Neuropathology and Applied Neurobiology
Wiley Online Library : Neuropathology and Applied Neurobiology

  • Creutzfeldt-Jakob disease with the V203I mutation and M129V polymorphism of the prion protein gene (PRNP) and a 17 kDa prion protein fragment
  • A Huntington's disease phenocopy characterized by pallido-nigro-luysian degeneration with brain iron accumulation and p62-positive glial inclusions
  • The MTS vs. the ATP assay for in vitro chemosensitivity testing of primary glioma tumour culture
  • CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomas
    P. J. Kullar, D. M. Pearson, D. S. Malley, V. P. Collins and K. Ichimura (2010) Neuropathology and Applied Neurobiology36, 505–514 CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomasAims: Loss of both wild-type copies of the neurofibromatosis type 2 (NF2) gene is found in both sporadic and neurofibromatosis type 2-associated vestibular schwannomas (VS). Previous studies have identified a subset of VS with no loss or mutation of NF2. We hypothesized that methylation of NF2 resulting in gene silencing may play a role in such tumours. Methods: Forty sporadic VS were analysed by array comparative genomic hybridization using 1 Mb whole genome and chromosome 22 tile path arrays. The NF2 genes were sequenced and methylation of NF2 examined by pyrosequencing. Results: Monosomy 22 was the only recurrent change found. Twelve tumours had NF2 mutations. Eight tumours had complete loss of wild-type NF2, four had one mutated and one wild-type allele, 11 had only one wild-type allele and 17 showed no abnormalities. Methylation analysis showed low-level methylation in four tumours at a limited number of CpGs. No high-level methylation was found. Conclusions: This study shows that a significant proportion of sporadic VS (>40%) have unmethylated wild-type NF2 genes. This indicates that other mechanisms, yet to be identified, are operative in the oncogenesis of these VSs.

  • Human brain weight is correlated with expression of the ‘housekeeping genes’ beta-2-microglobulin (β2M) and TATA-binding protein (TBP)
    P. J. Harrison, L. M. Laatikainen, E. M. Tunbridge and S. L. Eastwood (2010) Neuropathology and Applied Neurobiology36, 498–504 Human brain weight is correlated with expression of the ‘housekeeping genes’ beta-2-microglobulin (β2M) and TATA-binding protein (TBP)Aims: Many variables affect mRNA measurements in post mortem human brain tissue. Brain weight has not hitherto been considered to be such a factor. This study examined whether there is any relationship between brain weight and mRNA abundance. Methods: We investigated quantitative real-time RT-PCR data for five ‘housekeeping genes’ using the 104 adult brains of the Stanley Microarray Consortium series. Eleven data sets were analysed, from cerebellum, hippocampus, and anterior cingulate cortex. We used a specified sequence of correlations, partial correlations and multiple regression analyses. Results: Brain weight correlated with the ‘raw’ (i.e. non-normalized) data for two mRNAs, β2-microglobulin and TATA-binding protein, measured in cerebellum and hippocampus, respectively. In hippocampus, the geometric mean of three housekeeping gene transcripts also correlated with brain weight. The correlations were significant after adjusting for age, sex and other confounders, and the effect of brain weight was confirmed using multiple regression. No correlations with brain weight were seen in the anterior cingulate cortex, nor for the other mRNAs examined. Conclusions: The findings were not anticipated; they need replication in another brain series, and a more systematic survey is indicated. In the interim, we suggest that quantitative gene expression studies in human brain should inspect for a potential influence of brain weight, especially as the affected transcripts are commonly used as reference genes for normalization purposes in studies of neurological and psychiatric disorders. The relationship of brain weight with β2-microglobulin mRNA may reflect the roles of major histocompatibility complex class I genes in synapse formation and plasticity.

