No description available.

As neuropatias periféricas hereditárias são um grupo heterogêneo de doenças relacionadas que afetam o sistema nervoso periférico. Elas podem ser classificadas de acordo com a velocidade de condução motora nos membros superiores (tipo 1 - CMT1, tipo 2 - CMT2 ou intermediário - iCMT), de acordo com o padrão de herança (autossômicas dominantes, autossômicas recessivas ou ligadas ao X) e quanto ao fenótipo de apresentação (neuropatias hereditária sensitivo e motora - CMT, neuropatia hereditária sensitiva - HSN ou neuropatia motora hereditária distal - dHMN). O uso das tecnologias de sequenciamento de nova geração (NGS) para diagnóstico de pacientes com neuropatia hereditária é particularmente eficiente uma vez que representa uma doença Mendeliana com mais de 90 genes diferentes relacionados. Foram avaliados 30 pacientes com diferentes subtipos de neuropatia hereditária (3 CMT1, 12 CMT2, 8 iCMT, 4 dHMN e 3 HSN). Foram identificadas 6 mutações (SH3TC2, GDAP1, MME, IGHMBP2, 2 AARS) e 7 variantes provavelmente patogênicas (KIF1A, DRP2, MME, MPZ, VRK1, SIGMAR1, FLVCR1). Com uma taxa de positividade de 43.3%. As variantes provavelmente patogênicas foram consideradas como a causa da apresentação fenotípica apresentada pelos pacientes baseado na frequência de variantes nos bancos de população normal, no efeito bioquímico das variantes sobre a estrutura proteica e pela análise in silico. No entanto, essas variantes necessitam de evidências adicionais que confirmem sua patogenicidade. Foram identificadas variantes novas nos genes MPZ, KIF1A, DRP2, IGHMBP2, VRK1, SIGMAR1 e FLVCR1 ampliando a variabilidade genotípica desses genes. A associação das mutações identificadas nos genes VRK1, KIF1A, IGHMBP2 e FLVRC1 permitiu a expansão dos fenótipos relacionados a esses genes. Mutações no gene VRK1 podem causar uma dHMN com sinais de liberação piramidal e envolviemento preferencial do compartimento posterior da perna. Transtorno do espectro autista pode ser observado em associação a mutações no gene KIF1A e mutações no gene FLVRC1 podem causar um fenótipo grave caracterizado por insensibilidade congénita a dor e acromutilações. Mutações no gene IGHMBP2 podem causar uma sobreposição entre os fenótipos SMARD1/CMT2S com disautonomia restrita ao trato gastro intestinal. Esse estudo demonstra que o uso de WES para o diagnóstico molecular de doenças geneticamente heterogêneas como as neuropatias hereditárias é uma ferramenta útil. / The hereditary peripheral neuropathies are a heterogeneous group of genetic disorders in which peripheral nervous system degeneration leads to weakness, atrophy and loss of sensation. It can be classified according motor conduction velocities in the upper limbs (type 1 - CMT1, type 2 - CMT2 or intermediate - iCMT), according to inheritance pattern (autosomal dominant, autosomal recessive or X linked) and according to the mainly group of fibres clinically involved (hereditary sensory and motor neuropathy - CMT, hereditary sensory neuropathy - HSN or distal hereditary motor neuropathy - dHMN). The use of next generation sequencing technologies (NGS) for the diagnosis of patients with genetic diseases is well established, as CMT is a Mendelian disease with more than 90 different related genes already reported. We evaluated 30 patients with all subtypes of hereditary neuropathy (3 CMT1, 12 CMT2, 8 iCMT, 4 dHMN and 3 HSN). Six mutations (SH3TC2, GDAP1, MME, IGHMBP2, 2 AARS) and 7 likely pathogenic variants (KIF1A, DRP2, MME, MPZ, VRK1, SIGMAR1, FLVCR1) were detected, leading to a positive rate of 43.3%. Likely pathogenic variants were considered based on their frequency in normal population, in silico analysis and segregation with phenotype. Despite they have strong evidences to support their causative status further evidence of their pathogenicity is required. New variants were identified in the genes MPZ, KIF1A, DRP2, IGHMBP2, VRK1, SIGMAR1 and FLVCR1 amplifying their genotypic variability. The mutations identified in VRK1, KIF1A, IGHMBP2 and FLVRC1 expanded their phenotype spectrum. Mutations in the VRK1 gene may cause dHMN with upper motor neuron signs. Autistic spectrum disorder may be observed in association with mutations in the KIF1A gene and mutations in the FLVRC1 gene may cause a severe phenotype characterized by congenital insensitivity to pain and acromutilations. Mutations in the IGHMBP2 gene may cause an overlap between SMARD1 and CMT2S phenotypes with organ specific dysautonomia. This study demonstrates that WES is a powerful tool for molecular diagnosis of hereditary neuropathies. Additionally, this study provides new information on the mutations in the VRK1, KIF1A and FLVRC1 genes by adding new mutations and increasing the phenotypic variability of the neuropathies associated with these genes.This study demonstrates WES is a powerful tool for molecular diagnosis of hereditary neuropathies.

