Anti-contactin-associated protein-2 encephalitis: relevance of antibody titres, presentation and outcome.

BACKGROUND AND PURPOSE: To clarify the relevance of titres of IgG antibodies against contactin-associated protein-2 (CASPR2) in diagnosing anti-CASPR2 encephalitis and to describe features and outcomes. METHODS: This was a retrospective analysis of 64 patients with CASPR2 antibodies, categorized independently as 'autoimmune encephalitis' or 'other disease'. Logistic regression methods were performed to identify potential predictors of 'autoimmune encephalitis' in addition to CASPR2 antibodies. RESULTS: An upfront CASPR2 antibody serum titre cut-off at ≥1:200 had a diagnostic sensitivity of 85% and a specificity of 81%. Logistic regression analyses indicated that, in addition to titre, encephalitic magnetic resonance imaging (MRI) was a significant predictor of 'autoimmune encephalitis' (Nagelkerke's R2 = 0.81, P < 0.001) with high sensitivity (84%) and very high specificity (100%). Patients with CASPR2 antibodies and an estimated probability of >70% of having anti-CASPR2 encephalitis (n = 22) had limbic encephalitis (n = 18, one patient plus ataxia), Morvan syndrome (n = 2) or a hyperkinetic movement disorder (n = 2). Median modified Rankin score (mRS) at diagnosis was 3 (range 1-4). Twenty patients were male; median age was 64 (range 54-75) years; 5/15 patients with cerebrospinal fluid data had intrathecal CASPR2 antibody synthesis, and 12/19 with follow-ups >3 months (median 12 months, range 4-43 months) improved by ≥1 mRS point resulting in a median mRS of 2 (range 0-6; one death; all but one having received immunotherapy); and 2/15 patients with follow-up MRI developed hippocampal atrophy. CONCLUSIONS: Only higher CASPR2 serum antibody titres indicate anti-CASPR2 encephalitis, and diagnostic accuracy increases if MRI findings are considered. Anti-CASPR2 encephalitis has characteristic features and a favourable outcome with immunotherapy.

Risk prediction of very early recurrence, death and progression after acute ischaemic stroke.

BACKGROUND AND PURPOSE: Early recurrent strokes lead to extended hospitalization and high number of complications. We investigated three stroke scores, the Essen Stroke Risk Score (ESRS), the ABCD(2) and the Recurrence Risk Estimator at 90 days (RRE-90) for their prognostic value to predict early recurrent stroke, death and progressive stroke. METHODS: Clinical and radiological data from 1727 consecutive patients with ischaemic stroke, being admitted to the stroke unit, were evaluated retrospectively. Predictive value of stroke scores was tested for early recurrence within 7 days, death and progressive stroke expressed as observational risk and area under the receiver operator curve (AUROC). RESULTS: Early recurrent stroke occurred in 56 patients (3.2%), 40 patients (2.3%) died within the first 7 days and 125 patients (7.2%) had a progressive stroke. ESRS was not predictive for early recurrence, death or progressive stroke. ABCD(2) score was predictive for death (P<0.01; χ(2); AUROC, 0.65; 0.58-0.72), and progressive stroke (P<0.001; χ(2); AUROC, 0.70; 0.66-0.74). RRE-90 predicted early recurrent stroke (P<0.001; χ(2); AUROC, 0.65; 0.58-0.73), early death (P<0.001; χ(2); AUROC, 0.72; 0.66-0.78) and progressive stroke (P<0.001; χ(2); AUROC, 0.66; 0.61-0.71). CONCLUSIONS: RRE-90 bears high potential to not only predict early recurrence but also death and progression after ischaemic stroke. ABCD(2) appears to be useful to predict risk of death and progression. These findings have relevant clinical implications for early triage of patients being admitted to stroke units.

[Differential diagnosis of Parkinson's syndromes--an overview]. / Differenzialdiagnostische Massnahmen bei Parkinsonsyndromen--eine Ubersicht.

With regard to aging of the general population non-neurologists are more frequently confronted with patients presenting with parkinsonian symptoms. The central task concerning treatment and prognosis in such cases is to differentiate idiopathic Parkinson's disease from secondary Parkinson syndromes and so called Parkinson-Plus-Syndromes. Besides clinical presentation neuroimaging techniques have been established in recent years, which can help with correct classification in early disease stages and difficult cases. In this article, we review the current clinical pathways for differential classification of parkinsonian symptoms with a special emphasis on clinical assessment and neuroimaging techniques like NMR and transcranial sonography.

