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IntroductionThis study aims to define the phenotypic and molecular spectrum of the two clinical forms of β-galactosidase (β-GAL) deficiency, GM1-gangliosidosis and mucopolysaccharidosis IVB (Morquio disease type B, MPSIVB).MethodsClinical and genetic data of 52 probands, 47 patients with GM1-gangliosidosis and 5 patients with MPSIVB were analysed.ResultsThe clinical presentations in patients with GM1-gangliosidosis are consistent with a phenotypic continuum ranging from a severe antenatal form with hydrops fetalis to an adult form with an extrapyramidal syndrome. Molecular studies evidenced 47 variants located throughout the sequence of the GLB1 gene, in all exons except 7, 11 and 12. Eighteen novel variants (15 substitutions and 3 deletions) were identified. Several variants were linked specifically to early-onset GM1-gangliosidosis, late-onset GM1-gangliosidosis or MPSIVB phenotypes. This integrative molecular and clinical stratification suggests a variant-driven patient assignment to a given clinical and severity group.ConclusionThis study reports one of the largest series of b-GAL deficiency with an integrative patient stratification combining molecular and clinical features. This work contributes to expand the community knowledge regarding the molecular and clinical landscapes of b-GAL deficiency for a better patient management.

Elevated serum chitotriosidase (CHITO) is an indication of macrophage activation, and its capacity have been explored as a marker of inflammation in a number of disease states. For over a decade, CHITO plasma levels have been used by clinicians as a biomarker of inflammation in the lysosomal disease, Gaucher disease, including monitoring response to therapies in patients with Gaucher disease type I. Although it is becoming increasingly recognized that inflammation is a prominent component of many lysosomal diseases, the relation of CHITO levels to disease burden has not been well-characterized in the large majority of lysosomal diseases. Moreover, the role of CHITO in lysosomal diseases that affect the central nervous system (CNS) has not been systematically studied. In this study, one hundred and thirty-four specimens of CSF and serum were collected from 34 patients with lysosomal diseases affecting the CNS. This study included patients with GM1-gangliosidosis, GM2-gangliosidosis, mucopolysaccharidoses (MPS), multiple sulfatase deficiency and Gaucher disease. CHITO levels in the CSF were significantly higher in patients with more rapidly progressing severe neurological impairment: GM1-gangliosidosis vs MPS (p

Satellite glial cells (SGCs) of dorsal root ganglia (DRG) react in response to various injuries in the nervous system. This study investigates reactive changes within SGCs in a murine model for GM1‐gangliosidosis (GM1). DRG of homozygous β‐galactosidase‐knockout mice and homozygous C57BL/6 wild‐type mice were investigated performing immunostaining on formalin‐fixed, paraffin‐embedded tissue. A marked upregulation of glial fibrillary acidic protein (GFAP), the progenitor marker nestin and Ki67 within SGCs of diseased mice, starting after 4 months at the earliest GFAP, along with intracytoplasmic accumulation of ganglioside within neurons and deterioration of clinical signs was identified. Interestingly, nestin‐positive SGCs were detected after 8 months only. No changes regarding inwardly rectifying potassium channel 4.1, 2, 3‐cyclic nucleotide 3‐phosphodiesterase, Sox2, doublecortin, periaxin and caspase3 were observed in SGCs. Iba1 was only detected in close vicinity of SGCs indicating infiltrating or tissue‐resident macrophages. These results indicate that SGCs of DRG show phenotypical changes during the course of GM1, characterized by GFAP upregulation, proliferation and expression of a neural progenitor marker at a late time point. This points towards an important role of SGCs during neurodegenerative disorders and supports that SGCs represent a multipotent glial precursor cell line with high plasticity and functionality.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid &beta, galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent &beta, d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a &ldquo, strategic&rdquo, hydroxyl group. New compounds have revealed highly promising activities with a range of &beta, galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

GM1-gangliosidosis is caused by a reduced activity of β-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1−/− mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1−/− mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1−/− mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon–glial interactions and therapy trials of lysosomal storage diseases.

GM1-gangliosidosis is caused by a reduced activity of β-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1−/− mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1−/− mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1−/− mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon–glial interactions and therapy trials of lysosomal storage diseases.