Darbo tikslas: Išanalizuoti karvių bandos pieno kokybinę sudėtį ir patikslinti trūkinamų karvių profilaktikai taikomų medikamentų poveikį. Darbo uždaviniai: 1. Išanalizuoti karvių bandos pieno kokybinę sudėtį. 2. Tiriamojoje karvių fermoje reagentu CMT ištirti pasirinktų tyrimams karvių tešmens ketvirčius, prieš užtrūkinant ir po apsiveršiavimo praėjus 10dienų . 3. Atlikti bakteriologinius pieno tyrimus, prieš užtrūkinant ir po apsiveršiavimo praėjus 10dienų . 4. Nustatyti preparatų gydomąjį efektyvumą. Mokslinis - tiriamasis darbas buvo atliekamas 2010 – 2013 metais, privačiame ūkyje, Tauragės raj. Tyrimui buvo atrinktos 20 karvių, kurios buvo sugrupuotos į dvi grupes po 10 karvių. Grupėms buvo skirtas skirtingas gydymas, 1 –oji grupė gydyta preparatu „Rilexine 500“, 2 – oji grupė preparatu „OrbeSeal“. Prieš gydymą ir po gydymo buvo stebėtas SLS, pieno baltymų, riebalų, laktozės ir urėjos kiekio kitimas. Taip pat mastito sukėlėjų diagnozavimas, tešmens ketvirčių užkrėstumas. Išanalizavus bandos pieno kokybinę sudėtį, galima teigti, kad pieno kokybiniai rodikliai ima gerėti, po sėkmingo mastito gydymo. Prieš užtrūkinant abiejų grupių karvių teigiamai į CMT testą reagavo 55 % ketvirčių. Po veršiavimosi praėjus 10 dienų, pirmos grupės karvių į CMT teigiamai reagavo tik 10 %, o antros grupės karvių – 37,5 % ketvirčių. Pažeisti dažniau buvo galiniai ketvirčiai, lyginant su priekiniais. Prieš užtrūkinant 1 grupės karves, pagrindinė pieno mėginių mikroflora buvo sąlyginai... [toliau žr. visą tekstą] / Goal of the work: To analyze the qualitative composition of milk in the cow herd and effects of medications used for prevention of the termination of cow lactation. Tasks of the work: 1. To analyse the qualitative composition of milk in the cow herd. 2. Using the CMT reagent in the analysed cow farm, to examine the udder quarters of cows is selected for analysis, before termination of lactation and in 10 days after calving. 3. To perform bacteriological milk tests before termination of lactation and in 10 days after calving. 4. To determine the effectiveness of therapeutic preparations. The research work has been carried out in 2010–2013 in the private farm in Tauragė district. 20 cows were selected as the sample for research, and grouped into two groups of 10 cows. Groups received different treatment, the first group was treated with Rilexine 500, the second group with OrbeSeal preparation. Changes in the SCC, milk protein, fat, lactose and urea content were monitored before and after treatment along with diagnostics of mastitis pathogens and infection of the udder quarters. The analysis of the qualitative composition of the herd milk allows the state that the qualitative milk indicators start to improve after successful treatment of mastitis. Before termination of lactation, 55% quarters of both groups of cows showed positive response to the CMT test. 10 days after calving, only 10 % quarters of the first group of cows and 37.5% quarters of the second group of cows... [to full text]

No description available.

