Huntingtin coordinates dendritic spine morphology and function through cofilin-mediated control of the actin cytoskeleton.

Compelling evidence indicates that in Huntington's disease (HD), mutation of huntingtin (HTT) alters several aspects of early brain development such as synaptogenesis. It is not clear to what extent the partial loss of wild-type HTT function contributes to these abnormalities. Here we investigate the function of HTT in the formation of spines. Although larger spines normally correlate with more synaptic activity, cell-autonomous depletion of HTT leads to enlarged spines but reduced excitatory synaptic function. We find that HTT is required for the proper turnover of endogenous actin and to recruit AMPA receptors at active synapses; loss of HTT leads to LIM kinase (LIMK) hyperactivation, which maintains cofilin in its inactive state. HTT therefore influences actin dynamics through the LIMK-cofilin pathway. Loss of HTT uncouples spine structure from synaptic function, which may contribute to the ultimate development of HD symptoms.

Huntington's disease and brain development.

Huntington's disease is a rare inherited neurological disorder that generally manifests in mild-adulthood. The disease is characterized by the dysfunction and the degeneration of specific brain structures leading progressively to psychiatric, cognitive and motor disorders. The disease is caused by a mutation in the gene coding for huntingtin and, although it appears in adulthood, embryos carry the mutated gene from their development in utero. Studies based on mouse models and human stem cells have reported altered developmental mechanisms in disease conditions. However, does the mutation affect development in humans? Focusing on the early stages of brain development in human fetuses carrying the HD mutation, we have identified abnormalities in the development of the neocortex, the structure that ensure higher cerebral functions. Altogether, these studies suggests that developmental defects could contribute to the onset symptoms in adults, changing the perspective on disease and thus the health care of patients.

La maladie de Huntington est une maladie neurologique, rare et héréditaire, se manifestant généralement à l'âge adulte. Cette pathologie, caractérisée par la dysfonction et la dégénérescence de certaines structures cérébrales, conduit à des troubles psychiatriques, cognitifs et moteurs s'aggravant progressivement. La maladie est due à la mutation du gène codant pour la huntingtine et, bien qu'elle apparaisse à l'âge adulte, les embryons dès leur développement sont porteurs du gène muté. Les études, basées sur l'utilisation de modèles murins et de cellules souches humaines, montrent des mécanismes développementaux altérés en condition pathologique. Cependant, la mutation affecte-t-elle le développement chez l'Homme ? En nous intéressant aux stades précoces du développement cérébral de fœtus humains porteurs du gène muté, nous avons mis en évidence des anomalies du développement du néocortex, siège des grandes fonctions cérébrales. L'ensemble de ces travaux suggère que des défauts développementaux pourraient contribuer à l'apparition des symptômes adultes, changeant ainsi la vision de la maladie et de sa prise en charge.

Huntington's disease alters human neurodevelopment.

Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.

Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology.

In the developing cortex, projection neurons undergo multipolar-bipolar transition, radial-directed migration, and maturation. The contribution of these developmental steps to the structure of the adult cortex is not completely understood. Here, we report that huntingtin (HTT), the protein mutated in Huntington's disease, is enriched in polarizing projection neurons. The depletion of HTT in postmitotic projection neurons leads to the mislocalization of layer-specific neuronal populations in the mouse neocortex. HTT is required for the multipolar-bipolar transition of projection neurons and for the maintenance of their bipolar shape during their radial migration. HTT mediates these effects in vivo through the regulation of RAB11-dependent N-Cadherin trafficking. Importantly, HD pathological HTT alters RAB11-dependent neuronal migration. Finally, we show that the cortical defects resulting from the postmitotic loss of HTT specifically during embryonic development affect neuronal morphology at adulthood. Our data reveal a new HTT-RAB11-N-Cadherin pathway regulating multipolar-bipolar transition with direct implications for mature brain. VIDEO ABSTRACT.

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

The GSK3-MAP1B pathway controls neurite branching and microtubule dynamics.

The microtubule-associated protein MAP1B plays a key role in axon regeneration. We investigated the role of GSK3-mediated MAP1B phosphorylation in local fine-tuning of neurite branching and the underlying microtubule (MT) dynamics. In wildtype adult dorsal root ganglia (DRG) neurons, MAP1B phosphorylation is locally reduced at branching points, and branching dynamics from growth cones and distal neurite shafts is increased upon GSK3 inhibition. While map1b-/- neurites, that display increased branching, are not affected by GSK3 inhibition, transfection of map1b-/- neurons with full-length map1b-cDNA restores the wildtype branching phenotype, demonstrating that MAP1B is a key effector downstream of GSK3. Experiments in mutant mice lacking tyrosinated MTs indicate a preferential association of phospho-MAP1B with tyrosinated MTs. Interestingly, inhibition of GSK3-mediated MAP1B phosphorylation in map1b-cDNA-transfected fibroblasts protects both tyrosinated and acetylated MTs from nocodazole-induced depolymerization, while detyrosinated MTs are less abundant in the presence of MAP1B. Our data thus provide new insight into the molecular link between GSK3, MAP1B, neurite branching and MT stability regulation. We suggest that, at branching points, MAP1B undergoes a fine regulation of both its phosphorylation and sub-cellular amounts, in order to modulate the local balance between acetylated, detyrosinated, and tyrosinated microtubule pools.

Mutant huntingtin affects cortical progenitor cell division and development of the mouse neocortex.

