Rat Cerebellar Cultures Research Articles

Region-specific protective effects of monomethyl fumarate in cerebellar and hippocampal organotypic slice cultures following oxygen-glucose deprivation.

To date, apart from moderate hypothermia, there are almost no adequate interventions available for neuroprotection in cases of brain damage due to cardiac arrest. Affected persons often have severe limitations in their quality of life. The aim of this study was to investigate protective properties of the active compound of dimethyl fumarate, monomethyl fumarate (MMF), on distinct regions of the central nervous system after ischemic events. Dimethyl fumarate is an already established drug in neurology with known anti-inflammatory and antioxidant properties. In this study, we chose organotypic slice cultures of rat cerebellum and hippocampus as an ex vivo model. To simulate cardiac arrest and return of spontaneous circulation we performed oxygen-glucose-deprivation (OGD) followed by treatments with different concentrations of MMF (1-30 μM in cerebellum and 5-30 μM in hippocampus). Immunofluorescence staining with propidium iodide (PI) and 4',6-diamidine-2-phenylindole (DAPI) was performed to analyze PI/DAPI ratio after imaging with a spinning disc confocal microscope. In the statistical analysis, the relative cell death of the different groups was compared. In both, the cerebellum and hippocampus, the MMF-treated group showed a significantly lower PI/DAPI ratio compared to the non-treated group after OGD. Thus, we showed for the first time that both cerebellar and hippocampal slice cultures treated with MMF after OGD are significantly less affected by cell death.

Melatonin synthesized by activated microglia orchestrates the progression of microglia from a pro-inflammatory to a recovery/repair phenotype

Microglia, the sentinels of the central nervous system, are responsible for the surveillance and the innate defense against pathogen or danger/damage-associated molecular patterns. The response is fine-tuned to restrain pro-inflammatory responses, preserving neighboring cells. At the injured area, microglia temporarily shift to a pro-inflammatory phenotype (M1), followed by anti-inflammatory (M2) phenotypes. The duration and magnitude of the pro-inflammatory phase are finely regulated to avoid unnecessary loss of brain tissue. The present study shows that melatonin synthesized by microglia plays a key role in the transformation of M1 to M2 phenotypes. In a mixed rat cerebellar glia culture, the percentage of activated microglia did not vary significantly with the treatments, while the role of melatonin synthesized by microglia in promoting the end of the pro-inflammatory phase, and the initiation of the regulatory/phagocytic phases was inferred by using pharmacological tools. Total microglia were identified by the expression of CD11b/c, whereas positive to IBA-1 microglia were considered activated, independent of the phenotype. M1 and M2 phenotypes were distinguished with the biomarkers NOS-2 and ARG-1, as these enzymes act on the same substrate (L-arginine), producing pro-inflammatory (NO) or anti-inflammatory (polyamines and proline) end products, respectively. Luzindole, a blocker of melatonin receptors, impaired the conversion of M1 to M2 phenotypes and zymosan phagocytosis. Thus, melatonin content synthesized by cerebellar microglia determines the extension of the pro-inflammatory phase of defense response.

The Role of Mitochondria in Brain Cell Protection from Ischaemia by Differently Prepared Propolis Extracts

Mitochondria are both the primary targets and mediators of ischaemic damage in brain cells. Insufficient oxygen causes reactive oxygen species that damage the mitochondria, leading to the loss of functionality and viability of highly energy-demanding neurons. We have recently found that aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP) and ethanolic propolis extracts (EEP) can modulate mitochondria and ROS production in C6 cells of astrocytic origin. The aim of this study was to investigate the effect of the extracts on viability, mitochondrial efficiency and superoxide generation, and inflammatory cytokine release in primary rat cerebellar neuronal-glial cell cultures affected by ischaemia (mimicked by hypoxia +/− deoxyglucose). AqEP and Pg-AqEP (15–60 µg/mL of phenolic compounds, or PC) significantly increased neuronal viability in ischaemia-treated cultures, and this was accompanied by a reduction in mitochondrial superoxide levels. Less extended protection against ischaemia-induced superoxide production and death was exhibited by 2 to 4 µg/mL of PC EEP. Both Pg-AqEP and Ag-EP (but not EEP) significantly protected the cultures from hypoxia-induced elevation of TNF-α, IL-1β and IL-6. Only Pg-AqEP (but not AqEP or EEP) prevented hypoxia-induced loss of the mitochondrial basal and ATP-coupled respiration rate, and significantly increased the mitochondrial respiratory capacity. Summarising, the study revealed that hydrophilic propolis extracts might protect brain cells against ischaemic injury by decreasing the level of mitochondrial superoxide and preventing inflammatory cytokines, and, in the case of Pg-AqEP, by protecting mitochondrial function.

