New Insights of Phospholipase A2 Associated Neurodegeneration Phenotype Based on the Long-Term Follow-Up of a Large Hungarian Family.

INTRODUCTION: Phospholipase A2-associated Neurodegeneration (PLAN) is a group of neurodegenerative diseases associated with the alterations of PLA2G6. Some phenotype-genotype association are well known but there is no clear explanation why some cases can be classified into distinct subgroups, while others follow a continuous clinical spectrum. METHODS: Long-term neurological, and psychiatric follow-up, neuropathological, radiological, and genetic examinations, were performed in three affected girls and their family. RESULTS: Two 24-years old twins and their 22-years old sister harbored the p.P622S, and p.R600W mutation in PLA2G6. The age of onset and the most prominent presenting symptoms (gaze palsy, ataxia, dystonia, psychomotor regression indicated atypical neuroaxonal dystrophy (ANAD), however, optic atrophy, severe tetraparesis would fit into infantile neuroaxonal dystrophy (INAD). All siblings had hyperintensity in the globi pallidi and substantiae nigrae which is reported in ANAD, whereas it is considered a later neuroradiological marker in INAD. The slow progression, rigidity, bradykinesis, and the prominent psychiatric symptoms indicate PLA2G6-related dystonia-parkinsonism. Abnormal mitochondria, lipid accumulation and axonal spheroids were observed in the muscle and nerve tissue. Brain deposition appeared 6 years following the initial cerebellar atrophy. Mild MRI alterations were detected in the asymptomatic carrier parents. CONCLUSION: The colorful clinical symptoms, the slightly discordant phenotype, and the neuroimaging data in the family supports the view that despite the distinct definition of age-related phenotypes in PLAN, these are not strict disease categories, but rather a continuous phenotypic spectrum. The mild MRI alterations of the parents and the family history suggest that even heterozygous pathogenic variants might be associated with clinical symptoms, although systematic study is needed to prove this.

CANOMAD syndrome with respiratory failure.

CANOMAD (chronic ataxic neuropathy, ophthalmoplegia, M-protein agglutination, disialosyl antibodies) syndrome is a rare polyneuropathy. IgM paraproteins react with ganglioside-containing disialylated epitopes resulting in dorsal root ganglionopathy and B-lymphocyte infiltration of cranial and peripheral nerves. Clinical features include ataxia, slight muscle weakness, areflexia, sensory- and cranial nerve symptoms. Case studies have reported the efficacy of rituximab and intravenous immunoglobulin (IVIg) treatments. We present the case of a 57-year-old man, who had difficulty walking, with numbness and clumsiness in all limbs. He had areflexia, vibratory sensation loss and ataxia. Laboratory tests showed IgM monoclonal components and disialosyl antibodies in the serum. Nerve conduction studies indicated severe sensorimotor demyelinating polyneuroradiculopathy. Despite IVIg and rituximab treatments, the patient's disease course gradually worsened and he died of respiratory failure. Neuropathological examination revealed dorsal column- and dorsal root atrophy with mixed mononuclear cell infiltration. This article aims to draw attention to this syndrome, and the use of early potent immunosuppressive treatment to improve patients' quality of life.

A Glial-Neuronal Circuit in the Median Eminence Regulates Thyrotropin-Releasing Hormone-Release via the Endocannabinoid System.

Based on the type-I cannabinoid receptor (CB1) content of hypophysiotropic axons and the involvement of tanycytes in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis, we hypothesized that endocannabinoids are involved in the tanycyte-induced regulation of TRH release in the median eminence (ME). We demonstrated that CB1-immunoreactive TRH axons were associated to DAGLα-immunoreactive tanycyte processes in the external zone of ME and showed that endocannabinoids tonically inhibit the TRH release in this tissue. We showed that glutamate depolarizes the tanycytes, increases their intracellular Ca2+ level and the 2-AG level of the ME via AMPA and kainite receptors and glutamate transport. Using optogenetics, we demonstrated that glutamate released from TRH neurons influences the tanycytes in the ME. In summary, tanycytes regulate TRH secretion in the ME via endocannabinoid release, whereas TRH axons regulate tanycytes by glutamate, suggesting the existence of a reciprocal microcircuit between tanycytes and TRH terminals that controls TRH release.

