Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery.

Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors.

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adaptor MADD, potentially preventing the formation of the RAB3A-MADD-KIF1A/1Bß complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.

Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery.

Neurons rely on long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, and how efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule severing enzyme spastin in locally enhancing microtubule polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components, termed 'protosynapses', which are distributed along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new microtubule growth and synaptic vesicle precursor (SVP) pausing/retention. Disruption of neuronal spastin, either by CRISPRi-mediated depletion or transient overexpression, interrupts the localized enrichment of dynamic microtubule plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of microtubule polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors.

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1Bß complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.

TUBA1A tubulinopathy mutants disrupt neuron morphogenesis and override XMAP215/Stu2 regulation of microtubule dynamics.

Heterozygous, missense mutations in α- or ß-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation's impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.

Proteins are molecules made up of long chains of building blocks called amino acids. When a mutation changes one of these amino acids, it can lead to the protein malfunctioning, which can have many effects at the cell and tissue level. Given that human proteins are made up of 20 different amino acids, each building block in a protein could mutate to any of the other 19 amino acids, and each mutations could have different effects. Tubulins are proteins that form microtubules, thin tubes that help give cells their shape and allow them to migrate. These proteins are added or removed to microtubules depending on the cell's needs, meaning that microtubules can grow or shrink depending on the situation. Mutations in the tubulin proteins have been linked to malformations of varying severities involving the formation of ridges and folds on the surface of the brain, including lissencephaly, pachygyria or polymicrogyria. Hoff et al. wanted to establish links between tubulin mutations and the effects observed at both cell and tissue level in the brain. They focused on two mutations in the tubulin protein TUBA1A that affect the amino acid in position 409 in the protein, which is normally a valine. One of the mutations turns this valine into an amino acid called isoleucine. This mutation is associated with pachygyria, which leads to the brain developing few ridges that are broad and flat. The second mutation turns the valine into an alanine, and is linked to lissencephaly, a more severe condition in which the brain develops no ridges, appearing smooth. Hoff et al. found that both mutations interfere with the development of the brain by stopping neurons from migrating properly, which prevents them from forming the folds in the brain correctly. At the cellular level, the mutations lead to tubulins becoming harder to remove from microtubules, making microtubules more stable than usual. This results in longer microtubules that are harder for the cell to shorten or destroy as needed. Additionally, Hoff et al. showed that the mutant versions of TUBA1A have weaker interactions with a protein called XMAP215, which controls the addition of tubulin to microtubules. This causes the microtubules to grow uncontrollably. Hoff et al. also established that the magnitude of the effects of each mutation on microtubule growth scale with the severity of the disorder they cause. Specifically, cells in which TUBA1A is not mutated have microtubules that grow at a normal rate, and lead to typical brain development. Meanwhile, cells carrying the mutation that turns a valine into an alanine, which is linked to the more severe condition lissencephaly, have microtubules that grow very fast. Finally, cells in which the valine is mutated to an isoleucine ­ the mutation associated with the less severe malformation pachygyria ­ have microtubules that grow at an intermediate rate. These findings provide a link between mutations in tubulin proteins and larger effects on cell movement that lead to brain malformations. Additionally, they also link the severity of the malformation to the severity of the microtubule defect caused by each mutation. Further work could examine whether microtubule stabilization is also seen in other similar diseases, which, in the long term, could reveal ways to detect and treat these illnesses.

Cytoskeletal regulation guides neuronal trafficking to effectively supply the synapse.

The development and proper function of the brain requires the formation of highly complex neuronal circuitry. These circuits are shaped from synaptic connections between neurons and must be maintained over a lifetime. The formation and continued maintenance of synapses requires accurate trafficking of presynaptic and postsynaptic components along the axon and dendrite, respectively, necessitating deliberate and specialized delivery strategies to replenish essential synaptic components. Maintenance of synaptic transmission also requires readily accessible energy stores, produced in part by localized mitochondria, that are tightly regulated with activity level. In this review, we focus on recent developments in our understanding of the cytoskeletal environment of axons and dendrites, examining how local regulation of cytoskeletal dynamics and organelle trafficking promotes synapse-specific delivery and plasticity. These new insights shed light on the complex and coordinated role that cytoskeletal elements play in establishing and maintaining neuronal circuitry.

Bridging the Gap: The Importance of TUBA1A α-Tubulin in Forming Midline Commissures.

Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/ß-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in mammalian cells have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which leads to α-tubulin antibody promiscuity and makes genetic manipulation challenging. Here, we test mutant tubulin levels and assembly activity and analyze the impact of TUBA1A reduction on growth cone composition, neurite extension, and commissural axon architecture during brain development. We present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1A N102D (TUBA1A ND ), reduces TUBA1A protein levels and prevents incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1a ND/+ mice leads to grossly normal brain formation except a significant impact on axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a as a result of the Tuba1a ND mutation exhibit slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting stabilization of microtubules. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration and cortex development but not commissure formation, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

Tubulin mutations in brain development disorders: Why haploinsufficiency does not explain TUBA1A tubulinopathies.

