Neuronal Storage Disorders Research Articles

Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells.

Enzyme Replacement in Neuronal Storage Disorders in the Pediatric Population

In the past 15years, for select lysosomal storage diseases, there has been a shift from symptom management to disease modification in terms of treatment strategy, mainly related to use of enzyme replacement therapy (ERT). Yet the application of ERT is for very few diseases, and while beneficial, ERT does not represent a cure. For some disorders, the advent of ERT has made a dramatic impact, while for others, benefits have been much more modest. Understanding of the long-term effects as well as the appropriate time for initiation of ERT is under exploration in a number of diseases, while the feasibility of ERT is still being established for others. No definite effects of ERT on central nervous system manifestations of lysosomal storage diseases have been observed for any disease to date. New strategies, including intrathecal enzyme replacement, gene therapy and substrate reduction therapy are being developed in animal models and clinical trials, which hopefully will begin a new era of nervous system disease modification in neuronal storage disorders.

Dentatorubral‐pallidoluysian atrophy (DRPLA)

Dentatorubral-pallidoluysian atrophy (DRPLA) is a hereditary spinocerebellar degeneration. Despite the establishment of this disease in 1982, it has been pointed out that DRPLA has an unexplained aspect concerning its clinicopathological features; that is, the discrepancy between the variety of clinical manifestations and the uniformity of the brain lesions. The discovery of a causative gene mutation (abnormal expansion of the CAG repeat in DRPLA gene) triggered the development of novel neuropathology in DRPLA, which has suggested that polyglutamine-related pathogenesis involves a wide range of central nervous system regions far beyond the systems previously reported to be affected. It is now likely that DRPLA has an aspect of neuronal storage disorder and has multiple system degeneration, the lesion distribution of which varies depending on the CAG repeat sizes in the causative gene.

Serial 1H-MRS in GM2 gangliosidoses

GM2 gangliosidoses are a group of neuronal storage disorders caused by deficiency in the lysosomal enzyme hexosaminidase A. Clinically, the disease is marked by a relentless encephalopathy. Proton magnetic resonance spectroscopy (1H-MRS) provides in-vivo measurement of various brain metabolites including N-acetyl aspartate+N-acetyl aspartate glutamate (NAA), myo-inositol (mI), choline (Cho) and creatine (Cr). The NAA represents neuronal integrity while elevation in the mI reflects abnormal inflammation and gliosis in the brain tissue. An elevation in the Cho levels suggest cell membrane breakdown and demyelination. We report the clinical and laboratory data in two patients with GM2 gangliosidoses. Serial 1H-MRS evaluations were performed to drive metabolite ratios of NAA/Cr, mI/Cr and Cho/Cr. We acquired the data from four regions of interest (ROI) according to a standard protocol. The results documented a progressive elevation in mI/Cr in all four ROI in patient one and only one ROI (occipital gray matter) in patient 2. We also documented a decline in the NAA/Cr ratios in both cases in most ROI. These results were compared to six age-matched controls and confirmed statistically significant elevation in the mI in our cases. In conclusion, 1H-MRS alterations were suggestive of neuronal loss and inflammation in these patients. 1H-MRS may be a valuable tool in monitoring the disease progress and response to therapy in GM2 gangliosidoses. Elevation in the mI may prove to be more sensitive than the other metabolite alterations.

Different Molecular Mechanisms Leading to White Matter Hypomyelination in Infantile Onset Lysosomal Disorders

Hypomyelinating leukoencephalopathies may be related to a primary disturbance in the formation of myelin or may be caused by neuronal, oligodendrocytic or astrocytic dysfunction, leading to a failure of myelination. Abnormal myelination related to a direct metabolic damage on oligodendrocytes has been shown to occur in some animal models of lysosomal storage diseases. To demonstrate that cerebral white matter hypomyelination may occur also in humans affected by early-onset lysosomal storage diseases, we report three cases with infantile-onset lysosomal storage disorders (type 1 GM1 gangliosidosis, globoid cell leukodystrophy or Krabbe's disease, and type A Niemann-Pick disease) showing white matter hypomyelination. Hypomyelinating leukoencephalopathy may therefore represent a feature of lysosomal storage disorders with onset in the first months of life, when the process of myelination is particularly active, indicating that neuronal storage disorders may be primarily responsible for central nervous system hypomyelination.

