Arabidopsis cyclophilins direct intracellular transport of mobile mRNA via organelle hitchhiking.

Plants convert external cues into mobile mRNAs to synchronize meristematic differentiation with environmental dynamics. These mRNAs are selectively transported to intercellular pores, plasmodesmata (PD), for cell-to-cell movement. However, how plants recognize and deliver mobile mRNAs to PD remains unknown. Here we show that mobile mRNAs hitchhike on organelle trafficking to transport towards PD. Perturbed cytoskeleton organization or organelle trafficking severely disrupts the subcellular distribution of mobile mRNAs. Arabidopsis rotamase cyclophilins (ROCs), which are organelle-localized RNA-binding proteins, specifically bind mobile mRNAs on the surface of organelles to direct intracellular transport. Arabidopsis roc mutants exhibit phenotype alterations and disruptions in the transport of mobile mRNAs. These findings suggest that ROCs play a crucial role in facilitating the systemic delivery of mobile mRNAs. Our results highlight that an RNA-binding protein-mediated hitchhiking system is specifically recruited to orient plant mobile mRNAs for intercellular transport.

Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants.

BACKGROUND: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent protein-fused RBP and target mRNA conjugated with corresponding RNA recognition sequences allows for visualizing mRNAs by confocal microscopy. To minimize the background fluorescence in the cytosol, the nuclear localization sequence has been used to sequester the RBP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the visualization of nucleus-localized RNA and sometimes may interfere in detecting fluorescence signals in the cytosol, especially in cells with low signal-to-noise ratio. RESULTS: We eliminated the background fluorescence in the nucleus by using the split fluorescent protein-based approach. We fused two different RBPs with the N- or C-terminus of split fluorescent proteins (FPs). Co-expression of RBPs with the target mRNA conjugated with the corresponding RNA recognition sequences can bring split FPs together to reconstitute functional FPs for visualizing target mRNAs. We optimized the system with minimal background fluorescence and used the imaging system to visualize mRNAs in living plant cells. CONCLUSIONS: We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells.

In Vivo Imaging of Mobile mRNAs in Plants Using MS2.

Multicellular organisms rely on systemic signals to orchestrate diverse developmental and physiological programs. To transmit environmental stimuli that perceived in the leaves, plants recruit many mobile molecules including mobile mRNAs as systemic signals for interorgan communication. The mobile mRNAs provide an efficient and specific remote control system for plants to cope with environmental dynamics. Upon being transcribed in local tissues, mobile mRNAs are selectively targeted to plasmodesmata for cell-to-cell and long-distance translocation. The mRNA labeling system based on the RNA-binding protein MS2 provides a useful tool to investigate intracellular trafficking of mobile mRNAs in plants. Here we describe the detailed protocol to visualize intracellular trafficking of plant mobile mRNAs by using the MS2 live-cell imaging system.

Mobility of Antiflorigen and PEBP mRNAs in Tomato-Tobacco Heterografts.

Photoperiodic floral induction is controlled by the leaf-derived and antagonistic mobile signals florigen and antiflorigen. In response to photoperiodic variations, florigen and antiflorigen are produced in leaves and translocated through phloem to the apex, where they counteract floral initiation. Florigen and antiflorigen are encoded by a pair of homologs belonging to FLOWERING LOCUS T (FT)- or TERMINAL FLOWER1 (TFL1)-like clades in the phosphatidylethanolamine-binding domain protein (PEBP) family. The PEBP gene family contains FT-, TFL1-, and MOTHER OF FT AND TFL1 (MFT)-like clades. Evolutionary analysis suggests that FT- and TFL1-like clades arose from an ancient MFT-like clade. The protein movement of the PEBP family is an evolutionarily conserved mechanism in many plants; however, the mRNA movement of the PEBP family remains controversial. Here, we examined the mRNA movement of PEBP genes in different plant species. We identified a tobacco (Nicotiana sylvestris) CENTRORADIALIS-like1 gene, denoted NsCET1, and showed that NsCET1 is an ortholog of the Arabidopsis (Arabidopsis thaliana) antiflorigen ATC In tobacco, NsCET1 acts as a mobile molecule that non-cell-autonomously inhibits flowering. Grafting experiments showed that endogenous and ectopically expressed NsCET1 mRNAs move long distances in tobacco and Arabidopsis. Heterografts of tobacco and tomato (Solanum lycopersicum) showed that, in addition to NsCET1, multiple members of the FT-, TFL1-, and MFT-like clades of tobacco and tomato PEBP gene families are mobile mRNAs. Our results suggest that the mRNA mobility is a common feature of the three clades of PEBP-like genes among different plant species.

Selective Targeting of Mobile mRNAs to Plasmodesmata for Cell-to-Cell Movement.