Thyrotropin-Releasing-Hormone-Synthesizing Neurons of the Hypothalamic Paraventricular Nucleus Are Inhibited by Glycinergic Inputs.

Background: Glycine is a classical neurotransmitter that has role in both inhibitory and excitatory synapses. To understand whether glycinergic inputs are involved in the regulation of the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central controllers of the hypothalamic-pituitary-thyroid axis, the glycinergic innervation of the TRH neurons was studied in the hypothalamic paraventricular nucleus (PVN). Methods: Double-labeling immunocytochemistry and patch-clamp electrophysiology were used to determine the role of glycinergic neurons in the regulation of TRH neurons in the PVN. Anterograde and retrograde tracing methods were used to determine the sources of the glycinergic input of TRH neurons. Results: Glycine transporter-2 (GLYT2), a marker of glycinergic neurons, containing axons were found to establish symmetric type of synapses on TRH neurons in the PVN. Furthermore, glycine receptor immunoreactivity was observed in these TRH neurons. The raphe magnus (RMg) and the ventrolateral periaqueductal gray (VLPAG) were found to be the exclusive sources of the glycinergic innervation of the TRH neurons within the PVN. Patch-clamp electrophysiology using sections of TRH-IRES-tdTomato mice showed that glycine hyperpolarized the TRH neurons and completely blocked the firing of these neurons. Glycine also markedly hyperpolarized the TRH neurons in the presence of tetrodotoxin demonstrating the direct effect of glycine. In more than 60% of the TRH neurons, spontaneous inhibitory postsynaptic currents (sIPSCs) were observed, even after the pharmacological inhibition of glutamatergic and GABAergic neuronal transmission. The glycine antagonist, strychnine, almost completely abolished these sIPSCs, demonstrating the inhibitory nature of the glycinergic input of TRH neurons. Conclusions: These data demonstrate that TRH neurons in the PVN receive glycinergic inputs from the RMg and the VLPAG. The symmetric type of synaptic connection and the results of the electrophysiological experiments demonstrate the inhibitory nature of these inputs.

Genotypic and phenotypic spectrum of the most common causative genes of Charcot-Marie-Tooth disease in Hungarian patients.

Charcot-Marie-Tooth neuropathy (CMT) is a genetically and clinically heterogeneous group of neuromuscular disorders with an overall prevalence of 1 per 2500. Here we report the first comprehensive genetic epidemiology study of Hungarian CMT patients. 409 CMT1 and 122 CMT2 patients were enrolled and genetic testing of PMP22, GJB1, MPZ, EGR2 and MFN2 genes were performed routinely. NDRG1 and CTDP1 genes were screened only for founder mutations in Roma patients. Causative genetic mutations were identified in 67.2% of the CMT1 and in 33.6% of the CMT2 cases, which indicates an overall success rate of 59.9% in the study population. Considering all affected individuals, alterations were most frequently found in PMP22 (40.5%), followed by GJB1 (9.2%), MPZ (4.5%), MFN2 (2.5%), NDRG1 (1.5%), EGR2 (0.8%) and CTDP1 (0.8%). The phenotypic spectrum and the disease severity of the studied patients also varied broadly. Deafness and autoimmune disorders were more often associated with PMP22 duplication, while MFN2 and GJB1 mutations were frequently present with central nervous system abnormalities. Our study may be helpful in determining the strategy of genetic diagnostics in Hungarian CMT patients.

Localization of connexin 43 gap junctions and hemichannels in tanycytes of adult mice.