  • In this issue
  • Pathological demonstration of cervical spinal cord inflammatory vasculitis in a patient with spontaneous spinal epidural haematoma associated with systemic lupus erythematosus
  • 112th Meeting of the British Neuropathological Society
  • Limited expression of heparan sulphate proteoglycans associated with Aβ deposits in the APPswe/PS1dE9 mouse model for Alzheimer's disease
    N. M. Timmer, M. K. Herbert, J. W. Kleinovink, A. J. Kiliaan, R. M. W. de Waal and M. M. Verbeek (2010) Neuropathology and Applied Neurobiology36, 478–486 Limited expression of heparan sulphate proteoglycans associated with Aβ deposits in the APPswe/PS1dE9 mouse model for Alzheimer's diseaseAims: Alzheimer's disease (AD) is characterized by deposition of the amyloid beta (Aβ) peptide in brain parenchyma and vasculature. Several proteins co-deposit with Aβ, including heparan sulphate proteoglycans (HSPG). HSPG have been suggested to contribute to Aβ aggregation and deposition, and may influence plaque formation and persistence by stimulating Aβ fibrillization and by protecting Aβ against degradation. Mouse models for AD, expressing the human amyloid precursor protein (APP), produce Aβ deposits similar to humans. These models may be used to study disease pathology and to develop new therapeutic interventions. We aimed to investigate whether co-deposition of HSPG in AD brains can be replicated in the APPswe/PS1dE9 mouse model for AD and if a temporal association of HSPG with Aβ exists. Methods: We studied the co-deposition of several HSPG and of the glycosaminoglycan side chains of HSPG in the APPswe/PS1dE9 model at different ages by immunohistochemistry. Results: We found that, although APPswe/PS1dE9 mice did develop severe Aβ pathology with age, co-deposition of HS glycosaminoglycan chains and the various HSPG (agrin, perlecan and glypican-1) was scarce (<10–30% of the Aβ deposits were stained). Conclusions: Our data suggest that the molecular composition of Aβ deposits in the APPswe/PS1dE9 mouse, with respect to the several HSPG investigated in this study, does not accurately reflect the human situation. The near absence of HSPG in Aβ deposits in this transgenic mouse model may, in turn, hinder the translation of preclinical intervention studies from mice to men.

  • Ex vivo study of dentate gyrus neurogenesis in human pharmacoresistant temporal lobe epilepsy
    M. Paradisi, M. Fernández, G. Del Vecchio, G. Lizzo, G. Marucci, M. Giulioni, E. Pozzati, T. Antonelli, G. Lanzoni, G. P. Bagnara, L. Giardino and L. Calzà (2010) Neuropathology and Applied Neurobiology36, 535–550 Ex vivo study of dentate gyrus neurogenesis in human pharmacoresistant temporal lobe epilepsyAims: Neurogenesis in adult humans occurs in at least two areas of the brain, the subventricular zone of the telencephalon and the subgranular layer of the dentate gyrus in the hippocampal formation. We studied dentate gyrus subgranular layer neurogenesis in patients subjected to tailored antero-mesial temporal resection including amygdalohippocampectomy due to pharmacoresistant temporal lobe epilepsy (TLE) using the in vitro neurosphere assay. Methods: Sixteen patients were enrolled in the study; mesial temporal sclerosis (MTS) was present in eight patients. Neurogenesis was investigated by ex vivo neurosphere expansion in the presence of mitogens (epidermal growth factor + basic fibroblast growth factor) and spontaneous differentiation after mitogen withdrawal. Growth factor synthesis was investigated by qRT-PCR in neurospheres. Results: We demonstrate that in vitro proliferation of cells derived from dentate gyrus of TLE patients is dependent on disease duration. Moreover, the presence of MTS impairs proliferation. As long as in vitro proliferation occurs, neurogenesis is maintained, and cells expressing a mature neurone phenotype (TuJ1, MAP2, GAD) are spontaneously formed after mitogen withdrawal. Finally, formed neurospheres express mRNAs encoding for growth (vascular endothelial growth factor) as well as neurotrophic factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor). Conclusion: We demonstrated that residual neurogenesis in the subgranular layer of the dentate gyrus in TLE is dependent on diseases duration and absent in MTS.

