during rhythmical crawling movements in semi-intact preparations and target genetic knock-down for specific ion channels to selected motoneurons in an otherwise normal nervous system. This now allows for testing the specific functions of distinct ion channel proteins in selected neurons without affecting the rest of the network. See Kadas et al. (2015, J Physiol. 2015 Nov 15;593(22):4871-88), for an example of BK channel function in motoneurons.
Ion Channel Function in during development:
During recent years it has become clear that neural activity, and especially activity dependent calcium influx, is a natural partner of genetic programs during almost all steps of neuronal differentiation, from cell division to synaptogenesis and circuit refinement.
Drosophila as a Disease Model
We utilize the highly conserved nature of molecular pathways underlying brain development and cellular function to apply our research to topics directly relevant to public health. A number of genes that have been implicated in neurological diseases affect central neuron architecture and excitability. We have all tools at hand to test these genes in the Drosophila system by either manipulating the Drosophila orthologs, or by targeted expression of human genes in Drosophila. An immediate candidate molecule on our research plan is the Rett Syndrome related transcriptional regulator, MeCP2. When expressed in Drosophila, human MECP2 binds DNA, becomes phosphorylated in an activity dependent manner, interacts with known human orthologs, and affects dendritic patterning. We have recently published proof of principle of a Drosophila model to probe for molecular interactors and specific cellular functions of human MeCP2 (Vonhoff et al., 2012; FIGURE 3). In collaboration with our clinical partners in Phoenix, US (the Barrow Neurological Institute and Translational Genomic Research Institute, TGEN) we have recently identified novel MeCP2 targets by RNASeq, have used these to rescue MeCP2 induced cellular phenotypes in Drosophila, and have started validating their molecular and cellular functions in mouse primary neuron culture. We expect to provide new target molecules for developing future treatment strategies in humans. For an example see Williams et al., (2016, Neurobiol of Disease, in press).
The Mechanisms and Functions of Posttranscriptional Modifications of Voltage Gated Calcium Channels (Dr. Ryglewski)
Voltage gated calcium channels (VGCCs) are essential to multiple aspects of neuronal function. Depending on their gating properties, activation and inactivation kinetics, conductivity, and localization in different compartments or sub-compartments of neurons, they serve different functional roles, such as synaptic vesicle release, the generation of plateau potentials, the sculpting of action potential shape and firing patterns, amplification of synaptic input to dendrites and many more. Accordingly, calcium channel malfunction is implicated in numerous brain diseases. The specific functions of VGCCs in different types of neurons greatly outnumber the genes encoding such channels. Therefore, multiple additional mechanisms exist to regulate both the properties and the localization of these channels. By combination of genetic manipulation, patch clamp (FIGURE 4), and calcium imaging, we tackle the molecular mechanisms that underlie the generation of functionally distinct VGCCs from one gene as well as the resulting functional consequences.Vertebrate genomes encode 10 VGCC α-subunit genes that are categorized according to their properties: Cav1, Cav2, Cav3. Cav1.1-1.4 comprise L-type, Cav2.1-2.3 P-/Q-, N- and R-type, and Cav3.1-3.3 T-type VGCCs, the only ones that are truly low voltage activated (LVA). Cav1 and Cav2 are high voltage activated (HVA). Drosophila possesses only three VGCC genes that are homologous to the vertebrate VGCC families: Dmca1D (Cav1), Dmca1A (cacophony; Cav2), and DmαG (Cav3). However, we have shown that in the somatodendritic compartment of an identified Drosophila motoneuron the Cav2 homolog cacophony mediates at least two distinct HVA plus one LVA (Ryglewski et al., 2012) as well as presynaptic HVA calcium currents in axon terminals. This means that one gene, cacophony, underlies at least four distinct calcium currents. Electrophysiological and behavioral data suggest different functions for each of these currents.
We currently probe three mechanisms that may underlie the functional diversity and localization of cacophony calcium channels: (1) A-I pre-mRNA editing, (2) alternative splicing, and (3) the association with accessory β- and α2δ-subunits. Based on the analysis of these molecular mechanisms we will use targeted genetic manipulations in identified neurons to impair specific cacophony functions and assess the resulting cellular and behavioral consequences. Based on this work we expect a better understanding of the mechanisms and functions of post-transcriptional regulation of Cav2 calcium channels in the healthy CNS as well as the consequences that arise from mis-regulation.