Tanycytes are specialized glial cells lining the lateral walls and the floor of the third ventricle behind the optic chiasm. In addition to functioning as barrier cells, they also have an important role in the regulation of neuroendocrine axes and energy homeostasis. To determine whether tanycytes communicate with each other via Connexin 43 (Cx43) gap junctions, individual tanycytes were loaded with Lucifer yellow (LY) through a patch pipette. In all cases, LY filled a larger group of tanycytes as well as blood vessels adjacent to tanycyte processes. The Cx43-blocker, carbenoxolone, inhibited spreading of LY. The greatest density of Cx43-immunoreactive spots was observed in the cell membrane of α-tanycyte cell bodies. Cx43-immunoreactivity was also present in the membrane of ß-tanycyte cell bodies, but in lower density. Processes of both types of tanycytes also contained Cx43-immunoreactivity. At the ultrastructural level, Cx43-immunoreactivity was present in the cell membrane of all types of tanycytes including their ventricular surface, but gap junctions were more frequent among α-tanycytes. Cx43-immunoreactivity was also observed in the cell membrane between contacting tanycyte endfeet processes, and between tanycyte endfeet process and axon varicosities in the external zone of the median eminence and capillaries in the arcuate nucleus and median eminence. These results suggest that gap junctions are present not only among tanycytes, but also between tanycytes and the axons of hypophysiotropic neurons. Cx43 hemichannels may also facilitate the transport between tanycytes and extracellular fluids, including the cerebrospinal fluid, extracellular space of the median eminence and bloodstream.

Identification of a Novel GLA Gene Mutation, p.Ile239Met, in Fabry Disease With a Predominant Cardiac Phenotype.

Fabry disease (FD) is an X-linked inherited lysosomal storage disorder caused by mutations in the GLA gene, encoding for the enzyme α-galactosidase A. Although hundreds of mutations in the GLA gene have been described, many of them are variants of unknown significance. Here we report a novel GLA mutation, p.Ile239Met, identified in a large Hungarian three-generation family with FD. A 69 year-old female index patient with a clinical history of renal failure, hypertrophic cardiomyopathy, and 2nd degree AV block was screened for mutation in the GLA gene. Genetic screening identified a previously unreported heterozygous mutation in exon 5 of the GLA gene (c.717A>G; p.Ile239Met). Family screening indicated that altogether 6 family members carried the mutation (5 females, 1 male, average age: 55 ± 16 years). Three family members, including the index patient, manifested the cardiac phenotype of hypertrophic cardiomyopathy, while two other family members were diagnosed with left ventricular hypertrophy. Taking affection status as the presence of hypertrophic cardiomyopathy, left ventricular hypertrophy or elevated lyso-Gb3 levels, all affected family members carried the mutation. Linkage analysis of the family gave a two-point LOD score of 2.01 between the affection status and the p.Ile239Met GLA mutation. Lyso-Gb3 levels were elevated in all carrier family members (range: 2.4-13.8 ng/mL; upper limit of normal +2STD: ≤ 1.8 ng/mL). The GLA enzyme level was markedly reduced in the affected male family member (< 0.2 µmol/L/hour; upper limit of normal ± 2STD: ≥ 2.6 µmol/L/hour). We conclude that the p. Ile239Met GLA mutation is a pathogenic mutation for FD associated with predominant cardiac phenotype.

Three novel mutations and genetic epidemiology analysis of the Gap Junction Beta 1 (GJB1) gene among Hungarian Charcot-Marie-Tooth disease patients.

Pathogenic variants of the gap junction beta 1 (GJB1) gene are responsible for the Charcot-Marie-Tooth neuropathy X type 1 (CMTX1). In this study, we report the mutation frequency of GJB1 in 210 Hungarian CMT patients and the phenotype comparison between male and female CMTX1 patients. Altogether, 13 missense substitutions were found in the GJB1 gene. Among them, 10 have been previously described as pathogenic variants (p.Arg15Trp, p.Val63Ile, p.Leu89Val, p.Ala96Gly, p.Arg107Trp, p.Arg142Gln, p.Arg164Trp, p.Arg164Gln, p.Pro172Ala and p.Asn205Ser), while 3 were novel, likely pathogenic alterations (p.Val13Glu, p.Glu186Gly, p.Met194Ile). These variants were not present in controls and were predicted as disease causing by in silico analysis. The frequency of the variants was 6.7% in our cohort which refers to a common cause of hereditary neuropathy among Hungarian patients. In addition to the classical phenotype, CNS involvement was proved in 26.1% of the CMTX1 patients. GJB1 pathogenic alterations were found mainly in males but we also detected them in female probands. The statistical analysis of CMTX1 patients revealed a significant difference between the two genders regarding the age of onset, Charcot-Marie-Tooth neuropathy and examination scores.

