Gene-Based Approaches to Inherited Neurometabolic Diseases.

In the last decade, the gene therapy (GT) field experienced a renaissance, thanks to crucial understandings and innovations in vector design, stem cell manipulation, conditioning protocols, and cell/vector delivery. These efforts were successfully coupled with unprecedented clinical results of the trials employing the newly developed technology and with the novel establishment of academic-industrial partnerships. A renewed and strengthened interest is rising in the development of gene-based approaches for inherited neurometabolic disorders with severe neurological involvement. Inherited metabolic disorders are monogenetic diseases caused by enzymatic or structural deficiencies affecting the lysosomal or peroxisomal metabolic activity. The metabolic defect can primarily affect the central nervous system, leading to neuronal death, microglial activation, inflammatory demyelination, and axonal degeneration. This review provides an overview of the GT strategies currently under clinical investigation for neurometabolic lysosomal and peroxisomal storage diseases, such as adrenoleukodystrophy and metachromatic leukodystrophy, as well as novel emerging indications such as mucopolysaccharidoses, gangliosidoses, and neuronal ceroid lipofuscinoses, with a comprehensive elucidation of the main features and mechanisms at the basis of a successful GT approach for these devastating diseases.

Gangliosides and gangliosidoses: principles of molecular and metabolic pathogenesis.

Gangliosides are the main glycolipids of neuronal plasma membranes. Their surface patterns are generated by coordinated processes, involving biosynthetic pathways of the secretory compartments, catabolic steps of the endolysosomal system, and intracellular trafficking. Inherited defects in ganglioside biosynthesis causing fatal neurodegenerative diseases have been described so far almost exclusively in mouse models, whereas inherited defects in ganglioside catabolism causing various clinical forms of GM1- and GM2-gangliosidoses have long been known. For digestion, gangliosides are endocytosed and reach intra-endosomal vesicles. At the level of late endosomes, they are depleted of membrane-stabilizing lipids like cholesterol and enriched with bis(monoacylglycero)phosphate (BMP). Lysosomal catabolism is catalyzed at acidic pH values by cationic sphingolipid activator proteins (SAPs), presenting lipids to their respective hydrolases, electrostatically attracted to the negatively charged surface of the luminal BMP-rich vesicles. Various inherited defects of ganglioside hydrolases, e.g., of ß-galactosidase and ß-hexosaminidases, and of GM2-activator protein, cause infantile (with tetraparesis, dementia, blindness) and different protracted clinical forms of GM1- and GM2-gangliosidoses. Mutations yielding proteins with small residual catabolic activities in the lysosome give rise to juvenile and adult clinical forms with a wide range of clinical symptomatology. Apart from patients' differences in their genetic background, clinical heterogeneity may be caused by rather diverse substrate specificities and functions of lysosomal hydrolases, multifunctional properties of SAPs, and the strong regulation of ganglioside catabolism by membrane lipids. Currently, there is no treatment available for neuronal ganglioside storage diseases. Therapeutic approaches in mouse models and patients with juvenile forms of gangliosidoses are discussed.

Neurobiology and cellular pathogenesis of glycolipid storage diseases.

Disorders of lysosomal metabolism often involve the accumulation of specific types of glycolipid, particularly gangliosides, because of either degradative failure or other currently unknown mechanisms. Although the precise role of gangliosides in cells remains enigmatic, the presence of specific abnormalities secondary to ganglioside accumulation in lysosomal diseases has suggested important biological functions. Chief among these is the growth of new dendrites on particular classes of mature neurons secondary to an increase in GM2 ganglioside. That GM2 has also been shown to be elevated in normal immature neurons coincident with dendritic sprouting provides a compelling argument that this ganglioside plays a role in dendritic initiation. This discovery has led to the search for other regulators of dendritic differentiation that may in some way be linked to the expression and/or function of GM2 ganglioside. Principal candidates that have emerged include tyrosine kinase receptors, small GTPases and calcium/calmodulin-dependent protein kinase II. Understanding the mechanism underlying ectopic dendritogenesis in lysosomal diseases can be expected to generate significant insight into the control of dendritic plasticity in normal brain. The detrimental aspects of ganglioside accumulation in storage diseases as well as the potential link between gangliosides and dendritogenesis also provide a strong rationale for developing pharmacological means to manipulate ganglioside expression in neurons.

Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis.

Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses.

Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases.

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.

Mouse model of GM2 activator deficiency manifests cerebellar pathology and motor impairment.

The GM2 activator deficiency (also known as the AB variant), Tay-Sachs disease, and Sandhoff disease are the major forms of the GM2 gangliosidoses, disorders caused by defective degradation of GM2 ganglioside. Tay-Sachs and Sandhoff diseases are caused by mutations in the genes (HEXA and HEXB) encoding the subunits of beta-hexosaminidase A. The GM2 activator deficiency is caused by mutations in the GM2A gene encoding the GM2 activator protein. For degradation of GM2 ganglioside by beta-hexosamindase A, the GM2 activator protein must participate by forming a soluble complex with the ganglioside. In each of the disorders, GM2 ganglioside and related lipids accumulate to pathologic levels in neuronal lysosomes, resulting in clinically similar disorders with an onset in the first year of life, progressive neurodegeneration, and death by early childhood. We previously have described mouse models of Tay-Sachs (Hexa -/-) and Sandhoff (Hexb -/-) diseases with vastly different clinical phenotypes. The Hexa -/- mice were asymptomatic whereas the Hexb -/- mice were severely affected. Through gene disruption in embryonic stem cells we now have established a mouse model of the GM2 activator deficiency that manifests an intermediate phenotype. The Gm2a -/- mice demonstrated neuronal storage but only in restricted regions of the brain (piriform, entorhinal cortex, amygdala, and hypothalamic nuclei) reminiscent of the asymptomatic Tay-Sachs model mice. However, unlike the Tay-Sachs mice, the Gm2a -/- mice displayed significant storage in the cerebellum and defects in balance and coordination. The abnormal ganglioside storage in the Gm2a -/- mice consisted of GM2 with a low amount of GA2. The results demonstrate that the activator protein is required for GM2 degradation and also may indicate a role for the GM2 activator in GA2 degradation.

Spongecake and eggroll: two hereditary diseases in Drosophila resemble patterns of human brain degeneration.

Various neuronal degenerative diseases are characterized by late onset, relentless progression, and finally death. Many have a direct genetic basis; others are of still unknown etiological mechanisms [1,2]. The study of human neurodegenerative diseases is complicated by the difficulty of obtaining tissue samples at various stages of progression, especially early in the course of the disease. Since neurodegeneration occurs in many organisms [3-5], model organisms amenable to genetic and molecular techniques, such as the mouse, offer important advantages. Much less laborious and expensive are worms or flies, which have short generation times and can be rapidly screened for mutations. To investigate the use of the fly as a model system for identifying genes related to such diseases, we screened for mutants having reduced lifespan, then examined them for brain degeneration. We describe here two such mutants, each with a different pattern of degeneration as characterized by light and transmission electron microscopy. The brain of the aging spongecake mutant exhibits regionally specific, membrane-bound vacuoles similar to those seen in spongiform degenerations such as Creutzfeldt-Jakob disease [6,7]. The mutant eggroll develops dense, multilamellated structures in the brain, resembling ones found in lipid storage diseases such as Tay-Sachs [8].

Intracellular degradation of sulforhodamine-GM1: use for a fluorescence-based characterization of GM2-gangliosidosis variants in fibroblasts and white blood cells.

A novel fluorescent ganglioside, sulforhodamine-GM1 was administered into cells derived from carriers and patients with different subtypes of GM2 gangliosidosis, resulting from various mutations in the gene encoding the lysosomal enzyme hexosaminidase (Hex) A. The cells used were skin fibroblasts and white blood cells, i.e. lymphocytes, monocytes and macrophages. In the severe infantile form of the GM2 gangliosidosis, Tay-Sachs disease, the sulforhodamine-GM1 was hydrolyzed within the lysosomes to the corresponding sulforhodamine-GM2 which, because of lack of Hex A activity, was not further degraded. In comparison, in the cells derived from GM2 gangliosidoses carriers, as well as pseudodeficient and adult forms of GM2 gangliosidosis, the sulforhodamine-GM2 was further processed and sequentially degraded by the lysosomal glycosidases to sulforhodamine-ceramide. The latter was converted to sulforhodamine-sphingomyelin, which was secreted into the culture medium. The fluorescence of the sulforhodamine ceramide in cell extracts and/or sulforhodamine-sphingomyelin in the culture medium was quantified and related to parallel data obtained using cells of normal individuals. This permitted distinguishing between the various GM2 gangliosidoses subtypes and relating the intracellular hydrolysis of sulforhodamine-GM1 to the genotypes of the respective GM2 gangliosidoses variants.