The neuronal cytoskeleton performs incredible feats during nervous system development. Extension of neuronal processes, migration, and synapse formation rely on the proper regulation of microtubules. Mutations that disrupt the primary α-tubulin expressed during brain development, TUBA1A, are associated with a spectrum of human brain malformations. One model posits that TUBA1A mutations lead to a reduction in tubulin subunits available for microtubule polymerization, which represents a haploinsufficiency mechanism. We propose an alternative model for the majority of tubulinopathy mutations, in which the mutant tubulin polymerizes into the microtubule lattice to dominantly "poison" microtubule function. Nine distinct α-tubulin and ten ß-tubulin genes have been identified in the human genome. These genes encode similar tubulin proteins, called isotypes. Multiple tubulin isotypes may partially compensate for heterozygous deletion of a tubulin gene, but may not overcome the disruption caused by missense mutations that dominantly alter microtubule function. Here, we describe disorders attributed to haploinsufficiency versus dominant negative mechanisms to demonstrate the hallmark features of each disorder. We summarize literature on mouse models that represent both knockout and point mutants in tubulin genes, with an emphasis on how these mutations might provide insight into the nature of tubulinopathy patient mutations. Finally, we present data from a panel of TUBA1A tubulinopathy mutations generated in yeast α-tubulin that demonstrate that α-tubulin mutants can incorporate into the microtubule network and support viability of yeast growth. This perspective on tubulinopathy mutations draws on previous studies and additional data to provide a fresh perspective on how TUBA1A mutations disrupt neurodevelopment.

TUBA1A mutations identified in lissencephaly patients dominantly disrupt neuronal migration and impair dynein activity.

The microtubule cytoskeleton supports diverse cellular morphogenesis and migration processes during brain development. Mutations in tubulin genes are associated with severe human brain malformations known as 'tubulinopathies'; however, it is not understood how molecular-level changes in microtubule subunits lead to brain malformations. In this study, we demonstrate that missense mutations affecting arginine at position 402 (R402) of TUBA1A α-tubulin selectively impair dynein motor activity and severely and dominantly disrupt cortical neuronal migration. TUBA1A is the most commonly affected tubulin gene in tubulinopathy patients, and mutations altering R402 account for 30% of all reported TUBA1A mutations. We show for the first time that ectopic expression of TUBA1A-R402C and TUBA1A-R402H patient alleles is sufficient to dominantly disrupt cortical neuronal migration in the developing mouse brain, strongly supporting a causal role in the pathology of brain malformation. To isolate the precise molecular impact of R402 mutations, we generated analogous R402C and R402H mutations in budding yeast α-tubulin, which exhibit a simplified microtubule cytoskeleton. We find that R402 mutant tubulins assemble into microtubules that support normal kinesin motor activity but fail to support the activity of dynein motors. Importantly, the level of dynein impairment scales with the expression level of the mutant in the cell, suggesting a 'poisoning' mechanism in which R402 mutant α-tubulin acts dominantly by populating microtubules with defective binding sites for dynein. Based on our results, we propose a new model for the molecular pathology of tubulinopathies that may also extend to other tubulin-related neuropathies.

The α-Tubulin gene TUBA1A in Brain Development: A Key Ingredient in the Neuronal Isotype Blend.

Microtubules are dynamic cytoskeletal polymers that mediate numerous, essential functions such as axon and dendrite growth and neuron migration throughout brain development. In recent years, sequencing has revealed dominant mutations that disrupt the tubulin protein building blocks of microtubules. These tubulin mutations lead to a spectrum of devastating brain malformations, complex neurological and physical phenotypes, and even fatality. The most common tubulin gene mutated is the α-tubulin gene TUBA1A, which is the most prevalent α-tubulin gene expressed in post-mitotic neurons. The normal role of TUBA1A during neuronal maturation, and how mutations alter its function to produce the phenotypes observed in patients, remains unclear. This review synthesizes current knowledge of TUBA1A function and expression during brain development, and the brain malformations caused by mutations in TUBA1A.

The negatively charged carboxy-terminal tail of ß-tubulin promotes proper chromosome segregation.

Despite the broadly conserved role of microtubules in chromosome segregation, we have a limited understanding of how molecular features of tubulin proteins contribute to the underlying mechanisms. Here we investigate the negatively charged carboxy-terminal tail domains (CTTs) of α- and ß-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating ß-CTT causes elevated rates of chromosome loss and cell cycle delay. Complementary live-cell imaging and electron tomography show that ß-CTT is necessary to properly position kinetochores and organize microtubules within the assembling spindle. We identify a minimal region of negatively charged amino acids that is necessary and sufficient for proper chromosome segregation and provide evidence that this function may be conserved across species. Our results provide the first in vivo evidence of a specific role for tubulin CTTs in chromosome segregation. We propose that ß-CTT promotes the ordered segregation of chromosomes by stabilizing the spindle and contributing to forces that move chromosomes toward the spindle poles.

Novel α-tubulin mutation disrupts neural development and tubulin proteostasis.

Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.

Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails.

BACKGROUND: Microtubules (MTs) support diverse transport and force generation processes in cells. Both α- and ß-tubulin proteins possess carboxy-terminal tail regions (CTTs) that are negatively charged, intrinsically disordered, and project from the MT surface where they interact with motors and other proteins. Although CTTs are presumed to play important roles in MT networks, these roles have not been determined in vivo. RESULTS: We examined the function of CTTs in vivo by using a systematic collection of mutants in budding yeast. We find that CTTs are not essential; however, loss of either α- or ß-CTT sensitizes cells to MT-destabilizing drugs. ß-CTT, but not α-CTT, regulates MT dynamics by increasing frequencies of catastrophe and rescue events. In addition, ß-CTT is critical for the assembly of the mitotic spindle and its elongation during anaphase. We use genome-wide genetic interaction screens to identify roles for α- and ß-CTTs, including a specific role for ß-CTT in supporting kinesin-5/Cin8. Our genetic screens also identified novel interactions with pathways not related to canonical MT functions. CONCLUSIONS: We conclude that α- and ß-CTTs play important and largely discrete roles in MT networks. ß-CTT promotes MT dynamics. ß-CTT also regulates force generation in the mitotic spindle by supporting kinesin-5/Cin8 and dampening dynein. Our genetic screens identify links between α- and ß-CTT and additional cellular pathways and suggest novel functions.