Genetic background of the hereditary spastic paraplegia phenotypes in Hungary - An analysis of 58 probands.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases with progressive lower limb spasticity and weakness. The aim of this study is to determine the frequency of different SPG mutations in Hungarian patients, and to provide further genotype-phenotype correlations for the known HSP causing genes. METHODS: We carried out genetic testing for 58 probands with clinical characteristics of HSP. For historical reasons, three different approaches were followed in different patients: 1) Sanger sequencing of ATL1 and SPAST genes, 2) whole exome, and 3) targeted panel sequencing by next generation sequencing. RESULTS: Genetic diagnosis was established for 20 probands (34.5%). We detected nine previously unreported mutations with high confidence for pathogenicity. The most frequently affected gene was SPAST with pathogenic or likely pathogenic mutations in 10 probands. The most frequently detected variant in our cohort was the SPG7 p.Leu78*, observed in four probands. Altogether five probands were diagnosed with SPG7. Additional mutations were detected in SPG11, ATL1, NIPA1, and ABCD1. CONCLUSION: This is the first comprehensive genetic epidemiological study of patients with HSP in Hungary. Next generation sequencing improved the yield of genetic diagnostics in this disease group even when the phenotype was atypical. However, considering the frequency of the HSP-causing gene defects, SPG4, the most common form of the disease, should be tested first to be cost effective in this economic region.

[A rare case of thermoregulation disorder: Shapiro syndrome]. / Shapiro-szindróma, azaz a termoregulációs zavar ritka esete.

Anomalies of the corpus callosum are the most frequent malformations of the central nervous system. The triad of spontaneous periodic hypothermia and hyperhydrosis with the agenesis of corpus callosum is described as Shapiro syndrome. Shapiro syndrome is a very rare condition and it can occur in every age group. The presence of agenesis of corpus callosum is not a strict criteria of the syndrome; the most important presenting symptom is paroxysmal hypothermia. Although the definite cause of recurrent hypothermia is unknown, dysfunction of the hypothalamus is suspected. From therapeutic aspects, only supportive therapy is available. In this report the authors present the first Shapiro syndrome case diagnosed in Hungary. The main symptoms of the 21-year-old male patient were recurrent hyperhydrosis with hypothermia resulting in severe general malaise. The skull magnetic resonance imaging demonstrated agenesis of corpus callosum. The patient was treated with clonidine resulting in significant improvement of symptoms.

Diagnostic yield of five minutes compared to three minutes hyperventilation during electroencephalography.

PURPOSE: To investigate whether hyperventilation (HV) for 5min increases the diagnostic yield of electroencephalography (EEG) compared to 3min HV. METHODS: data were evaluated from 1084 consecutive patients, from three European centres, referred to EEG on suspicion of epilepsy. Seizures and interictal EEG abnormalities precipitated during the first 3min and during the last 2min of the HV period (totally 5min) were determined. RESULTS: Eight hundred seventy-seven patients (81%) completed 5min HV. Seizures were precipitated during the first 3min of HV in 21 patients, and during the last 2min in four more patients. Interictal EEG abnormalities were precipitated in the first 3min of HV in 16 patients, and during the last 2min in 7 more patients. Psychogenic nonepileptic seizures occurred in eight patients during the first 3min of HV and in two more patients during the last 2min. No adverse events occurred during the last 2min of HV, but eight patients (1%) stopped HV during the last 2min because they were not able to hyperventilate further. CONCLUSION: 16% of seizures and 30% of interictal EEG abnormalities triggered by HV occurred during the last 2min of HV, suggesting the clinical usefulness of prolonged hyperventilation for 5min. The vast majority of patients (99%) who are able to hyperventilate for 3min can complete 5min HV, without additional adverse events.

