Kinesin-5, a mitotic microtubule-associated motor protein, modulates neuronal migration

Kinesin-5 (also called Eg5 or kif11) is a homotetrameric motor protein that functions by modulating microtubule (MT)-MT interactions. In the case of mitosis, kinesin-5 slows the rate of separation of the half-spindles. In the case of the axon, kinesin-5 limits the frequency of transport of short MTs, and also limits the rate of axonal growth. Here we show that experimental inhibition of kinesin-5 in cultured migratory neurons results in a faster but more randomly moving neuron with a shorter leading process. As is the case with axons of stationary neurons, short MT transport frequency is notably enhanced in the leading process of the migratory neuron when kinesin-5 is inhibited. Conversely, overexpression of kinesin-5, both in culture and in developing cerebral cortex, causes migration to slow and even cease. Regions of anti-parallel MT organization behind the centrosome were shown to be especially rich in kinesin-5, implicating these regions as potential sites where kinesin-5 forces may be especially relevant. We posit that kinesin-5 acts as a "brake" on MT-MT interactions that modulates the advance of the entire MT apparatus. In so doing, kinesin-5 regulates the rate and directionality of neuronal migration and possibly the cessation of migration when the neuron reaches its destination.     

It cuts two ways: microtubule loss during Alzheimer disease

EMBO J 32: 2920–2937 10.1038/emboj.2013.207; published online September242013Microtubule loss from axons and dendrites is a key contributor to nervous system degeneration during Alzheimer disease. Previous evidence suggested a simple pathway by which tau dissociation from microtubules in the axon allows excess severing of microtubules by katanin. Now, new evidence has emerged for a more complex pathway by which abnormal tau invasion into dendrites, triggered by Aβ oligomers, results in excess severing of microtubules by spastin.Alzheimer disease (AD) is a member of a category of neurodegenerative disorders called tauopathies (Wang and Liu, 2008). These are diseases of the nervous system in which tau becomes abnormally phosphorylated, and thereby detaches from microtubules. As the microtubules lose tau, they diminish in number and density, and this loss of microtubule mass negatively impacts the capacity of the neuron to maintain axonal transport and synaptic connections. Terms such as disintegrate or ‘fall apart'' are often used to describe the effect on the microtubules as they lose tau, but to date there has been very little information on how this happens. There is no mechanistic evidence to support the view that the microtubules become less stable and simply disassemble by their normal dynamic properties.One possibility is that tau normally protects microtubules from being destroyed by various proteins in the axon that would otherwise cut them into pieces or in some other way break them down. This presumably reflects a physiological mechanism wherein the regulation of tau dissociation from the microtubule via signalling pathways controls when and where microtubule breakage normally occurs. When a pathological condition causes tau to detach from microtubules, they become extremely sensitive to such factors. In addition, there is strong evidence that the abnormal tau, whether soluble or filamentous, can elicit toxic gain-of-function effects on the axon (Wang and Liu, 2008).To make matters even more complex, AD is not a pure tauopathy. Beta amyloid (Aβ) accumulates abnormally in the brain during AD, and this prompts tau to become hyperphosphorylated and lose association with microtubules. However, the Aβ can also elicit microtubule loss, independent of tau dissociation from the microtubules. In AD, there is also a loss of microtubules from dendrites, and this introduces an additional degree of complexity. Tau is normally less enriched in dendrites than axons. In AD, tau invades dendrites abnormally through deregulation of its normal sorting mechanism, and this somehow leads to microtubule loss from dendrites (Zempel et al, 2010).Microtubule loss is a common end point of multiple pathways, some involving loss of tau function, others involving gain-of-function effects of abnormal tau, and still others working through tau-dependent Aβ toxicity. All of this is not to say that the effects on microtubules are the only reason or even the principal explanation for axonal degeneration in AD, but the loss of microtubules is an important contributor to nervous system degeneration. Preventing or reversing the effects on microtubules could help stave off degeneration and hence provide patients with additional years of cognitive health and better quality of life.Microtubule assembly and disassembly occur from the ends of a microtubule, mainly (and often exclusively) at the plus end of the microtubule in living cells. Proteins that regulate microtubule stability affect the rate of these dynamics at microtubule ends. In recent years, a great deal of attention has focused on a category of proteins, termed microtubule-severing proteins. These proteins are enzymes that yank at the microtubule anywhere along its length to pull out a tubulin subunit, and thereby ‘cut'' the microtubule by causing it to break into pieces (Roll-Mecak and Vale, 2008). If the microtubule is sufficiently stable in the region of the break, the parent microtubule is cut into two shorter microtubules that persist, with minimal disassembly of either of the two pieces. If a microtubule is severed in its more labile region, the breakage could cause a great deal of disassembly. If the tubulin being yanked is situated at one of the ends of a microtubule, the result would be a shortening of the microtubule from that end; that is, disassembly. Thus, microtubule severing in the axon can certainly lead to microtubule loss, either by cutting the polymer all the way to