Identification and characterization of Caenorhabditis elegans palmitoyl protein thioesterase1

Infantile neuronal ceroid lipofuscinosis (INCL; Batten disease) is a severe neurodegenerative disorder of childhood characterized by the accumulation of autofluorescent storage material in lysosomes. It is caused by mutation of the CLN1/PPT1 gene, which encodes the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1), but the mechanism of disease pathogenesis and substrates for the enzyme are unknown. Caenorhabditis elegans is a simple nematode worm, with a fully sequenced genome, which is easy to maintain and manipulate. It has a completely mapped cell lineage and nervous system and has already provided clues about the pathogenesis of several human neuronal and lysosomal storage disorders. We have identified and characterized a PPT1 homologue in C. elegans. We found that, although this gene was not essential for the animal's survival, its mutation resulted in a mild developmental and reproductive phenotype, affected the number and size of mitochondria, and resulted in an abnormality in mitochondrial morphology, possibly suggestive of a role for this organelle in INCL pathogenesis. This strain, deleted for ppt-1, potentially provides a model system for the study of PPT1 and the pathogenesis of INCL.

Quantification of mRNAs encoding proteins of the glycosphingolipid catabolism in mouse models of GM2 gangliosidoses and sphingolipid activator protein precursor (prosaposin) deficiency

We have investigated the mRNA amounts of six lysosomal proteins (β-hexosaminidase α- and β-subunit, sphingolipid activator protein precursor, GM2 activator protein, lysosomal sialidase, β-glucocerebrosidase) involved in the degradation of glycosphingolipids. We analyzed extracts from brain tissues of mouse models for lysosomal storage diseases, i.e., the GM2 gangliosidoses and the deficiency of the sphingolipid activator protein precursor (prosaposin). The mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. Although storage of the respective lysosomal proteins has been reported in human and mice, no increase of their mRNA amounts could be detected here. Our results indicate that there is no transcriptional upregulation of lysosomal proteins in the examined neuronal storage disorders.

Targeted Disruption of the Cln3 Gene Provides a Mouse Model for Batten Disease

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.

GM2 ganglioside as a regulator of pyramidal neuron dendritogenesis.

One of the most profound events in the life of a neuron in the mammalian CNS is the development of a characteristic dendritic tree, yet little is understood about events controlling this process. Pyramidal neurons of the cerebral cortex are known to undergo a single explosive burst of dendritic sprouting immediately after completing migration to the cortical mantle, and following maturation there is no evidence that new, primary dendrites are initiated. Yet in one group of rare genetic diseases--Tay-Sachs disease and related neuronal storage disorders--cortical pyramidal neurons undergo a second period of dendritogenesis. New dendritic membrane is generated principally at the axon hillock and in time is covered with normal-appearing spines and synapses. In our studies of normal brain development and storage diseases we consistently find one feature in common in cortical pyramidal neurons undergoing active dendritogenesis: They exhibit dramatically increased expression of GM2 ganglioside localized to cytoplasmic vacuoles within neuronal perikarya and proximal dendrites. There is also evidence that the increase in GM2 precedes dendritic spouting, and that after dendritic maturation is complete (in normal brain) the GM2 levels in neurons become substantially reduced. These findings are consistent with GM2 ganglioside playing a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.

Encephaloneuropathy with lysosomal zebra bodies and GM2 ganglioside storage

An 11-year-old girl died of a neuronal storage disorder that clinically was characterized by failure to thrive and muscular hypotonia from birth, with the subsequent evolution of motor neuron disease, epilepsy, and dementia. A wide range of metabolic disorders, including all forms of GM2 gangliosidosis, could be excluded. Electron microscopy demonstrated neuronal zebra body inclusions, and immunohistochemistry demonstrated that GM2 ganglioside was a major constituent of the storage material. We suggest that the patient died of a lysosomal storage disease that is clinically and biochemically different from Tay-Sachs disease, Sandhoff disease, and other GM2 gangliosidoses described previously. This case also further demonstrates that significant accumulation of GM2 ganglioside, which is crucial for dendritic formation, may occur in neuronal storage diseases lacking known defects in ganglioside catabolism.

