A methodology for specific disruption of microtubule polymerization into dendritic spines.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

A Methodology for Specific Disruption of Microtubules in Dendritic Spines.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Contributions of microtubule dynamics and transport to presynaptic and postsynaptic functions.

Microtubules (MT) are elongated, tubular, cytoskeletal structures formed from polymerization of tubulin dimers. They undergo continuous cycles of polymerization and depolymerization, primarily at their plus ends, termed dynamic instability. Although this is an intrinsic property of MTs, there are a myriad of MT-associated proteins that function in regulating MT dynamic instability and other dynamic processes that shape the MT array. Additionally, MTs assemble into long, semi-rigid structures which act as substrates for long-range, motor-driven transport of many different types of cargoes throughout the cell. Both MT dynamics and motor-based transport play important roles in the function of every known type of cell. Within the last fifteen years many groups have shown that MT dynamics and transport play ever-increasing roles in the neuronal function of mature neurons. Not only are neurons highly polarized cells, but they also connect with one another through synapses to form complex networks. Here we will focus on exciting studies that have illuminated how MTs function both pre-synaptically in axonal boutons and post-synaptically in dendritic spines. It is becoming clear that MT dynamics and transport both serve important functions in synaptic plasticity. Thus, it is not surprising that disruption of MTs, either through hyperstabilization or destabilization, has profound consequences for learning and memory. Together, the studies described here suggest that MT dynamics and transport play key roles in synaptic function and when disrupted result in compromised learning and memory.

A Single Transcript Knockdown-Replacement Strategy Employing 5' UTR Secondary Structures to Precisely Titrate Rescue Protein Translation.

One overarching goal of gene therapy is the replacement of faulty genes with functional ones. A significant hurdle is presented by the fact that under- or over-expression of a protein may cause disease as readily as coding mutations. There is a clear and present need for pipelines to translate experimentally validated gene therapy strategies to clinical application. To address this we developed a modular, single-transgene expression system for replacing target genes with physiologically expressed variants. In order to accomplish this, we first designed a range of 5' UTR "attenuator" sequences which predictably diminish translation of the paired gene. These sequences provide wide general utility by allowing control over translation from high expression, ubiquitous promoters. Importantly, we demonstrate that this permits an entirely novel knockdown and rescue application by pairing microRNA-adapted shRNAs alongside their respective replacement gene on a single transcript. A noteworthy candidate for this corrective approach is the degenerative and uniformly fatal motor neuron disease ALS. A strong proportion of non-idiopathic ALS cases are caused by varied mutations to the SOD1 gene, and as clinical trials to treat ALS are being initiated, it is important to consider that loss-of-function mechanisms contribute to its pathology as strongly as any other factor. As a generalized approach to treat monogenic diseases caused by heterogeneous mutations, we demonstrate complete and predictable control over replacement of SOD1 in stable cell lines by varying the strength of attenuators.

Double UP: A Dual Color, Internally Controlled Platform for in utero Knockdown or Overexpression.

In utero electroporation (IUE) is a powerful tool for testing the role of genes in neuronal migration and function, but this technique suffers from high degrees of variability. Such variability can result from inconsistent surgery, developmental gradients along both rostral-caudal and medial-lateral axes, differences within littermates and from one litter to another. Comparisons between control and experimental electroporations rely on section matching, which is inherently subjective. These sources of variability are cumulative, leading to difficult to interpret data and an increased risk of both false positives and false negatives. To address these limitations, we developed two tools: (1) a new plasmid, termed Double UP, which combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls, and (2) an automated program for unbiased and precise quantification of migration. In concert, these tools allow for more rigorous and objective experiments, while decreasing the mice, time, and reagents required to complete studies.

Novel α-Lipoic Acid/3-n-Butylphthalide Conjugate Enhances Protective Effects against Oxidative Stress and 6-OHDA Induced Neuronal Damage.

Neurodegenerative diseases are irreversible conditions that result in progressive degeneration and death of nerve cells. Although the underlying mechanisms may vary, oxidative stress is considered to be one of the major causes of neuronal loss. Importantly, there are still no comprehensive treatments to completely cure these diseases. Therefore, protecting neurons from oxidative damage may be the most effective therapeutic strategy. Here we report a neuroprotective effects of a novel hybrid compound (dlx-23), obtained by conjugating α-lipoic acid (ALA), a natural antioxidant agent, and 3-n-butylphthalide (NBP), a clinical anti-ischemic drug. Dlx-23 protected against neuronal death induced by both H2O2 induced oxidative stress in Cath.-a-differentiated (CAD) cells and 6-OHDA, a toxin model of Parkinson's disease (PD) in SH-SY5Y cells. These activities proved to be more potent than the parent compound (ALA) alone. Dlx-23 scavenged free radicals, increased glutathione levels, and prevented mitochondria damage. In addition, live imaging of primary cortical neurons demonstrated that dlx-23 protected against neuronal growth cone damage induced by H2O2. Taken together these results suggest that dlx-23 has substantial potential to be further developed into a novel neuroprotective agent against oxidative damage and toxin induced neurodegeneration.