subunits, inducing disassembly directly from microtubule ends or promoting disassembly as a secondary effect to the cutting.To date, the AAA enzymes katanin and spastin are the best studied of the microtubule-severing proteins (Yu et al, 2008). Spastin was originally identified as the product of the gene whose mutations are the chief cause of hereditary spastic paraplegia. Curiously, neurons express levels of spastin and katanin that are theoretically high enough to completely sever all of the microtubules in the neuron to subunits (Solowska et al, 2008), and yet this does not happen. Various regulatory mechanisms presumably keep the activities of the severing proteins in check. One of these mechanisms, in the case of katanin, is microtubule-bound tau, which protects the microtubule from being accessed by katanin (Qiang et al, 2006).Could microtubule loss in AD be due, at least in part, to excess microtubule severing due to deregulation of microtubule-severing proteins? We have posited that heightened severing of the microtubules by katanin, as the microtubules lose association with tau, is a contributing factor to the degradation of microtubules in the axons of AD patients (Sudo and Baas, 2011). A role for spastin in this pathway is questionable, because tau does not appear to protect microtubules against spastin as effectively as it does against katanin (Qiang et al, 2006). However, we now know that spastin is far from irrelevant to AD, as an exciting new article from the Mandelkow and Dawson laboratories implicates spastin in an entirely different pathway for microtubule loss in AD (Zempel et al, 2013). Whereas the katanin pathway is more relevant to axons, this new spastin pathway is more relevant to dendrites.In this new work, Zempel et al (2013) exposed mature primary neurons to oligomers of Aβ and observed microtubule breakdown in dendrites that had been invaded by tau. They found that the missorting of tau leads to an elevation of TTLL6 (Tubulin-Tyrosine-Ligase-Like-6) in dendrites, and this results in a marked increase in the polyglutamylation status of the microtubules. Because spastin has a strong preference for polyglutamylated microtubules, the microtubules become more sensitive to spastin-induced severing. Exactly why katanin is not a factor remains unclear, as polyglutamylation renders microtubules more sensitive to both of the severing proteins, not just spastin (Lacroix et al, 2010). Perhaps some of the tau that invades the dendrite is able to bind to microtubules and protect them from katanin, or perhaps katanin is less potent in dendrites because their microtubules are poorly acetylated, as katanin prefers acetylated microtubules to unacetylated ones (Sudo and Baas, 2011). Whatever the case, these new studies suggest that spastin, a protein whose mutations cause an entirely different neurodegenerative disease, is also a major factor in AD.What are the implications of these findings for AD treatment? In recent years, there have been encouraging results on rodent models for AD, in which behavioural improvement and enhanced neuronal vitality were observed when the animals were treated with drugs that stabilize microtubules against disassembly (Zhang et al, 2012). Such drugs are currently in clinical trials for AD (Barten et al, 2012). This strategy is based on the presumption that the microtubule loss that occurs in AD is due to destabilization of the microtubules. However, the results discussed here suggest that the primary cause of the microtubule loss could be something quite different, namely excess severing of microtubules. In this regard, it is relevant that both katanin and spastin seem to have a preference for severing stable microtubules (Lacroix et al, 2010; Sudo and Baas, 2011). Therefore, while treatment with a microtubule-stabilizing drug would mitigate disassembly that occurs as an aftereffect of microtubule severing, the severing events themselves would likely be increased (Figure 1). Heightened microtubule severing in axons and dendrites, even if the total levels of microtubule mass are preserved, could result in a gradual shift from a normal distribution of long and short microtubules to a predominance of microtubules too short to support sustained excursions of organelle transport. Long microtubules are also necessary as compression-bearing struts that prevent axons and dendrites from collapsing on themselves. We suspect that appropriate treatment regimes can be devised to prevent such dire consequences from happening, but we would advocate for the development of new drugs that inhibit microtubule-severing proteins. Such drugs may prove to be a better approach (on their own or in combination with a stabilizing drug) for preserving the fidelity of axonal and dendritic microtubules in AD patients. Given that the structure of the severing proteins is known, it may be straightforward to develop inhibitors, especially to their ATPase domains.Open in a separate windowFigure 1Microtubules in axons and dendrites consist of a stable region towards the minus end of the microtubule and a labile region towards the plus end, as well as a pool of free tubulin subunits. Microtubule severing is a normal event in the neuron, when tightly regulated. Abnormal (deregulated) microtubule severing is posited to account for microtubule loss in AD. Severing in the stable region of the microtubule would create two new microtubules, with fairly minimal disassembly of either one. Severing in the labile region of the microtubule would result in notably more disassembly. Severing at the end of the microtubule would result in disassembly. Because known microtubule-severing proteins favour the stable region of the microtubule, treatment of AD with a microtubule-stabilizing drug may mitigate disassembly that occurs as an aftereffect of the severing, but the severing events themselves would likely increase.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