Neuronal ceroid-lipofuscinosis--late-infantile or Jansky-Bielschowsky type--revisited.

The tissues from three patients with late-infantile NCL originally described by Max Bielschowsky became available to apply modern techniques such as fluorescence microscopy, electron microscopy and immunohistochemistry. While regular tinctorial preparations of the tissues documented a neuronal storage disorder in all three patients' tissues, the accumulated material proved to be autofluorescent, showed the ultrastructure of curvilinear lipopigments, and reacted strongly with an antibody against the subunit-C of mitochondrial ATP synthase, a major component of lipopigments in NCL and also with an antibody against sphingolipid activator proteins. Thus, these modern morphological techniques demonstrated that the originally described three siblings with late-infantile "amaurotic familial idiocy" really had neuronal ceroid-lipofuscinosis of the late-infantile or Jansky-Bielschowsky type, according to current diagnostic criteria. This type of archival study may also contribute to the mosaic of medical history.

GM2 ganglioside and pyramidal neuron dendritogenesis.

GM2 ganglioside, although scarce in normal adult brain, is the predominant ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both ganglioside and nonganglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and alpha-mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas other gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonganglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas nerurons in normal adult brain exhibit little or no staining for this ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 ganglioside precedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic trees of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.

Pathogenesis of brain dysfunction in Batten disease

Animal models of Batten disease and other neuronal storage disorders offer important opportunities to study the pathogenesis of brain dysfunction in this family of diseases. Although all of these conditions exhibit progressive intraneuronal storage, we have found that other aspects of the cellular pathology of Batten disease differ markedly from those of storage disorders caused by lysosomal hydrolase deficiencies. Likewise, lysosomal of cerebral cortex and other select brain regions, a prominent characteristic of Batten disease, does not occur in most other storage disorders. Our studies indicate that Batten disease has findings in common with human neurodegenerative diseases and that neuron death may be caused by excitotoxicity occurring secondary to the combined effects of suboptimal mitochondrial function and GABAergic (inhibitory) cell loss.

Gene replacement therapy in the central nervous system: Viral vector-mediated therapy of global neurodegenerative disease

AbstractThis target article describes the current state of global gene replacement in the brain using viral vectors and assesses possible solutions to some of the many problems inherent in gene therapy of the central nervous system (CNS). Gene replacement therapy in the CNS is a potential means of producing a stable expression of normal human proteins in deficient cells and thus curing certain genetically inherited enzyme deficiencies and metabolic diseases as well as cancers. The two major issues to be addressed in CNS gene replacement are the delivery of genetic material to the brain and the expression of recombinant genetic material in target cells within the CNS. Focal inoculation of recombinant virions or other genetic vectors has limitations in global CNS disease. A new approach is the blood-brain barrier (BBB) disruption technique developed in this laboratory, in which hypertonic mannitol transiently shrinks the BBB endothelium, allowing passage of high molecular weight compounds and even viruses. Gene therapy of the CNS will require a viral vector system that allows long-term, nontoxic gene expression in neurons or glial cells. Retroviral vectors have limitations in CNS gene replacement, although they are suitable for expressing recombinant genes in intracerebral grafts, or toxic genes in brain tumors. Using mutant neurotropic viruses with reduced neurotoxicity (such as defective herpes simplex virus type I [HSV-1], the HSV-1 amplicon vector system we have developed, or adenovirus mutants) has potential for direct treatment of neurons. Injecting these vectors into rodent brains can lead to stable expression of foreign genetic material in postmitotic neuronal cells. We discuss our BBB disruption delivery technique, our defective HSV-1 amplicon vector system, and our feline model for the neuronal lysosomal storage disorder Gm2-gangliosidosis (Sandhoff disease), which may prove to be a useful model system for CNS gene therapy.

