Jennifer I. Luebke

Exocytotic Ca2+ channels in mammalian central neurons

Intracellular Ca2+ initiates physiological events as diverse as gene transcription, muscle contraction, cell division and exocytosis. Predictably, the metabolic machinery that elicits and responds to changes in intracellular Ca2+ is correspondingly heterogeneous. This review focuses on one element of this complex web that is of particular importance to neurobiologists: identifying which members of the voltage-dependent Ca(2+)-channel superfamily are responsible for the Ca2+ that enters nerve terminals and elicits vesicular release of chemical transmitters.

Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus

N-type calcium channels play a dominant role in controlling synaptic transmission in many peripheral neurons. Transmitter release from mammalian central nerve terminals, however, is relatively resistant to the N channel antagonist omega-conotoxin GVIA. We studied the sensitivity of glutamatergic synaptic transmission in rat hippocampal slices to omega-conotoxin and to omega-Aga-IVA, a P channel antagonist. Both toxins reduced the amplitude of excitatory postsynaptic potentials in CA1 pyramidal neurons, but omega-Aga-IVA was the more rapid and efficacious. These results were corroborated by biochemical studies measuring subsecond, calcium-dependent [3H]glutamate release from hippocampal synaptosomes. Thus, at least two calcium channel types trigger glutamate release from hippocampal neurons, but P-type plays a more prominent role. Eliminating synaptic transmission in the CNS, therefore, may require inhibiting more than a single calcium channel type.

Changes in the structural complexity of the aged brain

Structural changes of neurons in the brain during aging are complex and not well understood. Neurons have significant homeostatic control of essential brain functions, including synaptic excitability, gene expression, and metabolic regulation. Any deviations from the norm can have severe consequences as seen in aging and injury. In this review, we present some of the structural adaptations that neurons undergo throughout normal and pathological aging and discuss their effects on electrophysiological properties and cognition. During aging, it is evident that neurons undergo morphological changes such as a reduction in the complexity of dendrite arborization and dendritic length. Spine numbers are also decreased, and because spines are the major sites for excitatory synapses, changes in their numbers could reflect a change in synaptic densities. This idea has been supported by studies that demonstrate a decrease in the overall frequency of spontaneous glutamate receptor‐mediated excitatory responses, as well as a decrease in the levels of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid and N‐methyl‐d‐aspartate receptor expression. Other properties such as γ‐aminobutyric acid A receptor‐mediated inhibitory responses and action potential firing rates are both significantly increased with age. These findings suggest that age‐related neuronal dysfunction, which must underlie observed decline in cognitive function, probably involves a host of other subtle changes within the cortex that could include alterations in receptors, loss of dendrites, and spines and myelin dystrophy, as well as the alterations in synaptic transmission. Together these multiple alterations in the brain may constitute the substrate for age‐related loss of cognitive function.

Depletion of microglia and inhibition of exosome synthesis halt tau propagation

Accumulation of pathological tau protein is a major hallmark of Alzheimers disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target.

Synapses are lost during aging in the primate prefrontal cortex

An electron microscopic analysis has been carried out on the effects of age on the numerical density of both excitatory (asymmetric) and inhibitory (symmetric) synapses in the neuropil of layers 2/3 and of layer 5 in area 46 from the frontal cortex of behaviorally tested rhesus monkeys. There is no change in the lengths of synaptic junctions with age or in the percentage distribution of synapses relative to the postsynaptic spines and dendritic shafts. However, in layers 2/3 there is an overall loss of about 30% of synapses from 5 to 30 years of age, and both asymmetric and symmetric synapses are lost at the same rate. In layer 5 the situation is different; the overall loss of synapses is only 20% and this is almost entirely due to a loss of asymmetric synapses, since there is no significant loss of symmetric synapses from this layer with age. When the synapse data are correlated with the overall cognitive impairment shown by the monkeys, it is found that there is a strong correlation between the numerical density of asymmetric synapses in layers 2/3 and cognitive impairment, with a weaker correlation between symmetric synapse loss and cognitive impairment. In layer 5 on the other hand there is no correlation between synapse loss and cognitive impairment. However synapse loss is not the only factor causing cognitive impairment, since in previous studies of area 46 we have found that age-related alteration in myelin in this frontal area also significantly contributes to cognitive decline. The synapse loss is also considered in light of earlier studies, which show that the frequency of spontaneous excitatory synaptic responses is reduced with age in layers 2/3 neurons.

