Maria Gabriela Sánchez

Inefficient clearance of myelin debris by microglia impairs remyelinating processes

Lampron et al. use a cuprizone mouse model of demyelination/remyelination to show that in CX3CR1-deficient mice, the clearance of myelin debris by microglia is impaired, affecting the integrity of axon and myelin sheaths.

Steroids-Dopamine Interactions in the Pathophysiology and Treatment of CNS Disorders: Steroids-Dopamine Interactions in CNS Disorders

Introduction: Dopamine cell loss is well documented in Parkinsons disease and dopamine hypofunction is proposed in certain depressive states. At the opposite, dopamine hyperactivity is an enduring theory in schizophrenia with extensive supporting evidence. Aims: This article reviews the sex differences in these diseases that are the object of many studies and meta‐analyses and could be explained by genetic differences but also an effect of steroids in the brain. This article then focuses on the extensive literature reporting on the effect of estrogens in these diseases and effects of the other ovarian hormone progesterone as well as androgens that are less documented. Moreover, dehydroepiandrosterone, the precursor of estrogens and androgens, shows effects on brain dopamine neurotransmission that are reviewed. To investigate the mechanisms implicated in the human findings, animal studies are reviewed showing effects of estrogens, progesterone, and androgens on various markers of dopamine neurotransmission under intact as well as lesioned conditions. Discussion: For possible future avenues for hormonal treatments in these central nervous system diseases, we discuss the effects of selective estrogen receptor modulators (SERMs), the various estrogen receptors and their specific drugs as well as progesterone drugs. Conclusion: Clinical and experimental evidence supports a role of steroid–dopamine interactions in the pathophysiology of schizophrenia, depression and Parkinsons disease. Specific steroidal receptor agonists and SERMs are available for endocrine and cancer treatments and could find other applications as adjunct treatments in central nervous system diseases.

Dark microglia: A new phenotype predominantly associated with pathological states.

The past decade has witnessed a revolution in our understanding of microglia. These immune cells were shown to actively remodel neuronal circuits, leading to propose new pathogenic mechanisms. To study microglial implication in the loss of synapses, the best pathological correlate of cognitive decline across chronic stress, aging, and diseases, we recently conducted ultrastructural analyses. Our work uncovered the existence of a new microglial phenotype that is rarely present under steady state conditions, in hippocampus, cerebral cortex, amygdala, and hypothalamus, but becomes abundant during chronic stress, aging, fractalkine signaling deficiency (CX3CR1 knockout mice), and Alzheimers disease pathology (APP‐PS1 mice). Even though these cells display ultrastructural features of microglia, they are strikingly distinct from the other phenotypes described so far at the ultrastructural level. They exhibit several signs of oxidative stress, including a condensed, electron‐dense cytoplasm and nucleoplasm making them as “dark” as mitochondria, accompanied by a pronounced remodeling of their nuclear chromatin. Dark microglia appear to be much more active than the normal microglia, reaching for synaptic clefts, while extensively encircling axon terminals and dendritic spines with their highly ramified and thin processes. They stain for the myeloid cell markers IBA1 and GFP (in CX3CR1‐GFP mice), and strongly express CD11b and microglia‐specific 4D4 in their processes encircling synaptic elements, and TREM2 when they associate with amyloid plaques. Overall, these findings suggest that dark microglia, a new phenotype that we identified based on their unique properties, could play a significant role in the pathological remodeling of neuronal circuits, especially at synapses. GLIA 2016;64:826–839

Oestrogen Receptors and Signalling Pathways: Implications for Neuroprotective Effects of Sex Steroids in Parkinson's Disease

Parkinson’s disease (PD) is an age‐related neurodegenerative disorder with a higher incidence in the male population. In the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD, 17β‐oestradiol but not androgens were shown to protect dopamine (DA) neurones. We report that oestrogen receptors (ER)α and β distinctly contribute to neuroprotection against MPTP toxicity, as revealed by examining the membrane DA transporter (DAT), the vesicular monoamine transporter 2 (VMAT2) and tyrosine hyroxylase in ER wild‐type (WT) and knockout (ERKO) C57Bl/6 male mice. Intact ERKOβ mice had lower levels of striatal DAT and VMAT2, whereas ERKOα mice were the most sensitive to MPTP toxicity compared to WT and ERKOβ mice and had the highest levels of plasma androgens. In both ERKO mice groups, treatment with 17β‐oestradiol did not provide neuroprotection against MPTP, despite elevated plasma 17β‐oestradiol levels. Next, the recently described membrane G protein‐coupled oestrogen receptor (GPER1) was examined in female Macaca fascicularis monkeys and mice. GPER1 levels were increased in the caudate nucleus and the putamen of MPTP‐monkeys and in the male mouse striatum lesioned with methamphetamine or MPTP. Moreover, neuroprotective mechanisms in response to oestrogens transmit via Akt/glycogen synthase kinase‐3 (GSK3) signalling. The intact and lesioned striata of 17β‐oestradiol treated monkeys, similar to that of mice, had increased levels of pAkt (Ser 473)/βIII‐tubulin, pGSK3 (Ser 9)/βIII‐tubulin and Akt/βIII‐tubulin. Hence, ERα, ERβ and GPER1 activation by oestrogens is imperative in the modulation of ER signalling and serves as a basis for evaluating nigrostriatal neuroprotection.

