Ph.D's in Press: anxiety, presynaptic scaffolding, epigenetics and more!

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Part 5 in an occasional feature, highlighting recently published articles featuring an author (or authors) who is a current member of the Stanford Neuroscience Ph.D program. (Part 1, Part 2Part 3, Part 4)* First off, 4th year student Sung-Yon Kim (Deisseroth lab) published his study of distinct subregions of the bed nucelus of the stria terminalis, demonstrating, using optogenetics that BNST neurons that project to distinct brain regions each implement independent features of anxiety. Additional Neuro PhD authors include: Christina Kim, Caitlin Mallory and Joanna Mattis.

Behavioural states in mammals, such as the anxious state, are characterized by several features that are coordinately regulated by diverse nervous system outputs, ranging from behavioural choice patterns to changes in physiology (in anxiety, exemplified respectively by risk-avoidance and respiratory rate alterations). Here we investigate if and how defined neural projections arising from a single coordinating brain region in mice could mediate diverse features of anxiety. Integrating behavioural assays, in vivo and in vitro electrophysiology, respiratory physiology and optogenetics, we identify a surprising new role for the bed nucleus of the stria terminalis (BNST) in the coordinated modulation of diverse anxiety features. First, two BNST subregions were unexpectedly found to exert opposite effects on the anxious state: oval BNST activity promoted several independent anxious state features, whereas anterodorsal BNST-associated activity exerted anxiolytic influence for the same features. Notably, we found that three distinct anterodorsal BNST efferent projections-to the lateral hypothalamus, parabrachial nucleus and ventral tegmental area-each implemented an independent feature of anxiolysis: reduced risk-avoidance, reduced respiratory rate, and increased positive valence, respectively. Furthermore, selective inhibition of corresponding circuit elements in freely moving mice showed opposing behavioural effects compared with excitation, and in vivo recordings during free behaviour showed native spiking patterns in anterodorsal BNST neurons that differentiated safe and anxiogenic environments. These results demonstrate that distinct BNST subregions exert opposite effects in modulating anxiety, establish separable anxiolytic roles for different anterodorsal BNST projections, and illustrate circuit mechanisms underlying selection of features for the assembly of the anxious state.

Kim et al (2013). Diverging neural pathways assemble a behavioural state from separable features in anxiety. Nature, 496(7444): 219-23.

Poh Hui Chia (Shen lab), who recently defended her thesis research, published her work on intramolecular regulation of presynaptic scaffold protein SYD-2/liprin-a.

SYD-2/liprin-α is a multi-domain protein that associates with and recruits multiple active zone molecules to form presynaptic specializations. Given SYD-2's critical role in synapse formation, its synaptogenic ability is likely tightly regulated. However, mechanisms that regulate SYD-2 function are poorly understood. In this study, we provide evidence that SYD-2's function may be regulated by interactions between its coiled-coil (CC) domains and sterile α-motif (SAM) domains. We show that the N-terminal CC domains are necessary and sufficient to assemble functional synapses while C-terminal SAM domains are not, suggesting that the CC domains are responsible for the synaptogenic activity of SYD-2. Surprisingly, syd-2 alleles with single amino acid mutations in the SAM domain show strong loss of function phenotypes, suggesting that SAM domains also play an important role in SYD-2's function. A previously characterized syd-2 gain-of-function mutation within the CC domains is epistatic to the loss-of-function mutations in the SAM domain. In addition, yeast two-hybrid analysis showed interactions between the CC and SAM domains. Thus, the data is consistent with a model where the SAM domains regulate the CC domain-dependent synaptogenic activity of SYD-2. Taken together, our study provides new mechanistic insights into how SYD-2's activity may be modulated to regulate synapse formation during development.

Chia et al (2013). Intramolecular regulation of presynaptic scaffold portein SYD-2/liprin-a. Molecular and Cellular Neuroscience, 56: 76-84.

In the category of reviews and commentaries, Jana Lim (Brunet lab) co-authored a review on transgenerational epigenetic inheritance.

It is textbook knowledge that inheritance of traits is governed by genetics, and that the epigenetic modifications an organism acquires are largely reset between generations. Recently, however, transgenerational epigenetic inheritance has emerged as a rapidly growing field, providing evidence suggesting that some epigenetic changes result in persistent phenotypes across generations. Here, we survey some of the most recent examples of transgenerational epigenetic inheritance in animals, ranging from Caenorhabditis elegans to humans, and describe approaches and limitations to studying this phenomenon. We also review the current body of evidence implicating chromatin modifications and RNA molecules in mechanisms underlying this unconventional mode of inheritance and discuss its evolutionary implications.

Lim and Brunet (2013). Bridging the transgenerational gap with epigenetic memory. Trends in Genetics, 29(3): 176-186.

Several Neuro PhD students were also second through n-th authors on papers. From the prolific Deisseroth lab, students Aslihan Selimeyoglu and Sung-Yon Kim are coauthors on a paper describing "a prefrontal cortex-brainstem neural projection that controls response to behavioral challenge".

