Paraquat disrupts KIF5A-mediated axonal mitochondrial transport in midbrain neurons and its antagonism by melatonin.

Paraquat (PQ) is a broad-spectrum herbicide used worldwide and is a hazardous chemical to human health. Cumulative evidence strengthens the association between PQ exposure and the development of Parkinson's disease (PD). However, the underlying mechanism and effective interventions against PQ-induced neurotoxicity remain unclear. In this study, C57BL/6 J mice were treated with PQ (i.p., 10 mg/kg, twice a week) and melatonin (i.g., 20 mg/kg, twice a week) for 8 weeks. Results showed that PQ-induced motor deficits and midbrain dopaminergic neuronal damage in C57BL/6 J mice were protected by melatonin pretreatment. In isolated primary midbrain neurons and SK-N-SH cells, reduction of cell viability, elevation of total ROS levels, axonal mitochondrial transport defects and mitochondrial dysfunction caused by PQ were attenuated by melatonin. After screening of expression of main motors driving axonal mitochondrial transport, data showed that PQ-decreased KIF5A expression in mice midbrain and in SK-N-SH cell was antagonized by melatonin. Using the in vitro KIF5A-overexpression model, it was found that KIF5A overexpression inhibited PQ-caused neurotoxicity and mitochondrial dysfunction in SK-N-SH cells. In addition, application of MTNR1B (MT2) receptor antagonist, 4-P-PDOT, significantly counteracted the protection of melatonin against PQ-induced neurotoxicity. Further, Kif5a-knockdown diminished melatonin-induced alleviation of motor deficits and neuronal damage against PQ in C57BL/6 J mice. The present study establishes a causal link between environmental neurotoxicants exposure and PD etiology and provides effective interventive targets in the pathogenesis of PD.

Increases in anterograde axoplasmic transport in neurons of the hyper-glutamatergic, glutamate dehydrogenase 1 (Glud1) transgenic mouse: Effects of glutamate receptors on transport.

The excitatory neurotransmitter glutamate has a role in neuronal migration and process elongation in the central nervous system (CNS). The effects of chronic glutamate hyperactivity on vesicular and protein transport within CNS neurons, that is, processes necessary for neurite growth, have not been examined previously. In this study, we measured the effects of lifelong hyperactivity of glutamate neurotransmission on axoplasmic transport in CNS neurons. We compared wild-type (wt) to transgenic (Tg) mice over-expressing the glutamate dehydrogenase gene Glud1 in CNS neurons and exhibiting increases in glutamate transmitter formation, release, and synaptic activation in brain throughout the lifespan. We found that Glud1 Tg as compared with wt mice exhibited increases in the rate of anterograde axoplasmic transport in neurons of the hippocampus measured in brain slices ex vivo, and in olfactory neurons measured in vivo. We also showed that the in vitro pharmacologic activation of glutamate synapses in wt mice led to moderate increases in axoplasmic transport, while exposure to selective inhibitors of ion channel forming glutamate receptors very significantly suppressed anterograde transport, suggesting a link between synaptic glutamate receptor activation and axoplasmic transport. Finally, axoplasmic transport in olfactory neurons of Tg mice in vivo was partially inhibited following 14-day intake of ethanol, a known suppressor of axoplasmic transport and of glutamate neurotransmission. The same was true for transport in hippocampal neurons in slices from Glud1 Tg mice exposed to ethanol for 2 h ex vivo. In conclusion, endogenous activity at glutamate synapses regulates and glutamate synaptic hyperactivity increases intraneuronal transport rates in CNS neurons.

Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism.

Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, ß, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1ß, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.

A novel orally active HDAC6 inhibitor T-518 shows a therapeutic potential for Alzheimer's disease and tauopathy in mice.

