Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.Unverricht-Lundborg-tyyppinen etenevä myoklonusepilepsia (EPM1) on peittyvästi periytyvä aivoja rappeuttava sairaus, jonka oireisiin kuuluvat epileptiset kohtaukset ja ärsykeherkät tahdottomat lihasnykäykset. Taudin oireet alkavat 6-15 vuoden iässä ja ovat eteneviä. EPM1:n taustalla ovat virheet kystatiini B (CSTB) -geenissä. Yleisin mutaatio on 12 emäksestä muodostuvan toistojakson monistuma CSTB-geenin säätelyalueella, minkä lisäksi tunnetaan yhdeksän muuta mutaatiota. CSTB-proteiini toimii tiettyjen valkuaisaineita pilkkovien entsyymien estäjänä, mutta sen tehtävää elimistössä ei tarkoin tunneta. Tässä väitöskirjatyössä tutkittiin normaalin CSTB-geenin ilmenemistä hiirellä ja ihmisellä, CSTB-proteiinin solulokalisaatiota sekä potilasmutaatioiden vaikutusta näihin. Kartoitimme ihmisen CSTB-geenin säätelyalueen ja osoitimme siihen paikantuvien transkriptiotekijöiden sitoutumisen. CSTB:n ilmeneminen väheni selvästi säätelyalueen sisältäessä toistojakson pidentymän sekä henkilöillä, joilla on normaalin muodon ja tautimutaation välinen muoto toistojakson pidentymästä. EPM1-potilaiden soluissa CSTB-geenin lähetti-RNA:n sekä proteiinin määrä oli merkittävästi alentunut. CSTB-proteiini paikantui jakautuvissa soluissa solunsisällä tumaan, sytoplasmaan ja lysosomeihin sekä erilaistuneissa soluissa pääasiallisesti sytoplasmaan. Potilasmutaatioiden seurauksena CSTB-proteiini menetti lokalisaation lysosomeihin. Tulosten mukaan solun erilaistumistaso vaikuttaa CSTB:n paikantumiseen ja lysosomaalinen sijainti on tarpeellinen sen oikean toiminnan kannalta. CSTB-geenissä havaittiin vaihtoehtoista silmikoitumista. CSTB2-muoto ilmeni laajasti, mutta sen osuus oli vain 0,4-4,1 % koko CSTB-geenin ilmentymisestä. Sen ilmeneminen oli alentunut potilassoluissa. Muut muodot näyttivät ilmenevän kudosspesifisesti. CSTB-geenin vaihtoehtoisen silmikoitumisen fysiologinen merkitys jäi epäselväksi. Havaitsimme hiiren Cstb-geenin ilmenevän laajasti ja olevan korkeinta 7-päivän ikäisessä sikiössä sekä kateenkorvassa. Keskushermostossa Cstb ilmeni isoaivokuorella, aivokuorukassa, talamuksessa ja hippokampuksessa, sekä pikkuaivojen Purkinje-solukerroksessa. Tulostemme perusteella CSTB ilmeneminen on voimakkaasti ajallisesti ja paikallisesti säädeltyä. Väitöskirjassani esitetyt tulokset luovat perustaa jatkotutkimuksille selvitettäessä CSTB-proteiinin normaalia toimintaa sekä sen puuttumisen ja virheellisen toiminnan merkitystä EPM1-taudin synnyssä

Biotransformation of Cyclodextrine-Complexed Semisynthetic Betulin Derivatives by Plant Cells

In this study, three semisynthetic betulonic acid-based compounds, 20(29)-dihydrolup-2-en[2,3- d ]isoxazol-28-oic acid, 1-betulonoylpyrrolidine, and lupa-2,20(29)-dieno[2,3- b ]pyrazin-28-oic acid, were studied in biotransformation experiments using Nicotiana tabacum and Catharanthus roseus cell suspension cultures. Biotransformation was performed using cyclodextrin to aid dissolving poorly water-soluble substrates. Several new derivatives were found, consisting of oxidized and glycosylated (pentose- and hexose-conjugated) products.Peer reviewe

Noggin null allele mice exhibit a microform of holoprosencephaly

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin−/− mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin−/− mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin−/− mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HP

Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.Peer reviewe

Lunapark deficiency leads to an autosomal recessive neurodevelopmental phenotype with a degenerative course, epilepsy and distinct brain anomalies

LNPK encodes a conserved membrane protein that stabilizes the junctions of the tubular endoplasmic reticulum network playing crucial roles in diverse biological functions. Recently, homozygous variants in LNPK were shown to cause a neurodevelopmental disorder (OMIM#618090) in four patients displaying developmental delay, epilepsy and nonspecific brain malformations including corpus callosum hypoplasia and variable impairment of cerebellum. We sought to delineate the molecular and phenotypic spectrum of LNPK-related disorder. Exome or genome sequencing was carried out in 11 families. Thorough clinical and neuroradiological evaluation was performed for all the affected individuals, including review of previously reported patients. We identified 12 distinct homozygous loss-of-function variants in 16 individuals presenting with moderate to profound developmental delay, cognitive impairment, regression, refractory epilepsy and a recognizable neuroimaging pattern consisting of corpus callosum hypoplasia and signal alterations of the forceps minor ('ear-of-the-lynx' sign), variably associated with substantia nigra signal alterations, mild brain atrophy, short midbrain and cerebellar hypoplasia/atrophy. In summary, we define the core phenotype of LNPK-related disorder and expand the list of neurological disorders presenting with the 'ear-of-the-lynx' sign suggesting a possible common underlying mechanism related to endoplasmic reticulum-phagy dysfunction