The prefrontal cortex (PFC) is thought to participate in high-level control of the generation of behaviours (including the decision to execute actions1); indeed, imaging and lesion studies in human beings have revealed that PFC dysfunction can lead to either impulsive states with increased tendency to initiate action2, or to amotivational states characterized by symptoms such as reduced activity, hopelessness and depressed mood3. Considering the opposite valence of these two phenotypes as well as the broad complexity of other tasks attributed to PFC, we sought to elucidate the PFC circuitry that favours effortful behavioural responses to challenging situations. Here we develop and use a quantitative method for the continuous assessment and control of active response to a behavioural challenge, synchronized with single-unit electrophysiology and optogenetics in freely moving rats. In recording from the medial PFC (mPFC), we observed that many neurons were not simply movement-related in their spike-firing patterns but instead were selectively modulated from moment to moment, according to the animal’s decision to act in a challenging situation. Surprisingly, we next found that direct activation of principal neurons in the mPFC had no detectable causal effect on this behaviour. We tested whether this behaviour could be causally mediated by only a subclass of mPFC cells defined by specific downstream wiring. Indeed, by leveraging optogenetic projection-targeting to control cells with specific efferent wiring patterns, we found that selective activation of those mPFC cells projecting to the brainstem dorsal raphe nucleus (DRN), a serotonergic nucleus implicated in major depressive disorder4, induced a profound, rapid and reversible effect on selection of the active behavioural state. These results may be of importance in understanding the neural circuitry underlying normal and pathological patterns of action selection and motivation in behaviour.

Warden et al (2013). A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature, 492 (428-432).

Also from the Deisseroth lab, Sung-Yon Kim, Kelly Zalocusky, Joanna Mattis and Logan Grosenick are all authors of the recently published article describing CLARITY, a novel method developed by lead author Kwanghun Chung for producing optically transparent tissue for the purpose of  tissue-intact imaging.

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.

Chung (2013). Structural and molecular interrogation of intact biological systems. Nature. doi:10.1038/nature12107

Georgia Panagiotakos is the second author on a paper published in PLOS One, showing the mechanisms underlying the production of the calcium channel associated transcriptional regulator (CCAT), which is encoded by the C-terminus of the voltage-gated calcium channel Cav1.2.

The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3′ end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.

Gomez-Ospina N, Panagiotakos G, Portmann T, Pasca SP, Rabah D, et al. (2013) A Promoter in the Coding Region of the Calcium Channel Gene CACNA1C Generates the Transcription Factor CCAT. PLoS ONE 8(4): e60526. doi:10.1371/journal.pone.0060526

Matt Figley (Gitler lab) is the third author on a paper in Nature Genetics, discussing the suppression of TDP-43 toxicity in ALS disease models by the inhibition of RNA lariat debranching enzyme.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43–mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

Armakola et al (2013). Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models. Nature Genetics, 44: 1302-1309.

And lastly, Daniel Kimmel, together with first-author Michael Greicius, co-authored a review on neuroimaging insights into network-based neurodegeneration.

Purpose of review: Convergent evidence from a number of neuroscience disciplines supports the hypothesis that Alzheimer's disease and other neurodegenerative disorders progress along brain networks. This review considers the role of neuroimaging in strengthening the case for network-based neurodegeneration and elucidating potential mechanisms.

Recent findings: Advances in functional and structural MRI have recently enabled the delineation of multiple large-scale distributed brain networks. The application of these network-imaging modalities to neurodegenerative disease has shown that specific disorders appear to progress along specific networks. Recent work applying theoretical measures of network efficiency to in-vivo network imaging has allowed for the development and evaluation of models of disease spread along networks. Novel MRI acquisition and analysis methods are paving the way for in-vivo assessment of the layer-specific microcircuits first targeted by neurodegenerative diseases. These methodological advances coupled with large, longitudinal studies of subjects progressing from healthy aging into dementia will enable a detailed understanding of the seeding and spread of these disorders.

Summary: Neuroimaging has provided ample evidence that neurodegenerative disorders progress along brain networks, and is now beginning to elucidate how they do so.

Greicius and Kimmel (2012). Neuroimaging insights into network-based neurodegeneration. Current Opinion in Neurology. 25(6): 727-734.

*Regarding the mechanics of this feature: This is purely through the magic of an ongoing My NCBI search for the names of Neuro PhD students. I wouldn’t be surprised if there were some false negatives in the data set. Neuro students – let me know if I’ve missed your paper, and I’ll gladly add it.


Astra Bryant

Astra Bryant is a graduate of the Stanford Neuroscience PhD program in the labs of Drs. Eric Knudsen and John Huguenard. She used in vitro slice electrophysiology to study the cellular and synaptic mechanisms linking cholinergic signaling and gamma oscillations – two processes critical for the control of gaze and attention, which are disrupted in many psychiatric disorders. She is a senior editor and the webmaster of the NeuWrite West Neuroblog