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Refractory epilepsy is the main neurological manifestation of Alpers' syndrome, a severe childhood-onset mitochondrial disease caused by bi-allelic pathogenic variants in the mitochondrial DNA (mtDNA) polymerase gamma gene (POLG). The pathophysiological mechanisms underpinning neuronal hyperexcitabilty leading to seizures in Alpers' syndrome remain unknown. However, pathological changes to reactive astrocytes are hypothesised to exacerbate neural dysfunction and seizure-associated cortical activity in POLG-related disease. Therefore, we sought to phenotypically characterise astrocytic pathology in Alpers' syndrome. We performed a detailed quantitative investigation of reactive astrocytes in post-mortem neocortical tissues from thirteen patients with Alpers' syndrome, eight neurologically normal controls and five sudden unexpected death in epilepsy (SUDEP) patients, to control for generalised epilepsy-associated astrocytic pathology. Immunohistochemistry to identify glial fibrillary acidic protein (GFAP)-reactive astrocytes revealed striking reactive astrogliosis localised to the primary visual cortex of Alpers' syndrome tissues, characterised by abnormal-appearing hypertrophic astrocytes. Phenotypic characterisation of individual GFAP-reactive astrocytes demonstrated decreased abundance of mitochondrial oxidative phosphorylation (OXPHOS) proteins and altered expression of key astrocytic proteins including Kir4.1 (subunit of the inwardly rectifying K + ion channel), AQP4 (astrocytic water channel) and glutamine synthetase (enzyme that metabolises glutamate). These phenotypic astrocytic changes were typically different from the pathology observed in SUDEP tissues, suggesting alternative mechanisms of astrocytic dysfunction between these epilepsies. Crucially, our findings provide further evidence of occipital lobe involvement in Alpers' syndrome and support the involvement of reactive astrocytes in the pathogenesis of POLG-related disease., (© 2023. The Author(s).)

TMEM119 and purinergic receptor P2Y12 (P2RY12), which are not expressed by recruited peripheral blood macrophages, are proposed to discriminate microglia from macrophages. Therefore, we investigated the distribution patterns of microglia and macrophages in 10 concentric lesions from four autopsied Baló's disease cases and one neuromyelitis optica spectrum disorder (NMOSD) case, using quantitative immunohistochemistry for the markers TMEM119, P2RY12, CD68, CD163 and GLUT5. Three cases with Baló's disease had distal oligodendrogliopathy (DO) showing preferential loss of myelin-associated glycoprotein and early active demyelination in the outermost demyelinating layer (termed DMY-MO). In DMY-MO with DO, TMEM119-positive activated microglia expressing upregulated GLUT5 but markedly downregulated P2RY12 were significantly increased. These activated microglia expressed inducible nitric oxide synthase. Oligodendrocytes and their precursors showed apoptotic-like nuclear condensation in DMY-MO. TMEM119-negative and CD68/CD163-positive macrophages were distributed throughout the lesion center of DMY-MO with DO and these cells demonstrated foamy morphology only in the inner portion but not in the outer portion. In concentric demyelinating lesions from another Baló's case and lamellar demyelinating lesions in an NMOSD case, which had late active demyelination without DO, the densities of TMEM119-, GLUT5- and P2RY12-positive microglia with ramified morphology were significantly increased in myelinated layers but not in demyelinating layers. In particular, in the NMOSD case, TMEM119-positive microglia were confined to the outer portion of the myelinated layers. CD68-positive macrophages with foamy morphology also expressing CD163 accumulated in myelinated as well as in demyelinated layers. These findings suggest that activated microglia expressing TMEM119 and GLUT5, but not P2RY12, are associated with apoptosis of oligodendrocytes in the leading edge of Baló's concentric lesions with DO, whereas TMEM119-, GLUT5- and P2RY12-positive microglia with ramified morphology are associated with myelin preservation in concentric lesions without DO in Baló's disease and NMOSD. These two types of microglia appear to play distinct roles in the formation of concentric lesions., (© 2020 International Society of Neuropathology.)

Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three-dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild-type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady-state kinetic measurements of phenylalanine activation and tRNA Phe aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease-related mutations in FARS2 gene., (© 2017 The Protein Society.)

Multiple sclerosis (MS), neuromyelitis optica (NMO), and Baló's disease (BD) are inflammatory demyelinating diseases of the CNS. We previously reported anti-aquaporin-4 (anti-AQP4) antibody-dependent AQP4 loss occurs in some NMO patients, while antibody-independent AQP4 astrocytopathy can occur in heterogeneous demyelinating conditions, including MS, NMO and BD. To investigate the relationship between astrocytopathy and demyelination, we focused on connexins (Cxs), which form gap junctions (GJs) between astrocytes and oligodendrocytes and maintain homeostasis in the CNS. We evaluated expression of astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 in autopsied materials from MS, NMO and BD patients. Astrocytic Cx43 and oligodendrocytic Cx32/Cx47 expressions were significantly diminished in both demyelinated and preserved myelin layers in all BD samples. In the leading edge of BD lesions, Cx43 and AQP4 loss preceded Cx32/Cx47 loss. Half of the NMO and MS samples showed preferential loss of astrocytic Cx43 expression in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte-oligodendrocyte GJs were lost. Cases with Cx43 loss were significantly associated with rapid disease progression, regardless of the disease phenotype. Pathologically, Cx43 loss was frequently accompanied by distal oligodendrogliopathy. Our findings suggest that Cx43 astrocytopathy can occur in MS, BD and NMO. Moreover, astrocytic Cx43 loss may be associated with disease aggressiveness and distal oligodendrogliopathy in demyelinating conditions. Early disruption of glial communications via GJs may cause loss of glia syncytium, thereby inducing oligodendroglial damage and myelin loss. Inhibition of Cx hemichannels and restoration of GJs may be a possible therapeutic target for demyelinating disorders., (© 2015 Japanese Society of Neuropathology.)