Accumulation of tau protein is a key pathology of age-related neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Those diseases are collectively termed tauopathies. Tau pathology is associated with axonal degeneration because tau binds to microtubules (MTs), a component of axon and regulates their stability. The acetylation state of MTs contributes to stability and histone deacetylase 6 (HDAC6) is a major regulator of MT acetylation status, suggesting that pharmacological HDAC6 inhibition could improve axonal function and may slow the progression of tauopathy. Here we characterize N-[(1R,2R)-2-{3-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-5-oxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}cyclohexyl]-2,2,3,3,3-pentafluoropropanamide (T-518), a novel, potent, highly selective HDAC6 inhibitor with clinically favorable pharmacodynamics. T-518 shows potent inhibitory activity against HDAC6 and superior selectivity over other HDACs compared with the known HDAC6 inhibitors in the enzyme and cellular assays. T-518 showed brain penetration in an oral dose and blocked HDAC6-dependent tubulin deacetylation at Lys40 in mouse hippocampus. A 2-week treatment restored impaired axonal transport and novel object recognition in the P301S tau Tg mouse, tauopathy model, while a 3-month treatment also decreased RIPA-insoluble tau accumulation. Pharmaceutical inhibition of HDAC6 is a potential therapeutic strategy for tauopathy, and T-518 is a particularly promising drug candidate.

Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): Mechanistic insights into MMAE ADC peripheral neuropathy.

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.

Paclitaxel increases axonal localization and vesicular trafficking of Nav1.7.

The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

Blocking retrograde axonal transport of autophagosomes contributes to sevoflurane-induced neuron apoptosis in APP/PS1 mice.

Autophagy, a crucial pathway for the degradation of proteins in eukaryotic cells, is linked to the development of Alzheimer's disease (AD), and the accumulated autophagosomes in the cells resulting in the death of cells. Sevoflurane can impair spatial learning and memory in mice with AD and lead to the apoptosis of nerve cells; however, the underlying mechanisms remain unknown. We aim to explore the effects and underlying mechanisms of sevoflurane in APPswe/PS1ΔE9 double-transgenic mice. 51 heterozygous APPswe/PS1ΔE9 double-transgenic mice were involved and divided into three groups, including control group, sham group and sevoflurane group. Morris water maze experiment was used to test the learning and memory abilities of mice, flow cytometry was conducted to detect apoptosis and mitochondrial membrane potential of brain cells in mice, transmission electron microscopy was used to observe the number of autophagosomes at the axon in mice, and western blot was carried out to detect the expression of Bax, Bcl-2, LC3II, P62, KIF3B and DIC proteins of brain cells in mice. In our study, we found that significantly longer escape latencies, fewer crossings of the platform and shorter time spent in the target quadrant of the morris water maze experiment in the sevoflurane group. Flow cytometry showed cellular apoptosis was increased and the membrane potential of the mitochondria was reduced of brain cells in the sevoflurane group. Transmission electron microscopy displayed that there was a remarkable upregulation of autophagosomes at the axon of brain cells in mice after treatment of sevoflurane. Western blot demonstrated that the expression of Bax, LC3II, P62 and KIF3B proteins were elevated, and the expression of Bcl-2 and DIC proteins were reduced in the sevoflurane group. Sevoflurane impaired acquisition learning and memory function, promoted the apoptosis of hippocampal neurons in APPswe/PS1ΔE9 double-transgenic mice, and the mechanism might be related to the activation of autophagy along with the disruption of autophagosomes retrograde transport in axons.

KIF5A-dependent axonal transport deficiency disrupts autophagic flux in trimethyltin chloride-induced neurotoxicity.