Amyloid-ß1-42 Disrupts Synaptic Plasticity by Altering Glutamate Recycling at the Synapse.

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disorders characterized by neuritic plaques containing amyloid-ß peptide (Aß) and neurofibrillary tangles. Evidence has been reported that Aß(1-42) plays an essential pathogenic role in decreased spine density, impairment of synaptic plasticity, and neuronal loss with disruption of memory-related synapse function, all associated with AD. Experimentally, Aß(1-42) oligomers perturb hippocampal long-term potentiation (LTP), an electrophysiological correlate of learning and memory. Aß was also reported to perturb synaptic glutamate (Glu)-recycling by inhibiting excitatory-amino-acid-transporters. Elevated level of extracellular Glu leads to activation of perisynaptic receptors, including NR2B subunit containing NMDARs. These receptors were shown to induce impaired LTP and enhanced long-term depression and proapoptotic pathways, all central features of AD. In the present study, we investigated the role of Glu-recycling on Aß(1-42)-induced LTP deficit in the CA1. We found that Aß-induced LTP damage, which was mimicked by the Glu-reuptake inhibitor TBOA, could be rescued by blocking the NR2B subunit of NMDA receptors. Furthermore, decreasing the level of extracellular Glu using a Glu scavenger also restores TBOA or Aß induces LTP damage. Overall, these results suggest that reducing ambient Glu in the brain can be protective against Aß-induced synaptic disruption.

[Retinal ganglion cell layer and visual function in patients with progressive external ophthalmoplegia caused by common mtDNA deletion]. / Retinalis idegrostréteg és látásfunkciók az mtDNS common deléciója által okozott progresszív ophthalmoplegia externa esetében.

AIM: Mitochondrial (mt) disorders are metabolic conditions with multiorgan involvement, which often cause neuroophtalmological symptoms. The aim of the study was to investigate the relation between progressive external ophthalmoplegia (PEO), visual pathway and mitochondrial DNA (mtDNA) mutations in patients younger than 55 years of age. METHODS: Five female patients (35 to 53 years of age) with mithochondrial disease were investigated. Automated threshold perimetry (Octopus G2 test), scanning laser polarimetry (GDx-VCC and GDx-ECC) and Fourier-domain optical coherence tomography (RTVue-100 OCT) were used in addition to detailed ophthalmological examination and evaluation of visually evoked potentials (VEP). Frequent mutations of the mtDNA were investigated in the patients' blood and muscle samples. RESULTS: PEO of various severity levels was found in all patients, using clinical tests. Genetic testing showed "common deletion" of mtDNA in all cases. For both eyes of 4 patients functional and structural ophthalmic tests had normal results. In one patient decreased visual acuity, reduced retinal nerve fiber layer thickness and prolonged L3 VEP latency time were found without optic disc damage and visual field deterioration. CONCLUSION: In 4 of our 5 patients with PEO due to common deletion of mtDNA retinal ganglion cells and visual function remained normal for a long period of life.

Abeta(1-42) enhances neuronal excitability in the CA1 via NR2B subunit-containing NMDA receptors.

Neuronal hyperexcitability is a phenomenon associated with early Alzheimer's disease. The underlying mechanism is considered to involve excessive activation of glutamate receptors; however, the exact molecular pathway remains to be determined. Extracellular recording from the CA1 of hippocampal slices is a long-standing standard for a range of studies both in basic research and in neuropharmacology. Evoked field potentials (fEPSPs) are regarded as the input, while spiking rate is regarded as the output of the neuronal network; however, the relationship between these two phenomena is not fully clear. We investigated the relationship between spontaneous spiking and evoked fEPSPs using mouse hippocampal slices. Blocking AMPA receptors (AMPARs) with CNQX abolished fEPSPs, but left firing rate unchanged. NMDA receptor (NMDAR) blockade with MK801 decreased neuronal spiking dose dependently without altering fEPSPs. Activating NMDARs by small concentration of NMDA induced a trend of increased firing. These results suggest that fEPSPs are mediated by synaptic activation of AMPARs, while spontaneous firing is regulated by the activation of extrasynaptic NMDARs. Synaptotoxic Abeta(1-42) increased firing activity without modifying evoked fEPSPs. This hyperexcitation was prevented by ifenprodil, an antagonist of the NR2B NMDARs. Overall, these results suggest that Abeta(1-42) induced neuronal overactivity is not dependent on AMPARs but requires NR2B.