Aim: Mitochondrial diseases affect about 1/5000-1/10000 in the population. Twenty percent of patients with mitochondrial disease show liver involvement. In contrast to current belief among most internists, these diseases do not only present in childhood., Methods: We present four cases of adults (three with Alpers-Huttenlocher syndrome and one with mitochondrial neurogastrointestinal encephalomyopathy), diagnosed between 2005 and 2010, in our university referral center., Result: We focus on the broad clinical spectrum of liver involvement in mitochondrial diseases and their diagnosis. Biochemical investigations are often found to be inconclusive, and genetic confirmation cannot always be obtained, leaving many patients without a final diagnosis. Evidence-based causal therapy is unavailable for most mitochondrial diseases and liver transplantation for this indication remains a controversial issue., Conclusion: For clinicians, it is important to consider the possibility of an underlying mitochondrial disorder when there is systemic involvement (more than one organ affected), a suggestive family history, or an elevated level of lactic acid in the blood or cerebrospinal fluid.

Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.

Extensive aquaporin-4 (AQP4) loss without perivascular deposition of either activated complement or immunoglobulins is a characteristic of Baló's disease. Our aim in this study was to investigate the relationship between astrocytopathy and demyelination in Baló's disease, focusing on connexins (Cx), which form gap junctions among glial cells and myelin. Autopsied specimens from four cases that provided seven actively demyelinating concentric lesions infiltrated with numerous CD68(+) macrophages were immunohistochemically examined for the astrocyte markers glial fibrillary acidic protein (GFAP), AQP4, Cx43, Cx30 and megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1). Specimens were also stained for oligodendrocyte/myelin markers, namely Cx32, Cx47, myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-specific protein (OSP) and Nogo-A. Serum samples from six patients that had undergone magnetic resonance imaging, confirming a diagnosis of Baló's disease, were assayed for the presence of anti-Cx43, -Cx32 and -AQP4 antibodies. Despite the presence of numerous GFAP- and MLC1-positive astrocytes, there was a marked decrease in the levels of Cx43, Cx32 and Cx47. At the leading edges, Cx43 and AQP4 were mostly absent despite positive GFAP, MLC1, Cx32, Cx47, MOG, MAG, and OSP immunoreactivity. Of the six Baló's disease patients, none were positive for anti-Cxs or -AQP4 antibodies. Baló's disease is characterized by extensive loss of Cxs and AQP4, and a lack of auto-antibodies to Cxs and AQP4. Loss of Cx43 and AQP4 in the presence of other oligodendrocyte/myelin proteins at the leading edges suggests the possibility that auto-antibody-independent astrocytopathy may contribute to disease pathology via the disruption of astrocyte-oligodendrocyte/myelin interactions.

Mitochondrial diseases are increasingly being recognized as causes of encephalopathy and intractable epilepsy. There is no gold-standard test for diagnosing mitochondrial disease, and the current diagnosis relies on establishing a consistent pattern of evidence from clinical data, neuroimaging, tissue biopsy, and biochemical, genetic, and other investigations. Experience in the diagnosis and treatment of patients with certain forms of mitochondrial disease, such as Alpers syndrome, is largely gained from case reports or small case series. The authors describe a case of Alpers syndrome due to POLG1 mutations, including serial neuroimaging and pathological investigations, to illustrate two main points: (1) Unique characteristics evident on serial diffusion-weighted imaging can be a valuable indicator of Alpers syndrome; and (2) abnormal lipid metabolism can be present in Alpers syndrome, which may need to be considered when using a ketogenic diet.

Selective aquaporin-4 (AQP4) loss and vasculocentric complement and immunoglobulin deposition are characteristic of neuromyelitis optica (NMO). We recently reported extensive AQP4 loss in demyelinated and myelinated layers of Baló's lesions without perivascular immunoglobulin and complement deposition. We aimed to reappraise AQP4 expression patterns in NMO and multiple sclerosis (MS). We evaluated AQP4 expression relative to glial fibrillary acidic protein, extent of demyelination, lesion staging (CD68 staining for macrophages), and perivascular deposition of complement and immunoglobulin in 11 cases with NMO and NMO spectrum disorders (NMOSD), five with MS and 30 with other neurological diseases. The lesions were classified as actively demyelinating (n = 66), chronic active (n = 86), chronic inactive (n = 48) and unclassified (n = 12). Six NMO/NMOSD and two MS cases showed preferential AQP4 loss beyond the demyelinated areas, irrespective of lesion staging. Five NMO and three MS cases showed AQP4 preservation even in actively demyelinating lesions, despite grave tissue destruction. Vasculocentric deposition of complement and immunoglobulin was detected only in NMO/NMOSD patients, with less than 30% of actively demyelinating lesions showing AQP4 loss. Our present and previous findings suggest that antibody-independent AQP4 loss can occur in heterogeneous demyelinating conditions, including NMO, Baló's disease and MS., (© 2011 The Authors. Brain Pathology © 2011 International Society of Neuropathology.)