Many plant mRNAs move from cell to cell or long distance to execute non-cell-autonomous functions. These mobile mRNAs traffic through the phloem to regulate many developmental processes, but despite the burgeoning discovery of mobile mRNAs, little is known about the mechanism underlying the intracellular sorting of these mRNAs. Here, we exploited a fluorescence-based mRNA labeling system, using the bacteriophage coat protein MS2, fused to GFP (MS2-GFP) and an MS2 recognition site in the RNA of interest, to visualize the intracellular trafficking of mobile mRNAs in living plant cells of Nicotiana benthamiana We first improved this system by using the nuclear localization sequence from FD, which substantially reduced the fluorescent background of MS2-GFP in the cytoplasm. The modified system allowed us to observe the cytoplasmic fluorescent foci dependent on MS2-binding sites. Coexpressing the MS2-GFP system with a virus movement protein, which is a plasmodesmata (PD)-localized nonspecific RNA-binding protein, targeted cytoplasmic fluorescent foci to the PD, suggesting that the cytoplasmic fluorescent foci contain mRNA and MS2-GFP. Our ex vivo RNA imaging revealed that mobile but not nonmobile mRNAs were selectively targeted to PD. Real-time images of intracellular translocation revealed that the translocation of mRNA and organelles in the transvacuolar strands may be governed by the same mechanism. Our study suggests that PD targeting of mRNA is a selective step in determining mRNA cell-to-cell movement of mRNAs.

Sudomotor innervation in transthyretin amyloid neuropathy: Pathology and functional correlates.

OBJECTIVE: Autonomic neuropathy is a major component of familial amyloid polyneuropathy (FAP) due to mutated transthyretin, with sudomotor failure as a common manifestation. This study aimed to investigate the pathology and clinical significance of sudomotor denervation. METHODS: Skin biopsies were performed on the distal leg of FAP patients with a follow-up duration of 3.8 ± 1.6 years. Sudomotor innervation was stained with 2 markers: protein gene product 9.5 (PGP 9.5), a general neuronal marker, and vasoactive intestinal peptide (VIP), a sudomotor nerve functional marker, followed by quantitation according to sweat gland innervation index (SGII) for PGP 9.5 (SGIIPGP 9.5) and VIP (SGIIVIP). RESULTS: There were 28 patients (25 men) with Ala97Ser transthyretin and late onset (59.9 ± 6.0 years) disabling neuropathy. Autonomic symptoms were present in 22 patients (78.6%) at the time of skin biopsy. The SGIIPGP 9.5 and SGIIVIP of FAP patients were significantly lower than those of age- and gender-matched controls. The reduction of SGIIVIP was more severe than that of SGIIPGP 9.5 (p = 0.002). Patients with orthostatic hypotension or absent sympathetic skin response at palms were associated with lower SGIIPGP 9.5 (p = 0.019 and 0.002, respectively). SGIIPGP 9.5 was negatively correlated with the disability grade at the time of skin biopsy (p = 0.004), and was positively correlated with the interval from the time of skin biopsy to the time of wheelchair usage (p = 0.029). INTERPRETATION: This study documented the pathological evidence of sudomotor denervation in FAP. SGIIPGP 9.5 was functionally correlated with autonomic symptoms, autonomic tests, ambulation status, and progression of disability.

Effect of glycemic control on sudomotor denervation in type 2 diabetes.

OBJECTIVE: Sudomotor symptoms are a common component of diabetic autonomic neuropathy, but the pathology of sudomotor innervation and its relationship with glycemic control have remained obscured. RESEARCH DESIGN AND METHODS: We enrolled 42 patients (26 males and 16 females aged 56.64 ± 12.67 years) with diabetic neuropathy defined by symmetric distally predominant sensory symptoms, abnormal nerve conduction studies, and reduced intraepidermal nerve fiber density in the leg. Skin biopsies of the distal leg were immunostained with antiprotein gene product 9.5 for nerve fibers and counterstained with Congo red for sweat glands. Sweat gland innervation index (SGII) was quantified with a new computerized area-based morphometric system. RESULTS: Protein gene product 9.5(+) nerve terminals surrounded secretory coils of the sweat glands in the skin of control subjects. Sudomotor denervation was present in diabetic patients, manifesting as depletion of periglandular nerve fibers with lower SGII compared with 42 age- and sex-matched control subjects (2.54 ± 1.87 vs. 4.68 ± 1.51%, P < 0.001). The SGII was correlated with HbA(1c) (P = 0.011) and was lower in patients with anhidrosis of the feet compared with those with normal sweating of the feet (0.82 ± 0.69 vs. 3.00 ± 1.81%, P = 0.001). Sudomotor denervation was concordant with cardiac autonomic dysfunction as assessed with reduced heart rate variability (P = 0.003). CONCLUSIONS: Sudomotor denervation is a significant presentation of diabetic neuropathy, and the SGII was associated with HbA(1c). A skin biopsy offers a structural assessment of sudomotor innervation.