[Lysosomal storage diseases with angiokeratoma corporis diffusum].

There are 6 well known lysosomal storage diseases which produce angiokeratoma corporis diffusum clinically. The clinical, histological, ultrastructural and biochemical characteristics are discussed. The best known angiokeratoma will be observed in patients with Fabry disease. Angiokeratoma in Fabry disease, however, may be much fewer than thought previously. Fucosidosis and galactosialidosis are next well known diseases to produce angiokeratoma. Approximately 50% of patients with these diseases have angiokeratoma. Recently reported Kanzaki disease, beta-mannosidosis and aspartylglucosaminuria will show angiokeratoma more or less extensively. Ultrastructurally Fabry disease only produce electron dense deposits in lysosomes and others electron lucent. These are summarized in Table 1 in the text.

Gangliosidosis in emus (Dromaius novaehollandiae).

A 6-month-old female emu (Dromaius novaehollandiae) died following acute central nervous system signs. Hematoxylin-and-eosin-stained sections revealed that neurons of the brain were distended with nonstaining 1-to-2-microns vacuoles. Ultrastructural examination of the affected neurons revealed numerous membranous cytoplasmic bodies (MCBs) similar in appearance to the MCBs seen in mammalian gangliosidoses. A full sibling of this emu was donated for study. This 7-month-old female emu was stunted compared with hatchmates. Neurologic examination revealed hypermetric gait, persistent head tremor, and mild ataxia. No gross lesions were evident at postmortem. Histopathologic and electron microscopic findings were similar to those in the index case in that swollen, pale neurons were present in the cerebrum, pons, medulla, cerebellum, spinal cord, spinal ganglia, autonomic ganglia, myenteric plexus, and ganglion cell layer of the retina. Analysis of brain gangliosides of the affected 7-month-old emu revealed 14- and 25-fold increases of GM1 and GM3 gangliosides, respectively, compared with control emus. The total brain ganglioside sialic acids were, on a wet weight basis, 519 micrograms/g (control A), 658 micrograms/g (control B), and 1800 micrograms/g (affected emu). The familial association seen with this condition suggests that emus are affected by an inherited disorder similar to mammalian gangliosidoses.

Lysosomal storage diseases in adults.

Most lysosomal storage disorders are known as pediatric diseases. In recent years late onset and adult forms of these disorders have been recognized. The adult form of a given lysosomal storage disorder differs from the childhood disease in several respects. Adult disorders are, with some exceptions, less common than the childhood diseases. The clinical picture is not only less severe, but often shows quite different clinical signs and symptoms than the early onset form. Metachromatic leucodystrophy, GM1 and GM2 gangliosidoses, Gaucher disease and aspartylglucosaminuria are presented as examples of lysosomal storage disorders manifesting as adult diseases. The differences of the early and late onset disorders are discussed in the light of recent results of molecular genetics, residual enzyme activity and pseudodeficiency.

Gangliosidosis GM1 Tipo I: forma infantil / Gangliosidosis GM1 Type I: infantile form

Se presenta un caso de Gangliosidosis GM1, tipo I, de diagnóstico tardío para llamar la atención sobre una enfermedad hereditaria. Se trata de una lactante de sexo femenino que a pesar de presentar los signos principales de la enfermedad no fue reconocida tempranamente. El diagnóstico se confirmó a los 6 meses por dosificación de la enzima beta-galactosidasa lisosomal en leucocitos. Se destaca la falla de la atención primaria y la necesidad de un diagnóstico seguro para poder realizar un adecuado consejo genético

Growth of ectopic dendrites on cortical pyramidal neurons in neuronal storage diseases correlates with abnormal accumulation of GM2 ganglioside.

Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.

Brain imaging in late-onset GM2 gangliosidosis.