Baló's disease is characterized by alternating rings of demyelination and preserved myelin. As additional multiple sclerosis (MS)-like lesions often coexist in Baló's cases, Baló's disease is regarded as a variant of MS. In demyelinated areas, many hypertrophic astrocytes are present in close contact with oligodendrocytes, which often show apoptotic features. In the outermost layer of preserved myelin, stress proteins involved in tissue preconditioning are abundant in oligodendrocytes. The peri-plaque perimeter is thus assumed resistant to subsequent attack, thereby leaving a layer of preserved myelin. In some cases, Baló's concentric rings develop step by step in a centrifugal direction, whereas many other cases show simultaneous enhancement of multiple rings. Therefore tissue preconditioning and successive ring formation does not fully describe the mechanism of the disease. We recently reported that in four Filipino Baló's patients, aquaporin-4 (AQP4) was extensively lost in glial fibrillary acidic protein-positive hypertrophic astrocytes, both in demyelinated and myelinated layers of all actively demyelinating lesions. None of six further patients with MRI-confirmed Baló's disease were seropositive for anti-AQP4 antibody. I propose that AQP4 astrocytopathy, in the absence of anti-AQP4 antibody, is characteristic of Baló's disease. This hypothesis should be tested in future experimental studies.

Background: DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes., Methods: mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE., Results: We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities., Conclusions: mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Baló's concentric sclerosis (BCS) is considered to be a rare variant of multiple sclerosis and characterized by alternating rings of demyelinated and preserved myelin layers. The mechanism underlying BCS remains to be elucidated. Recently, occurrence of concentric rings of Baló was described in the brainstem of a patient with neuromyelitis optica (NMO). Because selective loss of aquaporin-4 (AQP4) and vasculocentric deposition of complement and immunoglobulins are characteristic in NMO, we aimed to assess AQP4 expression in the concentric demyelinating lesions of BCS patients. We evaluated AQP4 expression relative to expression of another astrocytic marker (glial fibrillary acidic protein), the extent of demyelination, lesion staging and perivascular deposition of complement and immunoglobulin in four cases with BCS, and 30 individuals with other neurological diseases. All cases with BCS demonstrated extensive AQP4 loss in both demyelinated and myelinated layers of all actively demyelinating lesions, with perivascular lymphocytic cuffing of T cells, but no deposition of immunoglobulins or complement around vessels. These findings suggest that AQP4 loss occurs in heterogeneous demyelinating conditions, namely NMO and BCS. Furthermore, acute BCS lesions are characterized by extensive AQP4 loss without vasculocentric deposition of complement or immunoglobulin.

Mutations in the catalytic subunit of the mitochondrial DNA-polymerase gamma cause a wide spectrum of clinical disease ranging from infantile hepato-encephalopathy to juvenile/adult-onset spinocerebellar ataxia and late onset progressive external ophthalmoplegia. Several of these syndromes are associated with an encephalopathy that characteristically shows episodes of rapid neurological deterioration and the development of acute cerebral lesions. The purpose of this study was to investigate the nature, distribution and natural evolution of central nervous system lesions in polymerase gamma associated encephalopathy focusing particularly on lesions identified by magnetic resonance imaging. We compared radiological, electrophysiological and pathological findings where available to study potential mechanisms underlying the episodes of exacerbation and acute cerebral lesions. We studied a total of 112 magnetic resonance tomographies and 11 computed tomographies in 32 patients with polymerase gamma-encephalopathy, including multiple serial examinations performed during both the chronic and acute phases of the disease and, in several cases, magnetic resonance spectroscopy and serial diffusion weighted studies. Data from imaging, electroencephalography and post-mortem examination were compared in order to study the underlying disease process. Our findings show that magnetic resonance imaging in polymerase gamma-related encephalopathies has high sensitivity and can identify patterns that are specific for individual syndromes. One form of chronic polymerase gamma-encephalopathy, that is associated with the c.1399G > A and c.2243G > C mutations, is characterized by progressive cerebral and cerebellar atrophy and focal lesions of the thalamus, deep cerebellar structures and medulla oblongata. Acute encephalopathies, both infantile and later onset, show similar pictures with cortical stroke-like lesions occurring during episodes of exacerbation. These lesions can occur both with and without electroencephalographic evidence of concurrent epileptic activity, and have diffusion, spectroscopic and histological profiles strongly suggestive of neuronal energy failure. We suggest therefore that both infantile and later onset polymerase gamma related encephalopathies are part of a continuum.

