Antagonistic Roles of Tau and MAP6 in Regulating Neuronal Development.

Association of tau with microtubules causes them to be labile while association of MAP6 with microtubules causes them to be stable. As axons differentiate and grow, tau and MAP6 segregate from one another on individual microtubules, resulting in the formation of stable and labile domains. The functional significance of the yin/yang relationship between tau and MAP6 remains speculative, with one idea being that such a relationship assists in balancing morphological stability with plasticity. Here, using primary rodent neuronal cultures, we show that tau depletion has opposite effects compared to MAP6 depletion on the rate of neuronal development, the efficiency of growth cone turning, and the number of neuronal processes and axonal branches. Opposite effects to those of tau depletion were also observed on the rate of neuronal migration, in an in vivo assay, when MAP6 was depleted. When tau and MAP6 were together depleted from neuronal cultures, the morphological phenotypes negated one another. Although tau and MAP6 are multifunctional proteins, our results suggest that the observed effects on neuronal development are likely due to their opposite roles in regulating microtubule stability.

Modeling gain-of-function and loss-of-function components of SPAST-based hereditary spastic paraplegia using transgenic mice.

Hereditary spastic paraplegia (HSP) is a disease in which dieback degeneration of corticospinal tracts, accompanied by axonal swellings, leads to gait deficiencies. SPG4-HSP, the most common form of the disease, results from mutations of human spastin gene (SPAST), which is the gene that encodes spastin, a microtubule-severing protein. The lack of a vertebrate model that recapitulates both the etiology and symptoms of SPG4-HSP has stymied the development of effective therapies for the disease. hSPAST-C448Y mice, which express human mutant spastin at the ROSA26 locus, display corticospinal dieback and gait deficiencies but not axonal swellings. On the other hand, mouse spastin gene (Spast)-knockout (KO) mice display axonal swellings but not corticospinal dieback or gait deficiencies. One possibility is that reduced spastin function, resulting in axonal swellings, is not the cause of the disease but exacerbates the toxic effects of the mutant protein. To explore this idea, Spast-KO and hSPAST-C448Y mice were crossbred, and the offspring were compared with the parental lines via histological and behavioral analyses. The crossbred animals displayed axonal swellings as well as earlier onset, worsened gait deficiencies and corticospinal dieback compared with the hSPAST-C448Y mouse. These results, together with observations on changes in histone deacetylases 6 and tubulin modifications in the axon, indicate that each of these three transgenic mouse lines is valuable for investigating a different component of the disease pathology. Moreover, the crossbred mice are the best vertebrate model to date for testing potential therapies for SPG4-HSP.

KIFC1 Regulates the Trajectory of Neuronal Migration.

During neuronal migration, forces generated by cytoplasmic dynein yank on microtubules extending from the centrosome into the leading process and move the nucleus along microtubules that extend behind the centrosome. Scaffolds, such as radial glia, guide neuronal migration outward from the ventricles, but little is known about the internal machinery that ensures that the soma migrates along its proper path rather than moving backward or off the path. Here we report that depletion of KIFC1, a minus-end-directed kinesin called HSET in humans, causes neurons to migrate off their appropriate path, suggesting that this molecular motor is what ensures fidelity of the trajectory of migration. For these studies, we used rat migratory neurons in vitro and developing mouse brain in vivo, together with RNA interference and ectopic expression of mutant forms of KIFC1. We found that crosslinking of microtubules into a nonsliding mode by KIFC1 is necessary for dynein-driven forces to achieve sufficient traction to thrust the soma forward. Asymmetric bouts of microtubule sliding driven by KIFC1 thereby enable the soma to tilt in one direction or another, thus providing midcourse corrections that keep the neuron on its appropriate trajectory. KIFC1-driven sliding of microtubules further assists neurons in remaining on their appropriate path by allowing the nucleus to rotate directionally as it moves, which is consistent with how we found that KIFC1 contributes to interkinetic nuclear migration at an earlier stage of neuronal development.SIGNIFICANCE STATEMENT Resolving the mechanisms of neuronal migration is medically important because many developmental disorders of the brain involve flaws in neuronal migration and because deployment of newly born neurons may be important in the adult for cognition and memory. Drugs that inhibit KIFC1 are candidates for chemotherapy and therefore should be used with caution if they are allowed to enter the brain.

Trends in Distal Esophageal and Gastroesophageal Junction Cancer Care: The Dutch Nationwide Ivory Study.

OBJECTIVE: This study evaluated the nationwide trends in care and accompanied postoperative outcomes for patients with distal esophageal and gastro-esophageal junction cancer. SUMMARY OF BACKGROUND DATA: The introduction of transthoracic esophagectomy, minimally invasive surgery, and neo-adjuvant chemo(radio)therapy changed care for patients with esophageal cancer. METHODS: Patients after elective transthoracic and transhiatal esophagectomy for distal esophageal or gastroesophageal junction carcinoma in the Netherlands between 2007-2016 were included. The primary aim was to evaluate trends in both care and postoperative outcomes for the included patients. Additionally, postoperative outcomes after transthoracic and tran-shiatal esophagectomy were compared, stratified by time periods. RESULTS: Among 4712 patients included, 74% had distal esophageal tumors and 87% had adenocarcinomas. Between 2007 and 2016, the proportion of transthoracic esophagectomy increased from 41% to 81%, and neo-adjuvant treatment and minimally invasive esophagectomy increased from 31% to 96%, and from 7% to 80%, respectively. Over this 10-year period, postoperative outcomes improved: postoperative morbidity decreased from 66.6% to 61.8% ( P = 0.001), R0 resection rate increased from 90.0% to 96.5% (P <0.001), median lymph node harvest increased from 15 to 19 ( P <0.001), and median survival increased from 35 to 41 months ( P = 0.027). CONCLUSION: In this nationwide cohort, a transition towards more neo-adju-vant treatment, transthoracic esophagectomy and minimally invasive surgery was observed over a 10-year period, accompanied by decreased postoperative morbidity, improved surgical radicality and lymph node harvest, and improved survival.