Striatal Neurons Expressing D1 and D2 Receptors are Morphologically Distinct and Differently Affected by Dopamine Denervation in Mice

The loss of nigrostriatal dopamine neurons in Parkinson’s disease induces a reduction in the number of dendritic spines on medium spiny neurons (MSNs) of the striatum expressing D1 or D2 dopamine receptor. Consequences on MSNs expressing both receptors (D1/D2 MSNs) are currently unknown. We looked for changes induced by dopamine denervation in the density, regional distribution and morphological features of D1/D2 MSNs, by comparing 6-OHDA-lesioned double BAC transgenic mice (Drd1a-tdTomato/Drd2-EGFP) to sham-lesioned animals. D1/D2 MSNs are uniformly distributed throughout the dorsal striatum (1.9% of MSNs). In contrast, they are heterogeneously distributed and more numerous in the ventral striatum (14.6% in the shell and 7.3% in the core). Compared to D1 and D2 MSNs, D1/D2 MSNs are endowed with a smaller cell body and a less profusely arborized dendritic tree with less dendritic spines. The dendritic spine density of D1/D2 MSNs, but also of D1 and D2 MSNs, is significantly reduced in 6-OHDA-lesioned mice. In contrast to D1 and D2 MSNs, the extent of dendritic arborization of D1/D2 MSNs appears unaltered in 6-OHDA-lesioned mice. Our data indicate that D1/D2 MSNs in the mouse striatum form a distinct neuronal population that is affected differently by dopamine deafferentation that characterizes Parkinson’s disease.

Oestradiol modulation of serotonin reuptake transporter and serotonin metabolism in the brain of monkeys.

Serotonin (5‐hydroxytryptamine; 5‐HT) is an important brain neurotransmitter that is implicated in mental and neurodegenerative diseases and is modulated by ovarian hormones. Nevertheless, the effect of oestrogens on 5‐HT neurotransmission in the primate caudate nucleus, putamen and nucleus accumbens, which are major components of the basal ganglia, and the anterior cerebral cortex, mainly the frontal and cingulate gyrus, is not well documented. The present study evaluated 5‐HT reuptake transporter (SERT) and 5‐HT metabolism in these brain regions in response to 1‐month treatment with 17β‐oestradiol in short‐term (1 month) ovariectomised (OVX) monkeys (Macaca fascicularis). SERT‐specific binding was measured by autoradiography using the radioligand [3H]citalopram. Biogenic amine concentrations were quantified by high‐performance liquid chromatography. 17β‐Oestradiol increased SERT in the superior frontal cortex and in the anterior cingulate cortex, in the nucleus accumbens, and in subregions of the caudate nucleus of OVX monkeys. 17β‐Oestradiol left [3H]citalopram‐specific binding unchanged in the putamen, as well as the dorsal and medial raphe nucleus. 17β‐Oestradiol treatment decreased striatal concentrations of the precursor of 5‐HT, 5‐hydroxytryptophan, and increased 5‐HT, dopamine and 3‐methoxytyramine concentrations in the nucleus accumbens, caudate nucleus and putamen, whereas the concentrations of the metabolites 5‐hydroxyindoleacetic acid, 3,4‐dihydroxyphenylacetic acid and homovanillic acid remained unchanged. No effect of 17β‐oestradiol treatment was observed for biogenic amine concentrations in the cortical regions. A significant positive correlation was observed between [3H]citalopram‐specific binding and 5‐HT concentrations in the caudate nucleus, putamen and nucleus accumbens, suggesting their link. These results have translational value for women with low oestrogen, such as those in surgical menopause or perimenopause.

Effect of a chronic treatment with 17β-estradiol on striatal dopamine neurotransmission and the Akt/GSK3 signaling pathway in the brain of ovariectomized monkeys