Alterations in synaptic connectivity in cerebral cortex in neuronal storage disorders

AbstractNeuronal storage diseases are inborn errors of metabolism characterized by widespread intraneuronal storage and progressive neurological dysfunction, including mental retardation, movement disorders, and seizures. Most of these disorders are associated with specific defects in particular lysosomal enzymes. Moreover, a wide array of genetic defects has been documented. The immense progress made during the past three decades in understanding the primary molecular and enzymatic defects in this family of diseases stands in sharp contrast to the modest advances made in delineating pathogenetic mechanisms and treatment strategies. Although these disorders result from highly selective defects in metabolic pathways, they ultimately are complex conditions characterized by a plethora of seemingly unrelated secondary molecular and cellular abnormalities. Studies of children and animals affected by these diseases have revealed that significant changes in neuronal connectivity occur in the cerebral cortex. These include not only degenerative changes in axons and synapses of inhibitory neurons, but also a regrowth of dendrites and new synapse formation involving pyramidal neurons. The latter finding indicates that neurons in the cerebral cortex undergo significant “rewiring” during the height of the disease process. This regrowth of primary dendrites and formation of new synapses as part of a neurological disease is unprecedented. Understanding the causes and consequences of these changes in cortical synaptic connectivity in neuronal storage diseases may provide insight not only into the precise mechanisms underlying mental retardation in these diseases and their reversibility, but also into processes governing dendritic elaboration and synapse formation in the normal developing nervous system. © 1995 Wiley‐Liss, Inc.

Bone marrow transplantation corrects the enzyme defect in neurons of the central nervous system in a lysosomal storage disease.

Neuronal storage disorders are fatal neurodegenerative diseases of humans and animals that are caused by inherited deficiencies of lysosomal hydrolase activity. Affected individuals often appear normal at birth but eventually develop progressive neurologic symptoms including sensory and motor deficits, mental retardation, and seizures. We have examined efficacy of bone marrow transplantation as a means of enzyme replacement, using cats with the lysosomal storage disease alpha-mannosidosis. Treated animals showed little or no progression of neurologic signs 1-2 years after transplant, whereas untreated cats became severely impaired and reached endstage disease by 6 months of age. Increased lysosomal alpha-mannosidase activity was found in brain tissue of the treated animals, and electron microscopy revealed no evidence of lysosomal storage within most neurons. Histochemical localization of acidic alpha-D-mannoside mannohydrolase (EC 3.2. 1.24), using 5-bromo-4-chloro-3-indolyl alpha-D-mannopyranoside, showed that functional enzyme was present in neurons, glial cells, and cells associated with blood vessels. This study provides direct evidence that bone marrow transplantation as treatment for a neuronal storage disease can lead to significant levels of a missing lysosomal hydrolase within neurons of the central nervous system and to compensation for the genetic metabolic defect.

Neuroaxonal dystrophy in neuronal storage disorders: Evidence for major GABAergic neuron involvement

The formation of focal granular enlargements within axons (axonal spheroids or “torpedoes”; neuroaxonal dystrophy) is a well known phenomenon occurring in a variety of neurological diseases. The relative susceptibility of different types of neurons to this kind of axonal pathology, however, is largely unknown. An immunocytochemical study directed at localizing glutamic acid decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), in various CNS regions in feline models of lysosomal storage disorders has revealed vast numbers of axonal spheroids containing this enzyme. In some storage diseases (GM1 and GM2 gangliosidosis), GAD-immunoreactive spheroids were a common occurrence in many brain regions, whereas in other disorders these structures were more limited in distribution (α-mannosidosis), or were absent (mucopolysaccharidosis type I). Axonal spheroids unreactive for GAD were encountered in large numbers in subcortical white matter in GM2 gangliosidosis, but were infrequently observed in the other diseases. The incidence and distribution of GAD-immunoreactive spheroids in the various diseases under study were found to correlate closely with the type and degree of neurological deficits exhibited by affected animals. This study indicates that the neuroaxonal dystrophy occurring in some types of storage disorders commonly involves axons of GABAergic neurons and suggests that a resulting defect in neurotransmission in inhibitory circuits may be an important factor underlying brain dysfunction in this family of diseases.

Pathobiology of Neuronal Storage Disease

The neuronal storage disorders are a complex family of diseases characterized by an abnormal accumulation of unmetabolized substances within neuronal somata. A wide variety of enzymatic and molecular events underlies this storage process and these are most often known to be associated with the lysosomal system of affected cells. The storage process is seen primarily as a lysosomal event, although for some types of storage diseases—for example, neuronal ceroid lipofuscinosis, this linkage is not firmly established. A complete discussion of the pathobiology of these diseases would necessarily encompass all pathogenetic events spanning from the molecular level to clinical manifestations of disease. This chapter discusses the consequences of these primary metabolic disturbances at the level of individual neurons or of functional groups of neurons. It is an attempt to develop a framework for understanding those factors making specific contributions to the malfunction of neurons and to the generation of those clinical manifestations that are characteristic of these diseases.

