Welcome to the Hammarlund Lab
|Individual neurons sometimes continue working for the life of the animal. In other cases, their function is abrogated by injury, disease, or age-associated decline. Since damaged or dead neurons generally cannot be replaced, the continuing function of our nervous system depends on the ability of our individual neurons to survive for as long as we do: repairing damage, resisting disease, and maintaining function over the long term. Neurons are complex cells with an extended and fragile morphology. Each neuron must generate and maintain delicate balances in membrane potential, trafficking, and secretion to perform its function. How do neurons sometimes survive and continue to function for decades, and why do they sometimes fail?
We study the cell-biological mechanisms that modulate neuronal endurance. We use the model organism C. elegans, which allows us to analyze neuronal structure and function in adult animals, in vivo, with single-neuron resolution—an approach that is difficult in other systems. We develop novel molecular and genetic tools, which we use together with single-neuron laser axotomy, in vivo imaging, optogenetics, electron microscopy, and genetic analysis, to address two fundamental questions:
Answering these questions will provide fundamental insights into the mechanisms that attempt to maintain neuronal cellular and circuit function over time: when successful, allowing the brain to outlast the body; when unsuccessful, increasing susceptibility to cognitive decline and neurological disease. By understanding and manipulating these mechanisms we aim to prevent the decline of the nervous system.
|Axon Regeneration||Synaptic Transmission||Neuronal Aging|
|How do neurons recognize that they have been injured? What mechanisms link injury to the initiation of growth? What determines the regenerative potential of a neuron? How does regeneration rebuild functional circuits?||How do neurons maintain their ability to transmit information? What role do essential genes play at the synapse?||What accounts for the changes in neuronal function over time? How is aging in the nervous system regulated? Can neuronal aging be slowed or even reversed?|
The fact is the sweetest dream that labor knows. --Robert Frost
Our paper, "The RtcB RNA ligase is an essential component of the metazoan unfolded protein response", published in EMBO Reports PDF
- RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo.
Another great summer at the MBL Neurobiology course!
- Thanks to Prahatha and Michael!
Our paper, "Syndecan Promotes Axon Regeneration by Stabilizing Growth Cone Migration", published in Cell Reports link
- Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration. Using single-neuron laser axotomy and in vivo time-lapse imaging, we show that syndecan, a heparan sulfate (HS) proteoglycan, is required for growth cone function during axon regeneration in C. elegans. In the absence of syndecan, regenerating growth cones form but are unstable and collapse, decreasing the effective growth rate and impeding regrowth to target cells. We provide evidence that syndecan has two distinct functions during axon regeneration: (1) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and (2) an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein, independently of HS. Thus, syndecan is a regulator of a critical choke point in nervous system repair.
Our review (with Yishi Jin), Axon Regeneration in C. elegans, published in Current Opinion in Neurobiology PDF
- Single axon transection by laser surgery has made Caenorhabditis elegans a new model for axon regeneration. Multiple conserved molecular signaling modules have been discovered through powerful genetic screening. In vivo imaging with single cell and axon resolution has revealed unprecedented cellular dynamics in regenerating axons. Information from C. elegans has greatly expanded our knowledge of the molecular and cellular mechanisms of axon regeneration.
Really nice review of our paper on aging and regeneration, by Zhigang He and colleagues, published in Cell Metabolism PDF
- Axon regeneration capacity often declines with age. One might assume that loss of regeneration is an obvious consequence of organismal aging. However, in the latest issue of Neuron, Byrne et al. (2014) demonstrate that regeneration ability and aging are regulated cell-autonomously within neurons, and can be decoupled.
Our paper, "Insulin/IGF1 Signaling Inhibits Age-Dependent Axon Regeneration", published in Neuron PDF
- The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. Here we show that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2’s function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, we found that DAF-18/ PTEN inhibits regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR.
Our paper, "Axon Regeneration Genes Identified by RNAi Screening in C. elegans", published in Journal of Neuroscience PDF
- Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/beta-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of beta-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention.
Chris defends his thesis! Congratulations, Dr. Firnhaber!
Tyson defends his thesis! Congratulations, Dr. Edwards!
Our paper, "Neuron-Specific Feeding RNAi in C. elegans and Its Use in a Screen for Essential Genes Required for GABA Neuron Function", published in PLoS Genetics PDF
- Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.
Our paper, "Rapid and Permanent Neuronal Inactivation In Vivo via Subcellular Generation of Reactive Oxygen with the Use of KillerRed", published in Cell Reports PDF
- Inactivation of selected neurons in vivo can define their contribution to specific developmental outcomes, circuit functions, and behaviors. Here, we show that the optogenetic tool KillerRed selectively, rapidly, and permanently inactivates different classes of neurons in C. elegans in response to a single light stimulus, through the generation of reactive oxygen species (ROS). Ablation scales from individual neurons in single animals to multiple neurons in populations and can be applied to freely behaving animals. Using spatially restricted illumination, we demonstrate that localized KillerRed activation in either the cell body or the axon triggers neuronal degeneration and death of the targeted cell. Finally, targeting KillerRed to mitochondria results in organelle fragmentation without killing the cell, in contrast to the cell death observed when KillerRed is targeted to the plasma membrane. We expect this genetic tool to have wide-ranging applications in studies of circuit function and subcellular responses to ROS.