Trimethyltin chloride (TMT) is widely used as a constituent of fungicides and plastic stabilizers in the industrial and agricultural fields, and is generally acknowledged to have potent neurotoxicity, especially in the hippocampus; however, the mechanism of induction of neurotoxicity by TMT remains elusive. Herein, we exposed Neuro-2a cells to different concentrations of TMT (2, 4, and 8 µM) for 24 h. Proteomic analysis, coupled with bioinformatics analysis, revealed the important role of macroautophagy/autophagy-lysosome machinery in TMT-induced neurotoxicity. Further analysis indicated significant impairment of autophagic flux by TMT via suppressed lysosomal function, such as by inhibiting lysosomal proteolysis and changing the lysosomal pH, thereby contributing to defects in autophagic clearance and subsequently leading to nerve cell death. Mechanistically, molecular interaction networks of Ingenuity Pathway Analysis identified a downregulated molecule, KIF5A (kinesin family member 5A), as a key target in TMT-impaired autophagic flux. TMT decreased KIF5A protein expression, disrupted the interaction between KIF5A and lysosome, and impaired lysosomal axonal transport. Moreover, Kif5a overexpression restored axonal transport, increased lysosomal dysfunction, and antagonized TMT-induced neurotoxicity in vitro. Importantly, in TMT-administered mice with seizure symptoms and histomorphological injury in the hippocampus, TMT inhibited KIF5A expression in the hippocampus. Gene transfer of Kif5a enhanced autophagic clearance in the hippocampus and alleviated TMT-induced neurotoxicity in vivo. Our results are the first to demonstrate KIF5A-dependent axonal transport deficiency to cause autophagic flux impairment via disturbance of lysosomal function in TMT-induced neurotoxicity; manipulation of KIF5A may be a therapeutic approach for antagonizing TMT-induced neurotoxicity.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTB: actin beta; AGC: automatic gain control; ATG: autophagy-related; ATP6V0D1: ATPase H+ transporting lysosomal V0 subunit D1; ATP6V1E1: ATPase H+ transporting lysosomal V1 subunit E1; CA: cornu ammonis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; DCTN1: dynactin subunit 1; DG: dentate gyrus; DYNLL1: dynein light chain LC8-type 1; FBS: fetal bovine serum; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IPA: Ingenuity Pathway Analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; KIF5A: kinesin family member 5A; LAMP: lysosomal-associated membrane protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PBS: phosphate-buffered saline; PFA: paraformaldehyde; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PRM: parallel reaction monitoring; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SYP: synaptophysin; TAX1BP1: Tax1 binding protein 1; TMT: trimethyltin chloride; TUB: tubulin.

Irreversible incorporation of L-dopa into the C-terminus of α-tubulin inhibits binding of molecular motor KIF5B to microtubules and alters mitochondrial traffic along the axon.

L-dopa is the most effective drug used to date for management of Parkinson's disease symptoms. Unfortunately, long-term administration of L-dopa often results in development of motor disorders, including dyskinesias. Despite extensive research on L-dopa-induced dyskinesia, its pathogenesis remains poorly understood. We demonstrated previously that L-dopa can be post-translationally incorporated into the C-terminus of α-tubulin in living cells. In the present study, we investigated the effect of the presence of L-dopa-tubulin-enriched microtubules on mitochondrial traffic mediated by molecular motor KIF5B. Using biochemical approaches in combination with experiments on neuronal cell lines and mouse hippocampal primary cultures, we demonstrated that L-dopa incorporation into tubulin is irreversible. Transport of mitochondria along the axon was altered after L-dopa treatment of cells. In L-dopa-treated cells, mitochondria had reduced ability to reach the distal segment of the axon, spent more time in pause, and showed reduced velocity of anterograde movement. KIF5B motor, a member of the kinesin family involved in mitochondrial transport in neurons, showed reduced affinity for Dopa-tubulin-containing microtubules. Our findings, taken together, suggest that tyrosination state of tubulin (and microtubules) is altered by L-dopa incorporation into tubulin; the gradual increase in amount of altered microtubules affects microtubule functioning, impairs mitochondrial traffic and distribution, and this could be relevant in Parkinson's disease patients chronically treated with L-dopa.

Isotope-reinforced polyunsaturated fatty acids improve Parkinson's disease-like phenotype in rats overexpressing α-synuclein.