PubMedID: 30712135 Gangliosidoses (GM1 and GM2 gangliosidosis) are rare, autosomal recessive progressive neurodegenerative lysosomal storage disorders caused by defects in the degradation of glycosphingolipids. We aimed to investigate clinical, biochemical and molecular genetic spectrum of Turkish patients with infantile gangliosidoses and examined the potential role of serum aspartate transaminase levels as a biomarker. We confirmed the diagnosis of GM1 and GM2 gangliosidosis based on clinical findings with specific enzyme and/or molecular analyses. We retrospectively reviewed serum aspartate transaminase levels of patients with other biochemical parameters. Serum aspartate transaminase level was elevated in all GM1 and GM2 gangliosidosis patients in whom the test was performed, along with normal alanine transaminase. Aspartate transaminase can be a biochemical diagnostic clue for infantile gangliosidoses. It might be a simple but important biomarker for diagnosis, follow up, prognosis and monitoring of the response for the future therapies in these patients. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase β-galactosidase (β-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing β-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (β-gal−/−). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult β-gal−/− mice showed clearance of enzyme activity in the plasma and an increase in β-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating β-gal−/− mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated β-gal−/− mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed β-gal−/− mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in β-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active β-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to β-gal−/− mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in β-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).

GM1-gangliosidosis (MIM# 230500) and mucopolysaccharidosis IV (Morquio B, MIM# 230500) are autosomal-recessive diseases, which belong to the group of lysosomal storage disorders and are caused by changes in the structure of the same gene, GLB1. The mutations in GLB1 lead to deficiency in acid β-galactosidase, and, as a result, the accumulation of GM1-gangliosidosis and keratan sulphate in the patients’ lysosomes. Aim. To analyze the spectrum of mutations in GLB1 in Ukrainian patients with GM1 gangliosidosis and Morquio B disease. Results. We analyzed GLB1 in 25 Ukrainian patients with the diagnosis of GM1-gangliosidosis and one patient with Morquio B disease; 52 alleles were analyzed. Seventeen types of pathogenic mutations were identified, including 11 missense replacements, three deletions, one insertion and two mutations in the splicing site. The missense mutation p.His281Tyr (c.841C>T) in exon 8 was found to be the most common, as it was found in 19 out of 52 mutant alleles (36.5 %) of all the examined patients. The study allowed to identify six new mutations, which had not been found in any databases or previously described in scientific literature, including three deletions (c.699delG, c.833delG, c.1203_1205delTTA), two missense replacements (p.Gly243Arg, p.Gly262Ala) and one mutation in the splicing site (IVS12+8T>C). Conclusions. Our results can be used for a precise molecular diagnostics of patients with GM1-gangliosidosis and Morquio B disease and prenatal diagnostics in the high risk families. GM1-гангліозидоз (MIM# 230500) та мукополісахаридоз IV (Моркіо В, MIM# 230500) аутосомно-рецесивні захворювання, що відносяться до лізосомних хвороб накопичення та викликані змінами в структурі одного й того ж гену – GLB1. Результатом мутацій в гені GLB1 є дефіцит кислої β-галактозидази і, як наслідок, накопичення в лізосомах пацієнтів GM1-гангліозиду та кератансульфату. Мета. Проведення аналізу спектру мутацій в гені GLB1 серед пацієнтів з GM1-гангліозидозом та синдромом Моркіо В в Україні. Результати. При проведенні молекулярного аналізу гену GLB1 у 25 українських пацієнтів з діагнозом GM1-гангліозидоз та у 1 пацієнта з синдромом Моркіо В було проаналізовано 52 алелі, в яких виявлено 17 типів патогенних мутацій, серед яких 11 – місенс заміни, 3 – делеції, 1 – інсерція та 2 – мутації сайту слайсингу. Найчастішою виявилась місенс мутація p.His281Tyr (с.841С>T) у 8-му екзоні, яка була знайдена у 19 з 52 мутантних алелів (36,5 %) усіх обстежених пацієнтів. В ході дослідження було виявлено шість нових мутацій, які не були знайдені в електронних базах та не були описані раніше в літературі, серед яких 3 – делеції (c.699delG, c.833delG, с.1203_1205delTTA), 2 – місенс заміни (p.Gly243Arg, p.Gly262Ala) та 1 – заміна нуклеотиду в сайті сплайсингу (IVS12+8T>C). Висновки. Результати проведеного молекулярно-генетичного аналізу мутацій в гені GLB1 можуть бути використані для більш точної молекулярної діагностики хворих з GM1-гангліозидозом та синдромом Моркіо В та пренатальної діагностики в сім’ях високого ризику обтяжених даним захворюванням. GM1-ганглиозидоз (MIM # 230500) и мукополисахаридоз IV (Моркио В, MIM # 230500) аутосомно-рецессивные заболевания, относящиеся к лизосомным болезням накопления и вызванные изменениями в структуре одного и того же гена – GLB1. Результатом мутаций в гене GLB1 является дефицит кислой β-галактозидазы и, как следствие, накопление в лизосомах пациентов GM1-ганглиозида и кератансульфата. Цель. Проведение анализа спектра мутаций в гене GLB1 у пациентов с GM1-ганглиозидозом и синдромом Моркио В в Украине. Результаты. При проведении молекулярно-генетического анализа в гене GLB1 у 26 пациентов с GM1-ганглиозидозом и синдромом Моркио В в Украине было выявлено 52 аллеля, содержащих патогенные мутации. Наиболее часто встречалась миссенс мутация p.His281Tyr (с.841С>T) в 8 экзоне, которая была найдена у 19 из 52 мутантных аллелей (36,5 %) всех обследованных пациентов. В процессе исследования было обнаружено шесть новых патогенных мутаций, которые не были найдены в электронных базах и не описаны ранее в литературе, среди которых 3 – делеции (c.699delG, c.833delG, с.1203_1205delTTA), 2 – миссенс замены (p.Gly243Arg, p .Gly262Ala) и 1 – замена нуклеотида в сайте сплайсинга (IVS12 + 8T> C). Выводы. Результаты проведенного молекулярно-генетического анализа мутаций в гене GLB1 могут быть использованы для более точной молекулярной диагностики больных с GM1-ганглиозидозом и синдромом Моркио В и пренатальной диагностики в семьях высокого риска обремененных данным заболеванием.

GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid β-galactosidase (β-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1−/− cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes. Keywords: High-throughput imaging, GM1-gangliosidosis, Acid β-galactosidase, Cholera toxin B subunit

Background The gangliosidoses are rare inherited diseases that result in pathologic accumulation of gangliosides in the central nervous system and other tissues, leading to severe and progressive neurological impairment and early death in the childhood forms. No treatments are currently approved for the gangliosidoses, and development of treatments is impaired by limited understanding of the natural history of these diseases. Objective The objective of this study is to improve understanding of the juvenile gangliosidoses phenotypes and the late-infantile phenotypic subtype. Methods Through a prospective natural history study of subjects with juvenile GM1- and GM2-gangliosidosis, a timeline of clinical changes was developed for the classic juvenile phenotypes and the late-infantile phenotypes and results of serial neurodevelopmental testing was analyzed. Results Several candidate ‘outcome measures’ were identified: changes in ambulation and verbalization skills, the communication domain from neurodevelopmental testing and the caregiver-reported socialization domain. Conclusions The most common symptoms leading caregivers to seek a genetic diagnosis were changes in ambulation and verbalization.

GM1 ganglioside, a monosialic glycosphingolipid and a crucial component of plasma membranes, accumulates in lysosomal storage disorders, primarily in GM1 gangliosidosis. The development of biomarkers for simplifying diagnosis, monitoring disease progression and evaluating drug therapies is an important objective in research into neurodegenerative lysosomal disorders. With this in mind, we established fluorescent imaging and flow-cytometric methods to track changes in GM1 ganglioside levels in patients with GM1 gangliosidosis and in control cells. We also evaluated GM1 ganglioside content in patients’ cells treated with the commercially available Miglustat, a substrate inhibitor potentially suitable for the treatment of late-onset GM1 gangliosidosis. The flow-cytometric method proved to be sensitive, unbiased, and rapid in determining variations in GM1 ganglioside content in human lymphocytes derived from small amounts of fresh blood. We detected a strong correlation between GM1 ganglioside content and the clinical severity of GM1 gangliosidosis. We confirm the ability of Miglustat to act as a substrate reduction agent in the patients’ treated cells. As well as being suitable for diagnosing and managing patients with GM1 gangliosidosis this method could be useful in the diagnosis and management of other lysosomal diseases, such as galactosialidosis, Type C Niemann-Pick, and any other disease with pathologic variations of GM1 ganglioside.