Basic Fibroblast Growth Factor Elicits Formation of Interstitial Axonal Branches via Enhanced Severing of Microtubules

The formation of interstitial axonal branches involves the severing of microtubules at sites where new branches form. Here we wished to ascertain whether basic fibroblast growth factor (bFGF) enhances axonal branching through alterations in proteins involved in the severing of microtubules. We found that treatment of cultured hippocampal neurons with bFGF heightens expression of both katanin and spastin, which are proteins that sever microtubules in the axon. In addition, treatment with bFGF enhances phosphorylation of tau at sites expected to cause it to dissociate from microtubules. This is important because tau regulates the access of katanin to the microtubule. In live-cell imaging experiments, axons of neurons treated with bFGF displayed greater numbers of dynamic free ends of microtubules, as well as greater numbers of short mobile microtubules. Entirely similar enhancement of axonal branching, short microtubule transport, and frequency of microtubule ends was observed when spastin was overexpressed in the neurons. Depletion of either katanin or spastin with siRNA diminished but did not eliminate the enhancement in branching elicited by bFGF. Collectively, these results indicate that bFGF enhances axonal branch formation by augmenting the severing of microtubules through both a spastin-based mode and a katanin-based mode.     

Motor proteins regulate force interactions between microtubules and microfilaments in the axon

It has long been known that microtubule depletion causes axons to retract in a microfilament-dependent manner, although it was not known whether these effects are the result of motor-generated forces on these cytoskeletal elements. Here we show that inhibition of the motor activity of cytoplasmic dynein causes the axon to retract in the presence of microtubules. This response is obliterated if microfilaments are depleted or if myosin motors are inhibited. We conclude that axonal retraction results from myosin-mediated forces on the microfilament array, and that these forces are counterbalanced or attenuated by dynein-mediated forces between the microfilament and microtubule arrays.     

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Chromothripsis in Healthy Individuals Affects Multiple Protein-Coding Genes and Can Result in Severe Congenital Abnormalities in Offspring

Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a mother to her child. The chromothripsis in the mothers resulted in completely balanced rearrangements involving 8–23 breakpoint junctions across three to five chromosomes. Two mothers did not show any phenotypic abnormalities, although 3–13 protein-coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy-number changes in two children. This resulted in gene-dosage changes, which are probably responsible for the severe congenital phenotypes of these two children. In contrast, the third child, who has a severe congenital disease, harbored all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy-number changes. These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein-coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy individuals affects reproduction and is expected to substantially increase the risk of miscarriages, abortions, and severe congenital disease.