Neuron-specific overexpression of the co-chaperone Bcl-2-associated athanogene-1 in superoxide dismutase 1(G93A)-transgenic mice.

Bcl-2-associated athanogene-1 (BAG1) binds heat-shock protein 70 (Hsp70)/Hsc70, increases intracellular chaperone activity in neurons and proved to be protective in several models for neurodegeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. A common property shared by all mutant SOD1 (mtSOD1) species is abnormal protein folding and the propensity to form aggregates. Toxicity and aggregate formation of mutant SOD1 can be overcome by enhanced chaperone function in vitro. Moreover, expression of mtSOD1 decreases BAG1 levels in a motoneuronal cell line. Thus, several lines of evidence suggested a protective role of BAG1 in mtSOD1-mediated motoneuron degeneration. To explore the therapeutic potential of BAG1 in a model for ALS, we generated SOD1G93A/BAG1 double transgenic mice expressing BAG1 in a neuron-specific pattern. Surprisingly, substantially increased BAG1 protein levels in spinal cord neurons did not significantly alter the phenotype of SOD1G93A-transgenic mice. Hence, expression of BAG1 is not sufficient to protect against mtSOD1-induced motor dysfunction in vivo. Our work shows that, in contrast to the in vitro situation, modulation of multiple cellular functions in addition to enhanced expression of a single chaperone is required to protect against SOD1 toxicity, highlighting the necessity of combined treatment strategies for ALS.

Aggregopathy in neurodegenerative diseases: mechanisms and therapeutic implication.

Many neurodegenerative diseases such as Parkinson's, Alzheimer's, Huntington's and Lou Gehrig's disease are associated with the misfolding and aggregation of proteins. While the relevance of these aggregates for neuronal degeneration and their impact on cellular function is still a matter of debate, several experimental therapeutic approaches have been aimed at interfering with protein aggregation. In this review, we want to summarize the current understanding of aggregate formation and toxicity in neurodegenerative diseases with an emphasis on Parkinson's disease. Furthermore, we will discuss current treatment strategies in these diseases targeting aggregate formation and concurrent neuronal cell death in these diseases.

Mutant SOD1 detoxification mechanisms in intact single cells.

Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cell-by-cell-analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS.

[Programmed cell death in the retina. Molecular mechanisms and therapeutic strategies]. / Programmierter Zelltod in der Netzhaut. Molekulare Mechanismen und therapeutische Ansätze.

The cellular suicide program (apoptosis) is important not only for many physiological processes spanning from embryonal development to aging but also for the pathogenesis and the course of a wide spectrum of diseases. Among these disorders are chronic neurodegenerative diseases including acquired and inherited degeneration of photoreceptors as well as retinal ganglion cells during retinitis pigmentosa, macular degeneration, optic neuritis, or glaucoma. In the following review we will address general pro- and antiapoptotic signal transduction cascades, their relevance for retinal degeneration focussing on retinal ganglion cells, and new therapeutic strategies evolving from these findings.

Evaluation of cerebral perfusion deficit in stroke patients using new transcranial contrast imaging CPS technology--preliminary results.

BACKGROUND AND PURPOSE: Contrast-enhanced transcranial duplex sonography can be used to examine cerebral perfusion. This technique, however, is still faced with methodological problems. The aim of the present study is to evaluate cerebral perfusion deficit after administration of the contrast agent SonoVue in acute stroke patients using new contrast imaging software. METHODS: Ten subjects (6 male stroke patients and 4 healthy volunteers), were examined using transcranial duplex sonography (Acuson Sequoia 512 Ultrasound System) after a bolus injection of the contrast agent SonoVue. The transcranial examination was performed using transient response harmonic grey scale imaging with refill kinetics. The Sequoia ultrasonographic system was equipped with a new contrast harmonic imaging software "Cadence contrast pulse sequencing technology" (CPS). Triggered images with the mechanical index (MI) at 1.1 as well as continuous registration with MI at 0.28 were used for the evaluation of time intensity curves in several regions of interest. The sonographically imaged hypoperfused areas were compared with findings from MR imaging. RESULTS: In all healthy volunteers, the contrast-enhanced signal could be recognized well in the ipsi- and also in the contralateral hemisphere up to the skull crown. In stroke patients, the perfusion deficit in the area of the MCA could be detected ipsilaterally in all subjects using triggered registration. Additionally, the area of MCA infarction could also be visualized in two patients using contralateral insonation. The low MI continuous imaging was successful in three patients. For all patients, the ischaemic region corresponded well in shape and size with the findings from MR imaging. CONCLUSIONS: CPS enhances the possibility of perfusion-imaging in cerebral microcirculation and of perfusion-deficit-imaging in patients with cerebral ischaemia. Further studies with a larger number of patients should be carried out to improve this method.

Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways.

The bifunctional apoptosis regulator (BAR) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis. Immunoblot analysis of normal human tissues demonstrated high BAR expression in the brain, compared to low or absent expression in other organs. Immunohistochemical staining of human adult tissues revealed that the BAR protein is predominantly expressed by neurons in the central nervous system. Immunofluorescence microscopy indicated that BAR localizes mainly to the endoplasmic reticulum (ER) of cells. Overexpression of BAR in CSM 14.1 neuronal cells resulted in significant protection from a broad range of cell death stimuli, including agents that activate apoptotic pathways involving mitochondria, TNF-family death receptors, and ER stress. Downregulation of BAR by antisense oligonucleotides sensitized neuronal cells to induction of apoptosis. Moreover, the search for novel interaction partners of BAR identified several candidate proteins that might contribute to the regulation of neuronal apoptosis (HIP1, Hippi, and Bap31). Taken together, the expression pattern and functional data suggest that the BAR protein is involved in the regulation of neuronal survival.

Contribution of caspase-8 to apoptosis of axotomized rat retinal ganglion cells in vivo.

We have investigated the role of caspase-8 and its mode of activation during apoptosis of adult rat retinal ganglion cells (RGCs) in vivo. Retinal pro-caspase-8 expression was almost completely restricted to RGCs. Although caspase-8 is known to be involved in death-receptor-dependent apoptosis, measurable caspase-8 activity or even RGC death could be induced by neither tumor necrosis factor-alpha nor Fas ligand injections into unlesioned eyes. However, substantial caspase-8 activation could be detected after optic nerve transection as shown by a fluorogenic activity assay and Western blot analysis. Intravitreal injection of caspase-8 inhibitors significantly attenuated degeneration of RGCs and reduced the number of RGCs showing caspase-3 activation. A late peak of caspase-8 activity and additive protective effects of caspase-8 and -9 inhibition on axotomized RGCs place caspase-8 in our model rather late in the apoptosis cascade, possibly after the onset of mitochondrial dysfunction.

Bag1 is a regulator and marker of neuronal differentiation.

Bag 1 acts as a co-chaperone for Hsp70/Hsc70. We report here that stable over-expression of Bag1 in immortalized neuronal CSM14.1 cells prevents death following serum deprivation. Bag1 over-expression slowed the proliferative rate of CSM14.1 cells, resulted in increased levels of phospo-MAP kinases and accelerated neuronal differentiation. Immunocytochemistry revealed mostly nuclear localization of Bag1 protein in these cells. However, during differentiation in vitro, Bag1 protein shifted from predominantly nuclear to mostly cytosolic in CSM14.1 cells. To explore in vivo parallels of these findings, we investigated Bag1 expression in the developing mouse nervous system using immunohistochemical methods. Early in brain development, Bag1 was found in nuclei of neuronal precursor cells, whereas cytosolic Bag1 staining was observed mainly after completion of neuronal precursor migration and differentiation. Taken together, these findings raise the possibility that the Bag1 protein is expressed early in neurogenesis in vivo and is capable of modulating neuronal cell survival and differentiation at least in part from a nuclear location.

Modulation of metabotropic glutamate receptors fails to prevent the loss of adult rat retinal ganglion cells following axotomy or N-methyl-D-aspartate lesion in vivo.