Recurrent Disease After Esophageal Cancer Surgery: A Substudy of The Dutch Nationwide Ivory Study.

OBJECTIVE: This study investigated the patterns, predictors, and survival of recurrent disease following esophageal cancer surgery. BACKGROUND: Survival of recurrent esophageal cancer is usually poor, with limited prospects of remission. METHODS: This nationwide cohort study included patients with distal esophageal and gastroesophageal junction adenocarcinoma and squamous cell carcinoma after curatively intended esophagectomy in 2007 to 2016 (follow-up until January 2020). Patients with distant metastases detected during surgery were excluded. Univariable and multivariable logistic regression were used to identify predictors of recurrent disease. Multivariable Cox regression was used to determine the association of recurrence site and treatment intent with postrecurrence survival. RESULTS: Among 4626 patients, 45.1% developed recurrent disease a median of 11 months postoperative, of whom most had solely distant metastases (59.8%). Disease recurrences were most frequently hepatic (26.2%) or pulmonary (25.1%). Factors significantly associated with disease recurrence included young age (≤65 y), male sex, adenocarcinoma, open surgery, transthoracic esophagectomy, nonradical resection, higher T-stage, and tumor positive lymph nodes. Overall, median postrecurrence survival was 4 months [95% confidence interval (95% CI): 3.6-4.4]. After curatively intended recurrence treatment, median survival was 20 months (95% CI: 16.4-23.7). Survival was more favorable after locoregional compared with distant recurrence (hazard ratio: 0.74, 95% CI: 0.65-0.84). CONCLUSIONS: This study provides important prognostic information assisting in the surveillance and counseling of patients after curatively intended esophageal cancer surgery. Nearly half the patients developed recurrent disease, with limited prospects of survival. The risk of recurrence was higher in patients with a higher tumor stage, nonradical resection and positive lymph node harvest.

A cellular approach to understanding and treating Gulf War Illness.

Gulf War Illness (GWI), a disorder suffered by approximately 200,000 veterans of the first Gulf War, was caused by exposure to low-level organophosphate pesticides and nerve agents in combination with battlefield stress. To elucidate the mechanistic basis of the brain-related symptoms of GWI, human-induced pluripotent stem cells (hiPSCs) derived from veterans with or without GWI were differentiated into forebrain glutamatergic neurons and then exposed to a Gulf War (GW) relevant toxicant regimen consisting of a sarin analog and cortisol, a human stress hormone. Elevated levels of total and phosphorylated tau, reduced microtubule acetylation, altered mitochondrial dynamics/transport, and decreased neuronal activity were observed in neurons exposed to the toxicant regimen. Some of the data are consistent with the possibility that some veterans may have been predisposed to acquire GWI. Wistar rats exposed to a similar toxicant regimen showed a mild learning and memory deficit, as well as cell loss and tau pathology selectively in the CA3 region of the hippocampus. These cellular responses offer a mechanistic explanation for the memory loss suffered by veterans with GWI and provide a cell-based model for screening drugs and developing personalized therapies for these veterans.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish.

KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.

Hereditary spastic paraplegia: gain-of-function mechanisms revealed by new transgenic mouse.

Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.

Mitotic Motor KIFC1 Is an Organizer of Microtubules in the Axon.

KIFC1 (also called HSET or kinesin-14a) is best known as a multifunctional motor protein essential for mitosis. The present studies are the first to explore KIFC1 in terminally postmitotic neurons. Using RNA interference to partially deplete KIFC1 from rat neurons (from animals of either gender) in culture, pharmacologic agents that inhibit KIFC1, and expression of mutant KIFC1 constructs, we demonstrate critical roles for KIFC1 in regulating axonal growth and retraction as well as growth cone morphology. Experimental manipulations of KIFC1 elicit morphological changes in the axon as well as changes in the organization, distribution, and polarity orientation of its microtubules. Together, the results indicate a mechanism by which KIFC1 binds to microtubules in the axon and slides them into alignment in an ATP-dependent fashion and then cross-links them in an ATP-independent fashion to oppose their subsequent sliding by other motors.SIGNIFICANCE STATEMENT Here, we establish that KIFC1, a molecular motor well characterized in mitosis, is robustly expressed in neurons, where it has profound influence on the organization of microtubules in a number of different functional contexts. KIFC1 may help answer long-standing questions in cellular neuroscience such as, mechanistically, how growth cones stall and how axonal microtubules resist forces that would otherwise cause the axon to retract. Knowledge about KIFC1 may help researchers to devise strategies for treating disorders of the nervous system involving axonal retraction given that KIFC1 is expressed in adult neurons as well as developing neurons.

Knockdown of Fidgetin Improves Regeneration of Injured Axons by a Microtubule-Based Mechanism.

Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whether fidgetin knockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA-fidgetin on a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves. Fidgetin knockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regeneration in vivo after a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in which fidgetin was knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future.SIGNIFICANCE STATEMENT Here we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock down fidgetin from rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs, fidgetin knockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.