We describe a 3.5-year-old female with Alpers disease with a POLG genotype of p.A467T/p.G848S and with a lethal outcome. Laboratory investigation revealed elevated CSF neopterin, IL-6, IL-8, IFN-gamma, reduced CSF 5-methyltetrahydrofolate (5MTHF), and increased serum as well as CSF folate receptor blocking autoantibodies. Treatment with oral Leucovorine (5-formyl-tetrahydrofolate) was initiated at 0.25mg/kg bid, and later increased to 4mg/kg bid. Under treatment CSF levels of 5MTHF, seizure frequency and communicative abilities improved. Over a time span of 17months, CSF levels of IL-6 and IFN-gamma decreased, levels of folate receptor blocking autoantibodies continued to raise, whereas CSF IL-8 remained elevated 1500-fold above normal. The child died without apparent stress at the age of 5.5years. Alpers disease, a neurodegenerative disease usually presents in the first years of life as a progressive encephalopathy with multifocal myoclonic seizures, developmental regression, cortical blindness and early death. The underlying genetic defect has been attributed to mutations of the catalytic subunit of the mitochondrial DNA polymerase-gamma leading to an organ-specific mitochondrial DNA depletion syndrome with reduced activity of respiratory chain enzyme complexes in the brain and the liver. A curative therapy is not available. This case report of Alpers disease provides new insights into the pathophysiology of Alpers disease, where mitochondrial dysfunction in conjunction with inflammatory cytokines and blocking folate receptor autoantibodies may lead to a secondary cerebral folate deficiency syndrome. The treatment of the latter provides relief to the patient without stopping the underlying disease.

Introduction: Proton magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) yield different parameters for characterizing the evolution of a demyelinating white matter disease. The purpose was to elucidate biochemical and microstructural changes in Balo's concentric sclerosis lesions and to correlate the findings with the clinical course., Methods: Localized short-echo time MRS and DTI were performed over 6 years in a left occipital lesion of a female patient (age at onset 13.8 years) with Balo's concentric sclerosis. A right homonym hemianopsia persisted., Results: Metabolite patterns were in line with initial active demyelination followed by gliosis and partial recovery of neuroaxonal metabolites. Fractional anisotropy and mean diffusivity of tissue water remained severely altered. Fiber tracking confirmed a disruption in the geniculo-calcarine tract as well as involvement of the corpus callosum., Conclusion: MRS and DTI depict complementary parameters, but DTI seems to correlate better with clinical symptoms.

Multiple sclerosis is a chronic inflammatory disease, which leads to focal plaques of demyelination and tissue injury in the CNS. The structural and immunopathological patterns of demyelination suggest that different immune mechanisms may be involved in tissue damage. In a subtype of lesions, which are mainly found in patients with acute fulminant multiple sclerosis with Balo's type concentric sclerosis and in a subset of early relapsing remitting multiple sclerosis, the initial myelin changes closely resemble those seen in white matter stroke (WMS), suggesting a hypoxia-like tissue injury. Since mitochondrial injury may be involved in the pathogenesis of such lesions, we analysed a number of mitochondrial respiratory chain proteins in active lesions from acute multiple sclerosis and from WMS using immunohistochemistry. Functionally important defects of mitochondrial respiratory chain complex IV [cytochrome c oxidase (COX)] including its catalytic component (COX-I) are present in Pattern III but not in Pattern II multiple sclerosis lesions. The lack of immunohistochemically detected COX-I is apparent in oligodendrocytes, hypertrophied astrocytes and axons, but not in microglia. The profile of immunohistochemically detected mitochondrial respiratory chain complex subunits differs between multiple sclerosis and WMS. The findings suggest that hypoxia-like tissue injury in Pattern III multiple sclerosis lesions may be due to mitochondrial impairment.

We have identified compound heterozygous missense mutations in POLG1, encoding the mitochondrial DNA polymerase gamma (Pol gamma), in 7 children with progressive encephalopathy from 5 unrelated families. The clinical features in 6 of the children included psychomotor regression, refractory seizures, stroke-like episodes, hepatopathy, and ataxia compatible with Alpers-Huttenlocher syndrome. Three families harbored a previously reported A467T substitution, which was found in compound with the earlier described G848S or the W748S substitution or a novel R574W substitution. Two families harbored the W748S change in compound with either of 2 novel mutations predicted to give an R232H or M1163R substitution. Muscle morphology showed mitochondrial myopathy with cytochrome c oxidase (COX)-deficient fibers in 4 patients. mtDNA analyses in muscle tissue revealed mtDNA depletion in 3 of the children and mtDNA deletions in the 2 sibling pairs. Neuropathologic investigation in 3 children revealed widespread cortical degeneration with gliosis and subcortical neuronal loss, especially in the thalamus, whereas there were only subcortical neurodegenerative findings in another child. The results support the concept that deletions as well as depletion of mtDNA are involved in the pathogenesis of Alpers-Huttenlocher syndrome and add 3 new POLG1 mutations associated with an early-onset neurodegenerative disease.