La myéline est une gaine formée par l’enroulement de la membrane plasmique de la cellule de Schwann autour de l’axone dans le nerf périphérique. Lorsque cette gaine est détruite, on parle de démyélinisation, cela provoque de nombreuses maladies, dont les maladies de Charcot Marie Tooth (CMT) de type 1. Les maladies CMT sont héréditaires et atteignent le système nerveux périphérique. Les symptômes communs incluent : une faiblesse musculaire, une démarche maladroite, des troubles de l’équilibre et des pieds très cambrés ou très plats. Le type le plus fréquent est la forme autosomique dominante CMT1A.Une duplication du bras court du chromosome 17 contenant le gène PMP22 (Peripheral Myelin Protein 22) induit la CMT1A. La PMP22, une petite protéine exprimée par les cellules de Schwann, est donc en excès et entraine une démyélinisation. Il existe un modèle de rats transgéniques PMP22 (ou rats CMT1A) mimant cette pathologie humaine. Les rats CMT1A surexpriment la pmp22 de souris de façon hétérozygote. Jusqu’à présent, aucun remède n’existe pour les maladies CMT. Un des traitements envisageables est la thérapie génique. Le but de mon projet de thèse était d’étudier la validité et l'efficacité de la thérapie génique chez les rats CMT1A. La stratégie consiste à réduire la surexpression de la protéine PMP22 chez le rat CMT1A à l’aide d’ARNsh anti-PMP22. Pour ne pas être détruits par l’organisme et maintenir une expression longue, ces ARN sh-PMP22 sont transférés chez le rat grâce à des vecteurs viraux dérivés de virus adéno-associés, ou AAV (pour adeno-associated virus). Nous avons donc injecté un des différents sérotypes d'AAV,l'AAV9 exprimant les ARN sh-PMP22 de souris ainsi que la GFP comme marqueur des cellules infectées dans les nerfs sciatiques de rats CMT1A à l’âge de 6 jours ou 7 jours.Nous avons d’abord confirmé que les virus thérapeutiques infectaient une très large proportion de cellules de Schwann dans le nerf sciatique de rat CMT1A et ensuite que l’infection de ces cellules par les virus exprimant les ARN sh-PMP22 induisait une diminution significative de l’expression de la protéine PMP22. L'analyse du phénotype moteur des rats CMT1A traités avec les AAV9 exprimant les ARN sh-PMP22 montre que les rats CMT1A traités ne développent pas la maladie observée dans les contrôles. Également, les rats CMT1A présentent une hypoalgésie, un phénotype qui n’apparait pas dans les CMT1A traités avec les vecteurs thérapeutiques. Le traitement par thérapie génique empêche la réduction de la vitesse de conduction nerveuse observé dans les rats malades. Concernant la biodistribution des virus, 2,5 mois après le traitement, en dehors des nerfs sciatiques ou les virus ont été injectés, le virus était présent dans les muscles qui entourent le nerf et aussi dans quelques ganglion dorsaux. Pour la réponse immunitaire,les rats injectés, à seulement 2 exceptions près, n’ont pas développé de facteurs neutralisants anti-AAV9. Cette thérapie génique pourrait être utilisée dans les essais cliniques.Avant de passer aux études cliniques pour le traitement de la maladie CMT1A à l’aide d’AAV9 exprimant des ARN sh-PMP22 humain, la dose d’expression de ce ARN sh-PMP22 doit être très soigneusement déterminée car si la PMP22 est trop réduite, une autre maladie peut se développer, la neuropathie héréditaire avec hypersensibilité à la pression. Il est