Adaptive anti-oxidative responses to chronic exposure to stress-signaling molecule, oxidized cell-free DNA, in rat neural cells

Stress is an adaptation reaction to harmful environment changes. Both oxidized and non-oxidized cell-free DNA (oxo-cfDNA and no-cfDNA) liberating from damaged cells possesses a characteristic of stress-molecules. CfDNA molecules modified by oxidation exhibit more oxidative (increasing 8-oxodG in DNA) and antioxidative (enhanced transcription of NRF2 and NRF2-dependent genes) activities than no-cfDNA in non-neural cell cultures. Whether the oxo-cfDNA molecules affect brain cells under different regimens of treatment that may mimic multiple, chronically applied stressful insults remained unclear. We studied if multiple stimulations of nervous system cells with oxidized cfDNA may further augment the DNA oxidative damage by inducing 8-oxodG modifications, and increase antioxidant Hmox1 gene expression and the NRF2 protein level in a primary cell culture of rat cerebellum (RCC). We found that daily treatment of RCC with oxo-cfDNA for 3 days increased accumulation of 8-oxodG in intracellular DNA, activated transcription of Hmox1 gene and enhanced the NRF2 protein content. However, after eleven-day daily treatment, the opposite effect was observed, in which 8-oxodG level in intracellular DNA and Hmox1 gene expression and NRF2 protein level in cerebellar cells were significantly decreased. Therefore, under subchronic, 3-day regimen of treatment, oxidized cfDNA further augmented accumulation of 8-oxodG in cellular DNA and promoted activation of the antioxidant system whereas its 11-day chronic action decreased both activities presumably due to inducing tolerance to stress-signaling via overactivation and/or exhaustion of molecular targets of oxo-cfDNA in cerebellar cells.

Endocrine Disruptors Induced Distinct Expression of Thyroid and Estrogen Receptors in Rat versus Mouse Primary Cerebellar Cell Cultures.

The endocrine system of animals consists of fine-tuned self-regulating mechanisms that maintain the hormonal and neuronal milieu during tissue development. This complex system can be influenced by endocrine disruptors (ED)—substances that can alter the hormonal regulation even in small concentrations. By now, thousands of substances—either synthesized by the plastic, cosmetic, agricultural, or medical industry or occurring naturally in plants or in polluted groundwater—can act as EDs. Their identification and testing has been a hard-to-solve problem; Recent indications that the ED effects may be species-specific just further complicated the determination of biological ED effects. Here we compare the effects of bisphenol-A, zearalenone, and arsenic (well-known EDs) exerted on mouse and rat neural cell cultures by measuring the differences of the ED-affected neural estrogen- and thyroid receptors. EDs alters the receptor expression in a species-like manner detectable in the magnitude as well as in the nature of biological responses. It is concluded that the interspecies differences (or species specificity) in ED effects should be considered in the future testing of ED effects.

The Neuroprotective Effect of Forskolin and Its Influence of Mitochondrial Dysfunction in Neurons in Primary Rat Cerebellum Cultures

Activation of cyclic adenosine monophosphate (cAMP) synthesis is known to lead to triggering of neuroprotective signal cascades. It is therefore interesting to assess the effects of forskolin as an activator of adenylate cyclase and cAMP production on the neurotoxic action of glutamate in rat cerebellar neurons and to identify the participants in this signal cascade. This approach was used in experiments with a protein kinase A (PKA) inhibitor, the protein kinase C (PKC) blocker chelerythrine, and the calmodulin-dependent kinase II (CaMKII) blocker KN93, which showed that the neuroprotective effect of forskolin (1 μM) on long-term (24 h) exposure to glutamate (100 μM) involves PKA and CaMKII. Additional analysis of the dynamics of the development of mitochondrial dysfunction on exposure to glutamate showed that forskolin can prevent the drop in mitochondrial membrane potential in rat cerebellar neurons typical of excitotoxic stress.

