Cristiana Atzori

Abundant Tau Filaments and Nonapoptotic Neurodegeneration in Transgenic Mice Expressing Human P301S Tau Protein

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5–6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimers disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Defective Neurogenesis in Citron Kinase Knockout Mice by Altered Cytokinesis and Massive Apoptosis

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

LACK OF APOPTOSIS IN MICE WITH ALS

be detected by this method. However, sublimon-type templates give equally prominent products. Our findings imply that the background of sublimon-derived products generated from control templates makes LX-PCR unreliable as a sole diagnostic method for detecting deleted mtDNAs, except in the case of deletions representing a substantial fraction of mtDNA molecules in a given DNA preparation. We would thus recommend routine serial dilution of all DNA samples to test for the meaningful presence of deleted mtDNA molecules when using LX-PCR, and ideally the verification of all positive findings by Southern blot analysis, before a diagnostic conclusion is reached. Published claims, based exclusively on LX-PCR analysis, that deleted mtDNAs accumulate to high levels in aging and in many disease states , need to be critically re-evaluated in the light of our findings. Acknowledgments We thank M. Niittylahti and O.Lumme for technical assistance, and P. Rustin, I. Holt, S. Khogali and N.-G. Larsson for discussions. This work was supported by grants from the Finnish Academy, Muscular Dystrophy Group, Royal Society, Tampere University Hospital Medical Research Fund, Yrjö Jahnsson Foundation, Finnish Foundation of Alcohol Research and the Pirkanmaa Region Fund of the Finnish Cultural Foundation.

A Cell Cycle Alteration Precedes Apoptosis of Granule Cell Precursors in the weaver Mouse Cerebellum

A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.

S-100β protein is upregulated in astrocytes and motor neurons in the spinal cord of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss and astrogliosis. We studied the immunohistochemical expression of S-100β, a calcium-binding protein with both neurotrophic and neurotoxic activities, in the spinal cord of patients with ALS. Adjacent sections were processed with an in situ end-labeling technique for the demonstration of apoptosis-related DNA fragmentation. In controls, low expression of S-100β was found in astrocytes but not motor neurons. Compared to controls, S-100β was overexpressed in ALS. Most stained cells were reactive astrocytes, but a minority of motor neurons was also labeled. Neuronal labeling was unrelated to the presence of signs of atrophy/degeneration. S-100β expression was also unrelated to neuronal or glial apoptosis. S-100β upregulation in ALS spinal cord suggests that the protein might be involved in cellular defense mechanisms against oxidative stress.

Lack of caspase-dependent apoptosis in spinal motor neurons of the wobbler mouse

The mechanism of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is still unclear and the post-mortem analysis of samples from ALS patients does not permit a clarification of the early events of cell death occurring in ALS. Animal models of motor neuron degeneration represent a reliable tool to investigate the type of cell death. Attention was focused on the possible role of apoptosis in a spontaneous model of cervical spinal cord motor neuron degeneration, the wobbler mouse. Firstly, the rate of motor neuron loss occurring in the cervical spinal cord region of wobbler mice during different phases of symptoms progression was quantified by CholineAcetyltransferase (ChAT) immunohistochemistry. This was followed by a series of immunohistological studies to ascertain whether apoptosis was actually involved. ChAT immunostaining confirmed the severe loss of cholinergic neurons. Since the rate of motor neuron death is maximal in the first phase of the disease (from the 3rd to the 5th postnatal week), apoptotic markers were evaluated in 4-week-old wobbler mice. This study, carried out by examining a large number of cervical spinal cord sections from 20 affected animals and 20 healthy littermates, did not show either caspase activation or DNA fragmentation. These results strongly suggest that motor neuron death occurring in the wobbler mouse is not related to a caspase-dependent apoptotic mechanism.

Clearance of cerebrospinal fluid JCV DNA with mirtazapine in a patient with progressive multifocal leukoencephalopathy and sarcoidosis.

Progressive multifocal leukoencephalopathy (PML) is a severe encephalic demyelinating disease associated with JC virus (JCV) reactivation that occurs mostly in patients with immune disorders. Patients affected by sarcoidosis are at risk for developing PML both for leukocyte dysfunction and for receiving immunosuppressive medications: delayed diagnosis and high-dose corticosteroids are associated with a reduced survival. Although no specific treatment for PML exists, several therapeutic possibilities have been assessed with uncertain benefits (5HT2a receptor inhibitors are active in vitro against JCV): the cornerstone of sarcoidosis-associated PML is immunosuppressants withdrawal.We report the case of a female patient affected by systemic sarcoidosis for 30 years receiving low-dose corticosteroids (5 mg every other day). Due to memory impairment an MRI was performed showing three T2 hyperintense lesions involving white matter. Cerebrospinal fluid JCV PCR (845 copies/ml), neuropathological examination and immunohistochemistry (SV40 protein and JCV DNA positivity) on brain biopsy confirmed PML. Mirtazapine 15 mg was started while prednisone treatment was continued. 3 and 6 months later cognitive performances improved and brain MRIs were stable while cerebrospinal fluid JCV DNA was undetectable (6 months later). In conclusion the diagnosis of PML in patients with sarcoidosis is challenging given the overlapping presentation; the use of 5HT2a receptor antagonists deserves further studying in patients needing immunosuppressant drugs to control their dysimmune disease.

