Brainstem Chipmunk Sign: A Diagnostic Imaging Clue across All Subtypes of Alexander Disease.

BACKGROUND AND PURPOSE: While classic brain MR imaging features of Alexander disease have been well-documented, lesional patterns can overlap with other leukodystrophies, especially in the early stages of the disease or in milder phenotypes. We aimed to assess the utility of a new neuroimaging sign to help increase the diagnostic specificity of Alexander disease. MATERIALS AND METHODS: A peculiar bilateral symmetric hyperintense signal on T2-weighted images affecting the medulla oblongata was identified in an index patient with type I Alexander disease. Subsequently, 5 observers performed a systematic MR imaging review for this pattern by examining 55 subjects with Alexander disease and 74 subjects with other leukodystrophies. Interobserver agreement was assessed by the κ index. Sensitivity, specificity, and receiver operating characteristic curves were determined. RESULTS: The identified pattern was present in 87% of subjects with Alexander disease and 14% of those without Alexander disease leukodystrophy (P < .001), 3 with vanishing white matter, 4 with adult polyglucosan body disease, and 3 others. It was found equally in both type I and type II Alexander disease (28/32, 88% versus 18/21, 86%; P = .851) and in subjects with unusual disease features (2/2). Sensitivity (87.3%; 95% CI, 76.0%-93.7%), specificity (86.5%; 95% CI, 76.9%-92.5%), and interobserver agreement (κ index = 0.82) were high. CONCLUSIONS: The identified pattern in the medulla oblongata, called the chipmunk sign due to its resemblance to the face of this rodent, is extremely common in subjects with Alexander disease and represents a diagnostic tool that can aid in early diagnosis, especially in subjects with otherwise atypical MR imaging findings and/or clinical features.

Glial fibrillary acidic protein is pathologically modified in Alexander disease.

Here, we describe pathological events potentially involved in the disease pathogenesis of Alexander disease (AxD). This is a primary genetic disorder of astrocyte caused by dominant gain-of-function mutations in the gene coding for an intermediate filament protein glial fibrillary acidic protein (GFAP). Pathologically, this disease is characterized by the upregulation of GFAP and its accumulation as Rosenthal fibers. Although the genetic basis linking GFAP mutations with Alexander disease has been firmly established, the initiating events that promote GFAP accumulation and the role of Rosenthal fibers (RFs) in the disease process remain unknown. Here, we investigate the hypothesis that disease-associated mutations promote GFAP aggregation through aberrant posttranslational modifications. We found high molecular weight GFAP species in the RFs of AxD brains, indicating abnormal GFAP crosslinking as a prominent pathological feature of this disease. In vitro and cell-based studies demonstrate that cystine-generating mutations promote GFAP crosslinking by cysteine-dependent oxidation, resulting in defective GFAP assembly and decreased filament solubility. Moreover, we found GFAP was ubiquitinated in RFs of AxD patients and rodent models, supporting this modification as a critical factor linked to GFAP aggregation. Finally, we found that arginine could increase the solubility of aggregation-prone mutant GFAP by decreasing its ubiquitination and aggregation. Our study suggests a series of pathogenic events leading to AxD, involving interplay between GFAP aggregation and abnormal modifications by GFAP ubiquitination and oxidation. More important, our findings provide a basis for investigating new strategies to treat AxD by targeting abnormal GFAP modifications.

A retrospective observational cohort study of the anesthetic management and outcomes of pediatric patients with Alexander disease undergoing lumbar puncture or magnetic resonance imaging.

BACKGROUND: Alexander disease is a rare, progressive leukodystrophy, which predisposes patients to complications under general anesthesia due to clinical manifestations including developmental delay, seizures, dysphagia, vomiting, and sleep apnea. However, study of anesthetic outcomes is limited. AIMS: Our aim was to describe patient characteristics, anesthetic techniques, and anesthesia-related complications for Alexander disease patients undergoing magnetic resonance imaging and/or lumbar puncture at a quaternary-care children's hospital. METHODS: We performed a retrospective review of anesthetic outcomes in patients with Alexander disease enrolled in a prospective observational study. Included patients had diagnosed Alexander disease and underwent magnetic resonance imaging and/or lumbar puncture at our institution. We excluded anesthetics for other procedures or at outside institutions. Collected data included patient characteristics, anesthetic techniques, medications, and complications under anesthesia and in the subsequent 24 h. We performed descriptive statistics as appropriate. RESULTS: Forty patients undergoing 64 procedures met inclusion criteria. Fifty-six procedures (87.5%) required general anesthesia or monitored anesthesia care (MAC) and eight (12.5%) did not. The general anesthesia/MAC group tended to be younger than nonanesthetized patients (median age 6 years [IQR 3.8; 9] vs. 14.5 years [IQR 12.8; 17.5]). In both groups, dysphagia (78.6% vs. 87.5%, respectively), seizures (62.5% vs. 25%), and recurrent vomiting (17.9% vs. 25%) were frequently reported preprocedure symptoms. Inhalational induction was common (N = 48; 85.7%), and two (3.6%) underwent rapid sequence induction. Serious complications were rare, with no aspiration or seizures. Hypotension resolving with ephedrine occurred in eight cases (14.3%). One patient each (1.8%) experienced postprocedure emergence agitation or vomiting. Fifty-three (94.6%) were ambulatory procedures. No inpatients required escalation in acuity of care. CONCLUSIONS: In this single-center study, patients with Alexander disease did not experience frequent or irreversible complications while undergoing general anesthesia/MAC. Co-morbid symptoms were not increased postanesthesia. Some patients may not require anesthesia to complete short procedures.