  • Interferon (IFN) beta treatment induces major histocompatibility complex (MHC) class I expression in the spinal cord and enhances axonal growth and motor function recovery following sciatic nerve crush in mice
    R. G. Zanon, L. P. Cartarozzi, S. C. S. Victório, J. C. Moraes, J. Morari, L. A. Velloso and A. L. R. Oliveira (2010) Neuropathology and Applied Neurobiology36, 515–534 Interferon (IFN) beta treatment induces major histocompatibility complex (MHC) class I expression in the spinal cord and enhances axonal growth and motor function recovery following sciatic nerve crush in miceAims: Major histocompatibility complex (MHC) class I expression by neurones and glia constitutes an important pathway that regulates synaptic plasticity. The upregulation of MHC class I after treatment with interferon beta (IFN beta) accelerates the response to injury. Therefore the present work studied the regenerative outcome after peripheral nerve lesion and treatment with IFN beta, aiming at increasing MHC class I upregulation in the spinal cord. Methods: C57BL/6J mice were subjected to unilateral sciatic nerve crush and treatment with IFN beta. The lumbar spinal cords were processed for immunohistochemistry, in situ hybridization, Western blotting and RT-PCR, while the sciatic nerves were submitted for immunohistochemistry, morphometry and counting of regenerated axons. Motor function recovery was monitored using the walking track test. Results: Increased MHC class I expression in the motor nucleus of IFN beta-treated animals was detected. In the peripheral nerve, IFN beta-treated animals showed increased S100, GAP-43 and p75NTR labelling coupled with a significantly greater number of regenerated axons. No significant differences were found in neurofilament or laminin labelling. The morphological findings, indicating improvements in the regenerative process after IFN treatment were in line with the motor behaviour test applied to the animals during the recovery process. Conclusions: The present data reinforce the role of MHC class I upregulation in the response to injury, and suggest that IFN treatment may be beneficial to motor recovery after axotomy.

  • Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathy
    K. Bhaskar, G. A. Hobbs, S-H. Yen and G. Lee (2010) Neuropathology and Applied Neurobiology36, 462–477 Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathyAim: Tau protein is a prominent component of paired helical filaments in Alzheimer's disease (AD) and other tauopathies. While the abnormal phosphorylation of tau on serine and threonine has been well established in the disease process, its phosphorylation on tyrosine has only recently been described. We previously showed that the Src family non-receptor tyrosine kinases (SFKs) Fyn and Src phosphorylate tau on Tyr18 and that phospho-Tyr18-tau was present in AD brain. In this study, we have investigated the appearance of phospho-Tyr18-tau, activated SFK and proliferating cell nuclear antigen (PCNA) during disease progression in a mouse model of human tauopathy. Methods: We have used JNPL3, which expresses human tau with P301L mutation, and antibodies specific for phospho-Tyr18-tau (9G3), ser/thr phosphorylated tau (AT8), activated SFK and PCNA. Antibody staining was viewed by either epifluorescence or confocal microscopy. Results: Phospho-Tyr18-tau appeared concurrently with AT8-reactive tau as early as 4 months in JNPL3. Some 9G3-positive cells also contained activated SFKs and PCNA. We also investigated the triple transgenic mouse model of AD and found that unlike the JNPL3 model, the appearance of 9G3 reactivity did not coincide with AT8 in the hippocampus, suggesting that the presence of APP/presenilin influences tau phosphorylation. Also, Thioflavin S-positive plaques were 9G3-negative, suggesting that phospho-Tyr18-tau is absent from the dystrophic neurites of the mouse triple transgenic brain. Conclusions: Our results provide evidence for the association of tyrosine-phosphorylated tau with mechanisms of neuropathogenesis and indicate that SFK activation and cell cycle activation are also involved in JNPL3.

  • Endothelin-converting enzyme-1 in Alzheimer's disease and vascular dementia
    J. C. Palmer, P. G. Kehoe and S. Love (2010) Neuropathology and Applied Neurobiology36, 487–497 Endothelin-converting enzyme-1 in Alzheimer's disease and vascular dementiaAims: Alzheimer's disease (AD) is believed to be caused by the accumulation of amyloid beta (Aβ) peptide within the brain. Endothelin-converting enzyme-1 and 2 (ECE-1 and ECE-2) are expressed in endothelial cells and neurones, respectively, and both cleave ‘big endothelin’ to produce the vasoconstrictor endothelin-1 (ET-1). ECE-1 and ECE-2 also degrade Aβ. AD patients have regionally reduced microvascular blood flow in the brain, with impaired endothelium-dependent relaxation and cerebrovascular autoregulation, and abnormal production of ET-1 has been demonstrated in mice overexpressing amyloid precursor protein. We recently found ECE-2 mRNA and protein to be elevated in the brain in AD. In vitro, expression of ECE-2 was upregulated by Aβ. Our aims for this study were to examine expression of ECE-1 (which has 57% homology with ECE-2) in temporal cortex from patients with AD, vascular dementia (VaD) and controls. Methods: We examined the distribution of ECE-1 with immunohistochemistry, and measured ECE-1 mRNA by real-time polymerase chain reaction (PCR). ECE-1 protein levels were measured by western blot, and results analysed before and after adjustment for factor VIII-related antigen. Results: We showed ECE-1 to be in vascular endothelial cells. We did not find significant differences in ECE-1 mRNA or protein levels (either full-length ECE-1 or the soluble spliced variant, ECE-1sv) in AD or VaD compared with controls. Conclusions: Our findings suggest that any disease-specific contribution of ECE-1 to the accumulation of Aβ or reduction in local microvascular blood flow in AD or VaD is probably small, with abnormal production of ET-1 being more likely to reflect Aβ-mediated upregulation of ECE-2.