aussi important d’avoir un outil bien adapté qui permet d’évaluer l’efficacité du traitement. Aucun existant n’est assez fiable pour mesurer la myéline du nerf périphérique. Pour remédier à ce manque, nous avons testé la technique d'imagerie Coherent Anti-stokes Raman Scattering (CARS) en caractérisant avec succès les défauts de la myéline. Par conséquent, le CARS est une technique prometteuse permettant d’évaluer l’avancement des maladies de la myéline et l’efficacité de nouvelles thérapies pour les neuropathies périphériques démyélinisantes. / Myelin, a tissue synthesized by Schwann cells, covers and protects nerves. If damaged, it causes many demyelinating diseases such as the inherited peripheral nervous system disorder Charcot Marie Tooth or CMT type 1. CMT neuropathies display a large variability from one patient to another. Nevertheless, the most common symptoms include muscle weakness, an awkward way of walking (gait), equilibrium problem and highly arched or very flat feet. The most common subtype of CMT is an autosomal dominant disorder known as CMT1A. CMT1A is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene on the short arm of chromosome 17 (17p11.2) resulting in an excess of PMP22. This leads to demyelination. PMP22 is a small protein expressed by Schwann cells. There is still no cure for CMT diseases. One approach for a treatment is gene therapy. The aim of my thesis project was to deliver proof of principle for a gene therapy approach on a CMT1A rat model characterized by extra copies of mouse pmp22 gene (CMT1A rat). The treatment strategy consisted in reducing PMP22 overexpression in CMT1A rats with shRNA against PMP22. Viral vectors like adeno-associated virus (AAV having serotypes from1-10) are used to deliver shRNA in vivo so that they won’t be destroyed by the organism and for them to be long-lasting. Thus, we injected sciatic nerves of 6-7-day-old CMT1A rats with AAV9 expressing shRNA PMP22 with a GFP marker. We first confirmed that the virus highly transduced Schwann cells and that AAV9 shRNA PMP22 decreased PMP22 protein expression in CMT1A rats’ sciatic nerves. CMT1A rats treated with AAV9 shRNA PMP22 showed that they didn’t develop the motor phenotype seen in controls. Moreover, hypoalgesia observed in CMT1A rats was alleviated by treatment. In addition, gene therapy increased the reduced nerve conduction velocity found in CMT1A rats. Concerning safety, no viral off-targets were detected except in muscles close to the injection site (sciatic nerve) and in the dorsal root ganglions. Except for 2 rats, there was no immune response against AAV; no anti-AAV9 neutralizing factors. Consequently, this gene therapy could be used in clinical trials. Before moving to clinical studies, the minimal effective dosage should be very carefully defined because if PMP22 is completely deleted, another disease is caused: Hereditary Neuropathy with Pressure Palsies. It is also crucial to have a strong readout to evaluate the outcome of a treatment. However, no tool consistent enough exists for examining the peripheral nerve. Thus, we tested the label-free imaging technique Coherent Anti-stokes Raman Scattering (CARS) and successfully characterized myelination defects. Consequently, CARS could be used as a consistent outcome measure for developing new therapies for demyelinating peripheral neuropathies.