Both optic nerve (ON) transection and intraocular injection of N-methyl-D-aspartate (NMDA) are established lesion models to cause death of retinal ganglion cells (RGCs) in the adult rat. Excitotoxic effects via glutamate receptors resulting in secondary neuronal death are discussed as possible initiators in both types of RGC damage. We examined whether modulating glutamatergic transmission through metabotropic glutamate receptors rescues RGCs from lesion-induced degeneration in vivo. Unexpectedly, repeated intraocular injection of four different agonists/antagonists on the various subtypes of mGluRs did not decrease retinal damage in both lesion paradigms as revealed by measurement of visual performance and RGC survival. We conclude that activation/inactivation of retinal mGluRs does not play an important role for the initiation and execution of secondary RGC loss after ON transection and NMDA lesion in the adult rat.

Transection of the optic nerve in rats: studying neuronal death and survival in vivo.

Transection of the optic nerve (ON) in the adult rat, as a model of fiber tract lesion in the adult mammalian CNS, results in delayed, mainly apoptotic death of 80--90% of retinal ganglion cells (RGCs) within 14 days post-lesion. Because of good surgical accessibility of the retina and the optic nerve, the retino-tectal projection represents not only a convenient model to study the molecular mechanisms underlying neuronal death but also serves as a suitable system for investigating potential neuroprotective agents in vivo. In the present report, we provide a detailed protocol for this model including retrograde labeling of RGCs, ON lesion, assessment of the number of surviving neurons, and tissue preparation for several standard techniques like immunohistochemistry, reverse transcription--polymerase chain reaction (RT--PCR), enzyme assays and Immunoblot.

Brain-derived neurotrophic factor-mediated neuroprotection of adult rat retinal ganglion cells in vivo does not exclusively depend on phosphatidyl-inositol-3'-kinase/protein kinase B signaling.

The neurotrophin brain-derived neurotrophic factor (BDNF) serves as a survival, mitogenic, and differentiation factor in both the developing and adult CNS and PNS. In an attempt to identify the molecular mechanisms underlying BDNF neuroprotection, we studied activation of two potentially neuroprotective signal transduction pathways by BDNF in a CNS trauma model. Transection of the optic nerve (ON) in the adult rat induces secondary death of retinal ganglion cells (RGCs). Repeated intraocular injections of BDNF prevent the degeneration of RGCs 14 d after ON lesion most likely by inhibition of apoptosis. Here, we report that BDNF activates both protein kinase B (PKB) via a phosphatidyl-inositol-3'-kinase (PI-3-K)-dependent mechanism and the mitogen-activated protein kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2. Furthermore, we provide evidence that BDNF suppresses cleavage and enzymatic activity of the neuronal cell death effector caspase-3. Distinct from our recent study in which inhibition of the PI-3-K/PKB pathway attenuated the survival-promoting action of insulin-like growth factor-I on axotomized RGCs (Kermer et al., 2000), it does not in the case of BDNF. Thus, we assume that BDNF does not depend on a single signal transduction pathway exerting its neuroprotective effects on lesioned CNS neurons.

Insulin-like growth factor-I protects axotomized rat retinal ganglion cells from secondary death via PI3-K-dependent Akt phosphorylation and inhibition of caspase-3 In vivo.

Recently we have shown that the majority of retinal ganglion cells (RGCs) dies via activation of caspase-3 after transection of the optic nerve (ON) in the adult rat. In the present study we investigated whether insulin-like growth factor-I (IGF-I), an important factor in retinal development, prevents secondary death of RGCs after axotomy. Moreover, we studied potential intracellular mechanisms of IGF-mediated neuroprotection in more detail. Our results indicate that intraocular application of IGF-I protects RGCs from death after ON transection in a dose-dependent manner. We show reduced caspase-3 activity as one possible neuroprotective mechanism of IGF-I treatment in vivo. Caspase-3 mRNA expression remained unchanged. Because caspase inhibition can be mediated by Akt in vitro, we examined phosphorylation of Akt after axotomy and under IGF treatment. Western blot analysis revealed decreased Akt phosphorylation after axotomy without treatment and an increased phosphorylation of Akt under treatment with IGF-I. This strong increase could be reduced by simultaneous injection of wortmannin (WM), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-K). To prove the pathway suggested by these experiments as relevant for the in vivo situation, we assessed the number of RGCs 14 d after ON transection under a combined treatment strategy of IGF-I and WM. As expected, WM significantly reduced the neuroprotective effects of IGF-I. In summary, we show for the first time in vivo that IGF is neuroprotective via PI3-K-dependent Akt phosphorylation and by inhibition of caspase-3.

