Ying Jones

PPARγ Is Required for Placental, Cardiac, and Adipose Tissue Development

The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.

Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains

Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominately on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only ∼15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.

Disruption of dendritic translation of CaMKIIα impairs stabilization of synaptic plasticity and memory consolidation

Local protein translation in dendrites could be a means for delivering synaptic proteins to their sites of action, perhaps in a spatially regulated fashion that could contribute to plasticity. To directly test the functional role of dendritic translation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) in vivo, we mutated the endogenous gene to disrupt the dendritic localization signal in the mRNA. In this mutant mouse, the protein-coding region of CaMKIIalpha is intact, but mRNA is restricted to the soma. Removal of dendritic mRNA produced a dramatic reduction of CaMKIIalpha in postsynaptic densities (PSDs), a reduction in late-phase long-term potentiation (LTP), and impairments in spatial memory, associative fear conditioning, and object recognition memory. These results demonstrate that local translation is important for synaptic delivery of the kinase and that local translation contributes to synaptic and behavioral plasticity.

JNK1 Is Required for Maintenance of Neuronal Microtubules and Controls Phosphorylation of Microtubule-Associated Proteins

Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.

Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots

The importance of locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although light microscopy (LM) has been extensively used for mapping protein localization, many studies require the additional resolution of the electron microscope. Here we report the application of small nanocrystals (Quantum dots; QDs) to specifically and efficiently label multiple distinct endogenous proteins. QDs are both fluorescent and electron dense, facilitating their use for correlated microscopic analysis. Furthermore, QDs can be discriminated optically by their emission wavelength and physically by size, making them invaluable for multilabeling analysis. We developed pre-embedding labeling criteria using QDs that allows optimization at the light level, before continuing with electron microscopy (EM). We provide examples of double and triple immunolabeling using light, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.

Dicer and eIF2c are enriched at postsynaptic densities in adult mouse brain and are modified by neuronal activity in a calpain-dependent manner.

We have hypothesized that small RNAs may participate in learning and memory mechanisms. Because dendritic spines are important in synaptic plasticity and learning, we asked whether dicer, the rate‐limiting enzyme in the formation of small RNAs, is enriched within dendritic spines. In adult mouse brain, dicer and the RNA‐induced silencing complex (RISC) component eIF2c were expressed in the somatodendritic compartment of principal neurons and some interneurons in many regions, and dicer was enriched in dendritic spines and postsynaptic densities (PSDs). A portion of dicer and eIF2c were associated with each other and with fragile X mental retardation protein (FMRP), as assessed by co‐immunoprecipitation. Calpain I treatment of recombinant dicer or immunopurified brain dicer caused a marked increase in RNAse III activity. Purified PSDs did not exhibit RNAse III activity, but calpain caused release of dicer from PSDs in an enzymatically active form, together with eIF2c. NMDA stimulation of hippocampal slices, or calcium treatment of synaptoneurosomes, caused a 75 kDa dicer fragment to appear in a calpain‐dependent manner. The findings support a model whereby acute neuronal stimulation at excitatory synapses increases intracellular calcium, which activates calpain, which liberates dicer and eIF2c bound to PSDs. This supports the hypothesis that dicer could be involved in synaptic plasticity.

Modification of Postsynaptic Densities after Transient Cerebral Ischemia: A Quantitative and Three-Dimensional Ultrastructural Study

Abnormal synaptic transmission has been hypothesized to be a cause of neuronal death resulting from transient ischemia, although the mechanisms are not fully understood. Here, we present evidence that synapses are markedly modified in the hippocampus after transient cerebral ischemia. Using both conventional and high-voltage electron microscopy, we performed two- and three-dimensional analyses of synapses selectively stained with ethanolic phosphotungstic acid in the hippocampus of rats subjected to 15 min of ischemia followed by various periods of reperfusion. Postsynaptic densities (PSDs) from both area CA1 and the dentate gyrus were thicker and fluffier in postischemic hippocampus than in controls. Three-dimensional reconstructions of selectively stained PSDs created using electron tomography indicated that postsynaptic densities became more irregular and loosely configured in postischemic brains compared with those in controls. A quantitative study based on thin sections of the time course of PSD modification indicated that the increase in thickness was both greater and more long-lived in area CA1 than in dentate gyrus. Whereas the magnitude of morphological change in dentate gyrus peaked at 4 hr of reperfusion (140% of control values) and declined thereafter, changes in area CA1 persisted and increased at 24 hr of reperfusion (191% of control values). We hypothesize that the degenerative ultrastructural alteration of PSDs may produce a toxic signal such as a greater calcium influx, which is integrated from the thousands of excitatory synapses onto dendrites, and is propagated to the neuronal somata where it causes or contributes to neuronal damage during the postischemic phase.

Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function

The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication. During polar maturation, the PopZ‐centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. We propose that pole‐specific control of PopZ function co‐ordinates polar development and cell cycle progression by enabling independent assembly and tethering activities at the two cell poles.

Mimicking Melanosomes: Polydopamine Nanoparticles as Artificial Microparasols

A primary role of melanin in skin is the prevention of UV-induced nuclear DNA damage to human skin cells, where it serves to screen out harmful UV radiation. Melanin is delivered to keratinocytes in the skin after being excreted as melanosomes from melanocytes. Defects in melanin production in humans can cause diseases, many of which currently lack effective treatments due to their genetic origins (e.g., skin cancer, vitiligo, and albinism). The widespread prevalence of melanin-related diseases and an increasing interest in the performance of various polymeric materials related to melanin necessitates novel synthetic routes for preparing melanin-like materials. In this work, we prepared melanin-like nanoparticles (MelNPs) via spontaneous oxidation of dopamine, as biocompatible, synthetic analogues of naturally occurring melanosomes, and investigated their uptake, transport, distribution, and UV-protective capabilities in human keratinocytes. Critically, we demonstrate that MelNPs are endocytosed, undergo perinuclear aggregation, and form a supranuclear cap, or so-called microparasol in human epidermal keratinocytes (HEKa), mimicking the behavior of natural melananosomes in terms of cellular distribution and the fact that they serve to protect the cells from UV damage.

Subcellular localization and PKC-dependent regulation of the human lysophospholipase A/acyl-protein thioesterase in WISH cells.

Lysophospholipases play essential roles in keeping their multi-functional substrates, the lysophospholipids, at safe levels. Recently, a 25 kDa human lysophospholipase A (hLysoPLA I) that is highly conserved among rat, mouse, human and rabbit has been cloned, expressed and characterized and appears to hydrolyze only lysophospholipids among the various lipid substrates. Interestingly, this enzyme also displays acyl-protein thioesterase activity towards a G protein alpha subunit. To target the subcellular location of this hLysoPLA I, we have carried out immunocytochemical studies and report here that hLysoPLA I appears to be associated with the endoplasmic reticulum (ER) and nuclear envelope in human amnionic WISH cells and not the plasma membrane. In addition, we found that the hLysoPLA I can be up-regulated by phorbol 12-myristate 13-acetate (PMA) stimulation, a process in which phospholipase A(2) is activated and lysophospholipids are generated in WISH cells. Furthermore, the PMA-induced hLysoPLA I expression can be blocked by the protein kinase C (PKC) inhibitor Gö6976. The regulated expression of the LysoPLA/acyl-protein thioesterase by PKC may have important implications for signal transduction processes.