Lesions of Baló's concentric sclerosis are characterized by alternating layers of myelinated and demyelinated tissue. The reason for concentric demyelination in this variant of multiple sclerosis is unclear. In the present study we investigated the immunopathology in autopsy tissue of 14 patients with acute multiple sclerosis or fulminant exacerbations of chronic multiple sclerosis with Baló-type lesions in the CNS, focusing on the patterns of tissue injury in actively demyelinating lesions. We found that all active concentric lesions followed a pattern of demyelination that bears resemblances to hypoxia-like tissue injury. This was associated with high expression of inducible nitric oxide synthase in macrophages and microglia. At the edge of active lesions and, less consistently, in the outermost layer of preserved myelin, proteins involved in tissue preconditioning, such as hypoxia-inducible factor 1alpha and heat-shock protein 70, were expressed mainly in oligodendrocytes and to a lesser degree also in astrocytes and macrophages. Due to their neuroprotective effects, the rim of periplaque tissue, where these proteins are expressed, may be resistant to further damage in an expanding lesion and may therefore remain as a layer of preserved myelinated tissue.

Mitochondrial dysfunction of the energy generating system was suggested in two infants with progressive infantile poliodystrophy characterised by hypotonia, refractory epilepsy, visual impairment, psychomotor retardation, profound brain atrophy, hepatopathy, and increased levels of lactate in blood and cerebrospinal fluid. Histochemical and electron microscopic analyses of liver biopsies revealed cytochrome c oxidase deficiency, microvesicular steatosis, and enormous multiplication of mitochondria of various sizes. In the first patient, the quantitative Southern blot analyses in tissues obtained at autopsy demonstrated reduced content of mtDNA in the liver, brain, and fibroblasts (11 %, 15 %, and 25 % of the mean values in controls) while a normal content of mtDNA was found in muscle and heart. In the second patient, a reduced content of mtDNA was found in the muscle, liver, and brain (15 %, 10 %, and 30 %, respectively, of the mean values in controls). Biochemical studies in the first patient revealed decreased activities of all respiratory chain complexes except complex II in isolated liver mitochondria and decreased amounts of respiratory chain complexes I, III, IV and ATP synthase in liver and frontal cortex, but not in muscle, heart, and fibroblasts. In conclusions, mtDNA depletion associated with Alpers syndrome may be tissue specific.

Objective: Cytochrome c oxidase (COX) deficiency has been demonstrated in some patients with Alpers-Huttenlocher disease, but no genetic background has been identified. Our objective was to determine the molecular defect underlying the mitochondrial respiratory chain deficiency in a child with Alpers-Huttenlocher-like progressive cerebrohepatic disease., Methods: The entire coding region of mitochondrial DNA was analyzed by conformation-sensitive gel electrophoresis and sequencing. Biochemical and morphologic investigations were performed on tissue biopsy material, including oximetric and spectrophotometric analyses of oxidative phosphorylation, histochemistry, and electron microscopy., Results: Postmortem histologic examination revealed a marked loss of neurons in the olivary nuclei and a spongy change in the calcarine cortex, fatty infiltration and micronodular cirrhosis of the liver, and atrophic ovaries. A novel heteroplasmic 7706G>A mutation was found in the COX II gene. The median degree of the mutant heteroplasmy was 90% in 5 tissues examined but was lower in the blood of asymptomatic maternal relatives. The distribution of the mutant heteroplasmy was skewed to the left in single muscle fibers of the proband and her mother. The 7706G>A mutation converts a hydrophobic alanine in a conserved transmembrane segment to hydrophilic threonine., Conclusions: The 7706G>A mutation is pathogenic and may lead to impaired dioxygen transfer to the active site of COX. The clinical phenotype of this patient resembled that in Alpers-Huttenlocher disease, suggesting that analysis of mitochondrial DNA is worthwhile in patients with a progressive cerebrohepatic disease.

Background: Respiratory chain (RC) disorders are clinically, biochemically, and molecularly heterogeneous. The lack of standardized diagnostic criteria poses difficulties in evaluating diagnostic methodologies., Objective: To assess proposed adult RC diagnostic criteria that classify patients into "definite," "probable," or "possible" categories., Methods: The authors applied the adult RC diagnostic criteria retrospectively to 146 consecutive children referred for investigation of a suspected RC disorder. Data were collected from hospital, genetics, and laboratory records, and the diagnoses predicted by the adult criteria were compared with the previously assigned assessments., Results: The authors identified three major difficulties in applying the adult criteria:lack of pediatric-specific criteria; difficulty in segregating continuous data into circumscribed major and minor criteria; and lack of additivity of clinical features or enzyme tests. They therefore modified the adult criteria to allow for pediatric clinical and histologic features and for more sensitive coding of RC enzyme and functional studies. Reanalysis of the patients' data resulted in congruence between the diagnostic certainty previously assigned by the authors' center and that defined by the new general RC diagnostic criteria in 99% of patients., Conclusions: These general diagnostic criteria appear to improve the sensitivity of the adult criteria. They need further assessment in prospective clinical and epidemiologic studies.