Lipid peroxidation is a key to a portfolio of neurodegenerative diseases and plays a central role in α-synuclein (α-syn) toxicity, mitochondrial dysfunction and neuronal death, all key processes in the pathogenesis of Parkinson's disease (PD). Polyunsaturated fatty acids (PUFAs) are important constituents of the synaptic and mitochondrial membranes and are often the first molecular targets attacked by reactive oxygen species (ROS). The rate-limiting step of the chain reaction of ROS-initiated PUFAs autoxidation involves hydrogen abstraction at bis-allylic sites, which can be slowed down if hydrogens are replaced with deuteriums. In this study, we show that targeted overexpression of human A53T α-syn using an AAV vector unilaterally in the rat substantia nigra reproduces some of pathological features seen in PD patients. Chronic dietary supplementation with deuterated PUFAs (D-PUFAs), specifically 0.8% D-linoleic and 0.3% H-linolenic, produced significant disease-modifying beneficial effects against α-syn-induced motor deficits, synaptic pathology, oxidative damage, mitochondrial dysfunction, disrupted trafficking along axons, inflammation and DA neuronal loss. These findings support the clinical evaluation of D-PUFAs as a neuroprotective therapy for PD.

Stabilization of microtubules improves cognitive functions and axonal transport of mitochondria in Alzheimer's disease model mice.

One major pathological process in Alzheimer's disease is mediated by hyperphosphorylated tau, which includes altered microtubules (MTs) and functions associated with tau. A potential way to compensate for altered MT function is to use an MT stabilizer, such as epothilone D (EpoD). Previous studies have demonstrated improved cognitive functions and axonal transport by EpoD in tau-mutation mice. Here, we demonstrated that extended EpoD treatment also has beneficial effects on APP/PS1 double-transgenic mice, improving their motor and spatial memory, increasing key synaptic protein levels, while not affecting amyloid plaque density or level of tau phosphorylation. Interestingly, EpoD appears to improve the retrieval of formed memories. We also observed improved axonal transport of mitochondria in cultured neurons from APP/PS1 mice. In addition, higher level of perineuronal nets are found in APP/PS1 mice injected with EpoD, suggesting potential contributions of increased inhibition. Our results suggest potential therapeutic value of EpoD in treating Alzheimer's disease.

Of Mice and Monkeys: Neuroprotective Efficacy of the p38 Inhibitor BIRB 796 Depends on Model Duration in Experimental Glaucoma.

Glaucoma is a group of optic neuropathies associated with aging and sensitivity to intraocular pressure (IOP). Early progression involves retinal ganglion cell (RGC) axon dysfunction that precedes frank degeneration. Previously we demonstrated that p38 MAPK inhibition abates axonal dysfunction and slows degeneration in the inducible microbead occlusion model of glaucoma in rat. Here, we assessed the neuroprotective effect of topical eye delivery of the p38 MAPK inhibitor BIRB 796 in three models of glaucoma (microbead occlusion in rat and squirrel monkey and the genetic DBA/2 J mouse model) with distinct durations of IOP elevation. While BIRB 796 did not influence IOP, treatment over four weeks in rats prevented degradation of anterograde axonal transport to the superior colliculus and degeneration in the optic nerve. Treatment over months in the chronic DBA/2 J model and in the squirrel monkey model reduced expression and activation of p38 downstream targets in the retina and brain but did not rescue RGC axon transport or degeneration, suggesting the efficacy of BIRB 796 in preventing associated degeneration of the RGC projection depends on the duration of the experimental model. These results emphasize the importance of evaluating potential therapeutic compounds for neuroprotection in multiple models using elongated treatment paradigms for an accurate assessment of efficacy.

Activation of adenosine A3 receptor protects retinal ganglion cells from degeneration induced by ocular hypertension.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3 receptor (A3R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.

BDNF improves axon transportation and rescues visual function in a rodent model of acute elevation of intraocular pressure.

