Storage Bodies Research Articles

Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are inherited neurodegenerative lysosomal storage diseases caused by mutations in several different genes. Mutations in CLN5 cause a variant late-infantile human disease and some cases of juvenile and adult clinical disease. NCLs also occur in animals, and a flock of New Zealand Borderdale sheep with a CLN5 splice-site mutation has been developed for model studies. Dissociated mixed neural cells from CLN5-deficient foetal sheep brains contained no obvious storage bodies at plating but these accumulated rapidly in culture, mainly in microglial cells and also in neurons and astrocytes. Accumulation was very obvious after a week, as monitored by fluorescent microscopy and immunostaining for subunit c of mitochondrial ATP synthase. Photography at intervals revealed the dynamic nature of the cultures and a flow of storage bodies between cells, specifically the phagocytosis of storage-body containing cells by microglia and incorporation of the storage bodies into the host cells. No storage was observed in cultured control cells.Transduction of cell cultures with a lentiviral vector expressing a C-terminal Myc tagged CLN5 resulted in secretion of post-translationally glycosylated and processed CLN5. Transduction of CLN5-deficient cultures with this construct rapidly reversed storage body accumulation, to less than half in only six days. These results show that storage body accumulation is reversible with enzyme correction and support the use of these cultures for testing of therapeutics prior to whole animal studies.

Asociación del polimorfismo 3'UTR+62G>A en resistina con resistencia a la insulina, adiposidad y el índice adiponectina-resistina en población mexicana

Introduction: Insulin resistance (IR) is a disease with genetic susceptibility characterized by the increase in storage and irregular body fat distribution, and impaired production of adipokines. Objective: The objective was to investigate the relationship between 3’UTR+62G>A RETN gene polymorphism, with adiponectin-resistin index (AR index ), adiposity, and inmuno-metabolic markers. Methods: In this cross-sectional study, 260 individuals characterized as Mexican-Mestizo and classified in lean and overweight, and IR and without-IR, were included. Anthropometrics, body composition, body fat distribution and inflammation and metabolic markers were measured by routine methods, RETN 3’UTR+62G>A alleles were identified by PCR-RFLP and soluble insulin, total adiponectin and resistin were measured by ELISA methods. Results: The +62G allele frequencies for lean and overweight individuals were different P = 0.0343 (95.4% and 98.4%, respectively). The lean GA genotype carriers showed significant low measures of AR index , adiposity, and inmuno-metabolic markers, than the GG genotype carriers. We found differences between individuals with IR and without-IR: in AR index (P = 0.002), adiponectin (P = 0.002) and resistin levels (P = 0.033): 1.102 ± 0.03, 5.167 ± 0.36 µg/mL and 8.827 ± 0.42 ng/mL versus 1.336 ± 0.07, 3.577 ± 0.34 µg/mL and 10.480 ± 0.65 ng/mL. Showed correlations with inflammation markers, distribution and body fat storage (r = 0.262 to 0.414), P A polymorphism is associated with overweight. The presence of the +62A allele was associated with increase of total adiponectin, AR

Association of resistin gene 3'UTR+62G>A polymorphism with insulin resistance, adiposity and the adiponectin-resistin index in Mexican population

Insulin resistance (IR) is a disease with genetic susceptibility characterized by the increase in storage and irregular body fat distribution, and impaired production of adipokines. The objective was to investigate the relationship between 3'UTR+62G>A RETN gene polymorphism, with adiponectin-resistin index (ARindex), adiposity, and inmuno-metabolic markers. In this cross-sectional study, 260 individuals characterized as Mexican-Mestizo and classified in lean and overweight, and IR and without-IR, were included. Anthropometrics, body composition, body fat distribution and inflammation and metabolic markers were measured by routine methods, RETN 3'UTR+62G>A alleles were identified by PCR-RFLP and soluble insulin, total adiponectin and resistin were measured by ELISA methods. The +62G allele frequencies for lean and overweight individuals were different P=0.0343 (95.4% and 98.4%, respectively). The lean GA genotype carriers showed significant low measures of ARindex, adiposity, and inmuno-metabolic markers, than the GG genotype carriers. We found differences between individuals with IR and without-IR: in ARindex (P=0.002), adiponectin (P=0.002) and resistin levels (P=0.033): 1.102 ± 0.03, 5.167 ± 0.36 ug/mL and 8.827 ± 0.42 ng/mL versus 1.336 ± 0.07, 3.577 ± 0.34 ug/mL and 10.480 ± 0.65 ng/mL. Showed correlations with inflammation markers, distribution and body fat storage (r=0.262 to 0.414), P< 0.05. The present data suggest that in a Mexican-mestizo population the RETN +62G>A polymorphism is associated with overweight. The presence of the +62A allele was associated with increase of total adiponectin, ARindex, resistin levels, metabolic markers and body fat storage. ARindex can be an early indicator of insulin resistance.