Alpers-Huttenlocher syndrome, considered a mitochondrial disease, combines encephalopathy and liver failure. An 11-year-old boy with Alpers-Huttenlocher syndrome underwent conventional MR imaging, diffusion-weighted imaging, and proton MR spectroscopy. Diffusion-weighted imaging showed cytotoxic edema interpreted as acute-phase encephalopathy. MR spectroscopy revealed a lactate peak in the cortex that appeared abnormal on diffusion-weighted images, possibly representing respiratory deficiency with anaerobic metabolism. MR spectroscopy proved to be more sensitive regarding lactate detection than did neurometabolic examination of serum and CSF. A reduced N-acetylaspartate-creatine ratio was detected in both the cortex that appeared abnormal and the cortex that appeared normal on the diffusion-weighted images, indicating neuronal damage that was widespread, even beyond the boundaries of conventional MR imaging changes.

We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse-sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.

Pelizaeus-Merzbacher disease is a rare, sex-linked recessive, dysmyelinating disease of the central nervous system that has been associated with mutations in the myelin proteolipid protein (PLP) gene. Only 25% of patients studied with Pelizaeus-Merzbacher disease have exonic mutations in this gene, the underlying cause of the disease in the remaining patients is unknown. The PLP gene encodes two major alternatively spliced transcripts called PLP and DM20. PLP messenger RNA is specifically expressed in central nervous system tissue, whereas DM20 messenger RNA is found in central nervous system, cardiac, and other tissues. We studied cultured skin fibroblasts from 2 brothers with Pelizaeus-Merzbacher disease who exhibited no detectable exonic mutation of the PLP gene. Examination of RNA from these cells showed that the level of DM20 messenger RNA is elevated sixfold relative to male control skin fibroblasts. An unrelated female carrier, also with no detectable exonic mutation, showed a threefold increase in DM20 messenger RNA in cultured skin fibroblasts. Our findings suggest that in some patients, Pelizaeus-Merzbacher disease is caused by overexpression of PLP gene transcripts, and that in these families a 50% increase of DM20 messenger RNA in females, relative to the increase in affected males, can identify a female carrier.

The neuropathology of a clinically well-documented case of the neonatal progeroid syndrome Wiedemann-Rautenstrauch is described. The most striking feature was a nearly complete absence of mature myelin in the brain. When immunohistochemistry for myelin basic protein was applied, some subcortical nerve fibres were accompanied by immature myelin sheaths. The neuropathology corresponds exactly to that of Pelizaeus-Merzbacher disease (Seitelberger type). Furthermore, this morphology, with the presence of myelin basic protein in the absence of mature myelin sheaths is reminiscent of the early stages of myelination in the newborn. From a brief review of the literature on Wiedemann-Rautenstrauch syndrome, we conclude, that the neuropathology of the syndrome is heterogeneous, and that there is relationship between the progeroid aspect and pathological myelination.

Clinical, neuropathological and molecular genetic studies in a 9 month old boy with Pelizaeus-Merzbacher disease are described. The principal clinical features were developmental delay, nystagmus, stridor and seizures. Both brain and spinal cord showed almost complete absence of stainable central myelin, while cranial and spinal root myelin was preserved. Probes for cDNA in the boy and his asymptomatic mother indicated an increase in the dosage of proteolipid protein gene (of at least twofold) compared with controls.

Canavan disease (CD) is a rare autosomal recessive disorder characterized by macrocephaly and progressive leukodystrophy. Up to now biopsy or necropsy were required to define the diagnosis. Recently the disease has been related to N-acetylaspartic aciduria and deficiency of aspartoacylase, an enzyme possibly involved in the myelin synthesis. These biochemical findings have provided a diagnostic marker for the disease. We report a new case of infantile CD in which the demonstration of N-acetylaspartic aciduria and a marked deficiency of aspartoacylase activity confirmed the diagnosis.

alpha B-Crystallin is a major protein component of Rosenthal fibers, which massively accumulate in the brains of patients suffering from Alexander's disease. To examine whether or not accumulation of alpha B-crystallin is due to any abnormality of the gene structures, we determined the sequence of the alpha B-crystallin gene in two cases of pathologically confirmed Alexander's disease. Direct sequencing of the promoter and coding regions of the alpha B-crystallin gene in patients revealed them to have a normal sequence. Northern blotting showed a single alpha B-crystallin mRNA species expressed in the Alexander's disease brain.

The phosphorylation of alpha-crystallin B was studied in homogenates of autopsy samples of brain tissue from patients with Alexander's disease, a condition characterized by over-expression of this protein. After incubation in the presence of [gamma-32P]ATP and cAMP the homogenates were analyzed by two-dimensional electrophoresis, (isoelectric focusing followed by SDS-PAGE). Three major polypeptides having the same molecular weight as bovine lens alpha-crystallin B and pIs 7.1, 6.9 and 6.7 were detected in the Coomassie blue stained gels. These three polypeptides were recognized by an alpha-crystallin B-specific antiserum in Western blots. The polypeptides with pIs 7.1 and 6.7 co-migrated in isoelectric focusing gels with bovine lens alpha B and its phosphorylated form alpha Bp, respectively. Radioautography of the two-dimensional gels demonstrated the presence of 32P in the most acidic polypeptide. The results demonstrate the occurrence of alpha B phosphorylation in Alexander's disease brain tissue.