  • Fusion between human mesenchymal stem cells and rodent cerebellar Purkinje cells
    Aims We explored whether cellular fusion and heterokaryon formation between human and rodent cells in the cerebellum of mice occurs after intravenous injection of human bone marrow-derived mesenchymal stem cells (MSCs). The influence of CNS inflammation on this process was also assessed. In addition we examined whether TNF-alpha and IFN-gamma, factors associated with inflammation, increase cellular fusion between human MSCs and rodent cerebellar neurons in vitro.Methods and results Human MSCs were intravenously injected into mice with experimental autoimmune encephalomyelitis (EAE) and control mice. After 22 days mouse Purkinje cells expressing human Golgi Zone were found within the Purkinje cell layer of the cerebellum, indicating that fusion and heterokaryon formation had occurred. The numbers of heterokaryons in the cerebellum were markedly increased in mice with EAE compared to control mice. Rodent cerebellar neuronal cells labelled with EGFP in vitro were co-cultured with human bone marrow-derived MSCs in the presence of TNF-alpha and/or IFN-gamma to determine their influence on fusion events. We found that fusion between MSCs and cerebellar neurons did occur in vitro and that the frequency of cellular fusion increased in the presence of TNF-alpha and/or IFN-gamma.Conclusions We believe that this is the first paper to define fusion and heterokaryon formation between human MSCs and rodent cerebellar neurons in vivo. We have also demonstrated that fusion between these cell populations occurs in vitro. These findings indicate that MSCs may be potential therapeutic agents for cerebellar diseases, and other neuroinflammatory and neurodegenerative disorders.

  • A series of Chinese patients with desminopathy associated with 6 novel and 1 reported mutations in the desmin gene
    Aims: Desminopathy is a hereditary cardiac and skeletal myopathy caused by mutations in the desmin gene. This study summarizes the clinical, myopathological and genetic features of a series of Chinese patients with desminopathy.Methods: Thirty-nine cases from 5 families with autosomal dominant inheritance and 2 sporadic cases were investigated. The majority of patients presented with mild myopathy and prominent cardiomyopathy. Fifteen of 16 deceased cases died of cardiac causes. Of the 25 patients alive, 24 patients developed cardiac abnormalities with disease progression. Muscle specimens from 9 patients were investigated in various morphological examinations. Gene sequencing and cell transfections were performed to determine whether the mutant desmin formed intermediate filaments.Results: Muscle biopsies revealed 5 cases with dystrophy-like patterns and amorphous material deposits; 4 other cases showed myopathy-like patterns with cytoplasmic bodies or nemaline bodies. Desmin and multiple proteins aggregated in the affected fibres. Six novel mutations and one previously reported mutation in the desmin gene were identified in the patients. All the mutant desmin genes except E457V produced multiple desmin-positive clumps or abnormal solid large aggregates in transfected cells.Conclusions: This study enlarges the spectrum of desmin mutations and geographic distribution of desminopathy. Although many novel mutations were identified in Chinese patients, the main clinical and myopathological findings were similar to those in Caucasian patients. Cardiac conduction abnormalities were prominent and usually appeared later than skeletal myopathy. The myopathology exhibited some heterogeneity among our patients, but the pathological changes were not indicative of the mutation location in the desmin gene.

