Esther de Graaff

A Point mutation in the FMR-1 gene associated with fragile X mental retardation.

The vast majority of patients with fragile X syndrome show a folate–sensitive fragile site at Xq27.3 (FRAXA) at the cytogenetic level, and both amplification of the (CGG)n repeat and hypermethylation of the CpG island in the 5′ fragile X gene (FMR–1) at the molecular level. We have studied the FMR–1 gene of a patient with the fragile X phenotype but without cytogenetic expression of FRAXA, a (CGG)n repeat of normal length and an unmethylated CpG island. We find a single point mutation in FMR–1 resulting in an Ne367Asn substitution. This de novo mutation is absent in the patients family and in 130 control X chromosomes, suggesting that the mutation causes the clinical abnormalities. Our results suggest that mutations in FMR–1 are directly responsible for fragile X syndrome, irrespective of possible secondary effects caused by FRAXA

Identification of delta/notch‐like epidermal growth factor‐related receptor as the Tr antigen in paraneoplastic cerebellar degeneration

Anti‐Tr is among the better described autoantibodies in paraneoplastic cerebellar degeneration (PCD) combined with Hodgkin lymphoma (HL); however, the Tr antigen remains unidentified.

TRIM46 Controls Neuronal Polarity and Axon Specification by Driving the Formation of Parallel Microtubule Arrays

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.

Liprin-α2 promotes the presynaptic recruitment and turnover of RIM1/CASK to facilitate synaptic transmission.

Liprin-α2 is required for the presynaptic recruitment and turnover of RIM1 and CASK, components of the release machinery, and facilitates synaptic output by regulating synaptic vesicle pool size.

NPHP4 Variants are Associated with Pleiotropic Heart Malformations

Rationale: Congenital heart malformations are a major cause of morbidity and mortality, especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs. Objective: To identify genetic mutations causing cardiac laterality defects. Methods and Results: We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double-outlet right ventricle, atrioventricular septal defects, and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified 8 additional missense variants that were absent or very rare in control subjects. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffers vesicle, which generate asymmetrical fluid flow necessary for normal L-R asymmetry. Conclusions: NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of Kupffers vesicle cilia and is required for global L-R patterning.

Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity

Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.

A role for Bicaudal-D2 in radial cerebellar granule cell migration

Bicaudal-D (BICD) belongs to an evolutionary conserved family of dynein adaptor proteins. It was first described in Drosophila as an essential factor in fly oogenesis and embryogenesis. Missense mutations in a human BICD homologue, BICD2, have been linked to a dominant mild early onset form of spinal muscular atrophy. Here we further examine the in vivo function of BICD2 in Bicd2 knockout mice. BICD2-deficient mice develop disrupted laminar organization of cerebral cortex and the cerebellum, pointing to impaired radial neuronal migration. Using astrocyte and granule cell specific inactivation of BICD2, we show that the cerebellar migration defect is entirely dependent upon BICD2 expression in Bergmann glia cells. Proteomics analysis reveals that Bicd2 mutant mice have an altered composition of extracellular matrix proteins produced by glia cells. These findings demonstrate an essential non-cell-autonomous role of BICD2 in neuronal cell migration, which might be connected to cargo trafficking pathways in glia cells.

Differential expression of liprin‐α family proteins in the brain suggests functional diversification

Liprin‐α proteins are major protein constituents of synapses and are important for the organization of synaptic vesicles and neurotransmitter receptors on their respective sides of the synapse. Although it is becoming apparent that the single liprin‐α gene in invertebrates is essential for synapse function, it is not known to what extent the four different liprin‐α homologs (liprin‐α1–4) in mammals are involved at synapses. We have designed specific antibodies against each of the four liprin‐α proteins and investigated their regional and cellular distribution in the brain. Here we show that all four liprin‐α proteins are present throughout the mature brain but have different regional distributions, which is highlighted by their differential localization in olfactory bulb, hippocampus, and cerebellar cortex. Double‐immunofluorescence staining indicates that different liprin‐α proteins are enriched in different synaptic populations but are also present at nonsynaptic sites. In particular, liprin‐α2 is preferentially associated with hippocampal mossy fiber endings in the CA3, whereas synapses in the molecular layers of the CA1 and dentate gyrus double‐labeled for liprin‐α3. The localization of liprin‐α2 and liprin‐α3 with excitatory synapses was confirmed in cultured primary hippocampal neurons. Liprin‐α4, which poorly co‐distributed with presynaptic markers in hippocampus, instead strongly co‐localized with VGLUT1 in the cerebellar molecular layer, suggesting its presence in parallel fiber‐Purkinje cell synapses. Finally, staining of cultured glial cells indicated that liprin‐α1 and liprin‐α3 are also associated with astrocytes. We conclude that liprin‐α family proteins might perform independent and specialized synaptic and nonsynaptic functions in different regions of the brain. J. Comp. Neurol. 519:3040–3060, 2011.