Combinatorial Preconditioning of Rat Brain Cultures with Subprotective Ethanol and Resveratrol Concentrations Promotes Synergistic Neuroprotection.

Preconditioning brain cultures with moderate concentrations of ethanol (EtOH) or trans-resveratrol (RES), key red wine constituents, can prevent amyloid-β (Aβ) neurotoxicity. Past studies have indicated that moderate EtOH activates synaptic N-methyl-D-aspartate receptors (NMDAR) that, in part, signal via protein kinase C (PKC) to increase protective antioxidant proteins such as peroxiredoxin-2 (Prx2). RES preconditioning also is reported to involve NMDAR and PKC. However, although moderate, the EtOH and RES concentrations used have been noticeably above circulating levels from two glasses of wine, a daily intake linked to reduced risk of cognitive decline among older social drinkers. Given their mechanistic parallels, we speculated that subprotective EtOH and RES concentrations in a combinatorial preconditioning paradigm might elicit synergistic neuroprotection. To examine this notion, rat cerebellar cultures were pretreated with 10mM EtOH (circulating concentration after ~ 2 drinks), 5μM RES, EtOH + RES combinatorially, or media alone (controls). After 3days, media were removed, and fresh media aliquots containing Aβ25-35 (25μM) were added. Assessing apoptosis 24h later with Hoescht 33342, neurodegeneration did not differ from controls in cultures separately preconditioned with 10mM EtOH or 5μM RES. However, apoptosis was prevented in combinatorially preconditioned cultures. Also, immunoblotting revealed elevated Prx2 levels due to combinatorial pretreatment that correlated with subsequent neuroprotection, whereas Prx2 was unchanged in separately pretreated cultures. Although the protective mechanisms require clarification, synergistically upregulated NMDAR-PKC-Prx2 (and other antioxidant proteins) is a reasonable component. These findings imply that EtOH + RES antioxidant synergy could be involved in neurobenefits attributed to low-moderate wine consumption.

N-Acetyl-L-cysteine in the Presence of Cu2+ Induces Oxidative Stress and Death of Granule Neurons in Dissociated Cultures of Rat Cerebellum.

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1mM) in the presence of Cu2+ (0.0005-0.001mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn2+ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn2+ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4h with Cu2+ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu2+. Incubation of cultures in a balanced salt solution in the presence of 0.01mM Cu2+ caused neuronal death already after 1h if the NAC concentration in the solution was within 0.005-0.05mM. NAC at higher concentrations (0.1-1mM) together with 0.01mM Cu2+ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.

Bisphenol A influences oestrogen- and thyroid hormone-regulated thyroid hormone receptor expression in rat cerebellar cell culture.

Thyroid hormones (THs) and oestrogens are crucial in the regulation of cerebellar development. TH receptors (TRs) mediate these hormone effects and are regulated by both hormone families. We reported earlier that THs and oestradiol (E2) determine TR levels in cerebellar cell culture. Here we demonstrate the effects of low concentrations (10-10 M) of the endocrine disruptor (ED) bisphenol A (BPA) on the hormonal (THs, E2) regulation of TRα,β in rat cerebellar cell culture. Primary cerebellar cell cultures, glia-containing and glia-destroyed, were treated with BPA or a combination of BPA and E2 and/or THs. Oestrogen receptor and TH receptor mRNA and protein levels were determined by real-time qPCR and Western blot techniques. The results show that BPA alone decreases, while BPA in combination with THs and/or E2 increases TR mRNA expression. In contrast, BPA alone increased receptor protein expressions, but did not further increase them in combination with THs and/or E2. The modulatory effects of BPA were mediated by the glia; however, the degree of changes also depended on the specific hormone ligand used. The results signify the importance of the regulatory mechanisms interposed between transcription and translation and raise the possibility that BPA could act to influence nuclear hormone receptor levels independently of ligand-receptor interaction.