Alexander disease with a novel GFAP insertion-deletion mutation mimicking progressive supranuclear palsy.

This report presents a case of Alexander disease showing clinical characteristics mimicking progressive supranuclear palsy (PSP). A 67-year-old woman complaining of motor disturbance exhibited severe atrophy of medulla, spinal cord, and midbrain tegmentum, as well as periventricular hyperintensity on cerebral MRI. Genetic analysis identified a novel in-frame deletion/insertion mutation in the exon 3 of the GFAP gene. Interestingly, neurological findings and decreased striatal uptake in dopamine transporter SPECT were suggestive of PSP. A novel GFAP gene mutation found in the present case may cause the unique clinical phenotype, which should be differentiated from PSP.

Plasma concentrations of glial fibrillary acidic protein, neurofilament light, and tau in Alexander disease.

INTRODUCTION: Alexander disease (AxD) is a rare leukodystrophy caused by dominant gain-of-function mutations in the gene encoding the astrocyte intermediate filament, glial fibrillary acidic protein (GFAP). However, there is an urgent need for biomarkers to assist in monitoring not only the progression of disease but also the response to treatment. GFAP is the obvious candidate for such a biomarker, as it is measurable in body fluids that are readily accessible for biopsy, namely cerebrospinal fluid and blood. However, in the case of ASOs, the treatment that is furthest in development, GFAP is the target of therapy and presumably would go down independent of disease status. Hence, there is a critical need for biomarkers that are not directly affected by the treatment strategy. METHODS: We explored the potential utility of biomarkers currently being studied in other neurodegenerative diseases and injuries, specifically neurofilament light protein (NfL), phosphorylated forms of tau, and amyloid-ß peptides (Aß42/40). RESULTS AND CONCLUSIONS: Here, we report that GFAP is elevated in plasma of all age groups afflicted by AxD, including those with adult onset. NfL and p-tau are also elevated, but to a much lesser extent than GFAP. In contrast, the levels of Aß40 and Aß42 are not altered in AxD.

Leukodystrophy with Macrocephaly, Refractory Epilepsy, and Severe Hyponatremia-The Neonatal Type of Alexander Disease.

INTRODUCTION: Alexander disease (AxD) is a rare neurodegenerative condition that represents the group of leukodystrophies. The disease is caused by GFAP mutation. Symptoms usually occur in the infantile age with macrocephaly, developmental deterioration, progressive quadriparesis, and seizures as the most characteristic features. In this case report, we provide a detailed clinical description of the neonatal type of AxD. METHOD: Next-Generation Sequencing (NGS), including a panel of 49 genes related to Early Infantile Epileptic Encephalopathy (EIEE), was carried out, and then Whole Exome Sequencing (WES) was performed on the proband's DNA extracted from blood. CASE DESCRIPTION: In the first weeks of life, the child presented with signs of increased intracranial pressure, which led to ventriculoperitoneal shunt implementation. Recurrent focal-onset motor seizures with secondary generalization occurred despite phenobarbital treatment. Therapy was modified with multiple anti-seizure medications. In MRI contrast-enhanced lesions in basal ganglia, midbrain and cortico-spinal tracts were observed. During the diagnostic process, GLUT-1 deficiency, lysosomal storage disorders, organic acidurias, and fatty acid oxidation defects were excluded. The NGS panel of EIEE revealed no abnormalities. In WES analysis, GFAP missense heterozygous variant NM_002055.5: c.1187C>T, p.(Thr396Ile) was detected, confirming the diagnosis of AxD. CONCLUSION: AxD should be considered in the differential diagnosis in all neonates with progressive, intractable seizures accompanied by macrocephaly.

