preclinical evidence from C57BL/6 mice

Chemotherapy-induced central nervous system (CNS) neurotoxicity presents an unmet medical need. Patients often report a cognitive decline in temporal correlation to chemotherapy, particularly for hippocampus-dependent verbal and visuo-spatial abilities. We treated adult C57Bl/6 mice with 12 × 20 mg kg−1 paclitaxel (PTX), mimicking clinical conditions of dose-dense chemotherapy, followed by a pulse of bromodesoxyuridine (BrdU) to label dividing cells. In this model, mice developed visuo-spatial memory impairments, and we measured peak PTX concentrations in the hippocampus of 230 nm l−1, which was sevenfold higher compared with the neocortex. Histologic analysis revealed a reduced hippocampal cell proliferation. In vitro, we observed severe toxicity in slowly proliferating neural stem cells (NSC) as well as human neuronal progenitor cells after 2 h exposure to low nanomolar concentrations of PTX. In comparison, mature post-mitotic hippocampal neurons and cell lines of malignant cells were less vulnerable. In PTX-treated NSC, we observed an increase of intracellular calcium levels, as well as an increased activity of calpain- and caspase 3/7, suggesting a calcium-dependent mechanism. This cell death pathway could be specifically inhibited with lithium, but not glycogen synthase kinase 3 inhibitors, which protected NSC in vitro. In vivo, preemptive treatment of mice with lithium prevented PTX-induced memory deficits and abnormal adult hippocampal neurogenesis. In summary, we identified a molecular pathomechanism, which invokes PTX-induced cytotoxicity in NSC independent of cell cycle status. This pathway could be pharmacologically inhibited with lithium without impairing paclitaxel’s tubulin-dependent cytostatic mode of action, enabling a potential translational clinical approach

Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC)-derived sensory neurons

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent, potentially irreversible adverse effect of cytotoxic chemotherapy often leading to a reduction or discontinuation of treatment which negatively impacts patients' prognosis. To date, however, neither predictive biomarkers nor preventive treatments for CIPN are available, which is partially due to a lack of suitable experimental models. We therefore aimed to evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for CIPN. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to axonal blebbing and a dose dependent decline of cell viability in clinically relevant IC(50) ranges, which was not observed for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. Paclitaxel treatment effects were less pronounced after 24 h but prominent when treatment was applied for 72 h. Global transcriptome analyses performed at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways. We further evaluated if known neuroprotective strategies can be reproduced in iPSC-DSN and observed protective effects of lithium replicating findings from rodent dorsal root ganglia cells. Comparing sensory neurons derived from two different healthy donors, we found preliminary evidence that these cell lines react differentially to neurotoxic drugs as expected from the variable presentation of CIPN in patients. In conclusion, iPSC-DSN are a promising platform to study the pathogenesis of CIPN and to evaluate neuroprotective treatment strategies. In the future, the application of patient-specific iPSC-DSN could open new avenues for personalized medicine with individual risk prediction, choice of chemotherapeutic compounds and preventive treatments

Generation of an NCS1 gene knockout human induced pluripotent stem cell line using CRISPR/Cas9

NCS1 (Neuronal calcium sensor protein 1) encodes a highly conserved calcium binding protein abundantly expressed in neurons. It modulates intracellular calcium homeostasis, calcium-dependent signaling pathways as well as neuronal transmission and plasticity. Here, we generated a NCS1 knockout human induced pluripotent stem cell (hiPSC) line using CRISPR-Cas9 genome editing. It shows regular expression of pluripotent markers, normal iPSC morphology and karyotype as well as no detectable off-target effects on top 6 potentially affected genes. This newly generated cell line constitutes a valuable tool for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological disease

Rationale and design of the prevention of paclitaxel-related neurological side effects with lithium trial – Protocol of a multicenter, randomized, double-blind, placebo-controlled proof-of-concept phase-2 clinical trial