Caspase-9: involvement in secondary death of axotomized rat retinal ganglion cells in vivo.

Recently we have shown that adult rat retinal ganglion cells (RGCs) die by apoptosis following optic nerve (ON) transection, activating caspase-3. In the present study, we report that caspase-9, known to be an important activator of caspase-3, becomes activated in the axotomized adult rat retina as revealed by immunoblot analysis and protease activity assays. Reduction of caspase-9 activity by repeated intraocular injection of specific inhibitors significantly prevented RGC death. Caspase-9 activity was effectively blocked by inhibitor treatment and by application of IGF-I and BDNF, neurotrophic factors which have been shown to be highly neuroprotective in this model. Taken together, our data suggest that caspase-9 plays a critical role in apoptosis induction in axotomized RGCs in vivo and is regulated under treatment with growth and survival factors. Thus, providing more insight into the mechanisms underlying neuronal death and survival following trauma might serve as a basis to improve future therapeutic strategies preventing or at least reducing the severe consequences of neuronal injury.

Transduction of axotomized retinal ganglion cells by adenoviral vector administration at the optic nerve stump: an in vivo model system for the inhibition of neuronal apoptotic cell death.

Axotomy of the rat optic nerve leads to apoptotic cell death of retinal ganglion cells (RGCs). We have used adenoviral vectors to transduce RGCs from the cut optic nerve stump, a paradigm in which only those neurons are transduced which are directly affected by the axonal lesion. Transgenes encoded by the vectors were p35 and CrmA, which are potent intracellular anti-apoptotic proteins. We found that p35, but not CrmA exerted significant rescue effects on RGCs 14 days after axotomy. Expression of the transgenes was driven by the murine CMV (MCMV) promoter. The respective mRNAs were detectable 7 days but not 14 days after transduction. Since surviving RGCs were present beyond the time-point of detectable transcription of the p35 transgene, we conclude that apoptosis has been efficiently inhibited. In addition, we observed that transduction with two control vectors without a transgene in E1 also resulted in a minor but significant RGC rescue, implicating neuroprotective effects due to adenoviral transduction itself. This system will be useful in dissecting the pathways leading to neuronal cell death after axonal lesions and in the evaluation of the important question whether the cellular suicide program can be reverted to survival by therapeutic gene delivery.

Both the neuronal and inducible isoforms contribute to upregulation of retinal nitric oxide synthase activity by brain-derived neurotrophic factor.

Although neurotrophins are best known for their trophic functions, growing evidence suggests that neurotrophins can also be neurotoxic, for instance by enhancing excitotoxic insults. We have shown recently that brain-derived neurotrophic factor (BDNF) limits its neuroprotective action on axotomized rat retinal ganglion cells (RGCs) by upregulating nitric oxide synthase (NOS) activity (Klöcker et al., 1998). The aim of the present study was to investigate this interaction of BDNF and NOS in the lesioned adult rat retina in more detail. We used NOS immunohistochemistry and NADPH-diaphorase (NADPH-d) reaction to characterize morphologically retinal NOS expression and activity. Using reverse transcription-PCR and Western blot analysis, we were able to identify the NOS isoforms being regulated. Six days after optic nerve lesion, we observed an increase in neuronal NOS (NOS-I) mRNA and protein expression in the inner retina. This did not lead to a marked increase in overall retinal NOS activity. Only RGC axons displayed strong de novo NADPH-d reactivity. In contrast, intraocular injection of BDNF resulted in a marked upregulation of NOS activity in NOS-I-immunoreactive structures, leaving the level of NOS-I expression unchanged. In addition, an induction of inducible NOS (NOS-II) was found after BDNF treatment. We identified microglial cells increasing in number and being activated by BDNF, which could serve as the cellular source of NOS-II. In summary, our data suggest that BDNF upregulates retinal NOS activity by both a post-translational regulation of NOS-I activity and an induction of NOS-II. These findings might be useful for developing pharmacological strategies to improve BDNF-mediated neuroprotection.