Mitochondrial brain proteome acetylation levels and behavioural responsiveness to amphetamine are altered in mice lacking Sirt3.

Post-translational modification of mitochondrial proteins represents one mechanism by which the functional activity of mitochondria can be regulated. In the brain, these modifications can influence the functional properties of different neural circuitries. Given that the sirtuin family member Sirt3 represents the primary protein deacetylase enzyme in mitochondria, we tested whether brain mitochondrial proteome acetylation would increase in male or female mice lacking Sirt3. Our results confirm that whole brain mitochondrial proteome acetylation levels are indeed elevated in both sexes of Sirt3-KO mice relative to controls. Consistently, we found the mitochondria of mouse embryonic fibroblast (MEF) cells derived from Sirt3-KO mice were smaller in size, and fewer in number than in wild-type MEFs, and that mitochondrial free calcium levels were elevated within the mitochondria of these cells. As protein acetylation can influence mitochondrial function, and changes in mitochondrial function have been linked to alterations in neural circuit function regulating motor activity and anxiety-like behavior, we tested whether Sirt3-deficient mice would display sensitized responsiveness to the stimulant amphetamine. Both male and female Sirt3-KO mice displayed hyper-locomotion and attenuated anxiety-like behavior in response to a dose of amphetamine that was insufficient to promote any behavioural responses in wild-type mice. Collectively, these results confirm that Sirt3 regulates mitochondrial proteome acetylation levels in brain tissue, and that the absence of Sirt3 increases the sensitivity of neural systems to amphetamine-induced behavioural responses.

The Role of SIRT3 in the Brain Under Physiological and Pathological Conditions.

Sirtuin enzymes are a family of highly seven conserved protein deacetylases, namely SIRT1 through SIRT7, whose enzymatic activities require the cofactor nicotinamide adenine dinucleotide (NAD+). Sirtuins reside in different compartments within cells, and their activities have been shown to regulate a number of cellular pathways involved in but not limited to stress management, apoptosis and inflammatory responses. Given the importance of mitochondrial functional state in neurodegenerative conditions, the mitochondrial SIRT3 sirtuin, which is the primary deacetylase within mitochondria, has garnered considerable recent attention. It is now clear that SIRT3 plays a major role in regulating a host of mitochondrial molecular cascades that can contribute to both normal and pathophysiological processes. However, most of the currently available knowledge on SIRT3 stems from studies in non-neuronal cells, and the consequences of the interactions between SIRT3 and its targets in the CNS are only beginning to be elucidated. In this review, we will summarize current advances relating to SIRT3, and explore how its known functions could influence brain physiology.

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; Naziroglu, 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.

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.

A Role for Diminished GABA Transporter Activity in the Cortical Discharge Phenotype of MeCP2-Deficient Mice.

Cortical network hyper-excitability is a common phenotype in mouse models lacking the transcriptional regulator methyl-CPG-binding protein 2 (MeCP2). Here, we implicate enhanced GABAB receptor activity stemming from diminished cortical expression of the GABA transporter GAT-1 in the genesis of this network hyper-excitability. We found that administering the activity-dependent GABAB receptor allosteric modulator GS-39783 to female Mecp2(+/-) mice at doses producing no effect in wild-type mice strongly potentiated their basal rates of spontaneous cortical discharge activity. Consistently, administering the GABAB receptor antagonist CGP-35348 significantly decreased basal discharge activity in these mice. Expression analysis revealed that while GABAB or extra-synaptic GABAA receptor prevalence is preserved in the MeCP2-deficient cortex, the expression of GAT-1 is significantly reduced from wild-type levels. This decrease in GAT-1 expression is consequential, as low doses of the GAT-1 inhibitor NO-711 that had no effects in wild-type mice strongly exacerbated cortical discharge activity in female Mecp2(+/-) mice. Taken together, these data indicate that the absence of MeCP2 leads to decreased cortical levels of the GAT-1 GABA transporter, which facilitates cortical network hyper-excitability in MeCP2-deficient mice by increasing the activity of cortical GABAB receptors.

Over-expression of the Sirt3 sirtuin Protects neuronally differentiated PC12 Cells from degeneration induced by oxidative stress and trophic withdrawal.

Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45kDa pro-form, which localized primarily within the cytosol, and a 30kDa processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen-glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.

Differential expression of sirtuin family members in the developing, adult, and aged rat brain.

The sirtuins are NAD(+)-dependent protein deacetylases and/or ADP-ribosyltransferases that play roles in metabolic homeostasis, stress response and potentially aging. This enzyme family resides in different subcellular compartments, and acts on a number of different targets in the nucleus, cytoplasm and in the mitochondria. Despite their recognized ability to regulate metabolic processes, the roles played by specific sirtuins in the brain-the most energy demanding tissue in the body-remains less well investigated and understood. In the present study, we examined the regional mRNA and protein expression patterns of individual sirtuin family members in the developing, adult, and aged rat brain. Our results show that while each sirtuin is expressed in the brain at each of these different stages, they display unique spatial and temporal expression patterns within the brain. Further, for specific members of the family, the protein expression profile did not coincide with their respective mRNA expression profile. Moreover, using primary cultures enriched for neurons and astrocytes respectively, we found that specific sirtuin members display preferential neural lineage expression. Collectively, these results provide the first composite illustration that sirtuin family members display differential expression patterns in the brain, and provide evidence that specific sirtuins could potentially be targeted to achieve cell-type selective effects within the brain.