Martin Paucar

Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice

Calcifications in the basal ganglia are a common incidental finding and are sometimes inherited as an autosomal dominant trait (idiopathic basal ganglia calcification (IBGC)). Recently, mutations in the PDGFRB gene coding for the platelet-derived growth factor receptor β (PDGF-Rβ) were linked to IBGC. Here we identify six families of different ancestry with nonsense and missense mutations in the gene encoding PDGF-B, the main ligand for PDGF-Rβ. We also show that mice carrying hypomorphic Pdgfb alleles develop brain calcifications that show age-related expansion. The occurrence of these calcium depositions depends on the loss of endothelial PDGF-B and correlates with the degree of pericyte and blood-brain barrier deficiency. Thus, our data present a clear link between Pdgfb mutations and brain calcifications in mice, as well as between PDGFB mutations and IBGC in humans.

Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export

Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.

Persistence of viral RNA in the brain of offspring to mice infected with influenza A/WSN/33 virus during pregnancy.

Epidemiological studies have indicated an association between influenza A virus infections during fetal life and neuropsychiatric diseases. To study the potential for influenza A virus infections to cause nervous system dysfunctions, we describe a mouse model using intranasal instillation of the mouse neuroadapted influenza A/WSN/33 strain in pregnant mice. Viral RNA and nucleoprotein were detected in fetal brains and the viral RNA persisted for at least 90 days of postnatal life. We have, thus, obtained evidence for transplacental passage of influenza virus in mice and the persistence of viral components in the brains of these animals into young adulthood.

7α-hydroxy-3-oxo-4-cholestenoic acid in cerebrospinal fluid reflects the integrity of the blood-brain barrier

There is a continuous flux of the oxysterol 27-hydroxycholesterol (27-OHC) from the circulation across the blood-brain barrier (BBB) into the brain. The major metabolite of 27-OHC in the brain is 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). We confirm a recent report describing the presence of this metabolite in cerebrospinal fluid (CSF) at a relatively high concentration. A simple and accurate method was developed for assay of 7-HOCA in CSF based on isotope dilution-mass spectrometry and use of 2H4-labeled internal standard. The concentration of this metabolite was found to be markedly increased in CSF from patients with a dysfunctional BBB. There was a high correlation between the levels of 7-HOCA in CSF and the CSF/serum albumin ratio. The concentration of 7-HOCA in CSF was not significantly affected by neurodegeneration. Our findings suggest that 7-HOCA could be used as a diagnostic marker for conditions with a dysfunctional BBB.

De Novo Mutations in Ataxin-2 Gene and ALS Risk

Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.

Expanding the ataxia with oculomotor apraxia type 4 phenotype.

Ataxia with oculomotor apraxia type 4 (AOA4) is an autosomal recessive (AR) disorder recently delineated in a Portuguese cohort and caused by mutations in the PNKP (polynucleotide kinase 3′-phosphatase) gene.1 AOA4 is a progressive, complex movement disorder that includes hyperkinetic features, eye movement abnormalities, polyneuropathy, varying degrees of cognitive impairment, and obesity. PNKP mutations were initially discovered to be the cause of the severe nonprogressive syndrome microcephaly, early-onset intractable seizures, and developmental delay (MCSZ).2 Here we describe a patient with compound heterozygous PNKP mutations presenting with an AOA4 phenotype. New features that we report include both mutations, presence of chorea, absence of oculomotor apraxia (OMA), and slow disease progression.

Hereditary spastic paraplegia type 5: Natural history, biomarkers and a randomized controlled trial

Spastic paraplegia type 5 (SPG5) is a rare subtype of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative disorders defined by progressive neurodegeneration of the corticospinal tract motor neurons. SPG5 is caused by recessive mutations in the gene CYP7B1 encoding oxysterol-7α-hydroxylase. This enzyme is involved in the degradation of cholesterol into primary bile acids. CYP7B1 deficiency has been shown to lead to accumulation of neurotoxic oxysterols. In this multicentre study, we have performed detailed clinical and biochemical analysis in 34 genetically confirmed SPG5 cases from 28 families, studied dose-dependent neurotoxicity of oxysterols in human cortical neurons and performed a randomized placebo-controlled double blind interventional trial targeting oxysterol accumulation in serum of SPG5 patients. Clinically, SPG5 manifested in childhood or adolescence (median 13 years). Gait ataxia was a common feature. SPG5 patients lost the ability to walk independently after a median disease duration of 23 years and became wheelchair dependent after a median 33 years. The overall cross-sectional progression rate of 0.56 points on the Spastic Paraplegia Rating Scale per year was slightly lower than the longitudinal progression rate of 0.80 points per year. Biochemically, marked accumulation of CYP7B1 substrates including 27-hydroxycholesterol was confirmed in serum (n = 19) and cerebrospinal fluid (n = 17) of SPG5 patients. Moreover, 27-hydroxycholesterol levels in serum correlated with disease severity and disease duration. Oxysterols were found to impair metabolic activity and viability of human cortical neurons at concentrations found in SPG5 patients, indicating that elevated levels of oxysterols might be key pathogenic factors in SPG5. We thus performed a randomized placebo-controlled trial (EudraCT 2015-000978-35) with atorvastatin 40 mg/day for 9 weeks in 14 SPG5 patients with 27-hydroxycholesterol levels in serum as the primary outcome measure. Atorvastatin, but not placebo, reduced serum 27-hydroxycholesterol from 853 ng/ml [interquartile range (IQR) 683-1113] to 641 (IQR 507-694) (-31.5%, P = 0.001, Mann-Whitney U-test). Similarly, 25-hydroxycholesterol levels in serum were reduced. In cerebrospinal fluid 27-hydroxycholesterol was reduced by 8.4% but this did not significantly differ from placebo. As expected, no effects were seen on clinical outcome parameters in this short-term trial. In this study, we define the mutational and phenotypic spectrum of SPG5, examine the correlation of disease severity and progression with oxysterol concentrations, and demonstrate in a randomized controlled trial that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in serum of SPG5 patients. We thus demonstrate the first causal treatment strategy in hereditary spastic paraplegia.