Graphical abstract Highlights ► N-Alkylation of 1-deoxy-D-galactonojirimycin with dansylamino terminated spacer arms provides powerful inhibitors. ► These enhance mutant lysosomal -galactosidase activities in GM1 gangliosidosis cell lines. ► Based on their fluorescence, they can serve as selective intracellular organellar probes., Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-d-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-d-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-d-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-d-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.

beta-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the beta-galactosidase gene cause the lysosomal storage disease G(M1)-gangliosidosis. In order to identify additional molecular changes associated with the presence of beta-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1(+/+), GLB1(+/-) and GLB1(-/-) fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total beta-galactosidase, beta-galactosidase variants and protective protein for beta-galactosidase gene (PPGB) in GLB1(+/-) and GLB1(-/-) compared to GLB1(+/+) fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant beta-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1(+/+), GLB1(+/-) and GLB1(-/-) genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

Recombinant gene delivery vehicles based on the replication-defective AAV have gained a preeminent position in the field of gene delivery to the brain. Efficient global gene delivery to the CNS is beneficial for the study of gene products is the entire CNS as well as for introducing and expressing genes encoding potential therapeutic or protective proteins in neuronal tissue. Although efficient global gene delivery to the CNS is an ambitious goal, recent scientific developments, as for instance the expansion of the repertoire of AAV vectors by the isolation of new serotypes and the creation of AAV vectors with chimeric capsids, might open up new avenues to achieve this goal. The overall aim of this thesis was to achieve efficient, scientifically and therapeutically relevant AAV- mediated gene delivery to the mouse brain. First, experiments were performed to assess the ability of different serotype AAV vectors to achieve efficient gene delivery to the CNS upon neonatal ICV injection. AAV8 was able to achieve most efficient, widespread delivery of transgenes. Second, the transduction properties of novel mosaic vectors incorporating capsid proteins from AAV1, AAV2, and AAV8 capsids are described. The transduction profiles of these vectors were compared to those of parental serotypes in mouse primary cortical cultures and in vivo after injection into the cerebral lateral ventricles of neonatal mice. Both in culture and in vivo, these mosaic vectors displayed additional properties. Third, the properties of both parental serotype vectors and mosaic vectors were assessed upon injection into the striatum of adult mice. The lysosomal storage disease GM1-gangliosidosis, an autosomal recessive deficiency of lysosomal acid β-galactosidase is biochemically characterized by accumulation of GM1-ganglioside in the central nervous system (CNS), and partially degraded glycoproteins, keratan sulfate and oligosaccharides in visceral organs. GM1-gangliosidosis is a proper candidate for gene therapy, as not all cells in the central nervous system have to be genetically modified to achieve therapeutic efficacy, because mechanisms for efficient secretion and uptake of lysosomal enzymes exist. Next, experiments were conducted to characterize the distribution of lysosomal β-galactosidase in the adult GM1-gangliosidosis mouse brain after AAV-mediated gene delivery. Several mechanisms of distribution were observed including: diffusion, axonal transport of the enzyme and axonal transport of mRNA. To evaluate the therapeutic potential of AAV- mediated gene delivery in neonatal GM1-gangliosidosis mice, an AAV vector encoding for β-galactosidase was injected into the cerebroventricles of neonatal GM1-gangliosidosis mice. which resulted in complete correction of enzymatic deficiency and lysosomal storage throughout the GM1-gangliosidosis mouse forebrain. Based on the results described in this thesis, AAV vectors can be considered highly promising vehicles for the delivery of therapeutically relevant transgenes to the CNS.

Recombinant gene delivery vehicles based on the replication-defective AAV have gained a preeminent position in the field of gene delivery to the brain. Efficient global gene delivery to the CNS is beneficial for the study of gene products is the entire CNS as well as for introducing and expressing genes encoding potential therapeutic or protective proteins in neuronal tissue. Although efficient global gene delivery to the CNS is an ambitious goal, recent scientific developments, as for instance the expansion of the repertoire of AAV vectors by the isolation of new serotypes and the creation of AAV vectors with chimeric capsids, might open up new avenues to achieve this goal. The overall aim of this thesis was to achieve efficient, scientifically and therapeutically relevant AAV- mediated gene delivery to the mouse brain. First, experiments were performed to assess the ability of different serotype AAV vectors to achieve efficient gene delivery to the CNS upon neonatal ICV injection. AAV8 was able to achieve most efficient, widespread delivery of transgenes. Second, the transduction properties of novel mosaic vectors incorporating capsid proteins from AAV1, AAV2, and AAV8 capsids are described. The transduction profiles of these vectors were compared to those of parental serotypes in mouse primary cortical cultures and in vivo after injection into the cerebral lateral ventricles of neonatal mice. Both in culture and in vivo, these mosaic vectors displayed additional properties. Third, the properties of both parental serotype vectors and mosaic vectors were assessed upon injection into the striatum of adult mice. The lysosomal storage disease GM1-gangliosidosis, an autosomal recessive deficiency of lysosomal acid β-galactosidase is biochemically characterized by accumulation of GM1-ganglioside in the central nervous system (CNS), and partially degraded glycoproteins, keratan sulfate and oligosaccharides in visceral organs. GM1-gangliosidosis is a proper candidate for gene therapy, as not all cells in the central nervous system have to be genetically modified to achieve therapeutic efficacy, because mechanisms for efficient secretion and uptake of lysosomal enzymes exist. Next, experiments were conducted to characterize the distribution of lysosomal β-galactosidase in the adult GM1-gangliosidosis mouse brain after AAV-mediated gene delivery. Several mechanisms of distribution were observed including: diffusion, axonal transport of the enzyme and axonal transport of mRNA. To evaluate the therapeutic potential of AAV- mediated gene delivery in neonatal GM1-gangliosidosis mice, an AAV vector encoding for β-galactosidase was injected into the cerebroventricles of neonatal GM1-gangliosidosis mice. which resulted in complete correction of enzymatic deficiency and lysosomal storage throughout the GM1-gangliosidosis mouse forebrain. Based on the results described in this thesis, AAV vectors can be considered highly promising vehicles for the delivery of therapeutically relevant transgenes to the CNS.

