Laurence C. Cantrill

Antibodies to surface dopamine-2 receptor in autoimmune movement and psychiatric disorders

Recent reports of autoantibodies that bind to neuronal surface receptors or synaptic proteins have defined treatable forms of autoimmune encephalitis. Despite these developments, many cases of encephalitis remain unexplained. We have previously described a basal ganglia encephalitis with dominant movement and psychiatric disease, and proposed an autoimmune aetiology. Given the role of dopamine and dopamine receptors in the control of movement and behaviour, we hypothesized that patients with basal ganglia encephalitis and other putative autoimmune basal ganglia disorders harboured serum autoantibodies against important dopamine surface proteins. Basal ganglia encephalitis sera immunolabelled live surface cultured neurons that have high expression of dopamine surface proteins. To detect autoantibodies, we performed flow cytometry cell-based assays using human embryonic kidney cells to express surface antigens. Twelve of 17 children (aged 0.4-15 years, nine males) with basal ganglia encephalitis had elevated immunoglobulin G to extracellular dopamine-2 receptor, compared with 0/67 controls. Immunofluorescence on wild-type mouse brain showed that basal ganglia encephalitis sera immunolabelled microtubule-associated protein 2-positive neurons in striatum and also in cultured striatal neurons, whereas the immunolabelling was significantly decreased in dopamine-2 receptor knock-out brains. Immunocytochemistry confirmed that immunoreactivity localized to the surface of dopamine-2 receptor-transfected cells. Immunoabsorption of basal ganglia encephalitis sera on dopamine-2 receptor-transfected human embryonic kidney cells decreased immunolabelling of dopamine-2 receptor-transfected human embryonic kidney cells, neurons and wild-type mouse brain. Using a similar flow cytometry cell-based assay, we found no elevated immunoglobulin G binding to dopamine 1, 3 or 5 receptor, dopamine transporter or N-methyl-d-aspartate receptor. The 12 dopamine-2 receptor antibody-positive patients with encephalitis had movement disorders characterized by parkinsonism, dystonia and chorea. In addition, the patients had psychiatric disturbance with emotional lability, attention deficit and psychosis. Brain magnetic resonance imaging showed lesions localized to the basal ganglia in 50% of the patients. Elevated dopamine-2 receptor immunoglobulin G was also found in 10/30 patients with Sydenhams chorea, 0/22 patients with paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection and 4/44 patients with Tourettes syndrome. No dopamine-1 receptor immunoglobulin G was detected in any disease or control groups. We conclude that assessment of dopamine-2 receptor antibodies can help define autoimmune movement and psychiatric disorders.

Effects of chilling on male gametophyte development in rice

Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post‐meiotic anthers. Anthers observed during tetrad release also have callose (1,3‐β‐glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity.

A collagen-hydroxyapatite scaffold allows for binding and co-delivery of recombinant bone morphogenetic proteins and bisphosphonates.

An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system.

CELL-TO-CELL COMMUNICATION VIA PLANT ENDOMEMBRANES

Cell‐to‐cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3,3′‐dihexyloxacarbocyanine iodide (DiOC6). No cell‐to‐cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.

Cellular organisation in meiotic and early post-meiotic rice anthers

We have used fluorescent, confocal laser and transmission electron microscopy (TEM) to examine cellular organisations, including callose (1,3‐β‐glucan) behaviour, in meiotic and early post‐meiotic rice anthers. These features are critical for pollen formation and provide information to better understand pollen sterility caused by abiotic stress in rice and other monocotyledonous species. Among organelles during meiosis, abundant plastids, mitochondria and nuclei of the anther cells show distinctive features. Chloroplasts in the endothecium store starch and indicate a potential for photosynthetic activity. During meiosis, the middle layer cells are markedly compressed and at the tetrad stage are either vacuolated or filled with degenerating electron‐opaque organelles. Viable mitochondria, stained with Rhodamine 123, are seen in the endothecium and tapetum, but the mitochondria in the middle layer are not stained during meiosis. The radial walls of the tapetum are disorganised and degenerating, indicating the formation of a syncytium; pro‐orbicules are located at the locular walls at the tetrad stage. Immunohistochemical studies show that the sporogenous cells are entirely enveloped by a thick callosic layer at early meiosis. Cell plate callose was assembled in a plane between the dyad cells. In the tetrads, however, callose formed only at the centre, showing that the tetrad microspores are not enveloped but separated by callose walls. Thick, undulating electron‐opaque walls around the tetrads indicate the beginning of exinous microspore wall differentiation.

Chilling to zero degrees disrupts pollen formation but not meiotic microtubule arrays in Triticum aestivum L.

Throughout the wheat-growing regions of Australia, chilling temperatures below 2 °C occur periodically on consecutive nights during the period of floral development in spring wheat (Triticum aestivum L.). In this study, wheat plants showed significant reductions in fertility when exposed to prolonged chilling temperatures in controlled environment experiments. Among the cultivars tested, the Australian cultivars Kite and Hartog had among the lowest levels of seed set due to chilling and their responses were investigated further. The developmental stage at exposure, the chilling temperature and length of exposure all influenced the level of sterility. The early period of booting, and specifically the +4 cm auricle distance class, was the most sensitive and corresponded to meiosis within the anthers. The response of microtubules to chilling during meiosis in Hartog was monitored, but there was little difference between chilled and control plants. Other abnormalities, such as plasmolysis and cytomixis increased in frequency, were associated with death of developing pollen cells, and could contribute to loss of fertility. The potential for an above-zero chilling sensitivity in Australian spring wheat varieties could have implications for exploring the tolerance of wheat flower development to chilling and freezing conditions in the field.

