James H. Eubanks

Sirtuin 3 rescues neurons through the stabilisation of mitochondrial biogenetics in the virally-expressing mutant α-synuclein rat model of parkinsonism

Parkinsons disease (PD) is a neurodegenerative movement disorder, which affects approximately 1-2% of the population over 60years of age. Current treatments for PD are symptomatic, and the pathology of the disease continues to progresses over time until palliative care is required. Mitochondria are key players in the pathology of PD. Genetic and post mortem studies have shown a large number of mitochondrial abnormalities in the substantia nigra pars compacta (SNc) of the parkinsonian brain. Furthermore, physiologically, mitochondria of nigral neurons are constantly under unusually high levels of metabolic stress because of the excitatory properties and architecture of these neurons. The protein deacetylase, Sirtuin 3 (SIRT3) reduces the impact subcellular stresses on mitochondria, by stabilising the electron transport chain (ETC), and reducing oxidative stress. We hypothesised that viral overexpression of myc-tagged SIRT3 (SIRT3-myc) would slow the progression of PD pathology, by enhancing the functional capacity of mitochondria. For this study, SIRT3-myc was administered both before and after viral induction of parkinsonism with the AAV-expressing mutant (A53T) α-synuclein. SIRT3-myc corrected behavioural abnormalities, as well as changes in striatal dopamine turnover. SIRT3-myc also prevented degeneration of dopaminergic neurons in the SNc. These effects were apparent, even when SIRT3-myc was transduced after the induction of parkinsonism, at a time point when cell stress and behavioural abnormalities are already observed. Furthermore, in an isolated mitochondria nigral homogenate prepared from parkinsonian SIRT3-myc infected animals, SIRT3 targeted the mitochondria, to reduce protein acetylation levels. Our results demonstrate that transduction of SIRT3 has the potential to be an effective disease-modifying strategy for patients with PD. This study also provides potential mechanisms for the protective effects of SIRT3-myc.

Three distinct neuronal phenotypes exist in embryonic rat hippocampal neurons cultured in basic fibroblast growth factor.

The possibility that neurons cultured in basic fibroblast growth factor (bFGF) are heterogeneous raises concerns about their subsequent use in gene transfection and transplantation studies. We have examined the fate of embryonic hippocampal neurons in bFGF culture, and now conclude that these cells are not only heterogeneous, but possess neurons of various stages of development. Morphological and immunocytochemical analysis reveal three distinct developmental classes of neurons are present in extended bFGF culture. This tripartite classification is supported by electrophysiological analysis, which reveals that upon depolarization, neurons with precursor and juvenile neuron morphologies are unable to fire action potentials. The third class of neurons, which resemble age-matched polarized neurons in standard serum culture, fired multiple action potentials indicative of a mature phenotype. These data show neurons at multiple developmental stages co-exist in bFGF culture, and provide an experimental basis for their classification.

ALA-PpIX mediated photodynamic therapy of malignant gliomas augmented by hypothermia

Background Malignant gliomas are highly invasive, difficult to treat, and account for 2% of cancer deaths worldwide. Glioblastoma Multiforme (GBM) comprises the most common and aggressive intracranial tumor. The study hypothesis is to investigate the modification of Photodynamic Therapy (PDT) efficacy by mild hypothermia leads to increased glioma cell kill while protecting normal neuronal structures. Methods Photosensitizer accumulation and PDT efficacy in vitro were quantified in various glioma cell lines, primary rat neurons, and astrocytes. In vivo studies were carried out in healthy brain and RG2 glioma of naïve Fischer rats. Hypothermia was induced at 1 hour pre- to 2 hours post-PDT, with ALA-PpIX accumulation and PDT treatments effects on tumor and normal brain PDT quantified using optical spectroscopy, histology, immunohistochemistry, MRI, and survival studies, respectively. Findings In vitro studies demonstrated significantly improved post-PDT survival in primary rat neuronal cells. Rat in vivo studies confirmed a neuroprotective effect to hypothermia following PpIX mediated PDT by T2 mapping at day 10, reflecting edema/inflammation volume reduction. Mild hypothermia increased PpIX fluorescence in tumors five-fold, and the median post-PDT rat survival time (8.5 days normothermia; 14 days hypothermia). Histology and immunohistochemistry show close to complete cellular protection in normal brain structures under hypothermia. Conclusions The benefits of hypothermia on both normal neuronal tissue as well as increased PpIX fluorescence and RG2 induced rat survival strongly suggest a role for hypothermia in photonics-based surgical techniques, and that a hypothermic intervention could lead to considerable patient outcome improvements.