Tau Accumulation Causes Mitochondrial Distribution Deficits in Neurons in a Mouse Model of Tauopathy and in Human Alzheimer's Disease Brain

Neurofibrillary tangles (NFT), intracellular inclusions of abnormal fibrillar forms of microtubule associated protein tau, accumulate in Alzheimers disease (AD) and other tauopathies and are believed to cause neuronal dysfunction, but the mechanism of tau-mediated toxicity are uncertain. Tau overexpression in cell culture impairs localization and trafficking of organelles. Here we tested the hypothesis that, in the intact brain, changes in mitochondrial distribution occur secondary to pathological changes in tau. Array tomography, a high-resolution imaging technique, was used to examine mitochondria in the reversible transgenic (rTg)4510, a regulatable transgenic, mouse model and AD brain tissue. Mitochondrial distribution is progressively disrupted with age in rTg4510 brain, particularly in somata and neurites containing Alz50-positive tau aggregates. Suppression of soluble tau expression with doxycycline resulted in complete recovery of mitochondrial distribution, despite the continued presence of aggregated tau. The effect on mitochondrial distribution occurs without concomitant alterations in neuropil mitochondrial size, as assessed by both array tomography and electron microscopy. Similar mitochondrial localization alterations were also observed in human AD tissue in Alz50+ neurons, confirming the relevance of tau to mitochondrial trafficking observed in this animal model. Because abnormalities reverted to normal if soluble tau was suppressed in rTg4510 mice, even in the continued presence of fibrillar tau inclusions, we suggest that soluble tau plays an important role in mitochondrial abnormalities, which likely contribute to neuronal dysfunction in AD.

Normal aging results in decreased synaptic excitation and increased synaptic inhibition of layer 2/3 pyramidal cells in the monkey prefrontal cortex

Executive system function, mediated largely by the prefrontal cortex (PFC), often declines significantly with normal aging in humans and non-human primates. The neural substrates of this decline are unknown, but age-related changes in the structural properties of PFC neurons could lead to altered synaptic signaling and ultimately to PFC dysfunction. The present study addressed this issue using whole-cell patch clamp assessment of excitatory and inhibitory postsynaptic currents (PSCs) in layer 2/3 pyramidal cells in in vitro slices of the PFC from behaviorally characterized young (< or =12 years old) and aged (> or =19 years old) rhesus monkeys. Behaviorally, aged monkeys were significantly impaired in performance on memory and executive system function tasks. Physiologically, the frequency of spontaneous glutamate receptor-mediated excitatory PSCs was significantly reduced in cells from aged monkeys, while the frequency of spontaneous GABAA receptor-mediated inhibitory PSCs was significantly increased. In contrast, there was no effect of age on the frequency, amplitude, rise time or decay time of action potential-independent miniature excitatory and inhibitory PSCs. The observed change in excitatory-inhibitory synaptic balance likely leads to significantly altered signaling properties of layer 2/3 pyramidal cells in the PFC with age.

Sensory neuron N-type calcium currents are inhibited by both voltage-dependent and -independent mechanisms

The voltage dependence of γ-aminobutyric-acid- and norepinephrine-induced inhibition of N-type calcium current in cultured embryonic chick dorsal-root ganglion neurons was studied with whole-cell voltage-clamp recording. The inhibitory action of the neurotransmitters was comprised of at least two distinct modulatory components, which were separable on the basis of their differential voltage dependence. The first component, which we term “kinetic slowing”, is associated with a slowing of the activation kinetics — an effect that subsides during a test pulse. The kinetic-slowing component is largely reversed at depolarized voltages (i.e., it is voltage-dependent). The second component, which we term “steady-state inhibition”, is by definition not associated with a change in activation kinetics and is present throughout the duration of a test pulse. The steady-state inhibition is not reversed at depolarized voltages (i.e., it is voltage-independent). Although the two components can be separated on the basis of their voltage dependence, they appear to be indistinguishable in their time courses for onset and recovery as well as their rates of desensitization following multiple applications of transmitter. Furthermore, neither component requires cell dialysis, as both are observed using perforated-patch as well as whole-cell recording configurations. The co-existence in nerve terminals of both voltage-dependent and -independent mechanisms to modulate calcium channel function could offer a means of differentially controlling synaptic transmission under conditions of low- and high-frequency presynaptic discharge.

The intersection of amyloid beta and tau in glutamatergic synaptic dysfunction and collapse in Alzheimer's disease

The synaptic connections that form between neurons during development remain plastic and able to adapt throughout the lifespan, enabling learning and memory. However, during aging and in particular in neurodegenerative diseases, synapses become dysfunctional and degenerate, contributing to dementia. In the case of Alzheimers disease (AD), synapse loss is the strongest pathological correlate of cognitive decline, indicating that synaptic degeneration plays a central role in dementia. Over the past decade, strong evidence has emerged that oligomeric forms of amyloid beta, the protein that accumulates in senile plaques in the AD brain, contribute to degeneration of synaptic structure and function. More recent data indicate that pathological forms of tau protein, which accumulate in neurofibrillary tangles in the AD brain, also cause synaptic dysfunction and loss. In this review, we will present the case that soluble forms of both amyloid beta and tau protein act at the synapse to cause neural network dysfunction, and further that these two pathological proteins may act in concert to cause synaptic pathology. These data may have wide-ranging implications for the targeting of soluble pathological proteins in neurodegenerative diseases to prevent or reverse cognitive decline.

The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klothos downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.