INTRODUCTION: Chemotherapy-induced polyneuropathy (CIPN) and post-chemotherapy cognitive impairment (PCCI) are frequent side effects of paclitaxel treatment. CIPN/PCCI are potentially irreversible, reduce quality of life and often lead to treatment limitations, which affect patients’ outcome. We previously demonstrated that paclitaxel enhances an interaction of the Neuronal calcium sensor-1 protein (NCS-1) with the Inositol-1,4,5-trisphosphate receptor (InsP3R), which disrupts calcium homeostasis and triggers neuronal cell death via the calcium-dependent protease calpain in dorsal root ganglia neurons and neuronal precursor cells. Prophylactic treatment of rodents with lithium inhibits the NCS1-InsP3R interaction and ameliorates paclitaxel-induced polyneuropathy and cognitive impairment, which is in part supported by limited retrospective clinical data in patients treated with lithium carbonate at the time of chemotherapy. Currently no data are available from a prospective clinical trial to demonstrate its efficacy. METHODS AND ANALYSIS: The PREPARE study will be conducted as a multicenter, randomized, double-blind, placebo-controlled phase-2 trial with parallel group design. N = 84 patients with breast cancer will be randomized 1:1 to either lithium carbonate treatment (targeted serum concentration 0.5–0.8 mmol/l) or placebo with sham dose adjustments as add-on to (nab-) paclitaxel. The primary endpoint is the validated Total Neuropathy Score reduced (TNSr) at 2 weeks after the last (nab-) paclitaxel infusion. The aim is to show that the lithium carbonate group is superior to the placebo group, meaning that the mean TNSr after (nab-) paclitaxel is lower in the lithium carbonate group than in the placebo group. Secondary endpoints include: (1) severity of CIPN, (2) amount and dose of pain medication, (3) cumulative dose of (nab-) paclitaxel, (4) patient-reported symptoms of CIPN, quality of life and symptoms of anxiety and depression, (5) severity of cognitive impairment, (6) hippocampal volume and changes in structural/functional connectivity and (7) serum Neurofilament light chain protein concentrations. ETHICS AND DISSEMINATION: The study protocol was approved by the Berlin ethics committee (reference: 21/232 – IV E 10) and the respective federal agency (Bundesinstitut für Arzneimittel und Medizinprodukte, reference: 61-3910-4044771). The results of the study will be published in peer-reviewed medical journals as well as presented at relevant (inter)national conferences. CLINICAL TRIAL REGISTRATION: [https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00027165], identifier [DRKS00027165]

Influence of bortezomib on cognition in patients and C57Bl/6 mice

The 26S proteasome inhibitor bortezomib is currently used to treat multiple myeloma, but also is effective in the treatment of antibody-mediated autoimmune disorders. One clinical concern is bortezomib’s toxicity towards the (central) nervous system. We used standardized neuropsychological testing to assess cognitive function in six patients with myasthenia gravis and systemic lupus erythematodes before and after treatment with a mean cumulative dose of 9.4 mg m-2 bortezomib (TAVAB study, NCT02102594) . Additionally, cognitive performance was measured in adult C57Bl/6 mice after treatment with a human equivalent cumulative dose of 15.6 mg m-2. Bortezomib concentrations were analyzed in the human cerebral spinal fluid (CSF) as well as the brain tissue and serum of adult C57Bl/6 mice at various time points after injection of 1.3 mg m-2 bortezomib with liquid chromatography-tandem mass spectrometry. Neither patients nor mice showed signs of cognitive impairment after bortezomib therapy. Bortezomib concentrations in the human CSF and murine brain tissue reached only 5-7% of serum concentrations with comparable concentrations measured in the hippocampus and the neocortex. Five-fold higher concentrations were needed to damage neuronal cells in vitro. In conclusion, penetration of the intact blood-brain-barrier by bortezomib is low. Overall, our data shows that bortezomib is a safe medication in terms of central nervous system toxicit

