Structure of the lens MP20 mediated adhesive junction.

Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C-termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.

11th Asia Oceania Human Proteome Organization Congress Report.

As the first in-person Asia Oceania Human Proteomics Organization (AOHUPO) congress since 2018, the 11th AOHUPO congress was an opportune time for the research community to reconnect and to renew friendships after the long period of restricted travel due to the global pandemic. Moreover, this congress was a great opportunity for the many AO regional proteomics and mass spectrometry scientists to meet in Singapore to exchange ideas and to present their latest findings. Cohosted by the Singapore Society for Mass Spectrometry and the Malaysian Proteomics Society and held in conjunction with the seventh Asia Oceania Agricultural Proteomics Organization Congress and Singapore Society for Mass Spectrometry 2023, the meeting featured both human and agricultural proteomics. Over five hundred scientists from the AO region converged on the MAX Atria @ Singapore EXPO, Changi, Singapore from May 8 to 10 for the main congress. The diverse program was made up of 64 invited speakers and panellists for seven plenary lectures, 27 concurrent symposia, precongress and postcongress workshops, and 174 poster presentations. The AOHUPO society were able to celebrate not only their 20th anniversary but also the outstanding academic research from biological and agricultural proteomics and related 'omics fields being conducted across the Asia-Oceania region.

Regulation of lens water content: Effects on the physiological optics of the lens.

The lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. Alterations to the refractive properties of the lens contribute to the process of emmetropisation in early childhood, and then the gradual loss in lens power that occurs throughout adulthood. In parallel to these changes to lens refractive power, age-dependent increases in lens stiffness and light scattering result in presbyopia and cataract, respectively. In recent years it has been confirmed that the lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the refractive properties and transparency of the lens. By actively regulating lens water content, the microcirculation system controls two key parameters, lens geometry and the gradient of refractive index, which together determine the refractive properties of the lens. Furthermore, by delivering nutrients and antioxidants to the lens nucleus, the microcirculation system maintains lens transparency by preventing crystallin aggregation. Interestingly, the solubility, intramolecular packing and refractive index increment of crystallin proteins can be modulated by the ability of crystallin proteins to dynamically bind water, a processed called syneresis. In a series of previous studies it has been shown that the application of external pressure to the lens can induce syneresis. Since it is now known that lens water transport generates a substantial internal hydrostatic pressure gradient, we speculate that the microcirculation is capable of regulating crystallin function by altering the amount of water bound to lens proteins in the nucleus, where the pressure gradient and protein concentrations are the highest. Here we present evidence for the links between lens transport, pressure, syneresis and protein function. Furthermore, because the lens pressure gradient can be regulated by intrinsic and extrinsic stimuli, we suggest mechanisms via which this integrative system can be used to effect the changes to the refractive and transparent properties of the lens that are observed across our lifetime.

Mapping Glucose Uptake, Transport and Metabolism in the Bovine Lens Cortex.

Purpose: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions. Methods: Ex vivo bovine lenses were incubated in artificial aqueous humour containing normoglycaemic stable isotopically-labelled (SIL) glucose (5 mM) for 5 min-20 h. Following incubations, lenses were frozen for subsequent matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) analysis using high resolution mass spectrometry. Manually dissected, SIL-incubated lenses were subjected to gas chromatography-mass spectrometry (GC-MS) to verify the identity of metabolites detected by MALDI-IMS. Normal, unincubated lenses were manually dissected into epithelium flat mounts and fibre cell fractions and then subjected to either gel-based proteomic analysis (Gel-LC/MS) to detect facilitative glucose transporters (GLUTs) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Indirect immunofluorescence and confocal microscopy of axial lens sections from unincubated fixed lenses labelled with primary antibodies specific for GLUT 1 or GLUT 3 were utilised for protein localisation. Results: SIL glucose uptake at 5 min was concentrated in the equatorial region of the lens. At later timepoints, glucose gradually distributed throughout the epithelium and the cortical lens fibres, and eventually the deeper lens nucleus. SIL glucose metabolites found in glycolysis, the sorbitol pathway, the pentose phosphate pathway, and UDP-glucose formation were mapped to specific lens regions, with distinct regional signal changes up to 20 h of incubation. Spatial proteomic analysis of the lens epithelium detected GLUT1 and GLUT3. GLUT3 was in higher abundance than GLUT1 throughout the epithelium, while GLUT1 was more abundant in lens fibre cells. Immunohistochemical mapping localised GLUT1 to epithelial and cortical fibre cell membranes. Conclusion: The major uptake site of glucose in the bovine lens has been mapped to the lens equator. SIL glucose is rapidly metabolised in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres, with low levels of metabolic activity observed in the nucleus.