The myelin basic protein (MBPi) accounts for the main encephalitogenic antigen of the demyelinating encephalopathy, but other myelin elements also be noted by some articles. We tried to determine the relationship between W1 protein and the demyelinating encephalopathies. The 2',3'-cyclic nucleotide 3'-phospho-diesterase(CNPase, a gift from Dr. Yasuzo Tuskada) monoclonal antibody was used as a probe to study the W1 protein level in 5 different demyelinating encephalopathies and 2 normal adult brains with immunocytochemical technique. Two different demyelinating types of W1 protein level were found out. Type 1 showed the W1 protein level parallel with demyelinated feature in general pathology whereas the type 2 showed demyelinated in the general pathology but the W1 protein level was normal in the immunocytochemical study. Multiple system degeneration, Binswanger's disease and postvaccinal encephalopathy of type B encephalitis belong to type 1 and multiple sclerosis and Balo's concentric sclerosis belong to type 2. These results might indicate the different pathogenesis of demyelinating encephalopathies.

Alpers disease consists of diffuse cerebral degeneration manifested as developmental delay, seizures, vomiting, and progressive neuromuscular deterioration, with liver disease and death. We report the clinical course of the liver disease, histologic progression of the hepatic lesions, and etiologic investigations in five patients (four girls, three kinships). All had grown and developed normally until seen at 6 to 36 months of age (mean 20 months), with vomiting (n = 5), progressive hypotonia (n = 3), or seizures (n = 2). All had been given anticonvulsants, including valproic acid in three. Liver disease was noted at a mean age of 35 months (range 9 to 67 months), with hepatomegaly (two patients), abnormal hepatic synthetic function (three) or transaminase values (three), and cirrhosis in one. Patients survived for a mean of 4.6 weeks (range 1 to 8 weeks) after the identification of liver disease; all died of hepatic failure. Results of evaluation for infectious and metabolic causes of liver disease and causes of degenerative neuromuscular disease were negative in all patients. Premortem liver biopsy specimens (n = 3) demonstrated an early lesion consisting of lobular disarray, microvesicular steatosis, periportal acute and chronic inflammation, and individual hepatocyte necrosis. Autopsy findings (n = 5) consisted of macrovesicular steatosis, massive hepatocyte dropout, and proliferation of bile ductular elements, with almost complete replacement of hepatocytes by proliferating bile ductular elements in two patients. Brain showed characteristic neuronal degeneration. We conclude that Alpers disease can be a cause of rapidly progressive liver failure in early childhood. Although the cause of this autosomal recessive disease is not known, it does not appear to be related to peroxisomal dysfunction.

The metabolism of [1-14C]lignoceric acid (C24:0) and [1-14C]tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal beta-oxidation (but normal peroxisomal numbers). Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from [1-14C]C24:0. However, the products of synthesis from acetate de novo (released by beta-oxidation), i.e. C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased. In contrast, cell lines from two patients with an unidentified lesion in peroxisomal beta-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and C18 fatty acids and an increase in carbon chain elongation. The relatively normal beta-oxidation activity of ALD cells appears to be related to low uptake of substrate, as a defect in beta-oxidation is apparent when measurements are performed on cell suspensions under high uptake conditions. Oxidation of [1-14C]C24:4 was relatively normal in ALD cells and in the cells from one PD patient but abnormal in those from the other. Our data suggest that, despite the deficiency in VLCFA CoA synthetase, ALD cells retain a near normal ability to oxidize both saturated and polyunsaturated VLCFA under some culture conditions. However, acetate released by beta-oxidation of the saturated VLCFA and, to a much lesser degree, the polyunsaturated VLCFA, appears to be used preferentially for the production of CO2 and water-soluble products, and acetate availability for fatty acid synthesis in other subcellular compartments is markedly decreased. It is likely that the increased carbon chain elongation of the saturated VLCFA which is also observed reflects the increased availability of substrate (C24:0) and/or an increase in microsomal elongation activity in ALD cells.

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.

In two siblings with limb-girdle muscle weakness and episodic headaches and vomiting from early childhood, progressive neurologic degeneration later developed, and both children died. In one child, corticosteroids induced improvement in both cerebral and muscular symptoms that lasted 1 year. This patient had elevated blood, urine, and spinal fluid lactate levels, together with increased cardiac output and oxygen consumption at rest. Several muscle fibers were characterized by a "ragged red" appearance with the trichrome stain. Subsarcolemmal and intermyofibrillar excess of mitochondrial oxidative enzyme reaction product was correlated with abnormal mitochondrial aggregates by electron microscopy. The brain revealed focal areas of cortical degeneration and necrosis with adjacent gliosis or edema. Ferrocalcific deposits were prominent in the globus pallidus. The other sibling had similar changes in the brain at autopsy. This familial multisystem disorder especially involving the brain, skeletal muscle, and heart appears to represent a defect in some mitochondrial oxidative mechanism.

Rosenthal fibers (RFs) are abnormal inclusions within astrocytes, characteristic of Alexander's disease. We have previously isolated a 22 kd protein component of RFs from Alexander's disease brain. By Western blotting, we detected its equivalent in several rat organs, with the highest level in heart, and in a human astrocytoma cell line (U-373MG). A cDNA library established from U-373MG was screened with an anti-RF protein antibody. A partial cDNA clone encoding the lens protein alpha B-crystallin was isolated. The anti-RF protein antibodies react with lens alpha B-crystallin. Furthermore, the distribution of alpha B-crystallin mRNA in rat organs is consistent with the Western blots. Therefore, alpha B-crystallin is not lens-specific and it can accumulate in large amounts in astrocytes in pathological conditions.