Dynamic microtubules at the synapse.

Microtubules (MTs) are a fundamental cytoskeletal component that give neurons structure and are the primary polymer system for long distance transport of cargo throughout the cytoplasm. Although neurons are highly polarized and their structure is often maintained throughout the life of an organism, MTs can remain dynamic in axons and dendrites, undergoing bouts of polymerization and depolymerization, referred to as dynamic instability. Furthermore, MTs can be nucleated outside of the centrosome or MT organizing center (MTOC) that is located in the cell body, allowing dynamic formation and branching of MT polymers throughout the neuron. Together, these recent findings point to a much more dynamic landscape of microtubules in developing and mature neurons than was previously appreciated. Here we will focus on recent studies that show MT dynamics are playing a role at the synapse, both post-synaptically in dendrites and pre-synaptically in axons.

Opposing functions of F-BAR proteins in neuronal membrane protrusion, tubule formation, and neurite outgrowth.

The F-BAR family of proteins play important roles in many cellular processes by regulating both membrane and actin dynamics. The CIP4 family of F-BAR proteins is widely recognized to function in endocytosis by elongating endocytosing vesicles. However, in primary cortical neurons, CIP4 concentrates at the tips of extending lamellipodia and filopodia and inhibits neurite outgrowth. Here, we report that the highly homologous CIP4 family member, FBP17, induces tubular structures in primary cortical neurons and results in precocious neurite formation. Through domain swapping and deletion experiments, we demonstrate that a novel polybasic region between the F-BAR and HR1 domains is required for membrane bending. Moreover, the presence of a poly-PxxP region in longer splice isoforms of CIP4 and FBP17 largely reverses the localization and function of these proteins. Thus, CIP4 and FBP17 function as an antagonistic pair to fine-tune membrane protrusion, endocytosis, and neurite formation during early neuronal development.

Single-neuronal cell culture and monitoring platform using a fully transparent microfluidic DEP device.

Dielectrophoresis using multi-electrode arrays allows a non-invasive interface with biological cells for long-term monitoring of electrophysiological parameters as well as a label-free and non-destructive technique for neuronal cell manipulation. However, experiments for neuronal cell manipulation utilizing dielectrophoresis have been constrained because dielectrophoresis devices generally function outside of the controlled environment (i.e. incubator) during the cell manipulation process, which is problematic because neurons are highly susceptible to the properties of the physiochemical environment. Furthermore, the conventional multi-electrode arrays designed to generate dielectrophoretic force are often fabricated with non-transparent materials that confound live-cell imaging. Here we present an advanced single-neuronal cell culture and monitoring platform using a fully transparent microfluidic dielectrophoresis device for the unabated monitoring of neuronal cell development and function. The device is mounted inside a sealed incubation chamber to ensure improved homeostatic conditions and reduced contamination risk. Consequently, we successfully trap and culture single neurons on a desired location and monitor their growth process over a week. The proposed single-neuronal cell culture and monitoring platform not only has significant potential to realize an in vitro ordered neuronal network, but also offers a useful tool for a wide range of neurological research and electrophysiological studies of neuronal networks.

Of microtubules and memory: implications for microtubule dynamics in dendrites and spines.

Microtubules (MTs) are cytoskeletal polymers composed of repeating subunits of tubulin that are ubiquitously expressed in eukaryotic cells. They undergo a stochastic process of polymerization and depolymerization from their plus ends termed dynamic instability. MT dynamics is an ongoing process in all cell types and has been the target for the development of several useful anticancer drugs, which compromise rapidly dividing cells. Recent studies also suggest that MT dynamics may be particularly important in neurons, which develop a highly polarized morphology, consisting of a single axon and multiple dendrites that persist throughout adulthood. MTs are especially dynamic in dendrites and have recently been shown to polymerize directly into dendritic spines, the postsynaptic compartment of excitatory neurons in the CNS. These transient polymerization events into dendritic spines have been demonstrated to play important roles in synaptic plasticity in cultured neurons. Recent studies also suggest that MT dynamics in the adult brain function in the essential process of learning and memory and may be compromised in degenerative diseases, such as Alzheimer's disease. This raises the possibility of targeting MT dynamics in the design of new therapeutic agents.

Transport of a kinesin-cargo pair along microtubules into dendritic spines undergoing synaptic plasticity.

Synaptic plasticity often involves changes in the structure and composition of dendritic spines. Vesicular cargos and organelles enter spines either by exocytosing in the dendrite shaft and diffusing into spines or through a kinesin to myosin hand-off at the base of spines. Here we present evidence for microtubule (MT)-based targeting of a specific motor/cargo pair directly into hippocampal dendritic spines. During transient MT polymerization into spines, the kinesin KIF1A and an associated cargo, synaptotagmin-IV (syt-IV), are trafficked in unison along MTs into spines. This trafficking into selected spines is activity-dependent and results in exocytosis of syt-IV-containing vesicles in the spine head. Surprisingly, knockdown of KIF1A causes frequent fusion of syt-IV-containing vesicles throughout the dendritic shaft and diffusion into spines. Taken together, these findings suggest a mechanism for targeting dendritic cargo directly into spines during synaptic plasticity and indicate that MT-bound kinesins prevent unregulated fusion by sequestering vesicular cargo to MTs.