  • A novel brain metastases model developed in immunodeficient rats closely mimics the growth of metastatic brain tumours in patients
    Aims: Brain metastasis is a common cause of mortality in cancer patients, and associated with poor prognosis. Our objective was to develop a clinically relevant animal model by transplanting human biopsy spheroids derived from metastatic lesions into brains of immunodeficient rats. Methods: 9 different patient brain metastases from 4 different primary cancers where implanted into brains of immunodeficient rats. The xenografts were compared to patient tumours by MR imaging, histochemistry, immunohistochemistry and DNA copy number analysis. Results: After transplantation, tumour growth was achieved in 7 out of 9 human brain metastases. Spheroids derived from 4 of the metastases initiated in the rat brains were further serially transplanted into new animals and a 100% tumour take was observed during second passage. 3 of the biopsies were implanted subcutaneously, where no tumour take was observed. The animal brain metastases exhibited similar radiological features as observed clinically. Histological comparisons between the primary tumours from the patients, the patient brain metastases and the derived xenografts showed striking similarities in histology and growth patterns. Also, immunohistochemistry showed a strong marker expression similarity between the patient tumours and the corresponding xenografts. DNA copy number analysis between the brain metastases, and the corresponding xenografts revealed strong similarities in gains and losses of chromosomal content. Conclusion: We have developed a representative in vivo model for studying the growth of human metastatic brain cancers. The model described represents an important tool to assess responses to new treatment modalities and for studying mechanisms behind metastatic growth in the central nervous system.

  • TDP-43 proteinopathy in familial motor neuron disease with TARDBP A315T mutation: a case report
  • Tumour vasculature and angiogenic profile of paediatric pilocytic astrocytoma; is it much different from glioblastoma?
    Aims:Pilocytic astrocytomas are the most frequent brain tumours in children. Because of their high vascularity, this study aimed to obtain insights into potential angiogenic related therapeutic targets in these tumours by characterization of the vasculature and the angiogenic profile. In this study 59 paediatric pilocytic astrocytomas were compared with 62 adult glioblastomas, as a prototype of tumour angiogenesis.Methods: Microvessel density (MVD), vessel maturity in terms of basement membrane and pericyte coverage, and turnover of both endothelial and tumour cells, and vascular endothelial growth factor (VEGF) expression were evaluated in tumour tissue, immunohistochemically stained with respectively CD34, collagen IV, SMA, Ki67/CD34, caspase-3/CD34, and VEGF(-A–D). As an indicator for vessel stability the angiopoietin (ANGPT)-1/ANGPT-2 balance was calculated using Real Time RT-PCR. Results: Pilocytic astrocytoma and glioblastoma showed similar fractions of vessels covered with basement membrane and pericytes. Overlapping ANGPT-1/ANGPT-2 balance and VEGF-A expression were found. Pilocytic astrocytoma had fewer but wider vessels compared with glioblastoma. Turnover of endothelial and tumour cells were relatively lower in pilocytic astrocytoma. Within pilocytic astrocytoma, higher ANGPT-1/ANGPT-2 balance was correlated with fewer apoptotic endothelial cells. Lower numbers of vessels were correlated with higher VEGF-A expression. Conclusions: Despite the fact that pilocytic astrocytoma showed a different vessel architecture compared with glioblastoma, a critical overlap in vessel immaturity/instability and the angiogenic profile was seen between both tumours. These findings suggest encouraging possibilities for targeting angiogenesis (for instance with anti-VEGF) as a therapeutic strategy in pilocytic astrocytoma.

  • Mutational analysis of D2HGDH and L2HGDH in brain tumours without IDH1 or IDH2 mutations
  • Combined NgR Vaccination and Neural Stem Cell Transplantation Promote Functional Recovery after Spinal Cord Injury in Adult Rats
    Aims: After spinal cord injury (SCI), there are many adverse factors at the lesion site such as glial scar, myelin-derived inhibitors, cell loss, and deficiency of neurotrophins that impair axonal regeneration. Therefore, combination therapeutic strategies might be more effective than a single strategy for promoting functional recovery after SCI. In the present study, we investigated whether a NgR vaccine, combined with neural stem cell (NSC) transplantation, could promote better functional recovery than when NgR vaccine or NSCs were used alone. Methods: Adult rats were immunized with NgR vaccine at 1 week after a contusive SCI at the thoracic level, and the NSCs, obtained from green fluorescent protein (GFP) transgenic rats, were transplanted into the injury site at 8 weeks post-injury. The functional recovery of the animals under various treatments was evaluated by three independent behavioral tests, i.e. BBB locomotor rating scale, footprint analysis, and grid walking. Results: The combined therapy with NgR vaccination and NSC transplantation protected more ventral horn motor neurons in the injured spinal cord and greater functional recovery than when they were used alone. Furthermore, NgR vaccination promoted migration of engrafted NSCs along the rostral-caudal axis of the injured spinal cords, and induced their differentiation into neurons and oligodendrocytes in vivo. Conclusions: The combination therapy of NgR vaccine and NSC transplantation exhibited significant advantages over any single therapy alone in this study. It may represent a potential new therapy for spinal cord injury.

