Jaana Tyynelä

Storage of saposins A and D in infantile neuronal ceroid-lipofuscinosis

We have isolated storage cytosomes from brain tissue of patients with infantile neuronal ceroid‐lipofuscinosis. The purified storage bodies were subjected to compositional analysis which revealed a high content of proteins, accounting for 43% of dry weight. Saposins A and D, also known as sphingolipid activator proteins (SAPs), were shown to constitute a major portion of the accumulated protein using gel electrophoresis and sequence analysis. This is the first time that saposins have been found to be stored in any form of neuronal ceroid‐lipofuscinosis.

Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na+-coupled Cl−–HCO3− exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.

Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry

Phospholipids (PL) in cerebral cortex from patients with infantile (INCL or CLN1) and juvenile (JNCL or CLN3) forms of neuronal ceroid‐lipofuscinosis (NCL) and controls were analysed by normal phase HPLC and on‐line electrospray ionization ion‐trap mass spectrometric detection (LC‐ESI‐MS). The method provided quantitative data on numerous molecular species of different PL classes, which are not achieved by using the conventional chromatographic methods. Compared with the controls, the INCL brains contained proportionally more phosphatidylcholine (PC), and less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Different molecular species of PC, PE, PS, phosphatidylinositol and sphingomyelin were quantified using multiple internal PL standards that differed in fatty acyl chain length and thus allowed correction for chain length dependency of instrument response. In INCL cortex, which had lost 65% of the normal PL content, the proportions of polyunsaturated molecular species, especially the PS and PE that contained docosahexaenoic acid (22:6n‐3), were dramatically decreased. The membranes may have adapted to this alteration by increasing the proportions of PL molecules substituted with monounsaturated and short‐chain fatty acids. Lysobisphosphatidic acid was highly elevated in the INCL brain and consisted mostly of polyunsaturated species. It is possible that changes in the composition of PL membranes accelerate progression of INCL by altering signalling and membrane trafficking in neurons.

Cell biology and function of neuronal ceroid lipofuscinosis-related proteins.

Neuronal ceroid lipofuscinoses (NCL) comprise a group of inherited lysosomal disorders with variable age of onset, characterized by lysosomal accumulation of autofluorescent ceroid lipopigments, neuroinflammation, photoreceptor- and neurodegeneration. Most of the NCL-related genes encode soluble and transmembrane proteins which localize to the endoplasmic reticulum or to the endosomal/lysosomal compartment and directly or indirectly regulate lysosomal function. Recently, exome sequencing led to the identification of four novel gene defects in NCL patients and a new NCL nomenclature currently comprising CLN1 through CLN14. Although the precise function of most of the NCL proteins remains elusive, comprehensive analyses of model organisms, particularly mouse models, provided new insight into pathogenic mechanisms of NCL diseases and roles of mutant NCL proteins in cellular/subcellular protein and lipid homeostasis, as well as their adaptive/compensatorial regulation at the transcriptional level. This review summarizes the current knowledge on the expression, function and regulation of NCL proteins and their impact on lysosomal integrity. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.

Variant Late Infantile Neuronal Ceroid-lipofuscinosis: Pathology and Biochemistry

The neuronal ceroid-lipofuscinoses (NCL) are among the most common inherited neurodegenerative disorders of childhood. The genomic defect causing a variant late infantile neuronal ceroid-lipofuscinosis (vLINCL, also called CLN-5 or variant Jansky-Bielschowsky disease) has recently been localized to chromosome 13q22, thus delineating this disease as a separate entity. This particular form of NCL is clinically well defined, but lacks pathomorphological and biochemical description. The present analyses indicate that subunit c of the mitochondrial ATP synthase is the major protein in vLINCL brain storage cytosomes. These cytosomes also contain minor amounts of sphingolipid activator proteins (SAPs). The immunohistological distribution of subunit c and SAPs in the central nervous system (CNS) and visceral tissues closely resembles that of classical LINCL. Thus, despite clinical differences and the fact that various forms of NCL are caused by different genetic defects, variant and classical LINCL as well as juvenile NCL are all characterized by pronounced lysosomal accumulation of the same hydrophobic protein, subunit c of the mitochondrial ATP synthase.

Northern Epilepsy: A Novel Form of Neuronal Ceroid‐Lipofuscinosis

Northern epilepsy is an autosomal recessive childhood onset epilepsy syndrome, clinically characterized by generalized tonic‐clonic seizures with onset at 5 to 10 years of age and subsequent slowly progressive mental deterioration. The patients may reach 50 or 60 years of age. A mutation responsible for the disease has recently been identified in a novel gene on chromosome 8p23, encoding a putative membrane protein with an unknown function. The present study, based on three autopsied patients, is the first neuropathological analysis of the disease, and showed intraneuronal accumulation of cytoplasmic autofluorescent granules. The granules were strongly stained by the Luxol fast blue, periodic acid‐Schiff, and Sudan black B methods in paraffin sections, and were immunoreactive for subunit c of the mitochondrial ATP synthase and sphingolipid activator proteins A and D. The intraneuronal storage was highly selective: the third layer of the isocortex and the hippocampal CA2, CA3, and CA4 sectors were severely affected, while other layers of the isocortex, the CA1 sector, and the cerebellar cortex were only minimally involved. The membrane‐bound storage cytosomes showed a curvilinear ultrastructure with admixture of some granular components. Western blotting and N‐terminal sequence analysis of purified storage material identified subunit c as the major component. These findings establish Northern epilepsy as a new form of neuronal ceroid‐lipofuscinosis with an exceptionally protracted course.

Enhanced activation of bound plasminogen on Staphylococcus aureus by staphylokinase

Activation of plasminogen (plg) to plasmin by the staphylococcal activator, staphylokinase (SAK), is effectively regulated by the circulating inhibitor, α2‐antiplasmin (α2AP). Here it is demonstrated that intact Staphylococcus aureus cells and solubilized staphylococcal cell wall proteins not only protected SAK‐promoted plg activation against the inhibitory effect of α2AP but also enhanced the activation. The findings suggest that the surface‐associated plg activation by SAK may have an important physiological function in helping staphylococci in tissue dissemination. Amino acid sequencing of tryptic peptides originating from the 59‐, 56‐ and 43‐kDa proteins, isolated as putative plg‐binding proteins, identified them as staphylococcal inosine 5′‐monophosphate dehydrogenase, α‐enolase, and ribonucleotide reductase subunit 2, respectively.

Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau In Vivo

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimers disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimers disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimers disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimers disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D–deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimers disease.

Synaptic changes in the thalamocortical system of cathepsin D-deficient mice: a model of human congenital neuronal ceroid-lipofuscinosis.

Cathepsin D (CTSD; EC 3.4.23.5) is a lysosomal aspartic protease, the deficiency of which causes early-onset and particularly aggressive forms of neuronal ceroid-lipofuscinosis in infants, sheep, and mice. Cathepsin D deficiencies are characterized by severe neurodegeneration, but the molecular mechanisms behind the neuronal death remain poorly understood. In this study, we have systematically mapped the distribution of neuropathologic changes in CTSD-deficient mouse brains by stereologic, immunologic, and electron microscopic methods. We report highly accentuated neuropathologic changes within the ventral posterior nucleus (ventral posteromedial [VPM]/ventral posterolateral [VPL]) of thalamus and in neuronal laminae IV and VI of the somatosensory cortex (S1BF), which receive and send information to the thalamic VPM/VPL. These changes included pronounced astrocytosis and microglial activation that begin in the VPM/VPL thalamic nucleus of CTSD-deficient mice and are associated with reduced neuronal number and redistribution of presynaptic markers. In addition, loss of synapses, axonal pathology, and aggregation of synaptophysin and synaptobrevin were observed in the VPM/VPL. These synaptic alterations are accompanied by changes in the amount of synaptophysin/synaptobrevin heterodimer, which regulates formation of the SNARE complex at the synapse. Taken together, these data reveal the somatosensory thalamocortical circuitry as a particular focus of pathologic changes and provide the first evidence for synaptic alterations at the molecular and ultrastructural levels in CTSD deficiency.

Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterized pathologically by neuronal accumulation of autofluorescent storage material and neurodegeneration. An ovine NCL form is caused by a recessive point mutation in the cathepsin D gene, which encodes a lysosomal aspartyl protease. This mutation results in typical NCL pathology with neurodegeneration and characteristic neuronal storage material. We have generated a Drosophila NCL model by inactivating the conserved Drosophila cathepsin D homolog. We report here that cathepsin D mutant flies exhibit the key features of NCLs. They show progressive neuronal accumulation of autofluorescent storage inclusions, which are also positive for periodic acid Schiff and luxol fast blue stains. Ultrastructurally, the storage material is composed of membrane-bound granular electron-dense material, similar to the granular osmiophilic deposits found in the human infantile and ovine congenital NCL forms. In addition, cathepsin D mutant flies show modest age-dependent neurodegeneration. Our results suggest that the metabolic pathway leading to NCL pathology is highly conserved during evolution, and that cathepsin D mutant flies can be used to study the pathogenesis of NCLs.