A KCNC3 mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking

The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). We detail the clinical phenotype of four SCA13 kindreds that confirm causation of the KCNC3R423H allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3R423H in Drosophila triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in Drosophila, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. We demonstrate that ocular co-expression of KCNC3R423H with Drosophila epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. Our results therefore confirm the KCNC3R423H allele as causative for SCA13, through a dominant negative effect on KCNC3WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology.

Novel hyperkinetic dystonia-like manifestation and neurological disease course of Swedish Gaucher patients☆

BACKGROUND Neuronopathic Gaucher disease type 3 (GD3) is frequent in northern Sweden, whereas GD1 is found throughout the country. In a nation-wide study, we examined neurological manifestations and clinical course in 12 patients with GD3 and 13 patients with GD1. METHODS The patients were evaluated by standardized neurological assessments. Every sixth month, the GD3 patients were rated with the modified Severity Scoring Tool. At baseline and at the 3years follow-up, patients underwent University of Pennsylvania Smell Identification Test, Montreal Cognitive Assessment and Hospital Anxiety and Depression Scale. When clinical signs were present, additional examinations were undertaken. RESULTS Marked clinical heterogeneity was evident in both GD3 and GD1 groups. Several GD3 patients had a hitherto unreported rapid and repetitive dystonia-like hyperkinetic movement disorder. Most patients with GD3 have abnormalities of horizontal gaze, ataxia and focal epilepsy, some also had cognitive impairment, anxiety and hyposmia. Six GD3 patients, all homoallelic for L444P GBA1 mutations, have lived beyond 40years of age; and none has developed Parkinsonism. Two of the GD1 patients suffer from Parkinsonism; mild to complete hyposmia was present in six GD3 and five GD1 patients. Neither the group of GD3 nor GD1 patients had detectable progression of their neurological manifestations. CONCLUSIONS These middle-aged and older Swedish GD3 or GD1 patients are clinically stable over time. However, we have identified unusual clinical features, discordant phenotypes and a hyperkinetic dystonia-like movement disorder which appears unique to this Swedish disease variant and expands the phenotype for GD.

A SLC20A2 gene mutation carrier displaying ataxia and increased levels of cerebrospinal fluid phosphate

Mutations in the SLC20A2 gene, encoding type III sodium-dependent phosphate transporter 2 (PiT-2), are themost common cause of primary familial brain calcifications (PFBC) [1,2]. The R467X mutation in SLC20A2 has been described only once in a young Japanese male with paroxysmal kinesigenic dyskinesia (PKD) responsive to carbamazepine [3]. This males mother was asymptomatic despite the presence of brain calcifications. Here we describe a Swedish 54 year-old female with symmetric brain calcifications affected by dementia, ataxia and supranuclear palsy (SNP). Her medical history consists of type 1 diabetes mellitus (T1DM)with poor glycemic control and complications (polyneuropathy and retinopathy), hypertension, obstructive sleep apnea (OSA) and cataracts. During childhood shewent through surgery for goiter. Her father was affected by tremor but was not available for evaluation. At age 42 insidious onset of personality change, impaired memory and a gait disorder was noticed. Her work performance as a nurse was questioned and she was dismissed from three jobs during the two years prior to evaluation. The patient became irritable and restless; these symptoms worsened at age 50 after an episode of diabetic ketoacidosis due to insulin pump malfunction. In addition to insulin, the patient was treated with metoprolol, felodipine and losartan. During the course of disease she reported persecutory delusions from time to time and lacked insight regardingher symptoms. These delusionswere somewhat responsive to reassurance; due to her unfavorablemetabolic profilewe refrained from treatment with neuroleptics. A CT-scan at age 50 revealed widespread and severe calcifications in the frontal and occipital cortex, basal ganglia, thalamus, cerebellum and midbrain including the substantia nigra (Figs. 1 and e-1). Her total calcification score (TCS)was 55 and remained unchanged 3 years later. Since age 53 the patient has been urine incontinent. Using coregistration, however [4], we found progression of brain calcifications in the cerebellum and thalamus (Tables e-1 and e-2). In addition, brain MRI demonstrated white matter hyperintensities outside of the calcified areas (Fig. e-2). Predominant gait ataxia, foot dystonia and SNP were evident at age 51 (Video). Her SARA score at age 51 was 5.5 and two years later 7.5. Significant impairment in executive functions, attention and working memory compatible with subcortical