  • Neonatal Rat Model of Intraventricular Haemorrhage and Post-Haemorrhagic Ventricular Dilatation with Long Term Survival into Adulthood
    Aims. Post-haemorrhagic ventricular dilatation (PHVD) is a significant problem in neonatal care, with sequelae extending beyond childhood. Its management is important in determining outcome. Although rodent hydrocephalus models have been developed, PHVD, as a specific entity with a distinct pathophysiology, has not been studied in a small animal model surviving to adulthood. Our objective is to evaluate survival, to adulthood, in our immature (7-day old, P7) neonatal rat model, and to analyse early motor reflexes and fine motor and cognitive function, and neuropathology, at 8-12 weeks.Methods. 66 rats underwent sequential bilateral stereotactic intraventricular haemorrhage (IVH); 36 more acted as controls. Staircase and radial maze evaluations were carried out at 7 –11 weeks; animals were sacrificed at 12 weeks. Post-mortem ventricular size and corpus callosum thickness were determined.Results. 76% of IVH animals developed PHVD; median (IQR) composite ventricular area was 3.46 mm2 (2.32 – 5.24). 16 (24%) animals demonstrated severe ventricular dilatation (area >5mm2). IVH animals failed to improve on the negative geotaxis test at 2 weeks. The staircase test did not identify any significant difference. On the radial maze animals with severe PHVD made more reference errors. Histopathology confirmed PHVD, ependymal disruption and extensive periventricular white matter injury. Median anterior corpus callosum thickness was significantly lower in IVH animals (0.35 mm) than in those not undergoing IVH (0.43 mm).Conclusion. Our P7 neonatal rat IVH model is suitable for long-term survival and replicates many of the morphological and some of the behavioural features seen in human PHVD.

  • Smad ubiquitination regulatory factor-2 in progressive supranuclear palsy
    Aims: Smad ubiquitination regulatory factor-2 (Smurf2) is an E3 ligase that belongs to the HECT domain ubiquitin ligase family. Smurf2 can interact with Smad proteins and promote their ubiquitin-dependent degradation, thereby controlling the cellular levels of these signaling mediators. Phosphorylated Smad2/3 (pSmad2/3) was recently identified in phosphorylated tau (phospho-tau) inclusions in patients with progressive supranuclear palsy (PSP). As Smurf2 is the E3 ligase of phosphorylated Smad2, we aimed at investigating the relationship among Smurf2, pSmad2/3, and phospho-tau in this study. Methods: The brains of 6 PSP and 3 control patients without neurological disorder were investigated by immunohistochemical analysis. Results: In the control subjects, Smurf2 immunoreactivity was not demonstrable in the neurons and glial cells, and that for pSmad2/3 was observed exclusively in neuronal and glial nuclei. In PSP patients, the pathognomonic neuronal and glial phospho-tau inclusions were immunopositive for both Smurf2 and pSmad2/3. The intensity of pSmad2/3 immuno-signals of neuronal and glial nuclei containing phospho-tau inclusions was less than that for the cells without the inclusions. Triple immunofluorescence staining for Smurf2, pSmad2/3, and phospho-tau revealed co-localization of these proteins within the neuronal and glial inclusions; and in some globose neurofibrillary tangles, the Smurf2 immunoreactivity appeared more centrally distributed than that of pSmad2/3 and phospho-tau. Conclusions: This is the first demonstration of the presence of Smurf2 immunoreactivity in the phospho-tau inclusions in PSP. These findings suggest that Smurf2 plays a significant role in the pathomechanism of PSP by causing abnormal redistribution of neuronal nuclear pSmad2/3 to the cytoplasm.

