HSPA9/Mortalin mediates axo-protection and modulates mitochondrial dynamics in neurons

Mortalin is a mitochondrial chaperone protein involved in quality control of proteins imported into the mitochondrial matrix, which was recently described as a sensor of neuronal stress. Mortalin is down-regulated in neurons of patients with neurodegenerative diseases and levels of Mortalin expression are correlated with neuronal fate in animal models of Alzheimer's disease or cerebral ischemia. To date, however, the links between Mortalin levels, its impact on mitochondrial function and morphology and, ultimately, the initiation of neurodegeneration, are still unclear. In the present study, we used lentiviral vectors to over- or under-express Mortalin in primary neuronal cultures. We first analyzed the early events of neurodegeneration in the axonal compartment, using oriented neuronal cultures grown in microfluidic-based devices. We observed that Mortalin down-regulation induced mitochondrial fragmentation and axonal damage, whereas its over-expression conferred protection against axonal degeneration mediated by rotenone exposure. We next demonstrated that Mortalin levels modulated mitochondrial morphology by acting on DRP1 phosphorylation, thereby further illustrating the crucial implication of mitochondrial dynamics on neuronal fate in degenerative diseases.

TSG101 negatively regulates mitochondrial biogenesis in axons

There is a tight association between mitochondrial dysfunction and neurodegenerative diseases and axons that are particularly vulnerable to degeneration, but how mitochondria are maintained in axons to support their physiology remains poorly defined. In an in vivo forward genetic screen for mutants altering axonal mitochondria, we identified tsg101. Neurons mutant for tsg101 exhibited an increase in mitochondrial number and decrease in mitochondrial size. TSG101 is best known as a component of the endosomal sorting complexes required for transport (ESCRT) complexes; however, loss of most other ESCRT components did not affect mitochondrial numbers or size, suggesting TSG101 regulates mitochondrial biology in a noncanonical, ESCRT-independent manner. The TSG101-mutant phenotype was not caused by lack of mitophagy, and we found that autophagy blockade was detrimental only to the mitochondria in the cell bodies, arguing mitophagy and autophagy are dispensable for the regulation of mitochondria number in axons. Interestingly, TSG101 mitochondrial phenotypes were instead caused by activation of PGC-1ɑ/Nrf2-dependent mitochondrial biogenesis, which was mTOR independent and TFEB dependent and required the mitochondrial fission–fusion machinery. Our work identifies a role for TSG101 in inhibiting mitochondrial biogenesis, which is essential for the maintenance of mitochondrial numbers and sizes, in the axonal compartment.

T3 Enters Axon Terminals of Mouse Cortical Neurons, Is Retrogradely Transported to the Cell Nucleus and Activates Gene Expression

Thyroid hormone (TH) is critical for brain development and function. T3 enters neurons through membrane transporters and reaches the cell nucleus where it binds to receptors (TR) to regulate gene transcription. However, neurons also express the type 3 deiodinase (D3), which is located in the cellular and nuclear membranes and inactivates T3. Here, we investigated the fate and biological impact of T3 that enters neurons through axons. Primary cortical neurons were isolated from E16.5 embryos of the TH action indicator (THAI) mice, which were engineered with a TH-responsive transgene where three copies of a T3-responsive element drive a luciferase (Luc) reporter. Neurons were seeded on a microfluidic device consisting of two independent compartments: (i) cellular, where about 70-90,000 cell bodies were located, and (ii) axonal, where a few hundred distal axons were located. Fluidic isolation of the compartments was monitored with Alexa Fluor 594 hydrazide. In the first set of experiments (repeated 3 times), 8-10-day old cultures were incubated for 48h with medium containing 1% charcoal-stripped serum (Tx-medium). Subsequently, 10nM T3 was added to the axonal compartment, and 24h later cell bodies were harvested and Luc mRNA measured by RT-qPCR. There was a 2.4 ± 0.7-fold increase in Luc mRNA levels, but the addition of 2uM Silychristin (MCT8 inhibitor) to the axonal compartment reduced T3 induction of Luc mRNA by 32 ± 4.2%. In the second set of experiments (repeated 3 times), 4.9 ± 2.2pM 125I-T3 (final concentration) was added to the cellular or axonal compartments. Medium was sampled and 125I-T3 and its metabolites were separated/quantified via UPLC linked to a flow scintillation detector. After 72h of adding 125I-T3 to the axonal compartment, about 0.73 % 125I-T3 (0.052 ± 0.025pM) was found in the cellular compartment. In addition, 3,3’-125I-T2 and 125I (0.011 ± 0.003 and 0.052 ± 0.023pM, respectively) were also detected. When 125I-T3 was added to the cellular compartment, about 1.6% 125I-T3 (0.048 ± 0.027pM), no metabolites, was detected in the axonal compartment. Only background radioactivity was detected in the opposing compartment when 125I-T3 was added in the absence of cells. We conclude that T3 can be taken up by neuronal axons, partly via MCT8, and transported retrogradely to the cell nucleus to initiate TH signaling. D3-generated T3 metabolites exit the cell body alongside with small amounts of intact T3. This pathway could explain how D2-generated T3 in tanycytes is taken up by TRH-secreting neurons to mediate negative T3 feedback. Anterograde T3 transport was also detected, the significance of which remains unknown.