Optic neuropathies lead to blindness; the common pathology is the degeneration of axons of the retinal ganglion cells. In this study, we used a rat model of retinal ischemia-reperfusion and a one-time intravitreal brain-derived neurotrophic factor (BDNF) injection; then we examined axon transportation function, continuity, physical presence of axons in different part of the optic nerve, and the expression level of proteins involved in axon transportation. We found that in the disease model, axon transportation was the most severely affected, followed by axon continuity, then the number of axons in the distal and proximal optic nerve. BDNF treatment relieved all reductions and significantly restored function. The molecular changes were more minor, probably due to massive gliosis of the optic nerve, so interpretation of protein expression data should be done with some caution. The process in this acute model resembles a fast-forward of changes in the chronic model of glaucoma. Therefore, impairment in axon transportation appears to be a common early process underlying different optic neuropathies. This research on effective intervention can be used to develop interventions for all optic neuropathies targeting axon transportation.

Multifunctional compounds lithium chloride and methylene Blue attenuate the negative effects of diisopropylfluorophosphate on axonal transport in rat cortical neurons.

Organophosphates (OPs) are valuable as pesticides in agriculture and for controlling deadly vector-borne illnesses; however, they are highly toxic and associated with many deleterious health effects in humans including long-term neurological impairments. Antidotal treatment regimens are available to combat the symptoms of acute OP toxicity, which result from the irreversible inhibition of acetylcholinesterase (AChE). However, there are no established treatments for the long-term neurological consequences of OP exposure. In addition to AChE, OPs can negatively affect multiple protein targets as well as biological processes such as axonal transport. Given the fundamental nature of axonal transport to neuronal health, we rationalized that this process might serve as a general focus area for novel therapeutic strategies against OP toxicity. In the studies described here, we employed a multi-target, phenotypic screening, and drug repurposing strategy for the evaluations of potential novel OP-treatments using a primary neuronal culture model and time-lapse live imaging microscopy. Two multi-target compounds, lithium chloride (LiCl) and methylene blue (MB), which are FDA-approved for other indications, were evaluated for their ability to prevent the negative effects of the OP, diisopropylfluorophosphate (DFP) on axonal transport. The results indicated that both LiCl and MB prevented DFP-induced impairments in anterograde and retrograde axonal transport velocities in a concentration dependent manner. While in vivo studies will be required to confirm our in vitro findings, these experiments support the potential of LiCl and MB as repurposed drugs for the treatment of the long-term neurological deficits associated with OP exposure (currently an unmet medical need).

Histone deacetylase 6 inhibition restores autophagic flux to promote functional recovery after spinal cord injury.

After spinal cord injury (SCI), the inhibitory molecules derived from scars at the lesion sites and the limited regenerative capacity of neuronal axons pose difficulties for the recovery after SCI. Remodeling of cytoskeleton structures including microtubule assembly and tubulin post-translational modification are widely accepted to play a crucial role in initiation of growth cone and regrowth of injured axon. Although increasing studies have focused on the association between tubulin acetylation and autophagy due to the role of tubulin acetylation in organelles and substances transport, there are no studies exploring the effect of tubulin acetylation on autophagy after spinal cord injury (SCI). Here, we found that histone deacetylase 6 (HDAC6) was significantly up-regulated after SCI, while inhibition of HDAC6 by Tubastatin A induced functional recovery after SCI. In view of enzyme-dependent and -independent mechanisms of HDAC6 to adjust diverse cellular processes, such as autophagy, the ubiquitin proteasome system and post-translational modification of tubulin, we mainly focused on the significance of HDAC6 in axonal regeneration and autophagy after SCI. Western blotting, Co-immunoprecipitation and immunofluorescence staining were conducted to showed that Tubastatin A treatment in nocodazole-treated cells and mice suffering from SCI prompted acetylation and stabilization of microtubules and thus restored transport function, which may contribute to restored autophagic flux and increased axonal length. Whereas inhibition of degradation of autolysosomes by bafilomycin A1 (Baf-A1) reversed functional recovery caused by Tubastatin A, revealing the association between tubulin acetylation and autophagy, which supports HDAC6 inhibition as a potential target for SCI treatment.