  • In sporadic inclusion-body myositis muscle fibres TDP-43-positive inclusions are less frequent and robust than p62-inclusions, and are not associated with paired helical filaments
  • Semliki Forest virus mediated gene therapy of the RG2 rat glioma
    Aims: Glioblastoma multiforme (GBM) is the most common and most malignant adult brain tumor. Despite numerous advances in cancer therapy there has been little change in the prognosis of GBM, which remains invariably fatal. We examined the Semliki Forest virus virus-like particle (SFV-VLP) expression system encoding interleukin-12 (IL-12) as a therapeutic intervention against the syngeneic RG2 rat glioma model. Methods: Glioma-bearing rats were treated with IL-12 encoding SFV VLPs via an implanted cannula. Animals were treated with 5x107 (low-dose) or 5x108 (high-dose) VLPs per treatment and the effect on glioma growth and survival assessed. Results: Low-dose treatment produced a 70% reduction in tumor volume, associated with a significant extension (20.45%) in survival, that was dependent upon IL-12 expression. High-dose treatment resulted in an 87% reduction in tumor volume, related to the oncolytic capacity of the SFV VLP system. VLP delivery to the CNS demonstrated the potential of the vector system to induce lethal pathology that was unrelated to replication-competent virus (RCV) or high-level IL-12 expression. Treatment-related death was pronounced in high-dose treated animals and appeared to be the result of inflammation, necrosis and oedema at the inoculation site. Conclusion: The efficacy of an IL-12 gene therapy approach for the treatment of the RG2 glioma model has been demonstrated in addition to the oncolytic capacity of the VLP vector system. Despite this, the broad tropism of the SFV based expression vector may limit use as a CNS gene therapy vector unless this inherent limitation can be overcome.

  • Sellar atypical teratoid/rhabdoid tumours in adults
  • Anti-inflammatory effects of pioglitazone on iron-induced oxidative injury in the nigrostriatal dopaminergic system
    Aims: Transition metals, oxidative stress and neuroinflammation have been proposed as part of a vicious cycle in CNS neurodegeneration. Our aim was to study the anti-inflammatory effect of pioglitazone, a peroxisome proliferative activated receptor (PPAR)-γ agonist, on iron-induced oxidative injury in rat brain. Methods: Intranigral infusion of ferrous citrate (iron) was performed on anaesthetized rats. Pioglitazone (20 mg/kg) was orally administered. Oxidative injury was investigated by measuring lipid peroxidation in the substantia nigra (SN) and dopamine content in the striatum. Western blot assay and DNA fragmentation were employed to study the involvement of α-synuclein aggregation, neuroinflammation as well as activation of endoplasmic reticulum (ER) and mitochondrial pathways in iron-induced apoptosis. Results: Intranigral infusion of iron time-dependently increased α-synuclein aggregation and heme oxygenase-1 (HO-1) levels. Furthermore, apoptosis was demonstrated by TUNEL-positive cells and DNA fragmentation in the iron-infused SN. Systemic pioglitazone was found to potentiate iron-induced elevation in nuclear PPAR-γ levels. However, pioglitazone inhibited iron-induced α-synuclein aggregation, elevations in interleukin (IL)-1β and IL-6 mRNA levels as well as increases in HO-1, cyclo-oxygenase II, nitric oxide synthase and ED-1 protein levels, an indicator of activated microglia. Moreover, iron-induced DNA laddering as well as activation of ER and mitochondrial pathways was attenuated by pioglitazone. In addition, pioglitazone decreased iron-induced elevation in lipid peroxidation in the infused SN and depletion in striatal dopamine level. Conclusions: Our results suggest that pioglitazone prevents iron-induced apoptosis via both ER and mitochondrial pathways. Furthermore, inhibition of α-synuclein aggregation and neuroinflammation may contribute to the pioglitazone-induced neuroprotection in CNS.