We describe brain CT and MRI characteristics of 10 patients with late-onset GM2 gangliosidosis. Cerebellar atrophy, particularly of the vermis, was a prominent feature in all patients with normal-appearing cerebral hemispheres. The severity of these findings did not correlate with the age of onset, disease duration, severity of neurologic impairment, or mode and distribution of the various clinical presentations. In particular, no cerebral abnormality was found by neuroimaging in seven patients with intellectual decline and in six patients with recurrent psychosis, while prominent cerebellar atrophy was present in the only patient who was free of cerebellar signs.

Canine GM1-gangliosidosis. A clinical, morphologic, histochemical, and biochemical comparison of two different models.

The clinical, morphologic, histochemical, and biochemical features of GM1-gangliosidosis in two canine models, English Springer Spaniel (ESS) and Portuguese Water Dog (PWD), have been compared. The disease onset, its clinical course, and survival period of the affected dogs were similar in both models. Skeletal dysplasia was noted radiographically at 2 months of age, whereas at 4 1/2 months of age there was progressive neurologic impairment. However, dwarfism and coarse facial features were seen only in ESS. Both models had similar deficiency in activity of lysosomal beta-galactosidase, but possessed a normal protein activator for GM1-beta-galactosidase. Both models stored GM1-ganglioside, asialo-GM1, and oligosaccharides in brain. Furthermore, only the PWD stored glycoproteins containing polylactosaminoglycans in visceral organs, and neither model stored them in the brain. Morphologically, both models demonstrated similar storage material in multiple tissues and cell types. The ultrastructure of the storage material was cell-type specific and identical in both models. However, some differences in the lectin staining pattern were noted. Our clinical, biochemical, and histochemical findings indicate that PWD and ESS may represent two different mutations of the beta-galactosidase gene. Moreover, the authors conclude that it is difficult, and inappropriate, to apply the human classification of GM1-gangliosidosis (i.e. infantile, juvenile, and adult forms) to these canine models.

An adult onset hexosaminidase A deficiency syndrome with sensory neuropathy and internuclear ophthalmoplegia.

A 42 year old man presented with a slowly progressive gait disturbance, generalised weakness, dysarthria, clumsiness and tremor of his hands, and involuntary jerks. Hexosaminidase A activity in plasma, leucocytes and fibroblasts was considerably reduced, establishing the diagnosis of GM2 gangliosidosis. Clinical examination showed two previously unreported features, a clinically evident sensory neuropathy and internuclear ophthalmoplegia.

Late-infantile type galactosialidosis. Histopathology of the retina and optic nerve.

We studied histopathologic findings from the retina and optic nerve of a patient with the late-infantile type of galactosialidosis and related them to clinical features of the condition. Markedly fewer ganglion cells were evident histopathologically using light microscopy. Results of histochemical studies demonstrated abnormal accumulation of lipid and proteinaceous material in the residual swollen ganglion cells. Marked loss of myelinated nerve fibers and thickening of the pial septum were also observed in the optic nerve. Both retinal ganglion cells and amacrine cells had intracytoplasmic inclusion bodies, but none were found in the optic nerve. These findings suggested that optic atrophy was induced by axonal wallerian degeneration secondary to retinal ganglion cell death. Although the fundus showed advanced optic nerve atrophy, a cherry red spot was not evident, possibly because of the marked decrease in ganglion cells in this case.

Prenatal lesions in an ovine fetus with GM1 gangliosidosis.

Sheep affected with ovine GM1 gangliosidosis are normal at birth and develop clinical signs, initially ataxia, commencing at approximately 5 months of age, which progresses rapidly to recumbency. Superovulation and embryo transfer techniques were applied to a flock of carrier sheep of ovine GM1 gangliosidosis to increase the numbers of carrier and affected animals. A recipient ewe with 3 at-risk fetuses died at 4 months of gestation (normal ovine gestation is 5 months), and spectrofluorimetric assay of cerebral lysosomal beta-galactosidase of the fetuses showed that 2 were carriers and one was an affected fetus. The affected fetus had marked cytoplasmic enlargement and vacuolization of central and peripheral nervous system neuronal soma and of hepatocytes and renal epithelial cells. Lectin histochemistry indicated abnormal storage of complex carbohydrates, with terminal saccharide moieties consisting of beta-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. This case underlines the need for prenatal initiation of therapy and also demonstrates that vacuolization alone is not the cause of clinical signs in this lysosomal storage disease in that clinical signs do not commence until at least 5 months after vacuolization is histologically apparent.