Design and Synthesis of Mercaptoacetamides as Potent, Selective, and Brain Permeable Histone Deacetylase 6 Inhibitors

A series of nonhydroxamate HDAC6 inhibitors were prepared in our effort to develop potent and selective compounds for possible use in central nervous system (CNS) disorders, thus obviating the genotoxicity often associated with the hydroxamates. Halogens are incorporated in the cap groups of the designed mercaptoacetamides in order to increase brain accessibility. The indole analogue 7e and quinoline analogue 13a displayed potent HDAC6 inhibitory activity (IC50, 11 and 2.8 nM) and excellent selectivity against HDAC1. Both 7e and 13a together with their ester prodrug 14 and disulfide prodrugs 15 and 16 were found to be effective in promoting tubulin acetylation in HEK cells. The disulfide prodrugs 15 and 16 also released a stable concentration of 7e and 13a upon microsomal incubation. Administration of 15 and 16in vivo was found to trigger an increase of tubulin acetylation in mouse cortex. These results suggest that further exploration of these compounds for the treatment of CNS disorders is warranted.

Long-term bFGF neuronal culture: reintroduction into serum medium yields neurons and non-neuronal cells with neuronal characteristics

The potential use of bFGF immortalized cells as hosts for delivering foreign genes into nervous tissue led us to examine the effect of maintaining, E-18 hippocampal neurons for extended periods in bFGF culture prior to transfer into a standard, serum-containing, medium. We found: (1) many, if not most, precursors seen in bFGF, mature into glia and not into primary neurons after medium exchange; (2) the electrophysiology of the neurons which do mature after medium transfer and replating, is similar to that of neurons in standard cultures; (3) extended culture in bFGF prior to cell harvesting and replating into standard medium generates neurons from the precursors that possess proper neuronal polarization, morphology, and electrophysiology; and (4) extended bFGF also induces the expression, on transfer into standard medium, of an additional cell type with a distinct non-neuronal morphology that stains with the neuronal marker MAP-2. These results illustrate the need for additional characterization of long-term growth factor effects on maintained progenitor cells prior to their use in gene therapy and transplantation.

Effects of Antiepileptic Drugs on Spontaneous Recurrent Seizures in a Novel Model of Extended Hippocampal Kindling in Mice

Epilepsy is a common neurological disorder characterized by naturally-occurring spontaneous recurrent seizures and comorbidities. Kindling has long been used to model epileptogenic mechanisms and to assess antiepileptic drugs. In particular, extended kindling can induce spontaneous recurrent seizures without gross brain lesions, as seen clinically. To date, the development of spontaneous recurrent seizures following extended kindling, and the effect of the antiepileptic drugs on these seizures are not well understood. In the present study we aim to develop a mouse model of extended hippocampal kindling for the first time. Once established, we plan to evaluate the effect of three different antiepileptic drugs on the development of the extended-hippocampal-kindled-induced spontaneous recurrent seizures. Male C57 black mice were used for chronic hippocampal stimulations or handling manipulations (twice daily for up to 70 days). Subsequently, animals underwent continuous video/EEG monitoring for seizure detection. Spontaneous recurrent seizures were consistently observed in extended kindled mice but no seizures were detected in the control animals. The aforementioned seizures were generalized events characterized by hippocampal ictal discharges and concurrent motor seizures. Incidence and severity of the seizures was relatively stable while monitored over a few months after termination of the hippocampal stimulation. Three antiepileptic drugs with distinct action mechanisms were tested: phenytoin, lorazepam and levetiracetam. They were applied via intra-peritoneal injections at anticonvulsive doses and their effects on the spontaneous recurrent seizures were analyzed 10–12 h post-injection. Phenytoin (25 mg/kg) and levetiracetam (400 mg/kg) abolished the spontaneous recurrent seizures. Lorazepam (1.5 mg/kg) decreased motor seizure severity but did not reduce the incidence and duration of corresponding hippocampal discharges, implicating its inhibitory effects on seizure spread. No gross brain lesions were observed in a set of extended hippocampal kindled mice submitted to histological evaluation. All these data suggests that our model could be considered as a novel mouse model of extended hippocampal kindling. Some limitations remain to be considered.

Continuous Monitoring via Tethered Electroencephalography of Spontaneous Recurrent Seizures in Mice

We describe here a simple, cost-effective apparatus for continuous tethered electroencephalographic (EEG) monitoring of spontaneous recurrent seizures in mice. We used a small, low torque slip ring as an EEG commutator, mounted the slip ring onto a standard mouse cage and connected rotary wires of the slip ring directly to animals implanted headset. Modifications were made in the cage to allow for a convenient installation of the slip ring and accommodation of animal ambient activity. We tested the apparatus for hippocampal EEG recordings in adult C57 black mice. Spontaneous recurrent seizures were induced using extended hippocampal kindling (≥95 daily stimulation). Control animals underwent similar hippocampal electrode implantations but no stimulations were given. Combined EEG and webcam monitoring were performed for 24 h daily for 5–9 consecutive days. During the monitoring periods, the animals moved and accessed water and food freely and showed no apparent restriction in ambient cage activities. Ictal-like hippocampal EEG discharges and concurrent convulsive behaviors that are characteristics of spontaneous recurrent seizures were reliably recorded in a majority of the monitoring experiments in extendedly kindled but not in control animals. However, 1–2 rotary wires were disconnected from the implanted headset in some animals after continuous recordings for ≥5 days. The key features and main limitations of our recording apparatus are discussed.