PI3K activation prevents Aß42-induced synapse loss and favors insoluble amyloid deposit formation.

Excess of Aß42 peptide is considered a hallmark of the disease. Here we express the human Aß42 peptide to assay the neuroprotective effects of PI3K in adult Drosophila melanogaster. The neuronal expression of the human peptide elicits progressive toxicity in the adult fly. The pathological traits include reduced axonal transport, synapse loss, defective climbing ability and olfactory perception, as well as lifespan reduction. The Aß42-dependent synapse decay does not involve transcriptional changes in the core synaptic protein encoding genes bruchpilot, liprin and synaptobrevin. All toxicity features, however, are suppressed by the coexpression of PI3K. Moreover, PI3K activation induces a significant increase of 6E10 and thioflavin-positive amyloid deposits. Mechanistically, we suggest that Aß42-Ser26 could be a candidate residue for direct or indirect phosphorylation by PI3K. Along with these in vivo experiments, we further analyze Aß42 toxicity and its suppression by PI3K activation in in vitro assays with SH-SY5Y human neuroblastoma cell cultures, where Aß42 aggregation into large insoluble deposits is reproduced. Finally, we show that the Aß42 toxicity syndrome includes the transcriptional shut down of PI3K expression. Taken together, these results uncover a potential novel pharmacological strategy against this disease through the restoration of PI3K activity.

Axonal transport defects and neurodegeneration: Molecular mechanisms and therapeutic implications.

Because of the extremely polarized morphology, the proper functioning of neurons largely relies on the efficient cargo transport along the axon. Axonal transport defects have been reported in multiple neurodegenerative diseases as an early pathological feature. The discovery of mutations in human genes involved in the transport machinery provide a direct causative relationship between axonal transport defects and neurodegeneration. Here, we summarize the current genetic findings related to axonal transport in neurodegenerative diseases, and we discuss the relationship between axonal transport defects and other pathological changes observed in neurodegeneration. In addition, we summarize the therapeutic approaches targeting the axonal transport machinery in studies of neurodegenerative diseases. Finally, we review the technical advances in tracking axonal transport both in vivo and in vitro.

ß-alanine supplementation induces taurine depletion and causes alterations of the retinal nerve fiber layer and axonal transport by retinal ganglion cells.

To study the effect of taurine depletion induced by ß-alanine supplementation in the retinal nerve fiber layer (RNFL), and retinal ganglion cell (RGC) survival and axonal transport. Albino Sprague-Dawley rats were divided into two groups: one group received ß-alanine supplementation (3%) in the drinking water during 2 months to induce taurine depletion, and the other group received regular water. After one month, half of the rats from each group were exposed to light. Retinas were analyzed in-vivo using Spectral-Domain Optical Coherence Tomography (SD-OCT). Prior to processing, RGCs were retrogradely traced with fluorogold (FG) applied to both superior colliculi, to assess the state of their retrograde axonal transport. Retinas were dissected as wholemounts, surviving RGCs were immunoidentified with Brn3a, and the RNFL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. ß-alanine supplementation decreases significantly taurine plasma levels and causes a significant reduction of the RNFL thickness that is increased after light exposure. An abnormal pNFH immunoreactivity in some RGC bodies, their proximal dendrites and axons, and a further diminution of the mean number of FG-traced RGCs compared with Brn3a+RGCs, indicate that their retrograde axonal transport is affected. In conclusion, taurine depletion causes RGC loss and axonal transport impairment. Finally, our results suggest that care should be taken when ingesting ß-alanine supplements due to the limited understanding of their potential adverse effects.