Chaperone therapy is a newly developed molecular approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. We found two valienamine derivatives, N-octyl-4-epi-β-valienamine (NOEV) and N-octyl-β-valienamine (NOV), as promising therapeutic agents for human β-galactosidase deficiency disorders (mainly GM1-gangliosidosis) and β-glucosidase deficiency disorders (Gaucher disease), respectively. We briefly reviewed the historical background of research in carbasugar glycosidase inhibitors. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently GM1-gangliosidosis model mice were developed and useful for experimental studies. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced β-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Furthermore, we executed computational analysis for prediction of molecular interactions between β-galactosidase and NOEV. Some preliminary results of computational analysis of molecular interaction mechanism are presented in this article. NOV also showed the chaperone effect toward several β-glucosidase gene mutations in Gaucher disease. We hope chaperone therapy will become available for some patients with GM1-gangliosidosis, Gaucher disease, and potentially other lysosomal storage diseases with central nervous system involvement.

It has been demonstrated that glycolipids of the globo-and ganglioseries accumulate in brains with GM1-gangliosidosis and Tay-Sachs disease （T-S dis.）, although they are not detected in differentiated normal brains and cannot be explained by representative glycosidase deficiencies. The present study was undertaken to elucidate the metabolic mechanism responsible for the glycolipid accumulation in brains with these two diseases, in comparison with control human brains at different stages of differentiation. A simple method for glycosyltransferase assay using ion-exchange chromatography was developed and employed for this purpose. 1） The activity of globotriaosylceramied （Gb3 Cer） synthase was hardly detectable in unaffected embryo brains, and seemed to be a rate-limiting enzyme of the synthesis of globoseries. However, enzyme activity was detected in infants, and showed significant levels in the two diseases （3 in 4 cases of GM1-gangliosidosis and all 3 cases of T-S dis.）. Globotetraosylceramide （Gb4Cer） synthase activity was low in the fetal stage and gradually increased in the normal infant. The brains with GM1-gangliosidosis and T-S dis. showed similar activity as that of the infant. These results endorsed the synthetic occurrence of globoseries glycolipids in the brains of the two diseases. 2） In the normal brains, activity of gangliotriaosylceramide （Gg3Cer） synthase was detected only in the early fetal period, while that of gangliotetraosylceramide （Gg4Cer） synthase was very difficult to ?detect in developing infant brains. All GM1-gangliosidosis brains showed higher levels of the two enzyme activities, expressing early embryonic level. The accumulation of Gg4Cer in the GM1-gang-liosidosis brains seemed to be caused not only by catabolic β-galactosidase deficiency, but also by a higher level of Gg4Cer synthase and sialidase which forms Gg4Cer from GM1. 3） Activities of GM3, GM2 and GM1 synthases showed high levels in unaffected embryonic stage and decreased thereafter except for GM3 synthase. In many cases of the two gangliosidoses, higher levels of these enzymes were detected when compared to those of age-matched controls. On the other hand, although GD3 synthase was much less active in developing infant brains and T-S brains, the enzyme was significantly elevated in all the GM1-gangliosidosis brains of the two subtypes, being characteristic of the diseased brains.

Article, 信州医学雑誌 32(1): 62-77(1984)