Cerebrospinal fluid biomarkers in patients with plasma HIV RNA below 20 copies/mL

Low level HIV‐1 CSF replication (CsfLLV) is often found even in patients with controlled plasma viraemia. The clinical consequences of such finding are uncertain; however, both symptomatic neurological disturbances and neurocognitive disorders may arise in the context of CSF‐escape. Two reports suggested that low level replication in the CSF may be associated with increased CSF neopterine although the impact on other markers of neuroinflammation/damage is currently unknown.

A previously undiagnosed case of Gerstmann-Sträussler-Scheinker disease revealed by PRNP gene analysis in patients with adult-onset ataxia

Ataxia is a frequently reported symptom in prion diseases (PD) and it is characteristic of Gerstmann‐Sträussler‐Scheinker syndrome (GSS), a genetic PD mainly related to the P102L mutation in the PRNP gene. Our aim was to screen for the P102L and other six known PRNP gene mutations (P105L, A117V, Y145X, E200K, D202N, and V210I) a group of 206 consecutive patients diagnosed with adult‐onset cerebellar ataxia of unknown origin. The patients, negative for the most common acquired and genetic forms, were analyzed using a combination of restriction endonuclease digestion and pyrosequencing; eight, affected by ataxia and cognitive dysfunction, were also sequenced for the PRNP gene. One patient resulted to be heterozygous for the P102L mutation. Retrospectively, the clinical picture was consistent with a “classical” GSS phenotype. In conclusion, the screening for the P102L mutation, or even the sequencing of the PRNP gene should be taken in consideration in patients with late‐onset ataxia (>50 years).

Diffuse leukoencephalopathy due to lymphomatosis cerebri: a clinicopathological report

Lymphomatosis cerebri (LC) is a rare variant of primary central nervous system lymphoma (PCNSL), characterized by diffuse infiltration of brain tissue by neoplastic cells without formation of discrete mass lesions [1, 2]. MRI appearance is usually consistent with a diffuse nonenhancing leukoencephalopathy [3]. A 57-year-old man came to our observation in May 2010 because of head trauma with transient loss of consciousness. Cranial CT evidenced a diffuse hypodensity of hemispheric white matter. Since the recent history of anorexia, confusion, and falls, the patient entered our neurologic ward. Neurologic examination disclosed mental slowness, bradykinesia, and mild ataxia. His previous medical history was unremarkable. Brain MRI confirmed a diffuse leukoencephalopathy involving cerebral hemispheres, hippocampi, corpus callosum, basal ganglia region, and midbrain (Fig. 1a–c). No contrast enhancement was present. Laboratory data, in particular hematograms and inflammatory profiles were unremarkable. Cerebrospinal fluid analysis evidenced raised protein concentration (75 mg/dl) with rare lymphocytes (1–2 elements/ll). Cytology was negative for malignant cells. Cytofluorometry ruled out clonal restriction. A discrete alteration of blood–brain barrier with no intrathecal IgG synthesis or oligoclonal bands was present. Microbiologic and virologic markers (including HIV testing) were all negative. A total body CT ruled out occult neoplasms or lymphoadenopathies. Moreover, no hypermetabolic areas were present on FDG-PET scans. An empiric treatment with high-dose systemic steroids (methylprednisolone 1 g/die per 10 days) was decided. The clinical picture significantly improved and the patient was discharged at home almost asymptomatic. In the following days (July 2010), the patient’s neurologic status gradually deteriorated. The patient was conducted again into our emergency department because of severe dyspnea and fever. Blood gas analysis evidenced marked hypoxia with hypocapnia. The suspect of pulmonary thromboembolism was confirmed by CT angiography. He was intubated and transferred to the intensive care unit. Anticoagulation was started. Brain MRI showed progression of white matter abnormalities and a punctiform area of contrast enhancement in left frontal lobe white matter (Fig. 1d). One week later, the patient expired because of a multiorgan failure syndrome. On autopsy, there was no macroscopic and histological evidence of systemic lymphoproliferative diseases or neoplasms. Brain gross appearance was unremarkable. Microscopy examination highlighted a diffuse infiltration of atypical cells, immunopositive for the CD20 antigen (Fig. 2a, b). Ki-67 positive cells were numerous (Fig. 2c). Neoplasm cells were evident in all sampled areas (cerebral cortex, hippocampi, white matter, basal ganglia, midbrain, and pons). Rare interspersed reactive CD3-positive T lymphocytes and moderate reactive gliosis were present, too. D. Imperiale (&) S. Taraglio C. Atzori R. Testi Centro Regionale Diagnosi ed Osservazione Malattie Prioniche DOMP, ASL To2, Via Cibrario72, 10144 Turin, Italy e-mail: daniele.imperiale@aslto2.piemonte.it