Yuko Hara

Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination. Myelin, a membranous sheet that surrounds the axons of neurons and facilitates the transmission of nerve impulses, is formed by oligodendrocytes in the central nervous system (CNS), and Schwann cells in the peripheral nervous system (PNS). Failure to myelinate, or demyelination, produces the symptoms of serious diseases such as leukodystrophies and multiple sclerosis. This study identifies a novel and essential regulator of myelin formation in the CNS: the transcriptional activator Krüppel-like factor-6 (Klf6). We show that Klf6 acts as a control point through which pro-myelinating signals regulate the development of oligodendrocytes from oligodendrocyte progenitor cells. Growth factors of the gp130 family are known to promote differentiation and survival of oligodendrocytes, via activation of the transcription factor Stat3, but the mechanistic details of how they do it remain unclear. Our findings show that Klf6 is induced in oligodendrocyte progenitors by gp130 growth factors, and promotes differentiation. Furthermore, oligodendrocyte progenitor cells in which Klf6 is inactivated do not mature, fail to form myelin, and eventually die. We also uncover that upon gp130 signaling, Klf6 activates the nuclear import factor importin-α5 (Impα5), a key controller of trafficking into the nucleus in the CNS. Notably, inactivation of Impα5 recapitulates the features of silencing Klf6, whereas Impα5 expression can partially rescue differentiation in Klf6-deficient oligodendrocyte progenitors. Finally, inactivation of Stat3 in oligodendrocyte progenitors produces defective Klf6 and Impα5 expression and myelination failure, strongly resembling the features produced by Klf6 inactivation. Collectively, these results establish a connection between gp130-Stat3 signaling and importin-based control of nuclear trafficking, which is essential for myelination in the CNS, and places Klf6 as a key coordinator of these events.

Humans and nonhuman primates are vulnerable to age- and menopause- related decline in working memory, a cognitive function reliant on area 46 of the dorsolateral prefrontal cortex (dlPFC). We showed previously that presynaptic mitochondrial number and morphology in monkey dlPFC neurons correlate with working memory performance. The current study tested the hypothesis that the types of synaptic connections these boutons form are altered with aging and menopause in rhesus monkeys and that these metrics may be coupled with mitochondrial measures and working memory. Using serial section electron microscopy, we examined the frequencies and characteristics of nonsynaptic, single-synaptic, and multisynaptic boutons (MSBs) in the dlPFC. In contrast to our previous observations in the monkey hippocampal dentate gyrus, where MSBs comprised ∼40% of boutons, the vast majority of dlPFC boutons were single-synaptic, whereas MSBs constituted a mere 10%. The frequency of MSBs was not altered by normal aging, but decreased by over 50% with surgical menopause induced by ovariectomy in aged monkeys. Cyclic estradiol treatment in aged ovariectomized animals restored MSB frequencies to levels comparable to young and aged premenopausal monkeys. Notably, the frequency of MSBs positively correlated with working memory scores, as measured by the average accuracy on the delayed response (DR) test. Furthermore, MSB incidence positively correlated with the number of healthy straight mitochondria in dlPFC boutons and inversely correlated with the number of pathological donut-shaped mitochondria. Together, our data suggest that MSBs are coupled to cognitive function and mitochondrial health and are sensitive to estrogen.SIGNIFICANCE STATEMENTMany aged menopausal individuals experience deficits in working memory, an executive function reliant on recurrent firing of prefrontal cortex (PFC) neurons. However, little is known about the organization of presynaptic inputs to these neurons and how they may be altered with aging and menopause. Multisynaptic boutons (MSBs) were of particular interest, because they form multiple synapses and can enhance coupling between presynaptic and postsynaptic neurons. We found that higher MSB frequency correlated with better working memory performance in rhesus monkeys. Additionally, aged surgically menopausal monkeys experienced a 50% loss of MSBs that was restored with cyclic estradiol treatment. Together, our findings suggest that hormone replacement therapy benefits cognitive aging, in part by retaining complex synaptic organizations in the PFC.

Estrogen facilitates higher cognitive functions by exerting effects on brain regions such as the prefrontal cortex and hippocampus. Estrogen induces spinogenesis and synaptogenesis in these two brain regions and also initiates a complex set of signal transduction pathways via estrogen receptors (ERs). Along with the classical genomic effects mediated by activation of ER α and ER β, there are membrane-bound ER α, ER β, and G protein-coupled estrogen receptor 1 (GPER1) that can mediate rapid nongenomic effects. All key ERs present throughout the body are also present in synapses of the hippocampus and prefrontal cortex. This review summarizes estrogen actions in the brain from the standpoint of their effects on synapse structure and function, noting also the synergistic role of progesterone. We first begin with a review of ER subtypes in the brain and how their abundance and distributions are altered with aging and estrogen loss (e.g., ovariectomy or menopause) in the rodent, monkey, and human brain. As there is much evidence that estrogen loss induced by menopause can exacerbate the effects of aging on cognitive functions, we then review the clinical trials of hormone replacement therapies and their effectiveness on cognitive symptoms experienced by women. Finally, we summarize studies carried out in nonhuman primate models of age- and menopause-related cognitive decline that are highly relevant for developing effective interventions for menopausal women. Together, we highlight a new understanding of how estrogen affects higher cognitive functions and synaptic health that go well beyond its effects on reproduction.

Significance: Human and nonhuman primates are vulnerable to age- and menopause-related decline in working memory, a cognitive function reliant on the energy-demanding excitation of prefrontal cortex (PFC) neurons. The number and strength of presynaptic boutons providing inputs to these PFC neurons regulate their excitability. We show that poor working memory in rhesus monkeys is associated with a higher incidence of presynaptic boutons harboring malformed, donut-shaped mitochondria that form abnormally small synaptic contacts. Surgically induced menopause results in working memory impairment and a concomitant increase in presynaptic donut-shaped mitochondria, both of which are reversed by estradiol treatment. Our findings suggest that hormone replacement therapy benefits cognitive aging, in part, by promoting mitochondrial and synaptic health in the PFC.

Rhesus monkeys provide a valuable model for studying the neurobiological basis of cognitive aging, because they are vulnerable to age-related memory decline in a manner similar to humans. In this study, young and aged monkeys were first tested on a well characterized recognition memory test (delayed nonmatching-to-sample; DNMS). Then, electron microscopic immunocytochemistry was performed to determine the subcellular localization of two proteins in the hippocampal dentate gyrus (DG): the GluA2 subunit of the glutamate AMPA receptor and the atypical protein kinase C ζ isoform (PKMζ). PKMζ promotes memory storage by regulating GluA2-containing AMPA receptor trafficking. Thus, we examined whether the distribution of GluA2 and PKMζ is altered with aging in DG axospinous synapses and whether it is coupled with memory deficits. Monkeys with faster DNMS task acquisition and more accurate recognition memory exhibited higher proportions of dendritic spines coexpressing GluA2 and PKMζ. These double-labeled spines had larger synapses, as measured by postsynaptic density area, than single-labeled and unlabeled spines. Within this population of double-labeled spines, aged monkeys compared with young expressed a lower density of synaptic GluA2 immunogold labeling, which correlated with lower recognition accuracy. Additionally, higher density of synaptic PKMζ labeling in double-labeled spines correlated with both faster task acquisition and better retention. Together, these findings suggest that age-related impairment in maintenance of GluA2 at the synapse in the primate hippocampus is coupled with memory deficits.