Axonal Compartment Of Neurons Research Articles

Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. Invitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted invivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and invivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

Pathological and physiological functional cross-talks of α-synuclein and tau in the central nervous system.

α-Synuclein and tau are abundant multifunctional brain proteins that are mainly expressed in the presynaptic and axonal compartments of neurons, respectively. Previous works have revealed that intracellular deposition of α-synuclein and/or tau causes many neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Despite intense investigation, the normal physiological functions and roles of α-synuclein and tau are still unclear, owing to the fact that mice with knockout of either of these proteins do not present apparent phenotypes. Interestingly, the co-occurrence of α-synuclein and tau aggregates was found in post-mortem brains with synucleinopathies and tauopathies, some of which share similarities in clinical manifestations. Furthermore, the direct interaction of α-synuclein with tau is considered to promote the fibrillization of each of the proteins in vitro and in vivo. On the other hand, our recent findings have revealed that α-synuclein and tau are cooperatively involved in brain development in a stage-dependent manner. These findings indicate strong cross-talk between the two proteins in physiology and pathology. In this review, we provide a summary of the recent findings on the functional roles of α-synuclein and tau in the physiological conditions and pathogenesis of neurodegenerative diseases. A deep understanding of the interplay between α-synuclein and tau in physiological and pathological conditions might provide novel targets for clinical diagnosis and therapeutic strategies to treat neurodegenerative diseases.

Axonal GABAA receptors depolarize presynaptic terminals and facilitate transmitter release in cerebellar Purkinje cells.

GABAA receptors have been described in the axonal compartment of neurons; contrary to dendritic GABAA receptors, axonal GABAA receptors usually induce depolarizing responses. In this study we describe the presence of functional axonal GABAA receptors in cerebellar Purkinje cells by using a combination of direct patch-clamp recordings from the axon terminals and laser GABA photolysis. In Purkinje cells, axonal GABAA receptors are depolarizing and induce an increase in neurotransmitter release that results in a change of short-term synaptic plasticity. These results contribute to our understanding of the cellular mechanisms of action of axonal GABAA receptors and highlight the importance of the presynaptic compartment in neuronal computation. In neurons of the adult brain, somatodendritic GABAA receptors (GABAA Rs) mediate fast synaptic inhibition and play a crucial role in synaptic integration. GABAA Rs are not only present in the somatodendritic compartment, but also in the axonal compartment where they modulate action potential (AP) propagation and transmitter release. Although presynaptic GABAA Rs have been reported in various brain regions, their mechanisms of action and physiological roles remain obscure, particularly at GABAergic boutons. Here, using a combination of direct whole-bouton or perforated patch-clamp recordings and local GABA photolysis in single axonal varicosities of cerebellar Purkinje cells, we investigate the subcellular localization and functional role of axonal GABAA Rs both in primary cultures and acute slices. Our results indicate that presynaptic terminals of PCs carry GABAA Rs that behave as auto-receptors; their activation leads to a depolarization of the terminal membrane after an AP due to the relatively high cytoplasmic Cl- concentration in the axon, but they do not modulate the AP itself. Paired recordings from different terminals of the same axon show that the GABAA R-mediated local depolarizations propagate substantially to neighbouring varicosities. Finally, the depolarization mediated by presynaptic GABAA R activation augmented Ca2+ influx and transmitter release, resulting in a marked effect on short-term plasticity. Altogether, our results reveal a mechanism by which presynaptic GABAA Rs influence neuronal computation.

A single intramuscular vaccination of mice with the HSV-1 VC2 virus with mutations in the glycoprotein K and the membrane protein UL20 confers full protection against lethal intravaginal challenge with virulent HSV-1 and HSV-2 strains.

Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 107 VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.

Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel “top-down” approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease

In Alzheimer disease (AD) and frontotemporal dementia the microtubule-associated protein Tau becomes progressively hyperphosphorylated, eventually forming aggregates. However, how Tau dysfunction is associated with functional impairment is only partly understood, especially at early stages when Tau is mislocalized but has not yet formed aggregates. Impaired axonal transport has been proposed as a potential pathomechanism, based on cellular Tau models and Tau transgenic mice. We recently reported K369I mutant Tau transgenic K3 mice with axonal transport defects that suggested a cargo-selective impairment of kinesin-driven anterograde transport by Tau. Here, we show that kinesin motor complex formation is disturbed in the K3 mice. We show that under pathological conditions hyperphosphorylated Tau interacts with c-Jun N-terminal kinase- interacting protein 1 (JIP1), which is associated with the kinesin motor protein complex. As a result, transport of JIP1 into the axon is impaired, causing JIP1 to accumulate in the cell body. Because we found trapping of JIP1 and a pathological Tau/JIP1 interaction also in AD brain, this may have pathomechanistic implications in diseases with a Tau pathology. This is supported by JIP1 sequestration in the cell body of Tau-transfected primary neuronal cultures. The pathological Tau/JIP1 interaction requires phosphorylation of Tau, and Tau competes with the physiological binding of JIP1 to kinesin light chain. Because JIP1 is involved in regulating cargo binding to kinesin motors, our findings may, at least in part, explain how hyperphosphorylated Tau mediates impaired axonal transport in AD and frontotemporal dementia.

Midline brain injury in the immature rat induces sustained cognitive deficits, bihemispheric axonal injury and neurodegeneration

Infants and children less than 4 years old suffer chronic cognitive deficits following mild, moderate or severe diffuse traumatic brain injury (TBI). It has been suggested that the underlying neuropathologic basis for behavioral deficits following severe TBI is acute brain swelling, subarachnoid hemorrhage and axonal injury. To better understand mechanisms of cognitive dysfunction in mild-moderate TBI, a closed head injury model of midline TBI in the immature rat was developed. Following an impact over the midline suture of the intact skull, 17-day-old rats exhibited short apnea times (3–15 s), did not require ventilatory support and suffered no mortality, suggestive of mild TBI. Compared to un-injured rats, brain-injured rats exhibited significant learning deficits over the first week post-injury ( p < 0.0005), and, significant learning ( p < 0.005) and memory deficits ( p < 0.05) in the third post-injury week. Between 6 and 72 h, blood–brain barrier breakdown, extensive traumatic axonal injury in the subcortical white matter and thalamus, and focal areas of neurodegeneration in the cortex and hippocampus were observed in both hemispheres of the injured brain. At 8 to 18 days post-injury, reactive astrocytosis in the cortex, axonal degeneration in the subcortical white matter tracts, and degeneration of neuronal cell bodies and processes in the thalamus of both hemispheres were observed; however, cortical volumes were not different between un-injured and injured rat brains. These data suggest that diffuse TBI in the immature rat can lead to ongoing degeneration of both cell soma and axonal compartments of neurons, which may contribute, in part, to the observed sustained cognitive deficits.

Differential Proteomics Analysis of Synaptic Proteins Identifies Potential Cellular Targets and Protein Mediators of Synaptic Neuroprotection Conferred by the Slow Wallerian Degeneration (Wlds) Gene

Non-somatic synaptic and axonal compartments of neurons are primary pathological targets in many neurodegenerative conditions, ranging from Alzheimer disease through to motor neuron disease. Axons and synapses are protected from degeneration by the slow Wallerian degeneration (Wld(s)) gene. Significantly the molecular mechanisms through which this spontaneous genetic mutation delays degeneration remain controversial, and the downstream protein targets of Wld(s) resident in non-somatic compartments remain unknown. In this study we used differential proteomics analysis to identify proteins whose expression levels were significantly altered in isolated synaptic preparations from the striatum of Wld(s) mice. Eight of the 16 proteins we identified as having modified expression levels in Wld(s) synapses are known regulators of mitochondrial stability and degeneration (including VDAC1, Aralar1, and mitofilin). Subsequent analyses demonstrated that other key mitochondrial proteins, not identified in our initial screen, are also modified in Wld(s) synapses. Of the non-mitochondrial proteins identified, several have been implicated in neurodegenerative diseases where synapses and axons are primary pathological targets (including DRP-2 and Rab GDP dissociation inhibitor beta). In addition, we show that downstream protein changes can be identified in pathways corresponding to both Ube4b (including UBE1) and Nmnat1 (including VDAC1 and Aralar1) components of the chimeric Wld(s) gene, suggesting that full-length Wld(s) protein is required to elicit maximal changes in synaptic proteins. We conclude that altered mitochondrial responses to degenerative stimuli are likely to play an important role in the neuroprotective Wld(s) phenotype and that targeting proteins identified in the current study may lead to novel therapies for the treatment of neurodegenerative diseases in humans.

Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain.

Hyperpolarization-activated cation currents (I(h)) contribute to various physiological properties and functions in the brain, including neuronal pacemaker activity, setting of resting membrane potential, and dendritic integration of synaptic input. Four subunits of the Hyperpolarization-activated and Cyclic-Nucleotide-gated nonselective cation channels (HCN1-4), which generate I(h), have been cloned recently. To better understand the functional diversity of I(h) in the brain, we examined precise immunohistochemical localization of four HCNs in the rat brain. Immunoreactivity for HCN1 showed predominantly cortical distribution, being intense in the neocortex, hippocampus, superior colliculus, and cerebellum, whereas those for HCN3 and HCN4 exhibited subcortical distribution mainly concentrated in the hypothalamus and thalamus, respectively. Immunoreactivity for HCN2 had a widespread distribution throughout the brain. Double immunofluorescence revealed colocalization of immunoreactivity for HCN1 and HCN2 in distal dendrites of pyramidal cells in the hippocampus and neocortex. At the electron microscopic level, immunogold particles for HCN1 and HCN2 had similar distribution patterns along plasma membrane of dendritic shafts in layer I of the neocortex and stratum lacunosum moleculare of the hippocampal CA1 area, suggesting that these subunits could form heteromeric channels. Our results further indicate that HCNs are localized not only in somato-dendritic compartments but also in axonal compartments of neurons. Immunoreactivity for HCNs often occurred in preterminal rather than terminal portions of axons and in specific populations of myelinated axons. We also found HCN2-immunopositive oligodendrocytes including perineuronal oligodendrocytes throughout the brain. These results support previous electrophysiological findings and further suggest unexpected roles of I(h) channels in the brain.

Serum cleaved Tau protein and neurobehavioral battery of tests as markers of brain injury in experimental bacterial meningitis

Brain injury due to bacterial meningitis affects multiple areas of the brain with a heterogeneous distribution generating a challenge to assess severity. Tau proteins are microtubular binding proteins localized in the axonal compartment of neurons. Brain injury releases cleaved Tau proteins (C-tau) into the extracellular space where they are transported to the cerebral spinal fluid. We hypothesized that C-tau crosses the blood–brain barrier during inflammation and that it can be detected in serum. The correlation between serum C-tau levels and the extent of the meningitic insult was examined. Furthermore, we studied whether the use of a subset of neurobehavioral tasks can assess the extent of brain injury after meningitis. The tests were chosen primarily for their ability to detect deficits in the acoustic system, low brain, reflexive responding, as well as for impaired motor coordination and the higher brain functions of learning and memory. A rat model of group B streptococcal meningitis with variable severity was utilized. At five days after bacterial inoculation followed by antibiotic therapy neurobehavioral tests were performed and serum C-tau and histologic samples of the brain were obtained. Our study shows that during meningitis C-tau appears in serum and reflects the extent of neurologic damage. Neurobehavioral performance was altered after bacterial meningitis and could be correlated with histologic and biochemical markers of neurologic sequelae. We conclude that serum C-tau and a composite of neurobehavioral tests could become useful markers for assessing the severity of neurological damage in experimental bacterial meningitis.

The role of beta 2-subunit-containing nicotinic acetylcholine receptors in the brain explored with a mutant mouse.

