Development and Optimization of a Multilayer Rat Purkinje Neuron Culture

Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.publishedVersio

A method for dierentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96‐well microplates

The capacity of pluripotent stem cells both for self-renewal and to differentiate into any cell type have made them a powerful tool for studying human disease. Protocols for efficient differentiation towards cardiomyocytes using defined, serum-free culture medium combined with small molecules have been developed, but thus far, limited to larger formats. We adapted protocols for differentiating human pluripotent stem cells to functional human cardiomyocytes in a 96-well microplate format. The resulting cardiomyocytes expressed cardiac specific markers at the transcriptional and protein levels and had the electrophysiological properties that confirmed the presence of functional cardiomyocytes. We suggest that this protocol provides an incremental improvement and one that reduces the impact of heterogeneity by increasing inter-experimental replicates. We believe that this technique will improve the applicability of these cells for use in developmental biology and mechanistic studies of disease.publishedVersio

CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration

The pathogenesis of Yo‐mediated paraneoplastic cerebellar degeneration (PCD) is unclear. We applied cerebrospinal fluid and serum from PCD patients as well as CDR2 and CDR2L antibodies to neuronal tissue, cancer cell lines, and cells transfected with recombinant CDR2 and CDR2L to elucidate which is the major antigen of Yo antibodies. We found that Yo antibodies bound endogenous CDR2L, but not endogenous CDR2. However, Yo antibodies can bind the recombinant CDR2 protein used in routine clinical testing for these antibodies. Because Yo antibodies only bind endogenous CDR2L, we conclude that CDR2L is the major antigen of Yo antibodies in PCD.publishedVersio

Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase

BACKGROUND: Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-beta (Abeta) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice. METHODOLOGY/PRINCIPAL FINDINGS: We found that while endogenous Abeta concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Abeta deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Abeta degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice. CONCLUSIONS/SIGNIFICANCE: Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases

Plasticity changes in the lateral amygdala after kindling and alcohol withdrawal

Titel INHALTSVERZEICHNIS I EINLEITUNG 1 MATERIAL UND METHODEN 26 ERGEBNISSE 43 DISKUSSION 80 ZUSAMMENFASSUNG 100 SUMMARY 103 LITERATURVERZEICHNIS 106 ANHANG 128Ziel der hier vorgestellten Studie war der Vergleich von Änderungen in basaler Transmission, Kurzzeitplastizität (Doppelpulsreizung) und Langzeitplastizität (Langzeitpotenzierung (LTP), Langzeitdepression (LTD)) im lateralen Kern der Amygdala (LA) bei verschiedenen Tiermodellen der Temporallappenepilepsie (Amygdalakindling/ Pilocarpin-Behandlung) bzw. Alkoholentzug. Die Wistarratten wurden durch die tägliche Applikation von kurzen elektrischen Reizen über eine implantierte Elektrode in der linken basolateralen Amygdala gekindelt, die resultierenden Krampfanfälle wurden nach Racine klassifiziert. Feld-EPSPs (extrazelluläre Ableitungen) oder EPSPs (intrazelluläre Ableitungen) wurden in horizontalen Hirnschnittpräparaten von gekindelten Ratten (48 Stunden nach dem letzten induzierten epileptischen Anfall), von implantierten und nicht implantierten Kontrollen im LA abgeleitet. Die Kindlingprozedur führte zu einer Art Sättigung der Potenzierung, d.h. die LTP wurde in Abhängigkeit von der vorangehenden Anfallsanzahl signifikant reduziert. Eine ähnliche Depression der LA-LTP wurde auch bei Pilocarpin-behandelten Tieren beobachtet, die spontane epileptische Anfälle vor den in-vitro Experimenten entwickelten. Obwohl die Erregbarkeit in der LA in den gekindelten Ratten erhöht war, wurde Kurzzeitplastizität nicht beeinflußt. Auch die meisten Zellparameter der Pyramiden-ähnlichen Neurone waren durch Kindling nicht verändert. Interessanterweise führten Paradigmen (Thetapuls- bzw. Niederfrequenzstimulus), die in den nicht implantierten Kontrollen eine LTD der synaptischen Aktivität im LA verursachten, in den gekindelten Ratten zu einer LTP. Somit kann geschlußfolgert werden, daß Kindling eine Form von Metaplastizität darstellt. Um Mechanismen der beobachteten pathologischen Neuroplastizität bei den gekindelten Tieren aufzuklären, wurde partiell die GABAerge Transmission blockiert (100 nM SR95531) oder die glutamaterge Transmission erhöht (2 µM ATPA). Die partielle Blockade von GABAA-Rezeptoren führte in den extrazellulären Ableitungen zu einer signifikanten Erhöhung der LA-LTP in allen Tiergruppen, wobei die Kindling-induzierte Hemmung der LTP nicht komplett aufgehoben wurde. Der spezifische Kainat-GluR5-Agonist ATPA induzierte eine schwächere amygdaläre LTP in den Kontrollen. Bei den gekindelten Tieren erhöhte ATPA die LA- LTP (intra- und extrazellulär) so stark, daß die Kindling-induzierten Plastizitätsänderungen in der Amygdala kompensiert wurden. In Pilocarpin-behandelten Tieren hatte ATPA keinen Einfluß auf die LA-LTP. Ratten, mit einen einmaligen bzw. mehrmaligen Alkoholentzug zeigten eine LA-LTP-Depression ähnlich wie sie in den gekindelten Tieren beobachtet wurde. Diese Daten korrelierten mit einer Hemmung der Fähigkeit zur konditionierten Assoziation zwischen sensorischen und aversiven unkonditionierten Reizen. Die LTP-Depression im LA, die bei Tieren nach Kindling/Alkoholentzug auftrat, könnte die elektrophysiologische Grundlage der Gedächtnisverschlechterungen sein, welche nach Kindling im Tierversuch oder bei Patienten mit Temorallappenepilepsie bzw. nach Entzug zu beobachten ist.The aim of the present study was the comparison of changes in basal transmission, short term plasticity (paired pulse facilitation) and long-term plasticity (long-term potentiation (LTP), long-term depression (LTD)) in the lateral nucleus of the amygdala (LA) of different animal models of temporal lobe epilepsy (amygdala kindling and pilocarpine treatment) and alcohol withdrawal in rats. Rats were kindled through daily administration of brief electrical stimulations to the left basolateral nucleus of the amygdala, and resulting motor seizures were scored according to the Racine scale. Field EPSPs (extracellular recordings) or EPSPs (intracellular recordings) were recorded in the LA in horizontal slices derived from kindled rats 48 hours after the last induced seizure, and in slices from sham-implanted and non- implanted controls. Kindling produced some general saturation of potentiation since LTP in the LA was significantly reduced, the magnitude of which was dependent on the number of prior stage V seizures. A similar depression of LTP was also obtained in pilocarpine-treated animals which developed spontaneous seizures before the in-vitro experiments. Whereas in amygdala-kindled rats the excitability was increased in the amygdala, paired pulse facilitation was not influenced by the different treatments and most of the cell parameters of pyramidal-like neurons were unchanged in comparison to controls. Interestingly, stimulus paradigms (theta pulse stimulation or low frequency stimulation) which induced in non-implanted controls LTD of synaptic activity caused LTP in kindled rats. It can be concluded that kindling seems to represent a form of metaplasticity. To get a better insight in functional reasons of pathological neuroplasticity in kindled animals, we partially blocked the GABAergic transmission (SR 95531, 100 nM) or tried to enhance the glutamatergic transmission (ATPA, 2 µM). Partial blockade of GABAA receptors significantly facilitated the induction of LTP extracellularly recorded in all animal groups, but did not abolish the kindling-induced impairment of LTP completely. Whereas the specific kainate GluR5 agonist, ATPA, caused a weaker LTP in controls, it enhanced the magnitude of LTP (intracellularly and extracellularly recorded) in kindled animals. This enhancement of LTP could compensate for the kindling-induced plasticity changes in the amygdala. In pilocarpine-treated animals ATPA did not influence the magnitude of LTP. Rats which have undergone single or repeated alcohol withdrawal show a similar depression of LTP as demonstrated in kindled rats. These data correlated with an impairment of the ability to form conditioned associations between discrete stimuli and aversive unconditioned stimuli. This depression of LTP may be the electrophysiological basis for memory disturbances observed in animals after kindling or in patients with temporal lobe epilepsy and after alcohol withdrawal, respectively

Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis

Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs. Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration

Kindling-induced changes in plasticity of the rat amygdala and hippocampus

Temporal lobe epilepsy (TLE) is often accompanied by interictal behavioral abnormalities, such as fear and memory impairment. To identify possible underlying substrates, we analyzed long-term synaptic plasticity in two relevant brain regions, the lateral amygdala (LA) and the CA1 region of the hippocampus, in the kindling model of epilepsy. Wistar rats were kindled through daily administration of brief electrical stimulations to the left basolateral nucleus of the amygdala. Field potential recordings were performed in slices obtained from kindled rats 48 h after the last induced seizure, and in slices from sham-implanted and nonimplanted controls. Kindling resulted in a significant impairment of long-term potentiation (LTP) in both the LA and the CA1, the magnitude of which was dependent on the number of prior stage V seizures. Saturation of CA1-LTP, assessed through repeated spaced delivery of high-frequency stimulation, occurred at lower levels in kindled compared to sham-implanted animals, consistent with the hypothesis of reduced capacity of further synaptic strengthening. Furthermore, theta pulse stimulation elicited long-term depression in the amygdala in nonimplanted and sham-implanted controls, whereas the same stimulation protocol stimulation caused LTP in kindled rats. In conclusion, kindling differentially affects the magnitude, saturation, and polarity of LTP in the CA1 and LA, respectively, most likely indicating an activity-dependent mechanism in the context of synaptic metaplasticity

Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

Objective: Identify the subcellular location and potential binding partners of two cerebellar degeneration‐related proteins, CDR2L and CDR2, associated with anti‐Yo‐mediated paraneoplastic cerebellar degeneration. Methods: Cancer cells, rat Purkinje neuron cultures, and human cerebellar sections were exposed to cerebrospinal fluid and serum from patients with paraneoplastic cerebellar degeneration with Yo antibodies and with several antibodies against CDR2L and CDR2. We used mass spectrometry‐based proteomics, super‐resolution microscopy, proximity ligation assay, and co‐immunoprecipitation to verify the antibodies and to identify potential binding partners. Results: We confirmed the CDR2L specificity of Yo antibodies by mass spectrometry‐based proteomics and found that CDR2L localized to the cytoplasm and CDR2 to the nucleus. CDR2L co‐localized with the 40S ribosomal protein S6, while CDR2 co‐localized with the nuclear speckle proteins SON, eukaryotic initiation factor 4A‐III, and serine/arginine‐rich splicing factor 2. Interpretation: We showed that Yo antibodies specifically bind to CDR2L in Purkinje neurons of PCD patients where they potentially interfere with the function of the ribosomal machinery resulting in disrupted mRNA translation and/or protein synthesis. Our findings demonstrating that CDR2L interacts with ribosomal proteins and CDR2 with nuclear speckle proteins is an important step toward understanding PCD pathogenesis

CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration

The pathogenesis of Yo‐mediated paraneoplastic cerebellar degeneration (PCD) is unclear. We applied cerebrospinal fluid and serum from PCD patients as well as CDR2 and CDR2L antibodies to neuronal tissue, cancer cell lines, and cells transfected with recombinant CDR2 and CDR2L to elucidate which is the major antigen of Yo antibodies. We found that Yo antibodies bound endogenous CDR2L, but not endogenous CDR2. However, Yo antibodies can bind the recombinant CDR2 protein used in routine clinical testing for these antibodies. Because Yo antibodies only bind endogenous CDR2L, we conclude that CDR2L is the major antigen of Yo antibodies in PCD