Michelle C. Stone

Microtubules Have Opposite Orientation in Axons and Dendrites of Drosophila Neurons

In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus- and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, approximately 90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites.

Global Up-Regulation of Microtubule Dynamics and Polarity Reversal during Regeneration of an Axon from a Dendrite

We look inside neurons in vivo and identify major cytoskeletal rearrangements that allow a dendrite to become a regenerating axon.

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites.

BACKGROUND in many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, we have used this system to understand how cells can maintain dynamic arrays of polarized microtubules. RESULTS we find that growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, we show that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. CONCLUSIONS we conclude that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. We propose that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules.

Microtubules are organized independently of the centrosome in Drosophila neurons

BackgroundThe best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use Drosophila as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons.ResultsIn developing and mature neurons, centrioles were not surrounded by the core nucleation protein γ-tubulin. This suggests that the centrioles do not organize functional centrosomes in Drosophila neurons in vivo. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used DSas-4 centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment.ConclusionWe conclude that the centrosome is not a major site of microtubule nucleation in Drosophila neurons, and is not required for maintenance of neuronal microtubule organization in these cells.

Axon injury and stress trigger a microtubule-based neuroprotective pathway

Axon injury elicits profound cellular changes, including axon regeneration. However, the full range of neuronal injury responses remains to be elucidated. Surprisingly, after axons of Drosophila dendritic arborization neurons were severed, dendrites were more resistant to injury-induced degeneration. Concomitant with stabilization, microtubule dynamics in dendrites increased. Moreover, dendrite stabilization was suppressed when microtubule dynamics was dampened, which was achieved by lowering levels of the microtubule nucleation protein γ-tubulin. Increased microtubule dynamics and global neuronal stabilization were also activated by expression of expanded polyglutamine (poly-Q) proteins SCA1, SCA3, and huntingtin. In all cases, dynamics were increased through microtubule nucleation and depended on JNK signaling, indicating that acute axon injury and long-term neuronal stress activate a common cytoskeleton-based stabilization program. Reducing levels of γ-tubulin exacerbated long-term degeneration induced by SCA3 in branched sensory neurons and in a well established Drosophila eye model of poly-Q–induced neurodegeneration. Thus, increased microtubule dynamics can delay short-term injury-induced degeneration, and, in the case of poly-Q proteins, can counteract progressive longer-term degeneration. We conclude that axon injury or stress triggers a microtubule-based neuroprotective pathway that stabilizes neurons against degeneration.

Normal Spastin Gene Dosage Is Specifically Required for Axon Regeneration

Axon regeneration allows neurons to repair circuits after trauma; however, most of the molecular players in this process remain to be identified. Given that microtubule rearrangements have been observed in injured neurons, we tested whether microtubule-severing proteins might play a role in axon regeneration. We found that axon regeneration is extremely sensitive to levels of the microtubule-severing protein spastin. Although microtubule behavior in uninjured neurons was not perturbed in animals heterozygous for a spastin null allele, axon regeneration was severely disrupted in this background. Two types of axon regeneration-regeneration of an axon from a dendrite after proximal axotomy and regeneration of an axon from the stump after distal axotomy-were defective in Drosophila with one mutant copy of the spastin gene. Other types of axon and dendrite outgrowth, including regrowth of dendrites after pruning, were normal in heterozygotes. We conclude that regenerative axon growth is uniquely sensitive to spastin gene dosage.

Dendrite Injury Triggers DLK-Independent Regeneration

Axon injury triggers regeneration through activation of a conserved kinase cascade, which includes the dual leucine zipper kinase (DLK). Although dendrites are damaged during stroke, traumatic brain injury, and seizure, it is not known whether mature neurons monitor dendrite injury and initiate regeneration. We probed the response to dendrite damage using model Drosophila neurons. Two larval neuron types regrew dendrites in distinct ways after all dendrites were removed. Dendrite regeneration was also triggered by injury in adults. Next, we tested whether dendrite injury was initiated with the same machinery as axon injury. Surprisingly, DLK, JNK, and fos were dispensable for dendrite regeneration. Moreover, this MAP kinase pathway was not activated by injury to dendrites. Thus, neurons respond to dendrite damage and initiate regeneration without using the conserved DLK cascade that triggers axon regeneration.

Mitochondria and Caspases Tune Nmnat-Mediated Stabilization to Promote Axon Regeneration

Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, we investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury we found mitochondrial fission in dendrites was upregulated, and that reducing fission increased stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. We also identified caspases as negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, we found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, required Nmnat. Indeed Nmnat overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos were also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, we tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduced regeneration. In addition, overexpression of fos or JNK extended the timecourse of NP and dampened regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.

Spastin, atlastin, and ER relocalization are involved in axon but not dendrite regeneration.

A Drosophila model system is used to show that the hereditary spastic paraplegia proteins spastin and atlastin help axons but not dendrites regenerate. The endoplasmic reticulum concentrates at tips of regenerating axons but not dendrites, and this depends on spastin and atlastin.

Prevention of age-related spontaneous mammary tumors in outbred rats by late ovariectomy.

BACKGROUND Breast cancer prevention trials have shown that the antiestrogen tamoxifen inhibits development of estrogen receptor (ER)-positive tumors. In Sprague-Dawley rats, removal of ovarian function in young animals can reduce the incidence of spontaneous age-dependent mammary tumors. However, it is not known whether removal of ovaries late in life, before middle age onset, can still prevent mammary tumor development. METHODS In this study we used Hsd:Sprague-Dawley SD (Hsd) rats to determine the effect of late ovariectomy on mammary tumor development. Intact, sham-ovariectomized and ovariectomized rats were followed until 110 weeks of age, or over their life span. In some experiments, palpable tumors were surgically removed upon presentation. RESULTS Removal of ovaries before middle age onset ( approximately 5-7 months) inhibited development of spontaneous mammary tumors by 95%. Only one mammary tumor was observed in 19 late ovariectomized animals while 47 total tumors developed in 42 non-ovariectomized animals. Tumor incidence was reduced from 73.8 to 5.3% (relative risk=0.05, 95% CI=0.0072-0.354). The frequency of mammary carcinomas in non-ovariectomized virgin female rats was one in eight rats. Spontaneous rat carcinomas expressed ER and other biomarkers, such as cyclin D1. When palpable tumors were removed by surgical excision, tumor multiplicity increased from 0.76 to 1.61 tumors per rat. Surprisingly, ovariectomy increased the 110-week survival rate and maximum life span of Hsd rats. CONCLUSION Late ovariectomy prevents spontaneous mammary tumor development in Hsd rats. This animal model may be useful for evaluating novel interventions in breast cancer prevention.