A polyglutamine expansion in huntingtin (HTT) causes the specific death of adult neurons in Huntington's disease (HD). Most studies have thus focused on mutant HTT (mHTT) toxicity in adulthood, and its developmental effects have been largely overlooked. We found that mHTT caused mitotic spindle misorientation in cultured cells by altering the localization of dynein, NuMA, and the p150(Glued) subunit of dynactin to the spindle pole and cell cortex and of CLIP170 and p150(Glued) to microtubule plus-ends. mHTT also affected spindle orientation in dividing mouse cortical progenitors, altering the thickness of the developing cortex. The serine/threonine kinase Akt, which regulates HTT function, rescued the spindle misorientation caused by the mHTT, by serine 421 (S421) phosphorylation, in cultured cells and in mice. Thus, cortical development is affected in HD, and this early defect can be rescued by HTT phosphorylation at S421.

Shh signaling protects Atoh1 from degradation mediated by the E3 ubiquitin ligase Huwe1 in neural precursors.

Signaling networks controlled by Sonic hedgehog (SHH) and the transcription factor Atoh1 regulate the proliferation and differentiation of cerebellar granule neuron progenitors (GNPs). Deregulations in those developmental processes lead to medulloblastoma formation, the most common malignant brain tumor in childhood. Although the protein Atoh1 is a key factor during both cerebellar development and medulloblastoma formation, up-to-date detailed mechanisms underlying its function and regulation have remained poorly understood. Here, we report that SHH regulates Atoh1 stability by preventing its phosphodependent degradation by the E3 ubiquitin ligase Huwe1. Our results reveal that SHH and Atoh1 contribute to a positive autoregulatory loop promoting neuronal precursor expansion. Consequently, Huwe1 loss in mouse SHH medulloblastoma illustrates the disruption of this developmental mechanism in cancer. Hence, the crosstalk between SHH signaling and Atoh1 during cerebellar development highlights a collaborative network that could be further targeted in medulloblastoma.

Distinct roles of c-Jun N-terminal kinase isoforms in neurite initiation and elongation during axonal regeneration.

c-Jun N-terminal kinases (JNKs) (comprising JNK1-3 isoforms) are members of the MAPK (mitogen-activated protein kinase) family, activated in response to various stimuli including growth factors and inflammatory cytokines. Their activation is facilitated by scaffold proteins, notably JNK-interacting protein-1 (JIP1). Originally considered to be mediators of neuronal degeneration in response to stress and injury, recent studies support a role of JNKs in early stages of neurite outgrowth, including adult axonal regeneration. However, the function of individual JNK isoforms, and their potential effector molecules, remained unknown. Here, we analyzed the role of JNK signaling during axonal regeneration from adult mouse dorsal root ganglion (DRG) neurons, combining pharmacological JNK inhibition and mice deficient for each JNK isoform and for JIP1. We demonstrate that neuritogenesis is delayed by lack of JNK2 and JNK3, but not JNK1. JNK signaling is further required for sustained neurite elongation, as pharmacological JNK inhibition resulted in massive neurite retraction. This function relies on JNK1 and JNK2. Neurite regeneration of jip1(-/-) DRG neurons is affected at both initiation and extension stages. Interestingly, activated JNKs (phospho-JNKs), as well as JIP1, are also present in the cytoplasm of sprouting or regenerating axons, suggesting a local action on cytoskeleton proteins. Indeed, we have shown that JNK1 and JNK2 regulate the phosphorylation state of microtubule-associated protein MAP1B, whose role in axonal regeneration was previously characterized. Moreover, lack of MAP1B prevents neurite retraction induced by JNK inhibition. Thus, signaling by individual JNKs is differentially implicated in the reorganization of the cytoskeleton, and neurite regeneration.

Extensive structural remodeling of the injured spinal cord revealed by phosphorylated MAP1B in sprouting axons and degenerating neurons.

Spinal cord injury (SCI) results in loss of sensory and motor function because injured axons do not regenerate and neurons that die are not replaced. Nevertheless, there is evidence for spontaneous reorganization of spared pathways (i.e. sprouting) that could be exploited to improve functional recovery. The extent of morphological remodeling after spinal cord injury is, however, not understood. We have previously shown that a phosphorylated form of microtubule-associated protein-1B, MAP1B-P, is expressed by growing axons, but is detected in intact adult SC in fibers exhibiting a somatotopic distribution of myelinated sensory fibers. We now demonstrate that after adult SCI, MAP1B-P is up-regulated in other classes of axons. We used immunohistochemistry to show changing levels and distributions of MAP1B-P after a right thoracic hemisection of adult rat spinal cord. MAP1B-P labeling suggests rearrangements of the axonal circuitry that go well beyond previous descriptions. MAP1B-P-positive fibers are present in ectopic locations in gray matter in both dorsal and ventral horns and around the central canal. Double staining reveals that primary sensory and descending serotonergic and corticospinal axons are MAP1B-P positive. In white matter, high MAP1B-P expression is found on terminal enlargements near the injury, reflecting retraction of transected axons. MAP1B-P also accumulates in pre-apoptotic neuronal somata axotomized by the lesion, indicating association of MAP1B-P not only with axon extension and retraction, but also with neuronal degeneration. Finally, we provide evidence that MAP1B phosphorylation is correlated with activation of JNK MAP-kinase, providing a step towards unraveling the mechanisms of regulation of this plasticity-related cytoskeletal protein.