Yet another classy happy hour...thanks everyone!
End-of-Summer Bash. Good weather, great food, and yes, a slack line!
Genetics Happy Hour a la Hammarlund. Always classy!
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Our paper, "Neural Regeneration in Caenorhabditis elegans", published in Annual Review of Genetics
- Summary: Axon regeneration is a medically relevant process that can repair damaged neurons. This review describes current progress in understanding axon regeneration in the model organism Caenorhabditis elegans. Factors that regulate axon regeneration in C. elegans have broadly similar roles in vertebrate neurons. This means that using C. elegans as a tool to leverage discovery is a legitimate strategy for identifying conserved mechanisms of axon regeneration.
End of the 2012 Hammarlund Lab Summer Undergrad Program. Thanks to Will, Chukwuma, Kevin, and (amazing high school student) Austin!
Article about our Notch research published in Scientific American
Our meeting review, "Science in Suzhou: establishment and function of neural circuits", published in EMBO Reports! PDF
- Summary: The CSH Asia conference ‘Assembly, Plasticity, Dysfunction and Repair of Neural Circuits’ brought together developmental, cell, molecular and systems neuroscientists to discuss the establishment, function and plasticity of neural circuits.
Our paper, "Notch Signaling Inhibits Axon Regeneration", published in Neuron! PDF
- Summary: Many neurons have limited capacity to regenerate their axons after injury. Neurons in the mammalian central nervous system do not regenerate, and even neurons in the peripheral nervous system often fail to regenerate to their former targets. This failure is likely due in part to pathways that actively restrict regeneration; however, only a few factors that limit regeneration are known. Here, using single-neuron analysis of regeneration in vivo, we show that Notch/lin-12 signaling inhibits the regeneration of mature C. elegans neurons. Notch signaling suppresses regeneration by acting autonomously in the injured cell to prevent growth cone formation. The metalloprotease and gamma-secretase cleavage events that lead to Notch activation during development are also required for its activity in regeneration. Furthermore, blocking Notch activation immediately after injury improves regeneration. Our results define a postdevelopmental role for the Notch pathway as a repressor of axon regeneration in vivo.
Neuron Preview by Po, Calarco, and Zhen: PDF
Yale undergrad Trent Walradt joins the lab. Welcome Trent!
Happy Holidays from the Hammarlund lab! (Cookies by Rebecca Brown from the CNNR Bakeoff)
End of the 2011 Hammarlund Lab Summer Undergrad Program. Thanks to Jaimie, Quinn, Reba, and Yigit!
Sara passes her qualifying exams. Congratulations!
- Summary: Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response.
- We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint® pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.
Goodbye to new Yale Graduate and amazing lab member Ellie Hong!
December 9: Some of Marc's work from his first postdoc (Syntaxin N-terminal peptide motif is an initiation factor for the assembly of the SNARE–Sec1/Munc18 membrane fusion complex) published in PNAS
November 11: Chris passes his qualifying exam. Congratulations Chris!
November 5: Genetics Department Annual Retreat at Jiminy Peak. Faculty photo
October 19: Some of Chris' work from college (Modifiers of notch transcriptional activity identified by genome-wide RNAi) published in BMC Developmental Biology
August 12: Welcome postdoc Yasunori Saheki!
August 9: With a small tear in his eye, Dan heads west for a Visiting Assistant Professor position at Linfield College. Good Luck Dan!!
June 27-30: Neuronal Development, Synaptic Function, and Behavior C. elegans Topic Meeting.
June 15: Some of Alex's work from grad school (MADD-2, a Homolog of the Opitz Syndrome Protein MID1, Regulates Guidance to the Midline through UNC-40 in Caenorhabditis elegans) published in Developmental Cell
June 14: Welcome grad student Sara Kosmaczewski!
May 24: Welcome undergraduate Ellie Hong!
April 27: Tyson passes his qualifying exam. Congratulations Tyson!
October 6: Welcome postdoc Alexandra Byrne!
June 24-28: International C. elegans Meeting
June 10: "Imaging Axon Regeneration in vivo", Yale Microscopy Workshop
May 18: Welcome grad student Tyson Edwards!
May 7: Welcome grad student Christopher Firnhaber!
April 24: Arnold and Mabel Beckman Foundation Young Investigator Award.
Jan. 22: "Axon regeneration requires a conserved MAP kinase pathway" published in Science. PDF
Jan. 12: Welcome technician Laura Klein!
Dec. 12: Genetics Department Holiday Party
Dec. 11: CNNR Open House