Resolving the developmental distribution patterns of polyphenols and related primary metabolites in bilberry (Vaccinium myrtillus) fruit.

Bilberry (Vaccinium myrtillus) is a commercially important wild berry species, which accumulates high amounts of polyphenols, particularly anthocyanins, in the skin and flesh. Whilst a number of studies have quantified these phytochemicals in intact ripe bilberry fruit, we extend the current knowledge by investigating the spatial distribution of anthocyanin-associated polyphenols in fruit tissue, and study their links with primary metabolism during ripening. To address this, we used LC-MS and mass spectrometry imaging to measure and map primary and secondary metabolites in fruit. Correlation analysis showed that five sugars displayed strong positive correlations with anthocyanin accumulation, whereas all amino acids were negatively correlated. The accumulation patterns of polyphenols correlated in fruit skin and flesh, but altered with development. Finally, spatial segmentation analysis revealed that the chemical signatures of ripening first appear at defined regions under the skin and rapidly expand to encompass the entire fruit at the eating-ripe stage.

Hyperbaric oxygen as a model of lens aging in the bovine lens: The effects on lens biochemistry, physiology and optics.

Age related nuclear (ARN) cataracts in humans take years to form and so experimental models have been developed to mimic the process in animals as a means of better understanding the etiology of nuclear cataracts in humans. A major limitation with these animal models is that many of the biochemical and physiological changes are not typical of that seen in human ARN cataract. In this review, we highlight the work of Frank Giblin and colleagues who established an in vivo animal model that replicates many of the changes observed in human ARN cataract. This model involves exposing aged guinea pigs to hyperbaric oxygen (HBO), which by causing the depletion of the antioxidant glutathione (GSH) specifically in the lens nucleus, produces oxidative changes to nuclear proteins, nuclear light scattering and a myopic shift in lens power that mimics the change that often precedes cataract development in humans. However, this model involves multiple HBO treatments per week, with sometimes up to a total of 100 treatments, spanning up to eight months, which is both costly and time consuming. To address these issues, Giblin developed an in vitro model that used rabbit lenses exposed to HBO for several hours which was subsequently shown to replicate many of the changes observed in human ARN cataract. These experiments suggest that HBO treatment of in vitro animal lenses may serve as a more economical and efficient model to study the development of cataract. Inspired by these experiments, we investigated whether exposure of young bovine lenses to HBO for 15 h could also serve as a suitable acute model of ARN cataract. We found that while this model is able to exhibit some of the biochemical and physiological changes associated with ARN cataract, the decrease in lens power we observed was more characteristic of the hyperopic shift in refraction associated with ageing. Future work will investigate whether HBO treatment to age the bovine lens in combination with an oxidative stressor such as UV light will induce refractive changes more closely associated with human ARN cataract. This will be important as developing an animal model that replicates the changes to lens biochemistry, physiology and optics observed in human ARN cataracts is urgently required to facilitate the identification and testing of anti-cataract therapies that are effective in humans.

Partitioning and Spatial Distribution of Drugs in Ocular Surface Tissues.

Ocular drug absorption after eye drop instillation has been widely studied, but partitioning phenomena and spatial drug distribution are poorly understood. We investigated partitioning of seven beta-blocking drugs in corneal epithelium, corneal stroma, including endothelium and conjunctiva, using isolated porcine tissues and cultured human corneal epithelial cells. The chosen beta-blocking drugs had a wide range (-1.76-0.79) of n-octanol/buffer solution distribution coefficients at pH 7.4 (Log D7.4). In addition, the ocular surface distribution of three beta-blocking drugs was determined by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) after their simultaneous application in an eye drop to the rabbits in vivo. Studies with isolated porcine corneas revealed that the distribution coefficient (Kp) between the corneal epithelium and donor solution showed a positive relationship and good correlation with Log D7.4 and about a 50-fold range of Kp values (0.1-5). On the contrary, Kp between corneal stroma and epithelium showed an inverse (negative) relationship and correlation with Log D7.4 based on a seven-fold range of Kp values. In vitro corneal cell uptake showed a high correlation with the ex vivo corneal epithelium/donor Kp values. Partitioning of the drugs into the porcine conjunctiva also showed a positive relationship with lipophilicity, but the range of Kp values was less than with the corneal epithelium. MALDI-IMS allowed simultaneous detection of three compounds in the cornea, showed data in line with other experiments, and revealed uneven spatial drug distribution in the cornea. Our data indicate the importance of lipophilicity in defining the corneal pharmacokinetics and the Kp values are a useful building block in the kinetic simulation models for topical ocular drug administration.