Neste trabalho foram realizados experimentos de soldagem circunferencial em tubos de aço inoxidável super duplex UNS S32750, com diâmetros de 19,05 mm e 48,20 mm. Foram executadas soldas utilizando-se diversos parâmetros de soldagem num equipamento MIG com controle CMT&reg Cold Metal Transfer. Os cordões de solda foram avaliados por inspeção visual e dimensional, além dos ensaios de tração e microdureza Vickers, bem como a análise microestrutural em conjunto com análise de precipitação de fases, a qual foi realizada em acordo com a prática A da norma ASTM A923, e ensaio de corrosão conforme a prática A da norma ASTM G48 em conjunto com a norma ASTM A923. Os resultados indicaram que a soldagem do tubo com diâmetro de 19,05 mm apresentou cordão de solda com dimensões inaceitáveis conforme norma, tendo sido esta condição atribuída a utilização de um elevado diâmetro do arame para as condições (parâmetros) usadas de soldagem. A soldagem do tubo com diâmetro de 48,20 mm apresentou falta de penetração nas condições empregadas, quando soldado pelo processo CMT&reg convencional. No caso da utilização do CMT&reg combinado com arco pulsado, em condições que geraram maior aporte de calor durante a soldagem, assim obteve-se penetração total da junta e acabamento superficial adequado. Os resultados indicaram que a soldagem utilizando o processo CMT&reg combinado com arco pulsado, nas condições (parâmetros) empregados geraram bom acabamento superficial, aliado propriedades mecânicas compatíveis, atendendo exigências de normas, bem como uma microestrutura balanceada e alta resistência à corrosão. / This study carried out circumferential welding experiments in UNS S32750 Super Duplex Stainless Steel tubes using diameters of 19,05 mm and 48,20 mm. Welds were performed using various welding parameters on a MIG machine with Cold Metal Transfer&reg CMT control. The weld joints were evaluated by visual and dimensional inspection in addition to the Vickers microhardness and traction tests, as well as the microstructural analysis in conjunction with phase precipitation analysis, which was performed according to practice A of ASTM A923, and corrosion test in accordance with practice A of ASTM G48 in conjunction with ASTM A923. The results indicated that welds performed in pipes with a diameter of 19.05 mm showed a weld joint with unacceptable dimensions according to the standard, this condition being attributed the use of a high wire diameter for the welding conditions used. Welding performed for pipes with a diameter of 48.20 mm showed a lack of penetration under the conditions employed when welded by the conventional CMT&reg process. In the case of the use of CMT&reg combined with pulsed arc, under conditions that generated greater heat input during welding, this resulted in total penetration of the joint and adequate surface finish. The results indicated that welding using the CMT&reg process combined with pulsed arc, under the conditions (parameters) employed generated good surface finish, combined mechanical properties, meeting standards requirements, as well as a balanced microstructure and high resistance to corrosion.

Neste trabalho foram realizados experimentos de soldagem circunferencial em tubos de aço inoxidável super duplex UNS S32750, com diâmetros de 19,05 mm e 48,20 mm. Foram executadas soldas utilizando-se diversos parâmetros de soldagem num equipamento MIG com controle CMT&reg Cold Metal Transfer. Os cordões de solda foram avaliados por inspeção visual e dimensional, além dos ensaios de tração e microdureza Vickers, bem como a análise microestrutural em conjunto com análise de precipitação de fases, a qual foi realizada em acordo com a prática A da norma ASTM A923, e ensaio de corrosão conforme a prática A da norma ASTM G48 em conjunto com a norma ASTM A923. Os resultados indicaram que a soldagem do tubo com diâmetro de 19,05 mm apresentou cordão de solda com dimensões inaceitáveis conforme norma, tendo sido esta condição atribuída a utilização de um elevado diâmetro do arame para as condições (parâmetros) usadas de soldagem. A soldagem do tubo com diâmetro de 48,20 mm apresentou falta de penetração nas condições empregadas, quando soldado pelo processo CMT&reg convencional. No caso da utilização do CMT&reg combinado com arco pulsado, em condições que geraram maior aporte de calor durante a soldagem, assim obteve-se penetração total da junta e acabamento superficial adequado. Os resultados indicaram que a soldagem utilizando o processo CMT&reg combinado com arco pulsado, nas condições (parâmetros) empregados geraram bom acabamento superficial, aliado propriedades mecânicas compatíveis, atendendo exigências de normas, bem como uma microestrutura balanceada e alta resistência à corrosão. / This study carried out circumferential welding experiments in UNS S32750 Super Duplex Stainless Steel tubes using diameters of 19,05 mm and 48,20 mm. Welds were performed using various welding parameters on a MIG machine with Cold Metal Transfer&reg CMT control. The weld joints were evaluated by visual and dimensional inspection in addition to the Vickers microhardness and traction tests, as well as the microstructural analysis in conjunction with phase precipitation analysis, which was performed according to practice A of ASTM A923, and corrosion test in accordance with practice A of ASTM G48 in conjunction with ASTM A923. The results indicated that welds performed in pipes with a diameter of 19.05 mm showed a weld joint with unacceptable dimensions according to the standard, this condition being attributed the use of a high wire diameter for the welding conditions used. Welding performed for pipes with a diameter of 48.20 mm showed a lack of penetration under the conditions employed when welded by the conventional CMT&reg process. In the case of the use of CMT&reg combined with pulsed arc, under conditions that generated greater heat input during welding, this resulted in total penetration of the joint and adequate surface finish. The results indicated that welding using the CMT&reg process combined with pulsed arc, under the conditions (parameters) employed generated good surface finish, combined mechanical properties, meeting standards requirements, as well as a balanced microstructure and high resistance to corrosion.