  • Genome Wide Polymorphism Analysis Demonstrates a Monoclonal Origin of Pilocytic Astrocytoma
  • Inhibition of microglial and astrocytic inflammatory responses by the immunosuppressant mycophenolate mofetil
    Aims: Nucleotide depletion induced by the immunosuppressant mycophenolate mofetil (MMF) has been shown to exert neuroprotective effects. It remains unclear whether nucleotide depletion directly counteracts neuronal demise or whether it inhibits microglial or astrocytic activation, thereby resulting in indirect neuroprotection.Methods: Effects of MMF on isolated microglial cells, astrocyte/microglial cell co-cultures, and isolated hippocampal neurons were analysed by immunocytochemistry, quantitative morphometry, and ELISA.Results: We found that: i) MMF suppressed lipopolysaccharide (LPS)-induced microglial secretion of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and nitric oxide (NO); ii) MMF suppressed LPS-induced astrocytic production of TNF-α but not of NO; iii) MMF strongly inhibited proliferation of both microglial cells and astrocytes; iv) MMF did not protect isolated hippocampal neurons from excitotoxic injury; and v) effects of MMF on glial cells were reversed after treatment with guanosineConclusions: Nucleotide depletion induced by MMF inhibits microglial and astrocytic activation. Microglial and astrocytic proliferation is suppressed by MMF-induced inhibition of the salvage pathway enzyme inosine monophosphate dehydrogenase. The previously observed neuroprotection after MMF treatment seems to be indirectly mediated, making this compound an interesting immunosuppressant in the treatment of acute central nervous system lesions.

  • Spatio-temporal DCC and netrin-1 expression in human fetal brain development
    Aims: DCC and its ligand netrin-1 are known as axonal guidance factors, being involved in angiogenesis, migration and survival of precursor cells in the embryonic mammalian CNS. So far, little is known about the distribution of those molecules in human CNS development.Methods: We investigated 22 human fetal brain specimens (12th and 28th week of gestation) for DCC and netrin-1 expression by means of immunohistochemistry, immunofluorescence and confocal laser microscopy. Statistical analysis was performed by applying a semiquantitative score, including staining intensity and frequency and correlated with fetal age.Results: DCC and netrin-1 were differentially expressed throughout the developing human fetal telencephalic and cerebellar cortical layers. Netrin-1 exhibited the highest levels in telencephalic germinal layers, whereas the strongest DCC immunoreactivity was seen in the developing cortical plate. Netrin-1 and DCC were predominantly present on cerebellar external granule layer cells. Distinct co-expression was seen in maturing fetal brainstem nuclei, cerebellar EGL and the choroid plexus. In contrast, endothelial cells showed strong netrin-1 expression with subsidiary DCC immunoreactivity. Pontine and telencephalic axonal fibre tracts also demonstrated strong netrin-1 expression.Conclusions: We show that DCC and netrin-1 are ubiquitously expressed in the human fetal brain, however both exhibiting a distinct spatio-temporal expression pattern. Together with the data from animal experiments, our findings might indicate also an important role for DCC and netrin-1 in human fetal CNS development.

  • EVALUATION OF POTENTIAL SYNERGISTIC ACTION OF A COMBINED TREATMENT WITH ALPHA-METHYL-PREDNISOLONE AND TAURINE ON THE MDX MOUSE MODEL OF DUCHENNE MUSCULAR DYSTROPHY
    Aims: Glucocorticoids are the sole drugs clinically used in Duchenne muscular dystrophy, in spite of the relevant side effects. Combination of glucocorticoids with synergistic drugs may be one strategy to lower doses and control side effects, meanwhile providing wider control of the complex pathology. This study is a pre-clinical evaluation of the effect of a combined treatment of α-methyl-prednisolone (PDN) with taurine, a safe aminoacid with positive effects on some pathology-related events.Methods: PDN (1 mg/kg/day i.p.) and taurine (1g/kg/day orally) were administered either alone or in combination, for 4-8 weeks to male dystrophic mdx mice chronically exercised on a treadmill. Effects were assessed in vivo and ex vivo with a variety of methodological approaches.Results:In vivo, each treatment significantly increased fore-limb strength, a marked synergistic effect being observed with the combination PDN+taurine. Ex vivo, PDN+taurine completely restored the mechanical threshold, an electrophysiological index of calcium homeostasis, of EDL myofibres and the benefit was greater than for PDN alone. In parallel, the over-activity of voltage-independent cation channels in dystrophic myofibres was reduced. No effects were observed on plasma levels of creatine kinase, while lactate dehydrogenase was decreased by taurine and, to a minor extent, by PDN + taurine. A similar histology profile was observed in PDN and PDN + taurine treated-muscles. PDN + taurine significantly increased taurine level in fast-twitch muscle and brain, by HPLC analysis.Conclusions: The combination PDN+taurine has additive actions on in vivo and ex vivo functional end-points, with less evident advantages on histopathology and biochemical markers of the disease.

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