Mass Spectrometry-Based Metabolomics to Investigate the Effect of Mechanical Shaking on Sauvignon Blanc Berry Metabolism.

Previous commercial studies carried out in New Zealand showed that mechanical shaking significantly reduced the incidence of Botrytis cinerea infection in wine grapes. However, the reasons behind this reduction are not well understood. Here, we employed a metabolomics approach to gain insights into the biochemical changes that occur in grape berries due to mechanical shaking. Berry samples were analyzed using three different analytical approaches including gas chromatography and mass spectrometry (MS), liquid chromatography and MS, and imaging mass spectrometry (IMS). Combined data provided a comprehensive overview of metabolic changes in grape berry, indicating the initiation of different stress mitigation strategies to overcome the effect of mechanical shaking. Berry primary metabolism was distinctly altered in the green berries in response to mechanical shaking, while secondary metabolism significantly changed in berries collected after veraison. Pathway analysis showed upregulation of metabolites related to nitrogen and lipid metabolism in the berries from shaken vines when compared with controls. From IMS data, we observed an accumulation of different groups of metabolites including phenolic compounds and amino and fatty acids in the areas near to the skin of berries from shaken vines. This observation suggests that mechanical shaking caused an accumulation of these metabolites, which may be associated with the formation of a protective barrier, leading to the reduction in B. cinerea infection in berries from mechanically shaken vines.

Applications of stable isotopes in MALDI imaging: current approaches and an eye on the future.

Matrix-assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) is now an established imaging modality with particular utility in the study of biological, biomedical and pathological processes. In the first instance, the use of stable isotopically labelled (SIL) compounds in MALDI-IMS has addressed technical barriers to increase the accuracy and versatility of this technique. This has undoubtedly enhanced our ability to interpret the two-dimensional ion intensity distributions produced from biological tissue sections. Furthermore, studies using delivery of SIL compounds to live tissues have begun to decipher cell, tissue and inter-tissue metabolism while maintaining spatial resolution. Here, we review both the technical and biological applications of SIL compounds in MALDI-IMS, before using the uptake and metabolism of glucose in bovine ocular lens tissue to illustrate the current limitations of SIL compound use in MALDI-IMS. Finally, we highlight recent instrumentation advances that may further enhance our ability to use SIL compounds in MALDI-IMS to understand biological and pathological processes. Graphical Abstract.

An imaging mass spectrometry atlas of lipids in the human neurologically normal and Huntington's disease caudate nucleus.

Huntington's disease (HD) is a fatal disorder associated with germline trinucleotide repeat expansions in the HTT gene and characterised by striatal neurodegeneration. No efficacious interventions are available for HD, highlighting a major unmet medical need. The molecular mechanisms underlying HD are incompletely understood despite its monogenic aetiology. However, direct interactions between HTT and membrane lipids suggest that lipidomic perturbations may be implicated in the neuropathology of HD. In this study, we employed matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) to generate a comprehensive, unbiased and spatially resolved lipidomic atlas of the caudate nucleus (CN) in human post-mortem tissue from neurologically normal (n = 10) and HD (n = 13) subjects. Fourier transform-ion cyclotron resonance mass spectrometry and liquid chromatography-tandem mass spectrometry were used for lipid assignment. Lipidomic specialisation was observed in the grey and white matter constituents of the CN and these features were highly conserved between subjects. While the majority of lipid species were highly conserved in HD, compared to age-matched controls, CN specimens from HD cases in our cohort spanning a range of neuropathological grades showed a lower focal abundance of the neuroprotective docosahexaenoic and adrenic acids, several cardiolipins, the ganglioside GM1 and glycerophospholipids with long polyunsaturated fatty acyls. HD cases showed a higher focal abundance of several sphingomyelins and glycerophospholipids with shorter monosaturated fatty acyls. Moreover, we demonstrate that MALDI-IMS is tractable as a primary discovery modality comparing heterogeneous human brain tissue, provided that appropriate statistical approaches are adopted. Our findings support further investigation into the potential role of lipidomic aberrations in HD.

