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.

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.

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.

Testosterone may increase selective attention to threat in young male macaques.

Animal studies indicate that sex hormones have widespread effects on the brain, cognition and emotion, but findings in humans are inconsistent. Well-controlled studies in nonhuman primates are crucial to resolve these discrepancies. In this study, we examined the effects of testosterone (T) on emotion in male rhesus monkeys. Six young adult males were tested on two emotional tasks during three hormonal conditions in a crossover design: when intact at baseline and when pharmacologically hypogonadal with add-back of T or placebo. The emotional tasks were the Approach-Avoidance task, which tested behavioral responses to three categories of objects (familiar, novel, and negative) and a Social Playback task which tested behavioral responses to scenes of unfamiliar conspecifics engaged in three types of social activities (neutral, positive, or negative). Following a 4-week baseline period, monkeys were treated with Depot Lupron, 200µg/kg before being randomly assigned to one of two treatment groups: Depot Lupron+Testosterone Enanthate (TE, 20mg/kg) or Depot Lupron+oil vehicle. In each treatment group, monkeys received one injection of Lupron and one injection of TE or one injection of Lupron and one injection of oil at the onset of a 4-week testing period, before crossing over to the alternate treatment for an additional 4weeks of testing. TE treatment had no effect on behavioral measures in the Approach-Avoidance task. For the Social Playback task, however, TE significantly increased watching time of video clips which depicted fights between unfamiliar conspecifics. The enhancing effect of T on watching time for negative social scenes is consistent with human data suggesting that T decreases aversion or facilitates approach to threatening social stimuli. Further studies are needed to understand the mechanisms by which T may mediate responsiveness to social threat in male primates.