Ethanol preconditioning of rat cerebellar cultures targets NMDA receptors to the synapse and enhances peroxiredoxin 2 expression

Epidemiological studies indicate that light-moderate alcohol (ethanol) consumers tend to have reduced risks of cognitive impairment and progression to dementia during aging. Exploring possible mechanisms, we previously found that moderate ethanol preconditioning (MEP, 20–30mM) of rat brain cultures for several days instigated neuroprotection against β-amyloid peptides. Our biochemical evidence implicated the NMDA receptor (NMDAR) as a potential neuroprotective “sensor”, specifically via synaptic NMDAR signaling. It remains unclear how ethanol modulates the receptor and its downstream targets to engender neuroprotection. Here we confirm with deconvolution microscopy that MEP of rat mixed cerebellar cultures robustly increases synaptic NMDAR localization. Phospho-activation of the non-receptor tyrosine kinases Src and Pyk2, known to be linked to synaptic NMDAR, is also demonstrated. Additionally, the preconditioning enhances levels of an antioxidant protein, peroxiredoxin 2 (Prx2), reported to be downstream of synaptic NMDAR signaling, and NMDAR antagonism with memantine (earlier found to abrogate MEP neuroprotection) blocks the Prx2 elevations. To further link Prx2 with antioxidant-based neuroprotection, we circumvented the ethanol preconditioning-NMDAR pathway by pharmacologically increasing Prx2 with the naturally-occurring cruciferous compound, 3H-1,2-dithiole-3-thione (D3T). Thus, D3T pretreatment elevated Prx2 expression to a similar extent as MEP, while concomitantly preventing β-amyloid neurotoxicity; D3T also protected the cultures from hydrogen peroxide toxicity. The findings support a mechanism that couples synaptic NMDAR signaling, Prx2 expression and augmented antioxidant defenses in ethanol preconditioning-induced neuroprotection. That this mechanism can be emulated by a cruciferous vegetable constituent suggests that such naturally-occurring “neutraceuticals” may be useful in therapy for oxidative stress-related dementias.

A new procedure for amyloid β oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca2+ entry and cell death in primary neurons

Oligomers of the amyloid β peptide (Aβo) are becoming the most likely neurotoxin in Alzheimer’s disease. Controversy remains on the mechanisms involved in neurotoxicity induced by Aβo and the targets involved. We have reported that Aβo promote Ca2+ entry, mitochondrial Ca2+ overload and apoptosis in cultured cerebellar neurons. However, recent evidence suggests that some of these effects could be induced by glutamate receptor agonists solved in F12, the media in which Aβo are prepared. Here we have tested the effects of different media on Aβo formation and on cytosolic Ca2+ concentration ([Ca2+]cyt) in rat cerebellar and hippocampal cell cultures. We found that Aβo prepared according to previous protocols but solved in alternative media including saline, MEM and DMEM do not allow oligomer formation and fail to increase [Ca2+]cyt. Changes in the oligomerization protocol and supplementation of media with selected salts reported to favor oligomer formation enable Aβo formation. Aβo prepared by the new procedure and containing small molecular weight oligomers increased [Ca2+]cyt, promoted mitochondrial Ca2+ overload and cell death in cerebellar granule cells and hippocampal neurons. These results foster a role for Ca2+ entry in neurotoxicity induced by Aβo and provide a reliable procedure for investigating the Ca2+ entry pathway promoted by Aβo.

Activity-Dependent Neurotrophin Signaling Underlies Developmental Switch of Ca2+Channel Subtypes Mediating Neurotransmitter Release

At the nerve terminal, neurotransmitter release is triggered by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs). During postnatal development, VGCC subtypes in the nerve terminal switch at many synapses. In immature rodent cerebella, N-type and P/Q-type VGCCs mediate GABAergic neurotransmission from Purkinje cells (PCs) to deep nuclear cells, but as animals mature, neurotransmission becomes entirely P/Q-type dependent. We reproduced this developmental switch in rat cerebellar slice culture to address the underlying mechanism. Chronic block of cerebellar neuronal activity with tetrodotoxin (TTX) in slice culture, or in vivo, reversed the switch, leaving neurotransmission predominantly N-type channel-dependent. Brain-derived neurotrophic factor or neurotrophin-4 rescued this TTX effect, whereas pharmacological blockade of neurotrophin receptors mimicked the TTX effect. In PC somata, unlike in presynaptic terminals, TTX had no effect on the proportion of Ca(2+) channel subtype currents. We conclude that neuronal activity activates the neurotrophin-TrkB signaling pathway, thereby causing the N-to-P/Q channel switch in presynaptic terminals.

Comparative neuronal uptake and cytotoxicity of anti-Hu and anti-Ri antibodies in rat cerebellar and hippocampal slice cultures

Background: Anti-Hu and anti-Ri are paraneoplastic autoantibodies recognizing intracellular antigens present in essentially all neurons. At autopsy, brains of patients with anti-Hu antibody show neuronal destruction. In contrast, anti-Ri antibody is less clearly associated with neuronal death, and patients with anti-Ri antibodies may respond to treatment. We have demonstrated that anti-Yo antibodies, associated with paraneoplastic cerebellar degeneration, accumulate intracellularly in cerebellar Purkinje cells in slice cultures of rat cerebellum and that antibody accumulation is followed by cell death.

Conserved cellular distribution of the glutamate receptors GluA2/3, mGlu1a and mGlu2/3 in isolated cultures of rat cerebellum

To what extent the intrinsic glutamatergic system of the cerebellum is able to keep normal features in the absence of mossy and climbing fibres, is at present not known. To answer this question, immunocytochemistry for light and high resolution electron microscopy was used to reveal the cellular and subcellular distribution of glutamate receptors in isolated cerebellar cultures. The localization of the ionotropic glutamate receptor subunits GluA2/3 and the metabotropic glutamate (mGlu) 1a and mGlu2/3 receptor subtypes was carried out in 0 to 9-day-old rat parasagittal slices developed in vitro for 20–40 days.The typical localization of GluA2/3, mGlu1a and mGlu2/3 observed in Purkinje cells, granule cells, Golgi cells and unipolar brush cells was maintained in the organotypic cultures. Furthermore, the subcellular distribution of mGlu1a showed the characteristic in vivo perisynaptic position in Purkinje cell dendritic spines receiving parallel fibre synapses.We conclude that the cellular and subcellular localization of the studied ionotropic and metabotropic glutamate receptors is not affected by the removal of the two extrinsic cerebellar glutamatergic inputs, the mossy and climbing fibres.

Suppression of thyroid hormone receptor-mediated transcription and disruption of thyroid hormone-induced cerebellar morphogenesis by the polybrominated biphenyl mixture, BP-6

Polybrominated biphenyls (PBBs) are polyhalogenated, bioaccumulative flame retardant chemicals, which have been used in a variety of consumer and household products. They were accidentally introduced into the food chain in Michigan in 1973 and have remained a source of health concern. Studies have shown that exposure to PBB may cause adverse neurotoxic effects. We therefore examined the effects of BP-6, a PBB mixture, on thyroid hormone (TH) receptor (TR)-mediated transcription, on TH-induced Purkinje cell dendritogenesis, and on TH-induced cerebellar granule cell neurite extension. Our study shows that BP-6 suppressed TR-mediated transcription in CV-1 cells. Mammalian two-hybrid studies revealed that BP-6 did not inhibit coactivator binding to TR nor did it recruit corepressors to TR. Further examination using the liquid chemiluminescent DNA pull down assay revealed partial dissociation of TR from TH response element (TRE). In primary rat cerebellar culture, BP-6 significantly suppressed TH-induced dendrite arborization of Purkinje cells, and in reaggregate rat granule cell culture, impaired TH-induced neurite extension of granule cells. Taken together, our results indicate that BP-6 may disrupt TH homeostasis and consequently impair normal neuronal development.