The basic biochemical defect of X-linked adrenoleukodystrophy (sudanophilic leukodystrophy, Schilder's disease) is unknown. To investigate reported abnormalities in cholesterol metabolism in vitro, we examined cultured skin fibroblasts of four patients and four normal control subjects. The kinetics of retention and accumulation of [14C]cholesterol by these cells was studied. After 3 days of exposure to tracer amounts of [14C]cholesterol, an apparent steady state between the medium and cellular cholesterol was established. The specific radioactivity expressed per mg of protein was similar for both Schilder and control fibroblasts. tafter labeling the pre-existing cellular cholesterol pool, the rate of loss of label was followed up for a 6-day period. About 23% and 14%, respectively, of the cellular radioactivity in both Schilder's disease and control cells were released into the medium after the consecutive change with fresh nonlabeled medium. No significant differences in [14C]cholesterol rates of uptake or release were observed between control and Schilder's disease fibroblasts. About 44% of the labeled cholesterol was present in an esterfied form after incubation in the presence of unheated serum in both Schilder's and control cultures.

Polarizing material has been reported in the liver of children with infantile Refsum's disease (IRD) and was absent in two patients with the cerebro-hepato-renal syndrome of Zellweger (CHRS). We examined in polarized light 15 liver biopsy and autopsy samples from six other patients with the cerebro-hepato-renal syndrome of Zellweger, two with the rhizomelic form of chondrodysplasia punctata (rCDP) and two with X-linked adrenoleukodystrophy (ALD), all conditions with deficient peroxisomes. Two types of birefringent inclusions were found in CHRS only: the first is transparent in bright field, the second appears as brown granules or rods, similar to lipofuscins. As in IRD large PAS-positive macrophage-like cells contain the transparent type. Electron microscopical investigation of these cells shows trilaminar structures within membrane-bound organelles. The two types were also seen in kidney and brown adipose tissue, the first type in pancreas, the second type in adrenal gland; no such was observed in myocardium or in thyroid gland (CHRS). No birefringent inclusions were present in rCDP and ALD. The nature of the inclusions is still unclear. An accumulation of the transparent polarizing material with increasing age of the patients is most likely.

Adrenoleucodystrophy (ALD) is a rare, X-linked inherited disease affecting the central nervous system. The clinical findings overlap those of other related progressive neurological diseases and complicate the diagnosis. In this article biochemical analyses of the very-long-chain fatty acid distribution in specimens obtained from 2 related patients clinically diagnosed as having ALD, which unequivocally confirmed the diagnosis, are described. Analyses of the long-chain fatty acids were performed on postmortem brain tissue samples from the one patient. In the other, who is in the terminal phase of the disease, the long-chain fatty acids were analysed in plasma, erythrocytes and fibroblasts from tissue culture. The limitations of the different analyses for biochemical identification of ALD are discussed.

Long-chain fatty acids in erythrocyte membrane phospholipids were analyzed in three patients with adrenoleukodystrophy (ALD) and four diseased controls with other neurologic diseases by high performance liquid chromatography (HPLC). HPLC chromatograms showed the increase of a very long-chain fatty acid C26:0 in glycerophospholipids of ALD erythrocyte membranes, which was not so prominent in sphingomyelin. The ratio of C26:0 to C22:0 increased in glycerophospholipids as well as sphingomyelin in ALD. These results suggest that ALD has a generalized abnormal metabolism of very long-chain saturated fatty acids.

We describe a 16-year-old boy who has a progressive dementia and seizures. On investigation, he was found to have a mitochondrial myopathy and elevated lactate levels in the blood and cerebrospinal fluid. His sister died at 18 years of age of a similar condition.

An autopsy study of an 11-year-old boy with the classic type of Pelizaeus-Merzbacher disease is presented. He developed normally until 5 years of age when he began to deteriorate with scanning speech and gait abnormality. Auditory brainstem responses were normally preserved. At the age of 11 years, 8 months, he died of pneumonia while in a vegetative state. The neuropathologic findings suggested a typical classic type of Pelizaeus-Merzbacher disease and biochemical analysis of cerebral white matter demonstrated a decreased ratio of long-chain fatty acids (greater than or equal to C19) to short- and medium-chain fatty acids (less than C19). These findings suggested defective myelin synthesis as the etiology of Pelizaeus-Merzbacher disease; comprehensive classifications of the disease are expected to include both pathologic and biochemical parameters.

Three major lipid fractions, i.e., cholesterol ester, triacylglycerol and glycerophospholipids, were investigated cultured fibroblasts to clarify whether very long-chain fatty acids were accumulated or not. Abnormally accumulated very long-chain fatty acids were confirmed in all three lipid fractions. Our results support our previous report that there may be a generalized abnormal metabolism of very long-chain fatty acids in adrenoleukodystrophy.