International Issues: Cross-border mobility of junior neurologists within and to the European Union.

OBJECTIVE: To assess the general interest in and motivation for cross-border mobility among residents and junior neurologists from member states of the European Union and neighboring countries. METHODS: Questionnaire-based paper survey among 118 participants of a neurology course. RESULTS: Ninety-seven (82%) participants returned the survey. Most of them had at one point considered relocating within or to the European Union for postgraduate education (87%) or employment (71%). Common motivations were superior prospects for clinical training (85%), resources at work and academic environment (both 80%), and remuneration (70%). Barely half of the surveyed intended to return to their home country. The attractiveness of Europe as a destination for migration was ranked over other continents. The most common reasons that reduce enthusiasm for relocation were the loss of family connection (55%) and uncertain future prospects (41%), whereas language barriers were less relevant (21%). CONCLUSION: There is keen interest of the upcoming generation of neurologists to relocate within and to the European Union. The motives include regional differences in training and career opportunities as well as economic welfare. Appropriate steps toward the harmonization of educational and career prospects are urgently required to ensure adequate provision of neurology service and patient care throughout Europe.

Generation of a highly active folding enzyme by combining a parvulin-type prolyl isomerase from SurA with an unrelated chaperone domain.

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.

[Inclusion body myositis--a rarely recognized disorder]. / Zárványtestes myositis--egy ritikán felismert betegség.

Inclusion body myositis is the most common disabling inflammatory myopathy in the elderly. It is more frequent in men and after the age of 50 years. Inflammatory and degenerative features coexist. There is a T-cell mediated autoimmunity driven by in situ clonally expanded cytotoxic CD8-positive T-cells invading non-necrotic muscle fibres expressing MHC-I antigen. The hallmarks of degeneration are the deposition of protein aggregates and the formation of vesicles. The course of the disease is slow and the diagnosis is usually set after several years. The muscle weakness and wasting is assymetric, affecting predominantly distal muscles of the upper extremity and proximal muscles of the legs. The signs and clinical course can be characteristic, but the diagnosis is established by muscle biopsy. There is currently no evidence based effective treatment for sIBM. Prednisone, azathioprine, methotrexate, cyclosporine and IFN-beta failed. Oxandrolon did not improve symptoms. Treatment with intravenous immunglobuline (IVIG) induced in some patients a transient improvement of swallowing and of muscle strenght, but the overall study results were negative. A T-cell depleting monoclonal antibody (alemtuzumab), in a small uncontrolled study slowed down disease progression for a six-month period. Repeated muscle biopsies showed the reduction of T-cells in the muscle and the suppression of some degeneration associated molecules. An effective therapeutic mean should act on both aspects of the pathomechanism, on the inflammatory and the degenerative processes as well.

Overexpression of Hsp27 ameliorates symptoms of Alzheimer's disease in APP/PS1 mice.

Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-ß (Aß) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimer's disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.

Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response.

The transcriptional regulator DntR, which previously has been isolated from bacterial strains capable of degrading 2,4-dinitrotoluene (DNT), was engineered in order to improve the ability to detect DNT. A directed evolution strategy was employed, where sequence diversity first was created by random mutagenesis in three subsequent rounds, followed by recombination of previously selected mutants. A gfp gene was used as a reporter for transcriptional activity mediated by DntR and cells with higher GFP expression after addition of DNT were sorted out using fluorescence-activated cell sorting (FACS). A DntR mutant, which displayed 10 times higher induction levels than wild-type DntR in response to DNT was isolated. This mutant still maintained low levels of gfp expression in the absence of DNT. The detection limit was ∼10 µM, a 25-fold improvement compared to wild-type DntR. The functional role of some substitutions found in this clone is discussed in the framework of the structural changes observed when comparing the recently determined structures of DntR with and without bound inducer ligand.