Corrigendum to “Sirtuin 3 rescues neurons through the stabilisation of mitochondrial biogenetics in the virally-expressing mutant α-synuclein rat model of parkinsonism” [Neurobiol. Dis. 106 (2018) 133–146]

Corrigendum to “Sirtuin 3 rescues neurons through the stabilisation of mitochondrial biogenetics in the virally-expressing mutant α-synuclein rat model of parkinsonism” [Neurobiol. Dis. 106 (2018) 133–146] Jacqueline A. Gleave, Lindsay R. Arathoon, Dennison Trinh, Kristin E. Lizal, Nicolas Giguère, James E.M. Barber, Zainab Najarali, M. Hassan Khan, Sherri L. Thiele, Mahin S. Semmen, James B. Koprich, Jonathan M. Brotchie, James H. Eubanks, Louis-Eric Trudeau, Joanne E. Nasha,

mRNA expression of transient receptor potential melastatin (TRPM) channels 2 and 7 in perinatal brain development

TRPM7 and TRPM2 are non‐specific cation channels of the Transient Receptor Potential channel superfamily. Each channel has gained attention for their potential to mediate oxidative and anoxic cell death (Rama and García, 2016; Nazıroğlu, 2011a; Abiria et al., 2017; Sun, 2017), however their physiological expression and roles in the developing brain remain poorly defined. We employed real‐time reverse transcription PCR to examine mRNA expression of TRPM7 and TRPM2 in the developing rat brain and brain‐specific cell types. We determined the temporal and spatial expression patterns at four developmental time points (postnatal day 7, 14, 21, and 90) in four critical regions of the brain (cortex, hippocampus, striatum, and cerebellum) and examined gene expression in neuronal, astrocytic, and microglial primary cell cultures. Our results revealed that TRPM7 mRNA expression peaks in the cortex at 2‐weeks after birth, and thus correlates most closely with a period of rat brain development associated with neurite outgrowth, which is heightened at 2‐weeks after birth. Our cell‐specific gene expression assays revealed that TRPM7 was expressed at equivalent levels in neurons, astrocytes, and microglia. Conversely, TRPM2 was most highly expressed in microglia with little expression in neurons and astrocytes. In the hippocampus and striatum, the expression profile of TRPM2 parallels the perinatal expression timeline for microglial infiltration and maturation in the rat brain. Microglial maturation is highest from the time of birth, up to 7‐days, but subsequently declines. The latter developmental expression profiles indicate a role for TRPM2 in microglial activation.

Contributions of the Hippocampal CA3 Circuitry to Acute Seizures and Hyperexcitability Responses in Mouse Models of Brain Ischemia

The hippocampal circuitry is widely recognized as susceptible to ischemic injury and seizure generation. However, hippocampal contribution to acute non-convulsive seizures (NCS) in models involving middle cerebral artery occlusion (MCAO) remains to be determined. To address this, we occluded the middle cerebral artery in adult C57 black mice and monitored electroencephalographic (EEG) discharges from hippocampal and neocortical areas. Electrographic discharges in the absence of convulsive motor behaviors were observed within 90 min following occlusion of the middle cerebral artery. Hippocampal discharges were more robust than corresponding cortical discharges in all seizure events examined, and hippocampal discharges alone or with minimal cortical involvement were also observed in some seizure events. Seizure development was associated with ipsilateral hippocampal injuries as determined by subsequent histological examinations. We also introduced hypoxia-hypoglycemia episodes in mouse brain slices and examined regional hyperexcitable responses ex vivo. Extracellular recordings showed that the hippocampal CA3 region had a greater propensity for exhibiting single/multiunit activities or epileptiform field potentials following hypoxic-hypoglycemic (HH) episodes compared to the CA1, dentate gyrus, entorhinal cortical (EC) or neocortical regions. Whole-cell recordings revealed that CA3 pyramidal neurons exhibited excessive excitatory postsynaptic currents, attenuated inhibitory postsynaptic currents and intermittent or repetitive spikes in response to HH challenge. Together, these observations suggest that hippocampal discharges, possibly as a result of CA3 circuitry hyperexcitability, are a major component of acute NCS in a mouse model of MCAO.