Quantitative diffusion imaging and genotype-by-sex interactions in a rat model of Alexander disease.

PURPOSE: The clinical diagnosis and classification of Alexander disease (AxD) relies in part on qualitative neuroimaging biomarkers; however, these biomarkers fail to distinguish and discriminate different subtypes of AxD, especially in the presence of overlap in clinical symptoms. To address this gap in knowledge, we applied neurite orientation dispersion and density imaging (NODDI) to an innovative CRISPR-Cas9 rat genetic model of AxD to gain quantitative insights into the neural substrates and brain microstructural changes seen in AxD and to potentially identify novel quantitative NODDI biomarkers of AxD. METHODS: Multi-shell DWI of age- and sex-matched AxD and wild-type Sprague Dawley rats (n = 6 per sex per genotype) was performed and DTI and NODDI measures calculated. A 3 × 2 × 2 analysis of variance model was used to determine the effect of genotype, biological sex, and laterality on quantitative measures of DTI and NODDI across regions of interest implicated in AxD. RESULTS: There is a significant effect of genotype in the amygdala, hippocampus, neocortex, and thalamus in measures of both DTI and NODDI brain microstructure. A genotype by biological sex interaction was identified in DTI and NODDI measures in the corpus callosum, hippocampus, and neocortex. CONCLUSION: We present the first application of NODDI to the study of AxD using a rat genetic model of AxD. Our analysis identifies alterations in NODDI and DTI measures to large white matter tracts and subcortical gray nuclei. We further identified genotype by sex interactions, suggesting a possible role for biological sex in the neuropathogenesis of AxD.

Microglia sense astrocyte dysfunction and prevent disease progression in an Alexander disease model.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.

Substitution of Glu to Lys at Codon 332 on the GFAP Gene Alone Is Causative for Adult-onset Alexander Disease.

A 57-year-old man whose mother had been pathologically diagnosed with Alexander disease (ALXDRD), presented with cerebellar ataxia, pyramidal signs, and mild dysarthria. Brain magnetic resonance imaging revealed typical ALXDRD alterations, such as atrophy of the medulla oblongata (MO) and cervical spinal cord, a reduced sagittal diameter of the MO, and garland-like hyperintensity signals along the lateral ventricular walls. A genetic analysis of GFAP by Sanger sequencing revealed a single heterozygous mutation of Glu to Lys at codon 332 (c.994G>A) in the GFAP gene. Our results newly confirmed that p.E332K alone is the pathogenic causative mutation for adult-onset ALXDRD.

Acquisition and Loss of Developmental Milestones and Time to Disease-Related Outcomes in Cerebral Alexander Disease.

Objective: To determine the ages at acquisition of developmental milestones, loss of motor function, and clinical symptoms in Alexander disease. Methods: Patients with confirmed cerebral Alexander disease were included. Data abstraction of developmental and disease-specific milestones was performed from medical records, physical exams, and questionnaires. Mixed effects logistic regression was used to determine if key clinical features were associated with milestone achievement, controlling for patient age. Results: 51 patients with cerebral/infantile Alexander disease were evaluated at a mean age of 10.96 years (range 2.29-31.08 years). Developmental milestones in Alexander disease were often achieved but delayed. Ambulation was achieved in 44 subjects (86%); 34 (67%) subjects walked independently (mean age 1.9 years, range 0.91-3.25 years) and an additional 10 (20%) subjects walked with assistance (mean age 3.9 years, range 1.8-8 years) but did not progress to independent ambulation. Developmental delay was the earliest and most prevalent symptom (N = 48 [94%], mean age 0.58 years), compared to an initial seizure (N = 41 [80%], mean age 2.80 years), and macrocephaly (N = 28 [55%], mean age 4.04 years), P < .0001 between these ages of onset. Loss of independent ambulation occurred in 11 of the 34 (32%) children who had acquired ambulation (range 3.41-15.10 years). Presence of seizures or macrocephaly did not predict the achievement or loss of ambulation. Conclusions: The clinical triad of developmental delay, seizures, and macrocephaly are not universally present in cerebral Alexander disease. Clinicians should have a high index of suspicion for Alexander disease in patients with mild delays and a first seizure.

Adult-onset Alexander disease among patients of Jewish Syrian descent.