Effects of high salt diet on spontaneous autoimmune polyneuropathy in mice

Excel file contains all obtained behavioural, electrophysiological and histological data from a study assessing the influence of a high salt diet on the occurrence of spontaneous autoimmune polyneuropathy in mice. Unsymptomatic female CD86-/- NOD mice were used and randomly assigned to a normal diet or high salt diet containing 4% NaCl in food and 1% in water. Mice were observed for their general wellbeing (weight) and development of a CIDP-like autoimmune polyneuropathy was documented by clinical score. The occurrence of paresis and loss of locomotor function was measured with RotaRod test. The sciatic nerves were analyzed with standard neurography measurements at the beginning and end of the dietary phase regarding demyelination and axonal loss. Data shows a prolonged onset of neuropathy with an attenuated loss of locomotor function due to a reduced demyelination. Electrophysiological data was confirmed with histological analysis showing reduced demyelination and neuroinflammation of peripheral nerves

Focal brain ischemia in mice does not cause electrophysiological signs of critical illness neuropathy

Background and aims: Critical illness polyneuropathy (CIP) is a common complication of severe systemic illness treated in intensive care medicine. Ischemic stroke leads to an acute critical injury of the brain with hemiparesis and immobilization and often requires extended medical treatment. In addition, stroke-induced sarcopenia contributes greatly to poor rehabilitation and immobilization and is characterized by denervation, atrophy and remodeling in the paralytic, but also to some extent the unaffected limb. In this pilot study, we asked whether signs of an underlying CIP-like phenotype occur after middle cerebral artery occlusion (MCAo) in mice. Methods: Focal brain ischemia was induced in adult male C57Bl/6 mice by a 60-minute MCAo followed by reperfusion. Nerve conduction function was measured in the ipsilateral sciatic nerve and muscle potentials were recorded in the ipsilateral gastrocnemius and quadriceps muscle with standard electroneurography/-myography on days 10, 22 and 44 after stroke. An additional crush-injury to the sciatic nerve was included in two sham-operated mice as internal positive control. Results: MCAo/ reperfusion induced functional deficits with a maximum Bederson score of 2 on day 3 and marked hemispheric brain atrophy of -27 % in histological analysis. No significant difference in large-fiber conduction function was found between MCAo and sham-operated animals, nor was there an increase in spontaneous electromyographic activity in muscles of animals in the MCAo group. In contrast, sham-operated mice with an additional crush injury of the sciatic nerve (sham+) developed marked abnormalities: reduction of the compound motor action potential amplitudes (CMAP) and motor conduction velocities (MCV) in combination with pathologic spontaneous activity (PSA), all of which had largely recovered by day 44. Conclusion: Mice showed no signs of systemic peripheral nerve dysfunction or axonal degeneration typical for a CIP-like phenotype after 60 min MCAo/ reperfusion

Influence of bortezomib on cognition in patients and C57Bl/6 mice

The 26S proteasome inhibitor bortezomib is currently used to treat multiple myeloma, but also is effective in the treatment of antibody-mediated autoimmune disorders. One clinical concern is bortezomib’s toxicity towards the (central) nervous system. We used standardized neuropsychological testing to assess cognitive function in six patients with myasthenia gravis and systemic lupus erythematodes before and after treatment with a mean cumulative dose of 9.4 mg m-2 bortezomib (TAVAB study, NCT02102594) . Additionally, cognitive performance was measured in adult C57Bl/6 mice after treatment with a human equivalent cumulative dose of 15.6 mg m-2. Bortezomib concentrations were analyzed in the human cerebral spinal fluid (CSF) as well as the brain tissue and serum of adult C57Bl/6 mice at various time points after injection of 1.3 mg m-2 bortezomib with liquid chromatography-tandem mass spectrometry. Neither patients nor mice showed signs of cognitive impairment after bortezomib therapy. Bortezomib concentrations in the human CSF and murine brain tissue reached only 5-7% of serum concentrations with comparable concentrations measured in the hippocampus and the neocortex. Five-fold higher concentrations were needed to damage neuronal cells in vitro. In conclusion, penetration of the intact blood-brain-barrier by bortezomib is low. Overall, our data shows that bortezomib is a safe medication in terms of central nervous system toxicityTHIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV