Robert Steinfeld

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC‐7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H+‐ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC‐7 or the H+‐ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC‐7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC‐7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron‐dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC‐3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H+‐ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H+‐ATPase and ClC‐7 can underlie human osteopetrosis.

Cathepsin D Deficiency Is Associated with a Human Neurodegenerative Disorder

Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D.

Folate receptor alpha defect causes cerebral folate transport deficiency: a treatable neurodegenerative disorder associated with disturbed myelin metabolism.

Sufficient folate supplementation is essential for a multitude of biological processes and diverse organ systems. At least five distinct inherited disorders of folate transport and metabolism are presently known, all of which cause systemic folate deficiency. We identified an inherited brain-specific folate transport defect that is caused by mutations in the folate receptor 1 (FOLR1) gene coding for folate receptor alpha (FRalpha). Three patients carrying FOLR1 mutations developed progressive movement disturbance, psychomotor decline, and epilepsy and showed severely reduced folate concentrations in the cerebrospinal fluid (CSF). Brain magnetic resonance imaging (MRI) demonstrated profound hypomyelination, and MR-based in vivo metabolite analysis indicated a combined depletion of white-matter choline and inositol. Retroviral transfection of patient cells with either FRalpha or FRbeta could rescue folate binding. Furthermore, CSF folate concentrations, as well as glial choline and inositol depletion, were restored by folinic acid therapy and preceded clinical improvements. Our studies not only characterize a previously unknown and treatable disorder of early childhood, but also provide new insights into the folate metabolic pathways involved in postnatal myelination and brain development.

Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma

Loss of folate receptor-α function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-α-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-α, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-α-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-α-positive exosomes. Loss of folate receptor-α-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-α-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-α-positive and -negative exosomes into mouse brains demonstrate folate receptor-α-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-α-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting.

RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection.

Congenital cytomegalovirus brain infection without symptoms at birth can cause a static encephalopathy with characteristic patterns of brain abnormalities. Here we show that loss-of-function mutations in the gene encoding the RNASET2 glycoprotein lead to cystic leukoencephalopathy, an autosomal recessive disorder with an indistinguishable clinical and neuroradiological phenotype. Congenital cytomegalovirus infection and RNASET2 deficiency may both interfere with brain development and myelination through angiogenesis or RNA metabolism.

The expanding clinical and genetic spectrum of ATP1A3-related disorders.

Objective: We aimed to delineate the clinical and genetic spectrum of ATP1A3-related disorders and recognition of a potential genotype-phenotype correlation. Methods: We identified 16 new patients with alternating hemiplegia of childhood (AHC) and 3 new patients with rapid-onset dystonia-parkinsonism (RDP) and included these as well as the clinical and molecular findings of all previously reported 164 patients with mutation-positive AHC and RDP in our analyses. Results: Major clinical characteristics shared in common by AHC and RDP comprise a strikingly asymmetric, predominantly dystonic movement disorder with rostrocaudal gradient of involvement and physical, emotional, or chemical stressors as triggers. The clinical courses include an early-onset polyphasic for AHC, a later-onset mono- or biphasic for RDP, as well as intermediate forms. Meta-analysis of the 8 novel and 38 published ATP1A3 mutations shows that the ones affecting transmembrane and functional domains tend to be associated with AHC as the more severe phenotype. The majority of mutations are located in exons 8, 14, 17, and 18. Conclusion: AHC and RDP constitute clinical prototypes in a continuous phenotypic spectrum of ATP1A3-related disorders. Intermediate phenotypes combining criteria of both conditions are increasingly recognized. Efficient stepwise mutation analysis of the ATP1A3 gene may prioritize those exons where current state of knowledge indicates mutational clusters.

Propionic acidemia: clinical course and outcome in 55 pediatric and adolescent patients

BackgroundPropionic acidemia is an inherited disorder caused by deficiency of propionyl-CoA carboxylase. Although it is one of the most frequent organic acidurias, information on the outcome of affected individuals is still limited.Study design/methodsClinical and outcome data of 55 patients with propionic acidemia from 16 European metabolic centers were evaluated retrospectively. 35 patients were diagnosed by selective metabolic screening while 20 patients were identified by newborn screening. Endocrine parameters and bone age were evaluated. In addition, IQ testing was performed and the patients’ and their families’ quality of life was assessed.ResultsThe vast majority of patients (>85%) presented with metabolic decompensation in the neonatal period. Asymptomatic individuals were the exception. About three quarters of the study population was mentally retarded, median IQ was 55. Apart from neurologic symptoms, complications comprised hematologic abnormalities, cardiac diseases, feeding problems and impaired growth. Most patients considered their quality of life high. However, according to the parents’ point of view psychic problems were four times more common in propionic acidemia patients than in healthy controls.ConclusionOur data show that the outcome of propionic acidemia is still unfavourable, in spite of improved clinical management. Many patients develop long-term complications affecting different organ systems. Impairment of neurocognitive development is of special concern. Nevertheless, self-assessment of quality of life of the patients and their parents yielded rather positive results.

Identification and Characterization of an Inborn Error of Metabolism Caused by Dihydrofolate Reductase Deficiency

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease.

Rapid and Simple Assay for the Determination of Tripeptidyl Peptidase and Palmitoyl Protein Thioesterase Activities in Dried Blood Spots

Neuronal ceroid lipofuscinoses constitute a group of at least eight inherited, progressive encephalopathies that are characterized by lipofuscin-like inclusions in various tissues and that have recently been classified as CLN1 to CLN8 according to their genetic defects (1). A form with mostly infantile manifestation (CLN1) and the classical late infantile form (CLN2) are caused by deficiencies of the lysosomal enzymes palmitoyl protein thioesterase 1 (PPT1) and tripeptidyl peptidase 1 (TPP1), respectively. The basic defects underlying the other forms are still ill-defined or unknown. To date, more than 30 mutations have been reported in the PPT1 and TPP1 genes, rendering molecular genetic analysis impractical as a primary means of diagnosis. Electron microscopy of characteristic cellular inclusions remains an important diagnostic method, but it is also tedious and not readily available. Enzymatic assays based on fluorescent substrates have recently been reported for PPT1 (2) and TPP1(3). These assays can also be used with isolated leukocytes. However, because few laboratories are experienced in diagnosing these rare disorders, blood samples must be sent to specialized centers by expensive express mail services to avoid loss of enzymatic activity. In our experience, samples for this kind of study frequently arrive in poor condition. As an alternative to the use of leukocytes, methods for determining several lysosomal enzymes from dried blood spots (DBS) have recently been described (4). DBS are also used routinely for enzyme measurements within newborn-screening programs for the detection of biotinidase and galactose-uridyltransferase deficiencies in neonates. DBS require only minute amounts of blood, are easy to …

Efficiency of long-term tetrahydrobiopterin monotherapy in phenylketonuria

Summary: Phenylketonuria, an inborn error of phenylalanine metabolism, occurs with a frequency of about 1 in 10 000 births and is treated with a strict dietary regimen. Recently, some patients with PKU have been found to show increased tolerance towards phenylalanine intake while receiving tetrahydrobiopterin (BH4) supplementation. We have treated two infants with BH4-responsive PKU with BH4 for more than 2 years. No additional dietary control was required to maintain blood phenylalanine concentrations in the desired range. Both children have shown normal development. Generally, our results suggest that BH4 treatment might be an option for some patients with mild PKU, as it frees them from dietary restrictions and thus improves their quality of life.