Further studies on ectopic dendrite growth and other geometrical distortions of neurons in feline GMl gangliosidosis

Systematic Golgi studies have been performed on major subcortical, diencephalic, brain stem and spinal cord regions from cats with the inherited neuronal storage disease, GM1 gangliosidosis. Resulting data have been compared with other Golgi studies of neuronal storage disorders in man and animals, including an earlier, more limited examination of this same disease model. These previous studies have shown that in human and feline gangliosidoses cortical pyramidal neurons undergo remarkable changes in soma-dendritic geometry. The latter include the formation of conspicuous cellular enlargements between somata and axonal initial segments (meganeurites) and the sprouting of secondary neuritic processes from this same region of the cell. Further, ultrastructural studies have revealed normal appearing synapses on the surface of this ectopically placed dendritic-like membrane. Results of the present study indicate that the distribution of meganeurites, secondary neurites and other geometrical distortions of neurons in GM1 gangliosidosis varies with cell type and brain region. This cell type-specific response to the metabolic error and subsequent storage could be categorized in three ways. Firstly, certain types of cells (e.g. multipolar neurons of the amygdala and claustrum) exhibited changes similar to those reported for cortical pyramidal neurons. That is, cells of these regions either displayed spine or neurite-bearing meganeurites, or enlarged axon hillocks which were covered with similar processes. Other types of neurons did not demonstrate ectopic neurite growth or spine-covered meganeurites, but did display prominent aspiny meganeurites (e.g. neurons of the superior colliculus, periaqueductal gray, hypothalamus and basal forebrain nuclei). A third category of neurons did not possess meganeurites or neurite growth but instead demonstrated massive somatic expansion which exceeded that observed in meganeurite-bearing cell types (e.g. certain brain stem and spinal cord neurons). These data have been compared with the more limited Golgi studies of other types of neuronal storage disorders and the same types of neurons appeared to respond in similar fashion across this spectrum of diseases. The data presented and discussed in this paper demonstrate three significant morphological events which occur in neurons as a result of lysosomal hydrolase deficiency. These are (i) storage, which occurs in all neurons but manifests as meganeurite formation or somatic enlargement depending on the cell type, (ii) axon hillock or meganeurite-associated spine and neurite growth, and (iii) new synapse formation on spine-covered meganeurites and on neurites. The independent occurrence of meganeurites and neurites is not consistent with the view that these morphological changes are simply different manifestations of a single defect in the regulation of neuronal surface membrane production, as has been suggested previously. Rather, this study offers the view that axon hillock neurites and meganeurite-associated neurites and spines are the morphological hallmark of increased axon hillock-asociated synapse formation, whereas meganeurites themselves simply manifest the storage process. Possible factors controlling the expression of these features of neurons are discussed.

Ectopic axon hillock-associated neurite growth is maintained in metabolically reversed swainsonine-induced neuronal storage disease

An experimentally induced and reversible model of a neuronal storage disease, swainsonine-induced feline alpha-mannosidosis, has been used to study the modifiability of ectopic, axon hillock-associated neurites and their new synaptic contacts. Earlier studies have fully documented that a variety of neuronal storage disorders are characterized by such changes in neuronal geometry and connectivity. Swainsonine administration was ended after 6 months of continuous treatment which had resulted in characteristic signs of alpha-mannosidosis. Studies of this animal 6 months after reversal showed that even though neuronal vacuolation and other CNS changes essentially normalized, ectopic neurites and their synaptic connections were still present and appeared similar to those of another animal which had been treated with swainsonine for the entire 12-month period. These results suggest that once initiated during the disease process, ectopic axon hillock-associated dendrites become an integral part of the soma-dendritic domain of affected neurons and may not be reversible. These findings may have relevance for current attempts to devise therapies involving enzyme replacement for individuals with inherited neuronal storage disease.