Seeding-Competent Tau in Gray Matter Versus White Matter of Alzheimer’s Disease Brain

Background: Neurofibrillary pathology of abnormally hyperphosphorylated tau spreads along neuroanatomical connections, underlying the progression of Alzheimer’s disease (AD). The propagation of tau pathology to axonally connected brain regions inevitably involves trafficking of seeding-competent tau within the axonal compartment of the neuron. Objective: To determine the seeding activity of tau in cerebral gray and white matters of AD. Methods: Levels of total tau, hyperphosphorylation of tau, and SDS- and β-mercaptoethanol–resistant high molecular weight tau (HMW-tau) in crude extracts from gray and white matters of AD frontal lobes were analyzed by immuno-blots. Tau seeding activity was quantitatively assessed by measuring RIPA buffer–insoluble tau in HEK-293FT/tau151-391 cells treated with brain extracts. Results: We found a comparable level of soluble tau in gray matter versus white matter of control brains, but a higher level of soluble tau in gray matter than white matter of AD brains. In AD brains, tau is hyperphosphorylated in both gray and white matters, with a higher level in the former. The extracts of both gray and white matters of AD brains seeded tau aggregation in HEK-293FT/tau151–391 cells but the white matter showed less potency. Seeding activity of tau in brain extracts was positively correlated with the levels of tau hyperphosphorylation and HMW-tau. RIPA-insoluble tau, but not RIPA-soluble tau, was hyperphosphorylated tau at multiple sites. Conclusion: Both gray and white matters of AD brain contain seeding-competent tau that can template aggregation of hyperphosphorylated tau, but the seeding potency is markedly higher in gray matter than in white matter.

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.

HSPA9/Mortalin Mediates Axo-Protection by Modulating Mitochondrial Dynamics in Neurons

Mortalin is a mitochondrial chaperone protein involved in quality control of proteins imported into the mitochondrial matrix, which was recently described as a sensor of neuronal stress. Mortalin is down-regulated in neurons of patients with neurodegenerative diseases and levels of Mortalin expression are correlated with neuronal fate in animal models of Alzheimer's disease or cerebral ischemia. To date, however, the links between Mortalin levels, its impact on mitochondrial function and morphology and, ultimately, the initiation of neurodegeneration, are still unclear. In the present study, we used lentiviral vectors to over- or under-express Mortalin in primary neuronal cultures. We first analyzed the early events of neurodegeneration in the axonal compartment, using oriented neuronal cultures grown in microfluidic-based devices. We observed that Mortalin down-regulation induced mitochondrial fragmentation and axonal damage, whereas its over-expression conferred protection against axonal degeneration mediated by oxidative stress. We next demonstrated that Mortalin levels modulated mitochondrial morphology by a direct action on DRP1 phosphorylation, thereby further illustrating the crucial implication of mitochondrial dynamics on neuronal fate in degenerative diseases.

Estimating intra-axonal axial diffusivity in the presence of fibre orientation dispersion

By analysing the diffusion MRI signal, we can infer information about the microscopic structure of the brain. Two parameters of interest - the intra-axonal axial diffusivity and fibre orientation dispersion - are potential biomarkers for very different aspects of the white matter microstructure, yet they are difficult to disentangle. The parameters covary such that, if one is not accurately accounted for, the other will be biased. In this work we use high b-value data to isolate the signal from the intra-axonal compartment and resolve any degeneracies with the extra-axonal compartment. In the high b-value regime, we then use a model of dispersed sticks to estimate the intra-axonal axial diffusivity and fibre orientation distribution on a voxelwise basis. Our results in in vivo, human data show an intra-axonal axial diffusivity of ~ 2.3 – 3 μm2/ms, where 3 μm2/ms is the diffusivity of free water at 37°C. The intra-axonal axial diffusivity is seen to vary considerably across the white matter. For example, in the corpus callosum we find high values in the genu and splenium, and lower values in the midbody. Furthermore, the axial diffusivity and orientation dispersion appear negatively correlated, behaviour which we show is consistent with the presence of fibre undulations but not consistent with a degeneracy between fanning fibres and axial diffusivity. Finally, we demonstrate that the parameter maps output from Neurite Orientation Dispersion and Density Imaging (NODDI) change substantially when the assumed axial diffusivity was increased from 1.7 to 2.5 or 3 μm2/ms.

Local Translation in Growth Cones and Presynapses, Two Axonal Compartments for Local Neuronal Functions

During neural development, growth cones, very motile compartments of tips of axons, lead axonal extension to the correct targets. Subsequently, presynapses, another axonal compartment with vigorous trafficking of synaptic vesicles, emerge to form functional synapses with postsynapses. In response to extracellular stimuli, the immediate supply of proteins by local translation within these two axonal compartments far from cell bodies confers high motility of growth cones and active vesicle trafficking in presynapses. Although local translation in growth cones and presynapses occurs at a very low level compared with cell bodies and even dendrites, recent progress in omics and visualization techniques with subcellular fractionation of these compartments has revealed the actual situation of local translation within these two axonal compartments. Here, the increasing evidence for local protein synthesis in growth cones and presynapses for axonal and synaptic functions has been reviewed. Furthermore, the mechanisms regulating local translation in these two compartments and pathophysiological conditions caused by dysregulated local translation are highlighted.

CNS Injury: Posttranslational Modification of the Tau Protein as a Biomarker

The ideal biomarker for central nervous system (CNS) trauma in patients would be a molecular marker specific for injured nervous tissue that would provide a consistent and reliable assessment of the presence and severity of injury and the prognosis for recovery. One candidate biomarker is the protein tau, a microtubule-associated protein abundant in the axonal compartment of CNS neurons. Following axonal injury, tau becomes modified primarily by hyperphosphorylation of its various amino acid residues and cleavage into smaller fragments. These posttrauma products can leak into the cerebrospinal fluid or bloodstream and become candidate biomarkers of CNS injury. This review examines the primary molecular changes that tau undergoes following traumatic brain injury and spinal cord injury, and reviews the current literature in traumatic CNS biomarker research with a focus on the potential for hyperphosphorylated and cleaved tau as sensitive biomarkers of injury.