Standardized test for anti-Tr/DNER in patients with paraneoplastic cerebellar degeneration

Objective: To determine sensitivity and specificity of a standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) for anti-Tr antibodies in comparison to a reference procedure. Methods: Delta/Notch-like epidermal growth factor-related receptor (DNER) was expressed in HEK293 and used as a substrate for RC-IFA. HEK293 control cells expressing CDR2/Yo and CDR2L as well as mock-transfected HEK293 cells were used as controls. Serum samples from 38 patients with anti-Tr antibodies (33 with paraneoplastic cerebellar degeneration [PCD] and Hodgkin lymphoma), 66 patients with anti-Tr–negative PCD, 53 patients with Hodgkin lymphoma without neurologic symptoms, 40 patients with rheumatic diseases, and 42 healthy blood donors were tested for anti-DNER reactivity in the RC-IFA. In addition, RC-IFA results were compared to those from a commercial tissue-based IFA using monkey cerebellum. Results: Using the RC-IFA, anti-DNER was detected in all anti-Tr–positive patients but in none of the controls (sensitivity 100%, 95% confidence interval [CI] 92.8%–100%; specificity 100%, 95% CI 98.7%–100%). In comparison, anti-Tr was not detected in 4 samples with low-titer autoantibodies using the commercial tissue-based assay. Preadsorption of sera with either recombinant full-length DNER or its extracellular domain selectively abolished anti-Tr reactivity. Conclusion: Anti-Tr antibodies bind to the extracellular domain of DNER and can be detected by RC-IFA using HEK293 cells expressing the recombinant receptor. The new method performs better than a frequently used commercial tissue-based indirect immunofluorescence assay (IFA) in samples with low-titer antibodies. Classification of evidence: This study provides Class II evidence that RC-IFA accurately detects anti-Tr as compared to conventional IFA.

Psychiatric phenomena as initial manifestation of encephalitis by anti-NMDAR antibodies

Objective Autoimmune encephalitis associated with autoantibodies against the N‐methyl‐d‐aspartate receptor (NMDAR) often presents with behavioural change. Our objective was to describe in detail the psychiatric presentation and pathways to care in order to aid the early diagnosis of NMDAR encephalitis. Methods Sera and cerebrospinal fluid (CSF) from patients with suspected NMDAR encephalitis were tested on HEK 293 cells transfected with the NR1 subunit of the NMDAR. Clinical information was obtained from the referring psychiatrists and neurologists and by review of the clinical records. Results Samples from 15 patients (13 female, 2 male, mean age 24 years, range 5–56 years) tested anti‐NMDAR positive. Twelve of the 15 patients (80%) presented with prominent psychiatric symptoms and 8 were initially referred to a psychiatric service. The most prominent initial psychiatric symptoms were anxiety in seven (47%), behavioural change (often bizarre) in six (40%) and agitation in five (33%). All patients developed psychiatric symptoms in the first 6 weeks of illness. Thirteen patients received psychotropic medications: antipsychotics in 12 and benzodiazepines in 11. Treating physicians considered the psychotropic medication not effective in 11 patients resulting in many drug switches. At nadir, all patients were in a very poor condition. However, eight patients (53%) recovered (almost) completely. Outcome tended to be better in patients who had received early immunotherapy or tumour removal. Conclusions Autoimmune encephalitis and anti‐NMDAR testing in serum and CSF should be considered in patients, especially young females, presenting with atypical psychiatric phenomena. Early diagnosis and treatment will likely improve the prognosis of NMDAR encephalitis.