Myriam Heiman

A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types

The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. Using bacterial artificial chromosome (BAC) transgenic mice that express EGFP-tagged ribosomal protein L10a in defined cell populations, we have developed a methodology for affinity purification of polysomal mRNAs from genetically defined cell populations in the brain. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. We find that even two morphologically indistinguishable, intermixed subclasses of medium spiny neurons display vastly different translational profiles and present examples of the physiological significance of such differences. This genetically targeted translating ribosome affinity purification (TRAP) methodology is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.

Application of a Translational Profiling Approach for the Comparative Analysis of CNS Cell Types

Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, translating ribosome affinity purification (TRAP) permits comprehensive studies of translated mRNAs in genetically defined cell populations after physiological perturbations. To establish the generality of this approach, we present translational profiles for 24 CNS cell populations and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole-tissue microarray studies and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of 16 transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of central nervous system structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well-known and previously uncharacterized neural cell populations.

Inhibition of mTOR Signaling in Parkinson’s Disease Prevents l-DOPA–Induced Dyskinesia

Dyskinetic side effects of a Parkinson’s disease medication may involve dopamine D1 receptor–mediated activation of mTORC1. Dyskinesia Relief In its role as a regulator of cell growth, the mammalian complex of rapamycin (mTOR) phosphorylates several proteins involved in protein synthesis, such as 4E-BP (eukaryotic initiation factor 4E binding protein) and S6K (p70 S6 kinase), in response to growth factors and nutrient availability. Santini et al. show that l-DOPA, the most commonly used medication to alleviate the immobility and rigidity (akinesia) characteristic of Parkinson’s disease (PD), also stimulates the rapamycin-sensitive mTOR complex 1 (mTORC1). In a mouse model of PD, l-DOPA treatment increased phosphorylation of several direct and indirect mTOR targets, including S6K, its substrate ribosomal protein S6 (S6), 4E-BP, and eukaryotic initiation factor 4E (eIF4E). These phosphorylation increases required the activity of dopamine D1 receptors and extracellular signal–regulated kinase (ERK). Furthermore, increased phosphorylation of S6K, S6, 4E-BP, and eIF4E correlated with stronger abnormal involuntary movements (AIMs), a measure of dyskinesia (a side effect of l-DOPA that limits its clinical use). Administration of rapamycin, which predominantly inhibits mTORC1, decreased the severity of AIMs without affecting the ability of l-DOPA to reduce akinesia. Thus, the mTORC1 signaling pathway could be targeted in PD patients suffering from the dyskinesia associated with l-DOPA treatment. Parkinson’s disease (PD), a disorder caused by degeneration of the dopaminergic input to the basal ganglia, is commonly treated with l-DOPA. Use of this drug, however, is severely limited by motor side effects, or dyskinesia. We show that administration of l-DOPA in a mouse model of Parkinsonism led to dopamine D1 receptor–mediated activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is implicated in several forms of synaptic plasticity. This response occurred selectively in the GABAergic medium spiny neurons that project directly from the striatum to the output structures of the basal ganglia. The l-DOPA–mediated activation of mTORC1 persisted in mice that developed dyskinesia. Moreover, the mTORC1 inhibitor rapamycin prevented the development of dyskinesia without affecting the therapeutic efficacy of l-DOPA. Thus, the mTORC1 signaling cascade represents a promising target for the design of anti-Parkinsonian therapies.

L-DOPA activates ERK signaling and phosphorylates histone H3 in the striatonigral medium spiny neurons of hemiparkinsonian mice

In the dopamine‐depleted striatum, extracellular signal‐regulated kinase (ERK) signaling is implicated in the development of l‐DOPA‐induced dyskinesia. To gain insights on its role in this disorder, we examined the effects of l‐DOPA on the state of phosphorylation of ERK and downstream target proteins in striatopallidal and striatonigral medium spiny neurons (MSNs). For this purpose, we employed mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoters for the dopamine D2 receptor (Drd2‐EGFP mice) or the dopamine D1 receptor (Drd1a‐EGFP mice), which are expressed in striatopallidal and striatonigral MSNs, respectively. In 6‐hydroxydopamine‐lesioned Drd2‐EGFP mice, l‐DOPA increased the phosphorylation of ERK, mitogen‐ and stress‐activated kinase 1 and histone H3, selectively in EGFP‐negative MSNs. Conversely, a complete co‐localization between EGFP and these phosphoproteins was observed in Drd1a‐EGFP mice. The effect of l‐DOPA was prevented by blockade of dopamine D1 receptors. The same pattern of activation of ERK signaling was observed in dyskinetic mice, after repeated administration of l‐DOPA. Our results demonstrate that in the dopamine‐depleted striatum, l‐DOPA activates ERK signaling specifically in striatonigral MSNs. This regulation may result in ERK‐dependent changes in striatal plasticity leading to dyskinesia.

Regulation of Alzheimer's disease amyloid-β formation by casein kinase I

Alzheimers disease (AD) is associated with accumulation of the neurotoxic peptide amyloid-β (Aβ), which is produced by sequential cleavage of amyloid precursor protein (APP) by the aspartyl protease β-secretase and the presenilin-dependent protease γ-secretase. An increase of casein kinase 1 (CK1) expression has been described in the human AD brain. We show, by using in silico analysis, that APP, β-secretase, and γ-secretase subunits contain, in their intracellular regions, multiple CK1 consensus phosphorylation sites, many of which are conserved among human, rat, and mouse species. Overexpression of constitutively active CK1ε, one of the CK1 isoforms expressed in brain, leads to an increase in Aβ peptide production. Conversely, three structurally dissimilar CK1-specific inhibitors significantly reduced endogenous Aβ peptide production. By using mammalian cells expressing the β C-terminal fragment of APP, it was possible to demonstrate that CK1 inhibitors act at the level of γ-secretase cleavage. Importantly, Notch cleavage was not affected. Our results indicate that CK1 represents a therapeutic target for prevention of Aβ formation in AD.

Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).

Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian CNS at a molecular level. To address this problem, we have recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell type–specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell type–specific mRNA profiles of any genetically defined cell type, and it has been used in organisms ranging from Drosophila melanogaster to mice and human cultured cells. Unlike other methodologies that rely on microdissection, cell panning or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and the potential artifacts that these treatments introduce) and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implement the TRAP methodology, which takes 2 d to complete once all materials are in hand.

Molecular adaptations of striatal spiny projection neurons during levodopa-induced dyskinesia

Significance Parkinsons disease is characterized by a set of motor features that depend on a loss of dopamine-producing cells in the midbrain. The most common pharmacotherapy for Parkinsons disease is dopamine replacement with levodopa administration. The majority of patients receiving this treatment develop debilitating abnormal involuntary movements, termed “levodopa-induced dyskinesia.” It is known that striatal projection neurons (SPNs) are involved in the genesis of levodopa-induced dyskinesia, but the genes involved in this process are not fully understood. We reveal the gene-expression profiles of different classes of SPNs during chronic levodopa administration. We correlate gene expression to mouse behavior, predicting which genes are most likely involved in the emergence of levodopa-induced dyskinesia, and which are thus potential targets for new antidyskinetic treatments. Levodopa treatment is the major pharmacotherapy for Parkinsons disease. However, almost all patients receiving levodopa eventually develop debilitating involuntary movements (dyskinesia). Although it is known that striatal spiny projection neurons (SPNs) are involved in the genesis of this movement disorder, the molecular basis of dyskinesia is not understood. In this study, we identify distinct cell-type–specific gene-expression changes that occur in subclasses of SPNs upon induction of a parkinsonian lesion followed by chronic levodopa treatment. We identify several hundred genes, the expression of which is correlated with levodopa dose, many of which are under the control of activator protein-1 and ERK signaling. Despite homeostatic adaptations involving several signaling modulators, activator protein-1–dependent gene expression remains highly dysregulated in direct pathway SPNs upon chronic levodopa treatment. We also discuss which molecular pathways are most likely to dampen abnormal dopaminoceptive signaling in spiny projection neurons, hence providing potential targets for antidyskinetic treatments in Parkinsons disease.

Strain-Specific Regulation of Striatal Phenotype in Drd2-eGFP BAC Transgenic Mice

Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists, providing a powerful means of dissecting complex neural circuits in the brain. Recently, it was reported that one popular line of these mice—mice possessing a BAC transgene with a D2 dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter—had abnormal striatal gene expression, physiology, and motor behavior. Unlike most of the work using BAC mice, this interesting study relied upon mice backcrossed on the outbred Swiss Webster (SW) strain that were homozygous for the Drd2-eGFP BAC transgene. The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported, SW mice were very sensitive to transgene expression. However, in more commonly used inbred strains of mice (C57BL/6, FVB/N) that were hemizygous for the transgene, the Drd2-eGFP BAC transgene did not alter striatal gene expression, physiology, or motor behavior. Thus, the use of inbred strains of mice that are hemizygous for the Drd2 BAC transgene provides a reliable tool for studying basal ganglia function.

Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons

Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R- or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R- and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.

Normal aging induces A1-like astrocyte reactivity

Significance In aging, the brain becomes vulnerable to injury and cognitive function declines, but the mechanisms responsible are unknown. Astrocytes, the most abundant class of glial cells, are vital for the proper function of the central nervous system, and impairment of astrocyte function has been implicated in disease. Here we perform RNA sequencing of astrocytes from different brain regions across the lifespan of the mouse to identify age-related transcriptional changes that could contribute to cognitive decline. We find that aged astrocytes take on a reactive phenotype characteristic of neuroinflammatory reactive astrocytes, and that microglia play a role in inducing astrocyte activation. The aging astrocyte RNA sequencing profiles provide an important new resource for future studies exploring the role of astrocytes in cognitive decline. The decline of cognitive function occurs with aging, but the mechanisms responsible are unknown. Astrocytes instruct the formation, maturation, and elimination of synapses, and impairment of these functions has been implicated in many diseases. These findings raise the question of whether astrocyte dysfunction could contribute to cognitive decline in aging. We used the Bac-Trap method to perform RNA sequencing of astrocytes from different brain regions across the lifespan of the mouse. We found that astrocytes have region-specific transcriptional identities that change with age in a region-dependent manner. We validated our findings using fluorescence in situ hybridization and quantitative PCR. Detailed analysis of the differentially expressed genes in aging revealed that aged astrocytes take on a reactive phenotype of neuroinflammatory A1-like reactive astrocytes. Hippocampal and striatal astrocytes up-regulated a greater number of reactive astrocyte genes compared with cortical astrocytes. Moreover, aged brains formed many more A1 reactive astrocytes in response to the neuroinflammation inducer lipopolysaccharide. We found that the aging-induced up-regulation of reactive astrocyte genes was significantly reduced in mice lacking the microglial-secreted cytokines (IL-1α, TNF, and C1q) known to induce A1 reactive astrocyte formation, indicating that microglia promote astrocyte activation in aging. Since A1 reactive astrocytes lose the ability to carry out their normal functions, produce complement components, and release a toxic factor which kills neurons and oligodendrocytes, the aging-induced up-regulation of reactive genes by astrocytes could contribute to the cognitive decline in vulnerable brain regions in normal aging and contribute to the greater vulnerability of the aged brain to injury.