Cellular organisation and differentiation of organelles in pre-meiotic rice anthers

Pre‐meiotic cellular organisation of rice anthers has a great significance in pollen formation. We have used a combination of confocal laser and transmission electron microscopy (TEM) to characterise and differentiate organelles in pre‐meiotic rice anthers. Along with the characteristic organelles in the cytoplasm the epidermal cells of the pre‐meiotic rice anther are coated on their outer surface by a conspicuous bi‐lamellate cuticle. Chloroplasts of the endothecium contain immature grana, thylakoids and also starch granules. These plastids clearly contain photosynthetic pigments as shown by autofluorescence in confocal microscope studies. Both confocal and TEM studies reveal clusters of mitochondria in the middle layer. The tapetum contains electron opaque ribosomes, bundles of mitochondria and plastids. The nuclei of the tapetum occupy a large volume of the cytoplasm indicating the onset of mitotic prophase. Intense Rhodamine 123 staining reveals that a major portion of the structurally indistinguishable organelles that were seen throughout the densely ribosomic cytoplasm of sporogenous cells are mitochondria.

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

To genuinely understand how complex biological structures function, we must integrate knowledge of their dynamic behavior and of their molecular machinery. The combined use of light or laser microscopy and electron microscopy has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, is often referred to as ‘correlative microscopy’. Correlative imaging allows researchers to gain additional novel structure–function information, and such information provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or ‘combined’) microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches. In this topical review, we provide a brief historical perspective of correlative microscopy and an in-depth overview of correlative sample-preparation and imaging methods presently available, including future perspectives on the trend towards integrative microscopy and microanalysis.

MeCP2 deficiency is associated with reduced levels of tubulin acetylation and can be restored using HDAC6 inhibitors

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by loss of function mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Despite the genetic cause being known in the majority of cases, the pathophysiology of the neurological phenotype of RTT is largely unknown. Tubulin and the microtubule network play an essential role in neuronal function whereby the acetylation state of microtubules dictates the efficiency of neuronal migration and differentiation, synaptic targeting and molecular motor trafficking of mRNA, high-energy mitochondria and brain-derived neurotrophic factor (BDNF)-containing vesicles. Recent reports have shown perturbations in tubulin and microtubule dynamics in MeCP2-deficient cells, suggesting a link between the aberrations of these cellular entities and the neurobiology of RTT. We have interrogated the functional state of the microtubule network in fibroblasts derived from two patients with RTT as well as cortical neurons from a RTT mouse model and observed a reduction in acetylated α-tubulin and an increase in the tubulin-specific deacetylase, histone deacetylase 6 (HDAC6). Furthermore, we show that inhibition of HDAC6 by Tubastatin A can restore tubulin acetylation levels. We also demonstrate microtubule instability in the RTT patient fibroblasts in response to nocodazole, which is progressively ameliorated in a mutation-dependent manner by Tubastatin A. We conclude that Tubastatin A is capable of counteracting the microtubule defects observed in MeCP2-deficient cells, which could in turn lead to the restoration of molecular trafficking along the microtubules and thus could be a potentially new therapeutic option for RTT.Key messageCells from MeCP2-deficient cells show reduced levels of acetylated α-tubulin.Cells from two patients and a RTT mouse model have increased levels of HDAC6 but not sirtuin 2 (SIRT2).Inhibition of HDAC6 by Tubastatin A increases the in vitro acetylation of α-tubulin.Inhibition of HDAC6 by Tubastatin A does not increase MECP2 expression.Cells from two patients show microtubule instability, which is ameliorated by Tubastatin A.

PTH(1-34) Treatment Increases Bisphosphonate Turnover in Fracture Repair in Rats.

Bisphosphonates (BP) are antiresorptive drugs with a high affinity for bone. Despite the therapeutic success in treating osteoporosis and metabolic bone diseases, chronic BP usage has been associated with reduced repair of microdamage and atypical femoral fracture (AFF). The latter has a poor prognosis, and although anabolic interventions such as teriparatide (PTH(1–34)) have been suggested as treatment options, there is a limited evidence base in support of their efficacy. Because PTH(1–34) acts to increase bone turnover, we hypothesized that it may be able to increase BP in turnover in the skeleton, which, in turn, may improve bone healing. To test this, we employed a mixture of fluorescent Alexa647‐labelled pamidronate (Pam) and radiolabeled 14C‐ZA (zoledronic acid). These traceable BPs were dosed to Wistar rats in models of normal growth and closed fracture repair. Rats were cotreated with saline or 25 μg/kg/d PTH(1–34), and the effects on BP liberation and bone healing were examined by X‐ray, micro‐CT, autoradiography, and fluorescent confocal microscopy. Consistent with increased BP remobilization with PTH(1–34), there was a significant decrease in fluorescence in both the long bones and in the fracture callus in treated animals compared with controls. This was further confirmed by autoradiography for 14C‐ZA. In this model of acute BP treatment, callus bone volume (BV) was significantly increased in fractured limbs, and although we noted significant decreases in callus‐bound BP with PTH(1–34), these were not sufficient to alter this BV. However, increased intracellular BP was noted in resorbing osteoclasts, confirming that, in principle, PTH(1–34) increases bone turnover as well as BP turnover.