Identification of STAT target genes in adipocytes

Adipocytes play important roles in lipid storage, energy homeostasis and whole body insulin sensitivity. Studies in the last two decades have identified the hormones and cytokines that activate specific STATs in adipocytes in vitro and in vivo. Five of the seven STAT family members are expressed in adipocyte (STATs 1, 3, 5A, 5B and 6). Many transcription factors, including STATs, have been shown to play an important role in adipose tissue development and function. This review will summarize the importance of adipocytes, indicate the cytokines and hormones that utilize the JAK-STAT signaling pathway in fat cells and focus on the identification of STAT target genes in mature adipocytes. To date, specific target genes have been identified for STATs, 1, 5A and 5B, but not for STATs 3 and 6.

Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci--such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis--to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality--and dysfunctionality--of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.

Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ~12 million people worldwide, with ~2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 μM) inhibited parasite growth in a dose-dependent manner (IC(50)=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3β-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3β-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.

The Research on the Funding Barriers of the Development Wood Carbon Sequestration Materials Industry

Global warming caused by CO2 and other greenhouse gas emissions has a direct impact of human existence, The current three sets of technological solutions corresponding to the global warming to develop low carbon economy such as improving energy efficiency and conservation, reducing the carbon content of energy, development and utilization of natural carbon sinks, are not only existence of selection bottleneck but also run hardly and cannot solve global warming in this century fundamentally. Wood carbon sequestration is a new global carbon pool based on technological innovation, combined with modern agriculture, created by means of industrialization. Wood carbon sequestration is not only a long-term carbon sequestration benefits storage body also can be a low-cost production resources or materials. To cultivate it to become an industry will not only become the new economic growth point for farmers to increase incomes, promote regional economic restructuring and industrial upgrading but also end the dependence of forest wood resources to human. This paper puts forward some corresponding countermeasures and suggestions on making reform on institutional level and the innovation of financial products to support the development of wood carbon sequestration materials industry, based on the financing obstacles to the development of wood carbon sequestration materials industry, form the significance of wood carbon sequestration materials development.

Identification of a New IgE-Binding Epitope of Peanut Oleosin That Cross-Reacts with Buckwheat

Peanut and buckwheat induce a severe allergic reaction, anaphylaxis, which is considered to be mediated by immunoglobulin E (IgE). We identified in this study a new IgE-binding epitope of the peanut allergen that cross-reacted with buckwheat. The phosphate-buffered saline-soluble fraction of buckwheat inhibited the binding between IgE and the peanut allergen. A cross-reactive peptide was isolated from the α-chymotrypsin hydrolysate of peanut. Based on the amino acid sequence and mass spectrometric analysis data, the peptide was identified as Ser-Asp-Gln-Thr-Arg-Thr-Gly-Tyr (SDQTRTGY); this sequence is identical to amino acids 2-9 in the N-terminal hydrophilic domain of oleosin 3 which is located on the surface of the lipid storage body. Synthetic SDQTRTGY was found to bind with IgE in the sera of all eight peanut-allergic patients tested. Since many foods of plant origin contain oleosin, the possibility of an anaphylactic cross-reaction in allergic patients should always be considered.

A One Base Pair Deletion in the Canine ATP13A2 Gene Causes Exon Skipping and Late-Onset Neuronal Ceroid Lipofuscinosis in the Tibetan Terrier

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5–7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71–84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies.

The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation

Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 µM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.

The formation, function and fate of protein storage compartments in seeds

Seed storage proteins (SSPs) have been studied for more than 250years because of their nutritional value and their impact on the use of grain in food processing. More recently, the use of seeds for the production of recombinant proteins has rekindled interest in the behavior of SSPs and the question how they are able to accumulate as stable storage reserves. Seed cells produce vast amounts of SSPs with different subcellular destinations creating an enormous logistic challenge for the endomembrane system. Seed cells contain several different storage organelles including the complex and dynamic protein storage vacuoles (PSVs) and other protein bodies (PBs) derived from the endoplasmic reticulum (ER). Storage proteins destined for the PSV may pass through or bypass the Golgi, using different vesicles that follow different routes through the cell. In addition, trafficking may depend on the plant species, tissue and developmental stage, showing that the endomembrane system is capable of massive reorganization. Some SSPs contain sorting signals or interact with membranes or with other proteins en route in order to reach their destination. The ability of SSPs to form aggregates is particularly important in the formation or ER-derived PBs, a mechanism that occurs naturally in response to overloading with proteins that cannot be transported and that can be used to induce artificial storage bodies in vegetative tissues. In this review, we summarize recent findings that provide insight into the formation, function, and fate of storage organelles and describe tools that can be used to study them.