Mapping glucose metabolites in the normal bovine lens: Evaluation and optimisation of a matrix-assisted laser desorption/ionisation imaging mass spectrometry method.

The spatial resolution of microdissection-based analytical methods to detect ocular lens glucose uptake, transport and metabolism are poor, whereas the multiplexing capability of fluorescence microscopy-based approaches to simultaneously detect multiple glucose metabolites is limited in comparison with mass spectrometry-based methods. To better understand lens glucose transport and metabolism, a more highly spatially resolved technique that maintains the fragile ocular lens tissue is required. In this study, a sample preparation method for matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) analysis of ocular lens glucose uptake and metabolism has been evaluated and optimised. Matrix choice, tissue preparation and normalisation strategy were determined using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS of bovine lens tissue and validation performed using gas chromatography-MS. An internal standard was applied concurrently with N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC) matrix to limit cracking of the fresh frozen lens tissue sections. MALDI IMS data were collected at a variety of spatial resolutions to detect both endogenous lens metabolites and stable isotopically labelled glucose introduced by ex vivo lens culture. Using this approach, initial steps in important metabolic processes that are linked to diabetic cataract formation were spatially mapped in the bovine lens. In the future, this method can be applied to study the dynamics of glucose uptake, transport and metabolic flux to aid in the study of diabetic lens cataract pathophysiology.

Age-dependent changes in glutathione metabolism pathways in the lens: New insights into therapeutic strategies to prevent cataract formation-A review.

Ocular tissues possess a robust antioxidant defence system to minimize oxidative stress and preserve tissue structure and function. Glutathione (GSH) is a powerful antioxidant and in the lens exists at unusually high concentrations. However, with advancing age, GSH levels deplete specifically in the lens centre initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering and age-related nuclear cataract. However, antioxidant supplementation has been shown to be ineffective in preventing or delaying cataract indicating that a better understanding of the delivery, uptake and metabolism of GSH in the different regions of the lens is required. This information is essential for the development of scientifically informed approaches that target the delivery of GSH to the lens nucleus, the region of the lens most affected by age-related cataract.

Detection of small molecule concentration gradients in ocular tissues and humours.

The eye is an elegant organ consisting of a number of tissues and fluids with specialised functions that together allow it to effectively transmit and transduce light input to the brain for visual perception. One key determinant of this integrated function is the spatial relationship of ocular tissues. Biomolecular distributions within the main ocular tissues cornea, lens, and retina have been studied extensively in isolation, yet the potential for metabolic communication between ocular tissues via the ocular humours has been difficult to visualise. To address this limitation, the current study presents a method to map spatial distributions of metabolites and small molecules in whole eyes, including ocular humours. Using a tape-transfer system and freeze-drying, the spatial distribution of ocular small molecules was investigated in mouse, rat, fish (black bream), and rabbit eyes using negative ion mode MALDI imaging mass spectrometry. Full-scan imaging was used for discovery experiments, while MS/MS imaging for identification and localisation was also demonstrated. In all eyes, metabolites such as glutathione and phospholipids were localised in the main ocular tissues. In addition, in rodent eyes, major metabolites were distributed relatively uniformly in ocular humours. In contrast, both uniform and spatially defined ocular metabolite distributions were observed in the black bream eye. Tissue and ocular humour distributions were reproducible, as demonstrated by the three-dimensional analysis of a mouse eye, and able to be captured with high spatial resolution analysis. The presented method could be used to further investigate the role of inter-tissue metabolism in ocular health, and to support the development of therapeutics to treat major ocular diseases.

MALDI imaging mass spectrometry of ß- and γ-crystallins in the ocular lens.

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of ß- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas ß-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.

Distribution of Small Molecular Weight Drugs into the Porcine Lens: Studies on Imaging Mass Spectrometry, Partition Coefficients, and Implications in Ocular Pharmacokinetics.