Quantitation of sudomotor innervation in skin biopsies of patients with diabetic neuropathy.

Previous assessments of the sudomotor system have depended on functional tests, and only a few studies document the pathologic findings of postganglionic nerve degeneration quantitatively and at the ultrastructural level. We developed a quantitative system of sudomotor innervation in skin biopsies of the distal leg by immunostaining of nerve fibers with anti-protein gene product 9.5 (PGP9.5) and by counterstaining with Congo red. A computerized area-based morphometric analysis was used to quantify the sweat gland innervation index (SGII), defined as the area of nerve fibers normalized to the area of sweat glands. This approach reduced the variations in measurements of sweat gland areas compared to the commonly used method by ∼5.6-fold (2.47% ± 2.54% vs 13.97% ± 14.24%, p < 0.001); hence, variations in SGII were also reduced. We examined 35 Type 2 diabetic patients (24 men and 11 women; mean age, 56.5 ± 12.8 years), with symmetrical length-dependent neuropathy and reduced intraepidermal nerve fiber density (0.76 ± 0.95 fibers/mm). By light and electron microscopy, PGP9.5-positive nerve terminals surrounded Congo red-positive sweat gland secretory coils in controls; these periglandular nerve terminals were either absent or markedly reduced in diabetic patients. Diabetic patients had lower SGII values than age- and sex-matched controls (2.60% ± 1.96% vs 4.84% ± 1.51%, p < 0.0001). The SGII values were lower in patients with anhidrosis of the feet versus those with normal sweating of the feet (0.89% ± 0.71% vs 3.10% ± 1.94%, p < 0.01). Thus, skin biopsy offers combined assessment of sudomotor innervation.

Differential regulation of neurotrophin S100B and BDNF in two rat models of depression.

Several clinical studies have demonstrated that serum brain-derived neurotrophic factor (BDNF) levels are decreased and serum S100B levels are increased in patients with major depression. In this study, we investigated whether these findings could be replicated in animal models of depression. We measured BDNF and S100B protein levels in the serum, prefrontal cortex, striatum and hippocampus of rats in models of depression, i.e., olfactory bulbectomy (OBX) and chronic unpredictable stress (CUS) models. Serum BDNF levels were significantly increased in the OBX rats, as were hippocampal BDNF levels in the CUS rats, in comparison with their respective controls. Significant increases in serum S100B levels were observed in both the OBX and CUS rats as compared with their respective controls; however, S100B levels were decreased in the prefrontal cortex of the CUS rats. No significant correlation was found between serum and regional brain S100B/BDNF levels. Our findings suggest that both of these animal models of depression, in which similar serum S100B level changes to those in depressed patients were observed, could be used as valid models to explore the role of S100B underlying major depression. Neither serum S100B nor BDNF levels reflect their levels in the brain, and changes in their levels in patients with neuropsychiatric diseases should be interpreted cautiously.

Depletion of peptidergic innervation in the gastric mucosa of streptozotocin-induced diabetic rats.

Autonomic neuropathy affecting the gastrointestinal system is a major presentation of diabetic neuropathy. Changes in the innervation of gastric mucosa or muscle layers can contribute to gastrointestinal symptoms. The present study investigated this issue by quantitatively analyzing the immunohistochemical patterns of the gastric innervation in rats with streptozotocin (STZ)-induced diabetes. In control rats, calcitonin gene-related peptide (CGRP) and substance P (SP) (+) nerve fibers appeared in the gastric mucosa and muscle layers. Double immunohistochemical staining showed that immunoreactivities for SP and CGRP were co-localized with a pan-neuronal marker protein gene product 9.5. Both SP (+) nerve fibers (p<0.001) and CGRP (+) nerve fibers (p<0.005) were decreased in the gastric mucosa within 4 weeks of diabetes; the reduction persisted throughout 24 weeks. Diabetic rats treated with insulin did not show decrease of SP or CGRP (+) fibers in the mucosa 4 weeks after STZ injection (p>0.05). There was no significant change in SP (+) nerve fibers (p>0.05) or CGRP (+) nerve fibers (p>0.05) of the gastric muscle layers. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression levels of SP and CGRP mRNA in the thoracic dorsal root ganglia were similar between diabetic and control animals (p>0.05). Qualitative and quantitative ultrastructural examinations on the gastric mucosa documented unmyelinated nerve degeneration. These results suggest the existence of gastric sensory neuropathy in STZ-induced diabetes, and this pathology provides a foundation for understanding diabetic gastropathy.