Myosin Va Is Required for P Body but Not Stress Granule Formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.

A metabolomic comparison of mouse models of the Neuronal Ceroid Lipofuscinoses

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of fatal inherited neurodegenerative diseases in humans distinguished by a common clinical pathology, characterized by the accumulation of storage body material in cells and gross brain atrophy. In this study, metabolic changes in three NCL mouse models were examined looking for pathways correlated with neurodegeneration. Two mouse models; motor neuron degeneration (mnd) mouse and a variant model of late infantile NCL, termed the neuronal ceroid lipofuscinosis (nclf) mouse were investigated experimentally. Both models exhibit a characteristic accumulation of autofluorescent lipopigment in neuronal and non neuronal cells. The NMR profiles derived from extracts of the cortex and cerebellum from mnd and nclf mice were distinguished according to disease/wildtype status. In particular, a perturbation in glutamine and glutamate metabolism, and a decrease in γ-amino butyric acid (GABA) in the cerebellum and cortices of mnd (adolescent mice) and nclf mice relative to wildtype at all ages were detected. Our results were compared to the Cln3 mouse model of NCL. The metabolism of mnd mice resembled older (6month) Cln3 mice, where the disease is relatively advanced, while the metabolism of nclf mice was more akin to younger (1-2months) Cln3 mice, where the disease is in its early stages of progression. Overall, our results allowed the identification of metabolic traits common to all NCL subtypes for the three animal models.

Protein body‐inducing fusions for high‐level production and purification of recombinant proteins in plants

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

Development of fasting abilities in subantarctic fur seal pups: balancing the demands of growth under extreme nutritional restrictions

1. Surviving prolonged food deprivation requires various metabolic adaptations such as energy and protein sparing, which can be highly conflicting with energy-demanding stages of an animal’s life history such as growth. 2. Due to the maternal attendance pattern, subantarctic fur seal (Arctocephalus tropicalis Gray) pups must repeatedly endure exceptionally long fasts of increasing duration throughout the 10-month lactation period. Little is known of (i) how these infants adapt to such extreme energetic constraints while sustaining growth and development; and (ii) the ecological implications of repeated prolonged fasting in early life in terms of offspring survival, maternal care and growth strategy in this species, as well as the evolutionary consequences of such life history trait. 3. Physiological responses to prolonged fasting and how they change with development throughout the pre-weaning period were investigated. Results show that beginning with their first fast, subantarctic fur seal pups are able to mobilize lipid reserves preferentially while conserving protein stores in response to nutritional deprivation. As pup age, profound changes in energy expenditure allow the implementation of an efficient strategy of fat storage and lean body mass preservation, which proves highly adaptive in the face of the low maternal provisioning rates experienced. 4. Despite increasing fasting durations, pup mortality decreased markedly throughout the maternal dependence period. Consistent with predictions, field measurements indicate that fasting endurance, although limited in early life, increases up to durations of nearly 3 months with age. Results suggest that the maternal provisioning strategy could be constrained by these ontogenetic changes in pup fasting abilities. 5. Furthermore, extreme energetic constraints and local density-dependent effects appear to exert a strong selective pressure upon the adoption of a convergent growth strategy between the sexes aiming to maximize fat storage and pre-weaning survival. 6. The issues of resulting trade-offs between pre- and post-weaning survival and the evolutionary consequences of extreme fasting abilities are also addressed.

The specific loss of GnRH-positive neurons from the hypothalamus of sheep with CLN6 neuronal ceroid lipofuscinosis occurs without glial activation and has only minor effects on reproduction

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are characterized by progressive neurodegeneration resulting in widespread brain atrophy. Each form is assumed to be the consequence of some universal intracellular event; however, time course studies on the cerebral cortex of a sheep model of the CLN6 form revealed distinct regional neurodegeneration preceded by regional glial activation, spreading from quite localized foci. Previous neurological investigations have concentrated on obviously affected cortical functions. This study investigated the impact of ovine CLN6 NCL on a subcortical structure and function, the discrete gonadotrophin-releasing hormone (GnRH) secreting neurons of the hypothalamus, and the effect of changes in the neuroendocrine system on reproductive efficiency and embryonic development. The number of immunopositive GnRH neurons in the hypothalamus and median eminence of affected sheep was reduced by 80%, but the rest of the hypothalamus showed no changes or atrophy. This specific loss of neuron type was not accompanied by either microglial or astrocyte activation, which was absent from the hypothalamus and was not associated with cell-type-specific storage body accumulation. Ovarian responsiveness to follicle stimulating hormone, ovulation rates, sperm production, fertilization rates, embryonic development, and reproductive efficiency were sub-par but reproduction was still functional. This remains when the sheep are profoundly blind. We conclude that physiological functionality and connectivity, not genotype, determine neuron fate in CLN6 NCL.

Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.

Morfoanatomia da plântula de Campomanesia xanthocarpa O. Berg. (Myrtaceae)

Campomanesia xanthocarpa (guabirobeira) é espécie arbórea, heliófita, ocorrendo desde o estado de Minas Gerais até o Rio Grande do Sul. Visando descrever a morfologia e anatomia da plântula, sementes foram coletadas e plantadas, utilizando-se vermiculita como substrato. Plântulas foram coletadas para análise morfológica desde a protrusão da radícula até a fase em que são emitidos os primeiros pares de eofilos. Para a análise anatômica foram coletadas plântulas com sete, 30 e 60 dias após a germinação. As amostras foram fixadas em glutaraldeído (1%) + formaldeído (4%), incluídas em historesina, seccionadas em micrótomo, e montadas de forma permanente. A plântula é epígea, fanerocotiledonar, com paracotilédones e eofilos simples, opostos. Nas plântulas com sete dias observa-se o início da formação do câmbio e as de 30 dias já apresentam crescimento secundário. O paracotilédone e o eofilo apresentam características morfoanatômicas semelhantes. O hipocótilo é o principal órgão de reserva, contendo grãos de amido, especialmente nas plântulas com sete dias. Foram registradas glândulas e células secretoras em todos os órgãos, exceto na raiz. A epiderme dos paracotilédones, eofilos e hipocótilo apresenta tricomas tectores unicelulares. Idioblastos com cristais são comuns no mesofilo do paracotilédone e do eofilo.

Macroautophagy is defective in mucolipin-1-deficient mouse neurons

Mucolipidosis type IV is a neurodegenerative lysosomal disease clinically characterized by psychomotor retardation, visual impairment, and achlorhydria. In this study we report the development of a neuronal cell model generated from cerebrum of Mcoln1 − / − embryos. Prior functional characterization of MLIV cells has been limited to fibroblast cultures gleaned from patients. The current availability of the mucolipin-1 knockout mouse model Mcoln1 −/− allows the study of mucolipin-1-defective neurons, which is important since the disease is characterized by severe neurological impairment. Electron microscopy studies reveal significant membranous intracytoplasmic storage bodies, which correlate with the storage morphology observed in cerebral cortex of Mcoln1 −/− P7 pups and E17 embryos. The Mcoln1 −/− neuronal cultures show an increase in size of LysoTracker and Lamp1 positive vesicles. Using this neuronal model system, we show that macroautophagy is defective in mucolipin-1-deficient neurons and that LC3-II levels are significantly elevated. Treatment with rapamycin plus protease inhibitors did not increase levels of LC3-II in Mcoln1 −/− neuronal cultures, indicating that the lack of mucolipin-1 affects LC3-II clearance. P62/SQSTM1 and ubiquitin levels were also increased in Mcoln1 −/− neuronal cultures, suggesting an accumulation of protein aggregates and a defect in macroautophagy which could help explain the neurodegeneration observed in MLIV. This study describes, for the first time, a defect in macroautophagy in mucolipin-1-deficient neurons, which corroborates recent findings in MLIV fibroblasts and provides new insight into the neuronal pathogenesis of this disease.

Glial fibrillary acidic protein is elevated in the lysosomal storage disease classical late-infantile neuronal ceroid lipofuscinosis, but is not a component of the storage material

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease of children caused by mutations in TPP1, the gene encoding the lysosomal protease tripeptidyl peptidase 1. LINCL is characterized by lysosomal accumulation of storage material of which only a single protein component, subunit c of mitochondrial ATP synthase, has been well established to date. Identification of other protein constituents of the storage material could provide useful insights into the pathophysiology of disease and the natural substrates for TPP1. We have therefore initiated a proteomic analysis of storage material in brain from a LINCL mouse model. One protein, GFAP (glial fibrillary acidic protein), was found to be elevated in the LINCL mice compared with normal controls in both isolated storage bodies and a lysosome-enriched subcellular fraction that contains storage material. To determine whether GFAP accumulates within the lysosome in LINCL, we examined its intracellular distribution using subcellular fractionation and morphological methods. These experiments demonstrate that GFAP is not a component of the storage material in LINCL, suggesting that reports of GFAP storage in other NCLs may need to be re-examined. A number of other proteins were elevated in the storage material and/or lysosome-enriched fraction from the LINCL mice, but it remains unclear whether these proteins are true constituents of the storage material or, like GFAP, whether they associate with this material upon purification.