Lens is the avascular tissue in the eye between the aqueous humor and vitreous. Drug binding to the lens might affect ocular pharmacokinetics, and the binding may also have a pharmacological role in drug-induced cataract and cataract treatment. Drug distribution in the lens has been studied in vitro with many compounds; however, the experimental methods vary, no detailed information on distribution between the lens sublayers exist, and the partition coefficients are reported rarely. Therefore, our objectives were to clarify drug localization in the lens layers and establish partition coefficients for a wide range of molecules. Furthermore, we aimed to illustrate the effect of lenticular drug binding on overall ocular drug pharmacokinetics. We studied the distribution of 16 drugs and three fluorescent dyes in whole porcine lenses in vitro with imaging mass spectrometry and fluorescence microscopy techniques. Furthermore, we determined lens/buffer partition coefficients with the same experimental setup for 28 drugs with mass spectrometry. Finally, the effect of lenticular binding of drugs on aqueous humor drug exposure was explored with pharmacokinetic simulations. After 4 h, the drugs and the dyes distributed only to the outermost lens layers (capsule and cortex). The lens/buffer partition coefficients for the drugs were low, ranging from 0.05 to 0.8. On the basis of the pharmacokinetic simulations, a high lens-aqueous humor partition coefficient increases drug exposure in the lens but does not significantly alter the pharmacokinetics in the aqueous humor. To conclude, the lens seems to act mainly as a physical barrier for drug distribution in the eye, and drug binding to the lens affects mainly the drug pharmacokinetics in the lens.

Age-related spatial differences of human lens UV filters revealed by negative ion mode MALDI imaging mass spectrometry.

Tryptophan-derived UV filters are predominantly found in the lenses of primates and humans. While protective against UV radiation, aging alters the complement and spatial distributions of human lens UV filters, and a role for UV filters has been suggested in age-related cataract formation. To establish how the spatial distributions of UV filters change in normal human lens aging, matrix assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) was utilised to map the locations and relative abundance of multiple UV filters simultaneously. Frozen human lenses were cryosectioned axially, and the 20 µm-thick sections coated with MALDI matrix via robotic sprayer and analysed using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS. While signal for many UV filters was detected throughout the lenses, signal intensity was generally highest in the central (embryonic) nucleus and decreased uniformly in outer (foetal, juvenile, adult) nuclear and cortical regions, and many UV filter signals declined with age. In contrast, two antioxidant-conjugated UV filters (Cys-3-OHKG and GSH-3-OHKG) were restricted to the lens nucleus and their relative signal increased with increasing lens age. The enhanced spatial resolution of MALDI-IMS over manual trephine dissection techniques and its multiplex capability allowed the spatial relationships between lens UV filters to be established and explored in relation to aging. Together these results confirmed that the complement of UV filters in each lens is dynamic and undergoes significant age-related changes. In the future, this information could be used to compare with other lens biomolecule changes to better understand the lens aging process and age-related cataract formation.

Subventricular zone lipidomic architecture loss in Huntington's disease.

The human subventricular zone (SVZ) has a defined cytological and neurochemical architecture, with four constituent laminae that act in concert to support its neurogenic activity. Lipidomic specialisation has previously been demonstrated in the neurologically normal human SVZ, with enrichment of functionally important lipid classes in each lamina. The SVZ is also responsive to neurodegenerative disorders, where thickening of the niche and enhanced proliferation of resident cells were observed in Huntington's disease (HD) brains. In this study, we hypothesised lipidomic changes in the HD SVZ. Using matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry, this analysis shows differences in the lipidomic architecture in the post-mortem Vonsattel grade III cases. Relative to matched, neurologically normal specimens (N = 4), the lipidomic signature of the HD SVZ (N = 4) was characterized by loss of sulfatides and triglycerides in the myelin layer, with an ectopic and focal accumulation of sphingomyelins and ceramide-1-phosphate observed in this lamina. A striking loss of lipidomic patterning was also observed in the ependymal layer, where the local abundance of phosphatidylinositols was significantly reduced in HD. This comprehensive spatially resolved lipidomic analysis of the human HD SVZ identifies alterations in lipid architecture that may shed light on the mechanisms of SVZ responses to neurodegeneration in HD. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Layer-specific lipid signatures in the human subventricular zone demonstrated by imaging mass spectrometry.