RAB23 regulates musculoskeletal development and patterning

RAB23 is a small GTPase which functions at the plasma membrane to regulate growth factor signaling. Mutations in RAB23 cause Carpenter syndrome, a condition that affects normal organogenesis and patterning. In this study, we investigate the role of RAB23 in musculoskeletal development and show that it is required for patella bone formation and for the maintenance of tendon progenitors. The patella is the largest sesamoid bone in mammals and plays a critical role during movement by providing structural and mechanical support to the knee. Rab23 ( -/- ) mice fail to form a patella and normal knee joint. The patella is formed from Sox9 and scleraxis (Scx) double-positive chondroprogenitor cells. We show that RAB23 is required for the specification of SOX9 and scleraxis double-positive patella chondroprogenitors during the formation of patella anlagen and the subsequent establishment of patellofemoral joint. We find that scleraxis and SOX9 expression are disrupted in Rab23 ( -/- ) mice, and as a result, development of the quadriceps tendons, cruciate ligaments, patella tendons, and entheses is either abnormal or lost. TGF beta-BMP signaling is known to regulate patella initiation and patella progenitor differentiation and growth. We find that the expression of TGF beta R2, BMPR1, BMP4, and pSmad are barely detectable in the future patella site and in the rudimentary tendons and ligaments around the patellofemoral joint in Rab23 ( -/- ) mice. Also, we show that GLI1, SOX9, and scleraxis, which regulate entheses establishment and maturation, are weakly expressed in Rab23 ( -/- ) mice. Further analysis of the skeletal phenotype of Rab23 ( -/- ) mice showed a close resemblance to that of Tgf beta 2 ( -/- ) mice, highlighting a possible role for RAB23 in regulating TGF beta superfamily signaling.Peer reviewe

A high-density association screen of 155 ion transport genes for involvement with common migraine

Free to read on publishers website The clinical overlap between monogenic Familial Hemiplegic Migraine (FHM) and common migraine subtypes, and the fact that all three FHM genes are involved in the transport of ions, suggest that ion transport genes may underlie susceptibility to common forms of migraine. To test this leading hypothesis, we examined common variation in 155 ion transport genes using 5257 single nucleotide polymorphisms (SNPs) in a Finnish sample of 841 unrelated migraine with aura cases and 884 unrelated non-migraine controls. The top signals were then tested for replication in four independent migraine case-control samples from the Netherlands, Germany and Australia, totalling 2835 unrelated migraine cases and 2740 unrelated controls. SNPs within 12 genes (KCNB2, KCNQ3, CLIC5, ATP2C2, CACNA1E, CACNB2, KCNE2, KCNK12, KCNK2, KCNS3, SCN5A and SCN9A) with promising nominal association (0.00041 < P < 0.005) in the Finnish sample were selected for replication. Although no variant remained significant after adjusting for multiple testing nor produced consistent evidence for association across all cohorts, a significant epistatic interaction between KCNB2 SNP rs1431656 (chromosome 8q13.3) and CACNB2 SNP rs7076100 (chromosome 10p12.33) (pointwise P = 0.00002; global P = 0.02) was observed in the Finnish case-control sample. We conclude that common variants of moderate effect size in ion transport genes do not play a major role in susceptibility to common migraine within these European populations, although there is some evidence for epistatic interaction between potassium and calcium channel genes, KCNB2 and CACNB2. Multiple rare variants or trans-regulatory elements of these genes are not ruled out

Abietane-type diterpenoid amides with highly potent and selective activity against Leishmania donovani and Trypanosoma cruzi

Dehydroabietylamine (1) was used as a starting material to synthesize a small library of dehydroabietyl amides by simple and facile methods, and their activities against two disease-causing trypanosomatids, namely, Leishmania donovani and Trypanosoma cruzi, were assayed. The most potent compound, 10, an amide of dehydroabietylamine and acrylic acid, was found to be highly potent against these parasites, displaying an IC50 value of 0.37 μM against L. donovani axenic amastigotes and an outstanding selectivity index of 63. Moreover, compound 10 fully inhibited the growth of intracellular amastigotes in Leishmania donovani-infected human macrophages with a low IC50 value of 0.06 μM. This compound was also highly effective against T. cruzi amastigotes residing in L6 cells with an IC50 value of 0.6 μM and high selectivity index of 58, being 3.5 times more potent than the reference compound benznidazole. The potent activity of this compound and its relatively low cytotoxicity make it attractive for further development in pursuit of better drugs for patients suffering from leishmaniasis and Chagas disease