Maintenance of neuronal polarity and regulation of cytoskeletal dynamics are vital during development and to uphold synaptic activity in neuronal networks. Here we show that soluble b-amyloid (Ab) disrupts actin and microtubule (MT) dynamics via activation of RhoA and inhibition of histone deacetylase 6 (HDAC6) in cultured hippocampal neurons. The contact of Ab with the extracellular membrane promotes RhoA activation, leading to growth cone collapse and neurite retraction, which might be responsible for hampered neuronal pathfinding and migration in Alzheimer's disease (AD). The inhibition of HDAC6 by Ab increases the level of heterodimeric acetylated tubulin and acetylated tau, both of which have been found altered in AD. We also find that the loss of HDAC6 activity perturbs the integrity of axon initial segment (AIS), resulting in mislocalization of ankyrin G and increased MT instability in the AIS concomitant with loss of polarized localization of tau and impairment of action potential firing.
Mutation or multiplication of the alpha-synuclein (Syn)-encoding gene is frequent cause of early onset Parkinson's disease (PD). Recent evidences point to the pathogenic role of excess Syn also in sporadic PD. Syn is a cytosolic protein, which has been shown to be released from neurons. Here we provide evidence that extracellular Syn induces an increase in surface-exposed glucose-related protein of 78 kDa (GRP78), which becomes clustered in microdomains of the neuronal plasma membrane. Upon interacting with Syn, GRP78 activates a signaling cascade leading to cofilin 1 inactivation and stabilization of microfilaments, thus affecting morphology and dynamics of actin cytoskeleton in cultured neurons. Downregulation of GRP78 abolishes the activity of exogenous Syn, indicating that it is the primary target of Syn. Inactivation of cofilin 1 and stabilization of actin cytoskeleton are present also in fibroblasts derived from genetic PD patients, which show a dramatic increase in stress fibers. Similar changes are displayed by control cells incubated with the medium of PD fibroblasts, only when Syn is present. The accumulation of Syn in the extracellular milieu, its interaction with the plasma membrane and Syn-driven clustering of GRP78 appear, therefore, responsible for the dysregulation of actin turnover, leading to early deficits in synaptic function that precede neurodegeneration.
Intersectin is a multifunctional protein that interacts with components of the endocytic and exocytic pathways, and it is also involved in the control of actin dynamics. Drosophila intersectin is required for viability, synaptic development, and synaptic vesicle recycling. Here, we report the characterization of intersectin function in Caenorhabditis elegans. Nematode intersectin (ITSN-1) is expressed in the nervous system, and it is enriched in presynaptic regions. The C. elegans intersectin gene (itsn-1) is nonessential for viability. In addition, itsn-1-null worms do not display any evident phenotype, under physiological conditions. However, they display aldicarb-hypersensitivity, compatible with a negative regulatory role of ITSN-1 on neurotransmission. ITSN-1 physically interacts with dynamin and EHS-1, two proteins involved in synaptic vesicle recycling. We have previously shown that EHS-1 is a positive modulator of synaptic vesicle recycling in the nematode, likely through modulation of dynamin or dynamin-controlled pathways. Here, we show that ITSN-1 and EHS-1 have opposite effects on aldicarb sensitivity, and on dynamin-dependent phenotypes. Thus, the sum of our results identifies dynamin, or a dynamin-controlled pathway, as a potential target for the negative regulatory role of ITSN-1.
During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion.
We demonstrate high-resolution photocross-linking of biodegradable poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) using UV excimer laser photocuring at 308 nm. The curing depth can be tuned in a micrometre range by adjusting the total energy dose (total fluence). Young's moduli of the scaffolds are found to be a few gigapascal, high enough to support bone formation. The results presented here demonstrate that the proposed technique is an excellent tool for the fabrication of stiff and biocompatible structures on a micrometre scale with defined patterns of high resolution in all three spatial dimensions. Using UV laser photocuring at 308 nm will significantly improve the speed of rapid prototyping of biocompatible and biodegradable polymer scaffolds and enables its production in a few seconds, providing high lateral and horizontal resolution. This short timescale is indeed a tremendous asset that will enable a more efficient translation of technology to clinical applications. Preliminary cell tests proved that PPF : DEF scaffolds produced by excimer laser photocuring are biocompatible and, therefore, are promising candidates to be applied in tissue engineering and regenerative medicine.
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