Self-assembled monolayers of a series of ω-(4‘-methyl-biphenyl-4-yl)-alkanethiols (CH3−C6H4−C6H4−(CH2) m −SH, m = 1−6) formed on polycrystalline gold and silver surfaces were characterized in detail by contact angle measurements, optical ellipsometry, X-ray photoelectron spectroscopy (XPS), reflection absorption infrared spectroscopy (IRRAS), and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The orientation of the biphenyl moiety, determined by combining the results from IRRAS and NEXAFS, exhibits a pronounced dependence on the number of methylene groups. Similar to n-alkanethiols an odd−even effect is observed which on silver is opposite to that on gold. For m = odd on gold and m = even on silver the arrangement of the aromatic moieties agrees well with the bulk structure of biphenyl, and the bonding of the thiols to the substrate is in agreement with an sp3 hybridization of the sulfur on gold and sp on silver, respectively. In the opposite case of m = even on gold and m = odd on silver, the biphenyl moieties adopt a significantly more canted orientation which, as a consequence, results in a lower coverage. The odd−even behavior of the coverage is in sharp contrast to that seen for n-alkanethiols. The experiments provide evidence that a significant driving force exists to pertain the sp3 and sp hybridization of sulfur on gold and silver, respectively. In the case of gold substrates the experimental results are in conflict with available bending potentials derived from ab initio calculations.

Background: Pathways involved in neuron-dependent GLT1 regulation in astrocytes remain to be characterized. Results: Neuronal microRNA 124a can be transferred into astrocytes through neuronal exosomes and significantly increases GLT1 protein expression in an indirect manner. Conclusion: Neuronal exosomal miRNA 124a is able to regulate astroglial GLT1 expression. Significance: We characterize a novel pathway in neuron-to-astrocyte communication and identify a microRNA that modulates GLT1 protein expression.

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Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1 + and GLT1 + cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1 + and GLT1 + astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.

SUMMARY The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia play a central role in the regulation of synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. The astroglial mechanisms underlying this essential neuron-glial communication are not known. Here we show that presynaptic terminals are sufficient and necessary for GLT1/EAAT2 transcriptional activation and have identified the molecular pathway that regulates astroglial responses to presynaptic input. Presynaptic terminals regulate astroglial GLT1/EAAT2 via kappa B-motif binding phosphoprotein (KBBP), the mouse homologue of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds to an essential element of GLT1/EAAT2 promoter. This neuron-stimulated factor is required for GLT1/EATT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling in vivo, by acute corticospinal tract transection, ricin-induced motor neuron death, or chronic neurodegeneration in amyotrophic lateral sclerosis (ALS) all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Our studies indicate that presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.

Astroglia play active and diverse roles in modulating neuronal/synaptic functions in the CNS. How these astroglial functions are regulated, especially by neuronal signals, remains largely unknown. Exosomes, a major type of extracellular vesicles (EVs) that originate from endosomal intraluminal vesicles (ILVs), have emerged as a new intercellular communication process. By generating cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) mice and immuno-EM/confocal image analysis, we found that neuronal CD63-GFP+ ILVs are primarily localized in soma and dendrites, but not in axonal terminals in vitro and in vivo. Secreted neuronal exosomes contain a subset of microRNAs (miRs) that is distinct from the miR profile of neurons. These miRs, especially the neuron-specific miR-124-3p, are potentially internalized into astrocytes. MiR-124-3p further up-regulates the predominant glutamate transporter GLT1 by suppressing GLT1-inhibiting miRs. Our findings suggest a previously undescribed neuronal exosomal miR-mediated genetic regulation of astrocyte functions, potentially opening a new frontier in understanding CNS intercellular communication.

The molecular signature and functional properties of astroglial subtypes in the adult CNS remain largely undefined. By using translational ribosome affinity purification followed by RNA-Seq, we profiled astroglial ribosome-associated (presumably translating) mRNAs in major cortical and subcortical brain regions (cortex, hippocampus, caudate-putamen, nucleus accumbens, thalamus, and hypothalamus) of BAC aldh1l1-translational ribosome affinity purification (TRAP) mice (both sexes). We found that the expression of astroglial translating mRNAs closely follows the dorsoventral axis, especially from cortex/hippocampus to thalamus/hypothalamus posteriorly. This region-specific expression pattern of genes, such as synaptogenic modulator sparc and transcriptional factors (emx2, lhx2, and hopx), was validated by qRT-PCR and immunostaining in brain sections. Interestingly, cortical or subcortical astrocytes selectively promote neurite growth and synaptic activity of neurons only from the same region in mismatched cocultures, exhibiting regionmatched astrocyte to neuron communication. Overall, these results generated new molecular signature of astrocyte types in the adult CNS, providing insights into their origin and functional diversity.

The low-energy electron-induced damage in self-assembled monolayers (SAMs) formed from ω-(4‘-methylbiphenyl-4-yl)alkanethiols CH3(C6H4)2(CH2) n SH (BPn, n = 0, 1, 4, 5, and 12) on gold substrates was studied. The pristine and heavily (8000 μC/cm2) irradiated films were characterized in detail by X-ray photoelectron spectroscopy, near-edge X-ray absorption fine structure spectroscopy, infrared reflection absorption spectroscopy, and advancing contact angle measurements. In contrast to SAMs of conventional alkanethiols but similar to pure aromatic thiol-derived systems, only minor damage is observed for the aliphatic−aromatic BPn films. In particular, the orientational order and anchoring to the substrate are retained upon the irradiation. At the same time, C−H bond scissions in the aromatic part occur, leading to a cross-linking between the neighboring biphenyl moieties. Whereas the general behavior of the BPn SAMs with respect to electron irradiation is qualitatively similar, the extent of the irradiation-induced changes depends on the packing of these systems. The densely packed BP1 and BP5 SAMs are much more stable with respect to electron bombardment than the less densely packed BP4 films. The relation between the packing density and the extent of the irradiation-induced changes seems to be a general phenomenon in monomolecular films, which provides a tool to tailor the reaction of these systems toward ionizing radiation for lithographic applications.