Neuronal nicotinic receptors comprise a family of pentameric oligomers made up of a combination of 10 different subunits. The beta 2 subunit has the widest pattern of expression in the brain and is thus likely to form a significant fraction of neuronal nicotinic receptors. Using mice lacking the beta 2 subunit, we have shown that nAChRs containing this subunit are responsible for most of the high-affinity binding sites for nicotine, cytisine, and epibatidine in the brain. Functional receptors containing the beta 2-subunit are found in the somatodendritic compartment as well as the axonal compartment of neurons. We have examined the contribution of these receptors to the effects of nicotine on the mesolimbic DA system, which mediates the reinforcing properties of many addictive drugs (including nicotine). Submicromolar doses of nicotine, corresponding to the concentrations of nicotine in vivo in self-administration paradigms, increased the firing rate of dopaminergic neurons in vitro in normal mice but not in mice lacking the beta 2 subunit. Consistently, systemic injection of nicotine induced an increase in extracellular dopamine in normal mice but not in mutant mice, and nicotine self-administration was reduced or suppressed in mutant mice. These data support the view that the beta 2-containing receptors are involved in mediating the reinforcing properties of nicotine.

Quantification of axonal damage in traumatic brain injury: affinity purification and characterization of cerebrospinal fluid tau proteins.

Diffuse axonal injury is a primary feature of head trauma and is one of the most frequent causes of mortality and morbidity. Diffuse axonal injury is microscopic in nature and difficult or impossible to detect with imaging techniques. The objective of the present study was to determine whether axonal injury in head trauma patients could be quantified by measuring levels of CSF tau proteins. Tau proteins are structural microtubule binding proteins primarily localized in the axonal compartment of neurons. Monoclonal antibodies recognizing the form of tau found in the CSF of head trauma patients were developed by differential CSF hybridoma screening using CSF from head trauma and control patients. Clones positive for head trauma CSF tau proteins were used to characterize this form of tau and for ELISA development. Using the developed ELISA, CSF tau levels were elevated >1,000-fold in head trauma patients (mean, 1,519 ng/ml of CSF) when compared with patients with multiple sclerosis (mean, 0.014 ng/ml of CSF; p < 0.001), normal pressure hydrocephalus (nondetectable CSF tau), neurologic controls (mean, 0.031 ng/ml of CSF; p < 0.001), or nonneurologic controls (nondetectable CSF tau; p < 0.001). In head trauma, a relationship between clinical improvement and decreased CSF tau levels was observed. These data suggest that CSF tau levels may prove a clinically useful assay for quantifying the axonal injury associated with head trauma and monitoring efficacy of neuroprotective agents. Affinity purification of CSF tau from head trauma patients indicated a uniform cleavage of approximately 18 kDa from all six tau isoforms, reducing their apparent molecular sizes to 30-50 kDa. These cleaved forms of CSF tau consisted of the interior portion of the tau sequence, including the microtubule binding domain, as judged by cyanogen bromide digestion. Consistent with these data, CSF cleaved tau bound taxol-polymerized microtubules, indicating a functionally intact microtubule binding domain. Furthermore, epitope mapping studies suggested that CSF cleaved tau proteins consist of the interior portion of the tau sequence with cleavage at both N and C terminals.

Ultrastructural localization of SNAP-25 within the rat spinal cord and peripheral nervous system.

Synaptosomal associated protein of 25 kDa (SNAP-25) has been implicated in the membrane fusion machinery of neurotransmitter release and axonal growth. Using immunocytochemistry, we have analyzed the distribution and ultrastructural localization of SNAP-25 in selected areas of the central and peripheral nervous systems of adult rats. We show that the protein is specifically expressed in the trans face of the Golgi apparatus and in the axonal compartment. In axons and nerve endings, SNAP-25 is localized to discrete areas of the membranes of most organelles such as the axoplasmic reticulum, the axolemma, the outer membrane of mitochondria and synaptic vesicles. This wide distribution of SNAP-25 suggests that the protein is involved in the fusion of membranes in the whole axonal compartment of neurons.

MRna in the Axonal Compartment of Neurons

In the last three decades it has become evident that the axonal compartment of neurons harbours a variety of RNA molecules, such as transfer RNA, ribosomal RNA and messenger RNA. The occurrence of these molecules would allow the axonal domain to sustain extrasomal protein synthesis. In this review the data will be presented that led to this conclusion. In addition to the mRNAs encoding cytoskeletal proteins, in axons of many peptidergic neurons transcripts encoding the endogenous neuropeptides have been shown to be present. Until this moment no conclusive evidence has been presented on the function(s) of these neuropeptide encoding mRNAs.