The present experiments sought the effect of chronic treatment with 17β-estradiol on striatal dopaminergic activity and the Akt/GSK3 signaling pathway in the brain of monkeys. Eight female monkeys (Macacca fascicularis) were ovariectomized (OVX) and a month later, half received a month treatment with 17β-estradiol and the other with vehicle. The DA transporter (DAT) was measured by autoradiography with [(125)I]RTI-121 and the vesicular DA transporter (VMAT(2)) with [(3)H]TBZ-OH at three rostro-caudal levels (anterior, middle and posterior) of the caudate nucleus and putamen subdivided in their lateral/medial, ventral/dorsal sub-regions. Specific binding to DAT was increased in all sub-regions of the caudate nucleus and the putamen of 17β-estradiol-treated compared to vehicle-treated monkeys whereas specific binding to VMAT(2) remained unchanged. We measured by Western blot the phosphorylated forms of Akt at serine 473 and threonine 308, GSK3β at serine 9 and tyrosine 216 and GSK3α at serine 21 in anterior, middle and posterior caudate nucleus and putamen. 17β-Estradiol treatment increased in all the caudate nucleus and putamen pAkt (Ser473)/βIII-tubulin, pGSK3β (Ser9)/βIII-tubulin and in putamen Akt/βIII-tubulin compared to vehicle-treated monkeys. In anterior and middle putamen, pAkt (Thr308)/βIII-tubulin was also increased in monkeys treated with 17β-estradiol. pGSK3β (Tyr216)/βIII-tubulin and pGSK3α (Ser21)/βIII-tubulin remained unchanged by the 17β-estradiol treatment. These results suggest that 17β-estradiol activates striatal DA neurotransmission in primates as reflected with increased DAT specific binding and downstream activation of Akt/GSK3 signaling. This supports a beneficial role of a chronic treatment with 17β-estradiol by increasing the activity of signaling pathways implicated in cell survival.

Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques.

A detailed protocol is provided here to identify amyloid Aβ plaques in brain sections from Alzheimers disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to β-pleated sheets found in dense core Aβ plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label β-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for Aβ (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing Aβ plaques at the right location. Importantly, Aβ plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

Estradiol and brain serotonin reuptake transporter in long-term ovariectomized parkinsonian monkeys

This study evaluated the effect of a one month 17β-estradiol treatment on brain serotonin (5-HT) reuptake transporter (SERT) in long-term ovariectomized (OVX) female monkeys (Macaca fascicularis) bearing a unilateral lesion with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injected directly into the left substantia nigra modeling Parkinson disease (PD). Ovariectomy and MPTP lesion were performed four years before the estrogen treatment to model postmenopausal PD patients. SERT was measured by autoradiography using the radioligand [(3)H]Citalopram. Specific binding to SERT decreased in anterior cerebral cortex, nucleus accumbens, caudate nucleus and putamen on the lesioned side of 17β-estradiol and vehicle-treated monkeys compared to the intact side. In caudate nucleus and putamen the lesioned-induced decrease of [(3)H]Citalopram specific binding was more extensive in anterior and middle than posterior parts. [(3)H]Citalopram specific binding was increased in the cortex anterior cingulate gyrus of monkeys treated with 17β-estradiol in both brain hemispheres and was unchanged in the other brain regions investigated including the raphe nucleus. Positive correlations between [(3)H]Citalopram specific binding and 5-HT as well as 5-HIAA concentrations (reported previously) were obtained in the caudate nucleus and putamen and a negative correlation between SERT binding and 5-HIAA/5-HT concentration ratio suggesting MPTP lesion-induced 5-HT neuronal loss and lower 5-HT neurotransmission controlling and decreasing SERT for homeostasis. 17β-estradiol treatment initiated four years after ovariectomy of monkeys modeling hormonal conditions of post-menopause shows that SERT still displays some responsiveness to estrogens as observed in the anterior cingulate cortex. These results support a role of estrogens in 5-HT activity in PD.

A dense cluster of D1+ cells in the mouse nucleus accumbens

The striatum is known to be largely composed of intermingled medium‐sized projection neurons expressing either the D1 or the D2 dopamine receptors. In the present study, we took advantage of the double BAC Drd1a‐TdTomato/Drd2‐GFP (D1/D2) transgenic mice to reveal the presence of a peculiar cluster of densely‐packed D1+ cells located in the shell compartment of the nucleus accumbens. This spherical cluster has a diameter of 110 µm and is exclusively composed by D1+ cells, which are all immunoreactive for the neuronal nuclear marker (NeuN). However, in contrast to other D1+ or D2+ striatal cells, those that form the accumbens cluster are devoid of calbindin (CB) and DARPP‐32, two faithful markers for striatal projection neurons. Using GAD‐GFP transgenic mice, we confirm the GABAergic nature of the D1+ clustered neurons. Intracellular injections from fixed brain slices indicate that these neurons are endowed with distinctive morphological features, including a small (5–6 µm), round cell body giving rise to a single primary dendrite that branches into two secondary processes. Single‐neuronal injections combined to electron microscopy reveal the existence of GAP junctions linking these D1+ cells. Based on their location, morphological characteristics and neurochemical phenotype, we conclude that the D1+ accumbens cluster form a highly compact group of small neurons distinct from the larger and more diffusely distributed D1+ or D2+ striatal projection neurons that surround it. This remarkable nucleus might play a crucial role in the limbic function of the murine striatum.