Beyond the cytoskeleton: The emerging role of organelles and membrane remodeling in the regulation of axon collateral branches.

The generation of axon collateral branches is a fundamental aspect of the development of the nervous system and the response of axons to injury. Although much has been discovered about the signaling pathways and cytoskeletal dynamics underlying branching, additional aspects of the cell biology of axon branching have received less attention. This review summarizes recent advances in our understanding of key factors involved in axon branching. This article focuses on how cytoskeletal mechanisms, intracellular organelles, such as mitochondria and the endoplasmic reticulum, and membrane remodeling (exocytosis and endocytosis) contribute to branch initiation and formation. Together this growing literature provides valuable insight as well as a platform for continued investigation into how multiple aspects of axonal cell biology are spatially and temporally orchestrated to give rise to axon branches. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1293-1307, 2016.

Physical models have gender-specific effects on student understanding of protein structure-function relationships.

Understanding how basic structural units influence function is identified as a foundational/core concept for undergraduate biological and biochemical literacy. It is essential for students to understand this concept at all size scales, but it is often more difficult for students to understand structure-function relationships at the molecular level, which they cannot as effectively visualize. Students need to develop accurate, 3-dimensional mental models of biomolecules to understand how biomolecular structure affects cellular functions at the molecular level, yet most traditional curricular tools such as textbooks include only 2-dimensional representations. We used a controlled, backward design approach to investigate how hand-held physical molecular model use affected students' ability to logically predict structure-function relationships. Brief (one class period) physical model use increased quiz score for females, whereas there was no significant increase in score for males using physical models. Females also self-reported higher learning gains in their understanding of context-specific protein function. Gender differences in spatial visualization may explain the gender-specific benefits of physical model use observed. © 2016 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 44(4):326-335, 2016.

Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions.

UNLABELLED: Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. SIGNIFICANCE STATEMENT: Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and spatially linked to attenuation of RF at sites of forward membrane protrusion. Importantly, clutching of RF is modulated by extracellular matrix (ECM) proteins and soluble axon guidance cues, suggesting that it may regulate axon guidance in vivo. Consistent with this notion, we found that RF rates of spinal neuron growth cones were slower in vivo than what was observed in vitro. Together, our study provides the best evidence that growth cone-ECM adhesions clutch RF locally to guide axons in vivo.

BAR-SH3 sorting nexins are conserved interacting proteins of Nervous wreck that organize synapses and promote neurotransmission.

Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.

Signaling to the microtubule cytoskeleton: an unconventional role for CaMKII.

Synaptic plasticity is a hallmark of the nervous system and is thought to be integral to higher brain functions such as learning and memory. Calcium, acting as a second messenger, and the calcium/calmodulin dependent kinase CaMKII are key regulators of neuronal plasticity. Given the importance of the actin and microtubule (MT) cytoskeleton in dendritic spine morphology, composition and plasticity, it is not surprising that many regulators of these cytoskeletal elements are downstream of the CaMKII pathway. In this review, we discuss the emerging role of calcium and CaMKII in the regulation of MTs and cargo unloading during synaptic plasticity.

Neurite guidance and three-dimensional confinement via compliant semiconductor scaffolds.

Neurons are often cultured in vitro on a flat, open, and rigid substrate, a platform that does not reflect well the native microenvironment of the brain. To address this concern, we have developed a culturing platform containing arrays of microchannels, formed in a crystalline-silicon nanomembrane (NM) resting on polydimethylsiloxane; this platform will additionally enable active sensing and stimulation at the local scale, via devices fabricated in the silicon. The mechanical properties of the composite Si/compliant substrate nanomaterial approximate those of neural tissue. The microchannels, created in the NM by strain engineering, demonstrate strong guidance of neurite outgrowth. Using plasma techniques, we developed a means to coat just the inside surface of these channels with an adhesion promoter (poly-d-lysine). For NM channels with openings larger than the cross-sectional area of a single axon, strong physical confinement and guidance of axons through the channels are observed. Imaging of axons that grow in channels with openings that approximate the size of an axon suggests that a tight seal exists between the cell membrane and the inner surface of the channel, mimicking a myelin sheath. Such a tight seal of the cell membrane with the channel surface would make this platform an attractive candidate for future neuronal repair. Results of measurements of impedance and photoluminescence of bare NM channels are comparable to those on a flat NM, demonstrating electrical and optical modalities of our platform and suggesting that this scaffold can be expanded for active sensing and monitoring of neuron cellular processes in conditions in which they exist naturally.