Growth factors deprivation induces a specific increase in PAR2 receptor mRNA expression in primary cerebellar cultures

We studied changes in the mRNA expression of protease receptors (PAR-1, PAR-2, PAR-3, and PAR-4) in primary neuroglial and glial cultures of rat cerebellum after a one hour deprivation of culture medium. Replacement of culture medium by saline induced an increase in the mRNA level of PAR-2 in cultures of both types, whereas the mRNA level of other receptors did not change. The maximum expression of PAR-2 was observed in 3 h after the deprivation and in 8 h PAR-2 mRNA returned to the basal level. Incubation of neuroglial cultures in the presence of PAR-2 agonist trypsin (10–100 nM) did not affect expression of PAR-2, however, it induced a specific decrease in the mRNA level of PAR-4. Replacement of trypsin by cathepsin B, which has substrate specificity that is similar to trypsin, did not have this effect. Thus, growth factor deprivation and treatment with trypsin had opposite effects on the mRNA expression of “trypsin” receptors, which may be related to functional role of PAR receptors in the brain.

Neuroprotective preconditioning of rat brain cultures with ethanol: potential transduction by PKC isoforms and focal adhesion kinase upstream of increases in effector heat shock proteins

Preconditioning rat hippocampal-entorhinocortical (HEC) slice or cerebellar cell cultures with moderate concentrations of ethanol (20-30 mm) neuroprotects against pro-inflammatory proteins such as HIV-1 glycoprotein 120 (gp120) or amyloid-β. The neuroprotective mechanism of ethanol is unclear, but it conceivably involves sensors→transducers→effectors, analogous to other preconditioning modalities. We initially found that the preconditioning augmented two likely heat shock protein (HSP) 'effectors', HSP70 and HSP27, and that precluding HSP upregulation abolished neuroprotection. Here we examined whether pro-survival kinases are transducers potentially leading to HSP effectors. In cerebellar cultures, protein kinase C (PKC) activity increased modestly after 2 days of 30 mm ethanol and was significantly induced after 6 days, when neuroprotection against gp120 becomes manifest. After 4 and particularly after 6 days of preconditioning, immunoblots showed highly elevated PKCε levels and moderately increased PKCα and PKCδ, accompanied by increased membrane translocation (activation) of these isoforms. Also, at the latter preconditioning duration, focal adhesion kinase (FAK), an important actin-associated kinase, and its Y397-phosphorylated form (p-FAK) were elevated, along with parallel increases in HSP27, S85p-HSP27 and HSP70. Furthermore, while confirming increased HSP27 and HSP70 in HEC slices ethanol-preconditioned for 6 days, we detected elevations in PKC isoforms, FAK, p-FAK and p-HSP27 in these organotypic cultures. Importantly, PKC inhibition with GF109203X suppressed FAK, HSP70 and HSP27 amplification/activation in ethanol-preconditioned cerebellar cultures, indicating that PKC is an upstream transducer of FAK and the HSP effectors. Neuroprotection associated with increases in HSP27/HSP70 from ethanol preconditioning entails upregulation/activation of PKC isoforms and FAK, the latter kinase implicating actin cytoskeletal prosurvival pathways in brain preconditioning.

Developmental neurotoxicants affect neuronal and glial differentiation and maturation in primary culture of rat cerebellum

Developmental neurotoxicants affect neuronal and glial differentiation and maturation in primary culture of rat cerebellum

Proteins conjugated to poly(butyl cyanoacrylate) nanoparticles as potential neuroprotective agents

Poly(butyl cyanoacrylate) (PBCA) nanoparticles (NPs) can penetrate blood-brain barrier providing the means for drug delivery to the central nervous system. Here, we study attachment of superoxide dismutase (SOD) and anti-glutamate N-methyl D-aspartate receptor 1 (NR1) antibody to PBCA NPs with the ultimate goal to design neuroprotective therapeutics for treatment of secondary spinal cord injury. Synthesis of monodispersed, ∼200 nm-diameter PBCA NPs was performed using polymerization at pH 2.0 with Dextran 70,000 as the stabilizer. Sulfo-HSAB spacers were used to covalently attach SOD and NR1 antibodies to the dextran-coated NPs. The prepared protein-NP conjugates possessed SOD activity and were capable of binding to rat cerebellar neurons. Thus, SOD and NR1 antibodies may be simultaneously attached to PBCA NPs while retaining at least a fraction of enzymatic activity and receptor-binding ability. The conjugates showed neuroprotective efficacy in vitro with rat cerebellar cell cultures challenged by superoxide.

Purkinje Cell Death After Uptake of Anti-Yo Antibodies in Cerebellar Slice Cultures

Paraneoplastic cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic cerebellar degeneration.