CMT4H est une forme de CMT démyélinisant, à transmission autosomique récessive, pour laquelle notre équipe a identifié le gène responsable. Il s'agit du gène FGD4, codant pour FRABIN, protéine de 766 AA possédant 5 domaines fonctionnels: le domaine FAB de liaison à l'actine, un domaine DH responsable de l'échange GDP/GTP, et trois domaines de liaison avec des polyphosphoinositides. C'est une RhoGEF, connue pour activer les RhoGTPases Cdc42 et Rac1.Mon projet de thèse vise à mieux comprendre les bases moléculaires et les mécanismes physiopatologiques qui sous-tendent CMT4H grace à l'étudie de modèles cellulaires et murins. Dans un premier temps, j'ai identifié deux nouvelles mutations dans le gène FGD4. J'ai pu démontrer l'impact fonctionnel des mutations p.Met298fs*8 et p.Ala172Glyfs*27 et une absence totale la protéine dans les fibroblastes de ces patients, donnant lieu à une augmentation de l'activation de Cdc42.L'étude d'un modèle murin d'ablation conditionnelle de FGD4 dans les cellules de Schwann, m'a permis de démontrer la présence d' anomalies myéliniques dans le nerf périphérique de ces souris, ainsi qu'une diminution de l'activation de Cdc42.J'ai également montré, que dans les fibroblastes, FRABIN était localisée au niveau des endomembranes et que l'endocytose semblait déficient dans des cellules de patients. Finalement, l'identification de SNX3,comme partenaire protéique de FRABIN constitue un argument supplémentaire fort en faveur du rôle de FRABIN dans le trafic membranaire.La poursuite de l'étude de nos modèles et de modèles iPS ,permettra de poursuivre l'exploration de ces mécanismes et de mieux comprendre la physiopathologie de la forme CMT4H. / Charcot-Marie-Tooth neuropathy type 4H (CMT4H) is an inherited, autosomal recessive, peripheral neuropathy characterized by demyelination of sensory-motor nerves and due to mutations in FGD4. FGD4 encodes FRABIN, a GDP/GTP nucleotide exchange factor (GEF), specific for the GTPase Cdc42, composed of five functional domains: an N-terminal F-actin binding (FAB) domain, one Dbl homology (DH) domain, two pleckstrin homology (PH) domains, and one cysteine-rich FYVE domain.The main goal of my project is to understand the mechanisms leading to the pathology in CMT4H. To this purpose, I studied both cellular and mouse models.First, molecular screening of FGD4 allowed us to identify two additional mutations in FGD4. We also demonstrated a complete absence of the 105 kDa FRABIN isoform in patients homozygous for splicing and frameshift mutations, which unexpectedly was related to abnormally high levels of Cdc42 activation.The study of a mouse model with conditional ablation of fgd4 in Schwann cells, that we have generated, demonstrates the presence of abnormal myelin outfoldings in sciatic nerves from KO mice, which might be linked to decreased levels of Cd42 in mouse sciatic nerves. Finally, altered recycling of transferrin receptors in patients, with complete absence of FRABIN described above, as well as the identification of SNX3, a protein involved in endosomal trafficking, as a partner for FRABIN are new elements that I provide in favour of a role for FRABIN in membrane and cellular trafficking.Still, there are many points to understand, notably the relation between the RhoGTPase and the endosomal pathways, and the study of our models will help answer these questions.