The subventricular zone is a key site of adult neurogenesis and is also implicated in neurodegenerative diseases and brain cancers. In the subventricular zone, cell proliferation, migration and differentiation of nascent stem cells and neuroblasts are regulated at least in part by lipids. The human subventricular zone is distinctly layered and each layer contains discrete cell types that support the processes of neuroblast migration and neurogenesis. We set out to determine the lipid signatures of each subventricular layer in the adult human brain (n = 4). We utilised matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry and liquid chromatography-mass spectrometry to characterise the lipidome of the subventricular zone, with histology and microscopy used for identifying anatomical landmarks. Our findings showed that the subventricular zone was rich in sphingomyelins and phosphatidylserines but deficient in phosphatidylethanolamines. The ependymal layer had an abundance of phosphatidylinositols, whereas the myelin layer was rich in sulfatides and triglycerides. The hypocellular layer showed enrichment of sphingomyelins. No discrete lipid signature was seen in the astrocytic ribbon. The biochemical functions of these lipid classes are consistent with the localisation we observed within the SVZ. Our study may, therefore, shed new light on the role of lipids in the regulation of adult neurogenesis.

Desacetyl-α-melanocyte stimulating hormone and α-melanocyte stimulating hormone are required to regulate energy balance.

OBJECTIVE: Regulation of energy balance depends on pro-opiomelanocortin (POMC)-derived peptides and melanocortin-4 receptor (MC4R). Alpha-melanocyte stimulating hormone (α-MSH) is the predicted natural POMC-derived peptide that regulates energy balance. Desacetyl-α-MSH, the precursor for α-MSH, is present in brain and blood. Desacetyl-α-MSH is considered to be unimportant for regulating energy balance despite being more potent (compared with α-MSH) at activating the appetite-regulating MC4R in vitro. Thus, the physiological role for desacetyl-α-MSH is still unclear. METHODS: We created a novel mouse model to determine whether desacetyl-α-MSH plays a role in regulating energy balance. We engineered a knock in targeted QKQR mutation in the POMC protein cleavage site that blocks the production of both desacetyl-α-MSH and α-MSH from adrenocorticotropin (ACTH1-39). RESULTS: The mutant ACTH1-39 (ACTHQKQR) functions similar to native ACTH1-39 (ACTHKKRR) at the melanocortin 2 receptor (MC2R) in vivo and MC4R in vitro. Male and female homozygous mutant ACTH1-39 (Pomctm1/tm1) mice develop the characteristic melanocortin obesity phenotype. Replacement of either desacetyl-α-MSH or α-MSH over 14 days into Pomctm1/tm1 mouse brain significantly reverses excess body weight and fat mass gained compared to wild type (WT) (Pomcwt/wt) mice. Here, we identify both desacetyl-α-MSH and α-MSH peptides as regulators of energy balance and highlight a previously unappreciated physiological role for desacetyl-α-MSH. CONCLUSIONS: Based on these data we propose that there is potential to exploit the naturally occurring POMC-derived peptides to treat obesity but this relies on first understanding the specific function(s) for desacetyl-α-MSH and α-MSH.

Novel roles for the lens in preserving overall ocular health.

Outside the traditional roles of the lens as an important refractive element and a UV filter, it was David Beebe's group that first demonstrated that the lens acts an oxygen sink that protects the tissues of the anterior segment of the eye from oxygen or oxygen metabolites. In this review, we follow on from this work, and present new evidence from our laboratory to demonstrate that the lens serves as a reservoir for the release of the antioxidant glutathione (GSH) into the aqueous humor to provide a source of GSH and/or its precursor amino acids to nearby tissues that interface with the aqueous humor, or to remove toxic metabolites from the eye via the aqueous outflow pathway. In addition to GSH release, our laboratory and others have shown that ATP is released from the lens under hyposmotic conditions to activate purinergic signalling pathways in an autocrine manner to alter lens function. In this review, we raise the idea that ATP and/or its subsequent degradation product adenosine may exert a paracrine function and influence purinergic signalling systems in other tissues to alter aqueous humor outflow. These new secondary roles indicate that the lens is not just a passive optical element, but a highly dynamic and active tissue that interacts with its neighbouring tissues, through modifying the environments in which these tissues function. We believe that the lens actively contributes to the ocular environment and as a consequence, removal of the lens would alter the functionality of neighbouring tissues. We speculate that a long term effect of lens removal may be to inadvertently increase the exposure of anterior tissues of the eye to oxidative stress due to elevated oxygen levels and a reduction in the availability of GSH and purinergic signalling molecules in the aqueous humor. Since cataract surgery is now being performed on younger patients due to our increasing diabetic population, over time, we predict these changes may increase the susceptibility of these tissues to oxidative stress and the incidence of subsequent ocular pathologies. If our view of the lens is correct, the actual loss of the biological lens may have longer term consequences for overall ocular health than currently appreciated.