Alexander disease (AxD) is a rare autosomal dominant leukodystrophy caused by heterozygous mutations in the glial fibrillary acid protein (GFAP) gene. The age of symptoms onset ranges from infancy to adulthood, with variable clinical and radiological manifestations. Adult-onset AxD manifests as a chronic and progressive condition, characterized by bulbar, motor, cerebellar, and other clinical signs and symptoms. Neuroradiological findings typically involve the brainstem and cervical spinal cord. Adult-onset AxD has been described in diverse populations but is rare in Israel. We present a series of patients diagnosed with adult-onset AxD from three families, all of Jewish Syrian descent. Five patients (4 females) were diagnosed with adult-onset AxD due to the heterozygous mutation c.219G > A, p.Met73Ile in GFAP. Age at symptoms onset ranged from 48 to 61 years. Clinical characteristics were typical and involved progressive bulbar and gait disturbance, followed by pyramidal and cerebellar impairment, dysautonomia, and cognitive decline. Imaging findings included medullary and cervical spinal atrophy and mostly infratentorial white matter hyperintensities. A newly recognized cluster of adult-onset AxD in Jews of Syrian origin is presented. This disorder should be considered in differential diagnosis in appropriate circumstances. Genetic counselling for family members is required in order to discuss options for future family planning.

Fetal-onset Alexander disease with radiological-neuropathological correlation.

Alexander disease is a leukodystrophy caused by mutations in the GFAP gene, primarily affecting the astrocytes. This report describes the prenatal and post-mortem neuroimaging findings in a case of genetically confirmed, fetal-onset Alexander disease with pathological correlation after termination of pregnancy. The additional value of fetal brain magnetic resonance imaging in the third trimester as a complementary evaluation tool to neurosonography is shown for suspected cases of fetal-onset Alexander disease. Diffuse signal abnormalities of the periventricular white matter in association with thickening of the fornix and optic chiasm can point towards the diagnosis. Furthermore, the presence of atypical imaging findings such as microcephaly and cortical folding abnormalities in this case broadens our understanding of the phenotypic variability of Alexander disease.

Adult-onset Alexander disease with brainstem and cervical cord enhancing lesions.

Leukodystrophies are a group of genetic diseases with diverse clinical features and prominent involvement of the central nervous system white matter. We describe a 27-year-old man who presented with a progressive neurological disease, and striking involvement of the brainstem and symmetrical white matter lesions on MR scanning. Having excluded several other causes of leukodystrophy, we confirmed Alexander disease when a genetic panel showed a probable pathogenic variant in GFAP: p.Leu359Pro. Clinicians should suspect Alexander disease in people with a progressive neurological motor decline who has pyramidal and bulbar signs and compatible neuroimaging.

Developmental Profiles in Children and Young Adults with Alexander Disease.

PURPOSE: The study aims to describe the developmental profile of children and young adults with Alexander disease [AxD] infantile form, analyzing their clinical features, adaptive behavior and neuropsychological skills. METHODS: Participants were eight children or young adults (Mean age = 11 years; SD = 6.86; range = 5-23) and their parents. A multi-method approach was adopted to assess participant competencies: (1) an online parent survey, (2) a semi-structured interview with parents, and (3) a direct assessment of the participant's neuropsychological skills. RESULTS: Only four parents and their children completed all measures, and a common developmental profile could not be identified. The participants experienced substantial impairment in gross-motor skills, memory and narrative macrostructure. Most parents reported a regressive trend in at least one area. CONCLUSIONS: The high individual variability and the regressive trend highlight the need for an accurate and periodic assessment of each individual's developmental profile.

STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease.

Alexander disease (AxD) is caused by mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament expressed by astrocytes in the central nervous system. AxD-associated mutations cause GFAP aggregation and astrogliosis, and GFAP is elevated with the astrocyte stress response, exacerbating mutant protein toxicity. Studies in mouse models suggest disease severity is tied to Gfap expression levels, and signal transducer and activator of transcription (STAT)-3 regulates Gfap during astrocyte development and in response to injury and is activated in astrocytes in rodent models of AxD. In this report, we show that STAT3 is also activated in the human disease. To determine whether STAT3 contributes to GFAP elevation, we used a combination of genetic approaches to knockout or reduce STAT3 activation in AxD mouse models. Conditional knockout of Stat3 in cells expressing Gfap reduced Gfap transactivation and prevented protein accumulation. Astrocyte-specific Stat3 knockout in adult mice with existing pathology reversed GFAP accumulation and aggregation. Preventing STAT3 activation reduced markers of reactive astrocytes, stress-related transcripts, and microglial activation, regardless of disease stage or genetic knockout approach. These results suggest that pharmacological inhibition of STAT3 could potentially reduce GFAP toxicity and provide a therapeutic benefit in patients with AxD.