Geólogo / El presente trabajo muestra la implementación de metodologías de trabajo en campo, microscopía y dataciones en circones para el estudio de las distintas fuentes de aporte y el contexto geodinámico del Complejo Metamórfico Trafún (CMT). Las metodologías consisten, en primera instancia, en una toma de datos geológicos en terreno, tales como litologías, estructuras y toma de muestras. La metodología siguiente consiste en un trabajo de microscopía óptica para estudiar la petrografía de las muestras, se estudia la mineralogía metamórfica y sus relaciones deformacionales, junto al estudio del protolito de las rocas. Se separaron en 3 tipos de rocas, metapelitas, metapsammitas y metaruditas. La información obtenida de las metapsammitas, mediante conteo modales, permite obtener diagramas de clasificación tectónica. De las metaruditas se muestrean variados clastos los cuales son descritos microscópicamente. La última metodología consiste en la obtención de edades U-Pb en circones, tanto detríticos para una metapsammita, como ígneos de un clasto de granitoide en la metarudita. Obteniendo edades de 372 Ma. para el clasto intrusivo, y edad de máxima depositación de 357 Ma. para la metapsammita junto con un fuerte aporte desde fuentes devónicas y ordovícicas.

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance signaling lipid important for the maintenance of the endomembrane system and selected membrane trafficking pathways. In yeast, in response to hyperosmotic stress, PI(3,5)P2 levels rise more than 20-fold in 5 minutes, and return to near basal levels in 30 minutes. This transient change suggests that PI(3,5)P2 levels are tightly regulated and may be involved in signaling a response to stress. In yeast, PI(3,5)P2 is synthesized through phosphorylation of PI(3)P by the PI(3)P 5-kinase Fab1. Loss of PI(3,5)P2 in yeast causes swollen vacuoles, defective retrograde trafficking from the vacuole, defective vacuole acidification, and mis-localization of a subset of vacuole lumenal proteins. In yeast, Vac14 is a regulator of PI(3,5)P2 levels. Mammalian Vac14 and Fab1 are found in the same complex. To study the physiological significance of PI(3,5)P2, a mouse strain was generated with the Vac14 gene disrupted by a gene-trap genomic insertion. Vac14 protein was not detectable in mutant mice. In fibroblasts cultured from the mutant mice, PI(3,5)P2 and PI(5)P are decreased to 42% and 44% of the corresponding wild-type levels, respectively. The mutant mouse brains exhibit spongiform-like morphology. Cytoplasmic vacuoles are found in neuronal cell bodies of the olfactory bulb, trigeminal ganglion, and dorsal root ganglion. Non-neural tissues appear largely normal. Similar vacuoles are also found in cultured neurons and fibroblasts. In fibroblasts, these vacuoles are formed from swelling of late endosomes/lysosomes. Some early endosomes are also enlarged. A population of cation-independent mannose-6-phosphate receptor (CI-M6PR), which recycles between endosomes and the trans-Golgi network (TGN), is trapped in early and late endosomes, indicating a block in endosome-to-TGN trafficking. These results suggest that: 1) Neurons are acutely sensitive to loss of PI(3,5)P2. 2) In mammals, PI(3,5)P2 is required for the morphology of late endosomes/lysosomes and retrograde trafficking from endosomes to the TGN. The first conclusion is supported by another mouse strain with a retro-transposon inserted in the Fig4 gene. Fig4 is another regulator of PI(3,5)P2 levels. Similar neurodegeneration was observed in the Fig4 mutant mice.