Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42, during which time global cellular reactive oxygen species increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ.

Alzheimer's disease (AD) is the most common type of dementia. Amyloid β (Aβ) plaques, tau-containing neurofibrillary tangles, and neuronal loss leading to brain atrophy are pathologic hallmarks of AD. Given the importance of early diagnosis, extensive efforts have been undertaken to identify diagnostic and prognostic biomarkers for AD. Circulating extracellular vesicles (EVs) provide a platform for "liquid biopsy" biomarkers for AD. Here, we characterized the RNA contents of plasma EVs of age-matched individuals who were cognitively normal (healthy controls (HC)) or had mild cognitive impairment (MCI) due to AD or had mild AD dementia (AD). Using RNA sequencing analysis, we found that mitochondrial (mt)-RNAs, including MT-ND1-6 mRNAs and other protein-coding and non-coding mt-RNAs, were strikingly elevated in plasma EVs of MCI and AD individuals compared with HC. EVs secreted from cultured astrocytes, microglia, and neurons after exposure to toxic conditions relevant to AD pathogenesis (Aβ aggregates and H2O2), contained mitochondrial structures (detected by electron microscopy) and mitochondrial RNA and protein. We propose that in the AD brain, toxicity-causing mitochondrial damage results in the packaging of mitochondrial components for export in EVs and further propose that mt-RNAs in plasma EVs can be diagnostic and prognostic biomarkers for MCI and AD.

Information processing and storage in the brain rely on AMPA-receptors (AMPARs) and their context-dependent dynamics in synapses and extra-synaptic sites. We found that distribution and dynamics of AMPARs in the plasma membrane are controlled by Noelins, a three-member family of conserved secreted proteins expressed throughout the brain in a cell-type-specific manner. Noelin tetramers tightly assemble with the extracellular domains of AMPARs and interconnect them in a network-like configuration with a variety of secreted and membrane-anchored proteins including Neurexin1, Neuritin1, and Seizure 6-like. Knock out of Noelins1-3 profoundly reduced AMPARs in synapses onto excitatory and inhibitory (inter)neurons, decreased their density and clustering in dendrites, and abolished activity-dependent synaptic plasticity. Our results uncover an endogenous mechanism for extracellular anchoring of AMPARs and establish Noelin-organized networks as versatile determinants of constitutive and context-dependent neurotransmission.

Olfactomedin 1 (Olfm1) is a secreted glycoprotein that is preferentially expressed in neuronal tissues. Here we show that deletion of exons 4 and 5 from the Olfm1 gene, which encodes a 52 amino acid long region in the N-terminal part of the protein, increased neonatal death and reduced body weight of surviving homozygous mice. Magnetic resonance imaging analyses revealed reduced brain volume and attenuated size of white matter tracts such as the anterior commissure, corpus callosum, and optic nerve. Adult Olfm1 mutant mice demonstrated abnormal behavior in several tests including reduced marble digging, elevated plus maze test, nesting activity and latency on balance beam tests as compared with their wild-type littermates. The olfactory system was both structurally and functionally disturbed by the mutation in the Olfm1 gene as shown by functional magnetic resonance imaging analysis and a smell test. Deficiencies of the olfactory system may contribute to the neonatal death and loss of body weight of Olfm1 mutant. Shotgun proteomics revealed 59 candidate proteins that co-precipitated with wild-type or mutant Olfm1 proteins in postnatal day 1 brain. Olfm1-binding targets included GluR2, Cav2.1, teneurin-4 and Kidins220. Modified interaction of Olfm1 with binding targets led to an increase in intracellular Ca(2+) concentration and activation of ERK1/2, MEK1 and CaMKII in the hippocampus and olfactory bulb of Olfm1 mutant mice compared with their wild-type littermates. Excessive activation of the CaMKII and Ras-ERK pathways in the Olfm1 mutant olfactory bulb and hippocampus by elevated intracellular calcium may contribute to the abnormal behavior and olfactory activity of Olfm1 mutant mice.

It has been reported that dietary energy restriction, including intermittent fasting (IF), can protect heart and brain cells against injury and improve functional outcome in animal models of myocardial infarction (MI) and stroke. Here we report that IF improves glycemic control and protects the myocardium against ischemia-induced cell damage and inflammation in rats. Echocardiographic analysis of heart structural and functional variables revealed that IF attenuates the growth-related increase in posterior ventricular wall thickness, end systolic and diastolic volumes, and reduces the ejection fraction. The size of the ischemic infarct 24 h following permanent ligation of a coronary artery was significantly smaller, and markers of inflammation (infiltration of leukocytes in the area at risk and plasma IL-6 levels) were less, in IF rats compared to rats on the control diet. IF resulted in increased levels of circulating adiponectin prior to and after MI. Because recent studies have shown that adiponectin can protect the heart against ischemic injury, our findings suggest a potential role for adiponectin as a mediator of the cardioprotective effect of IF.

Olfactomedin 2 (Olfm2) is a secretory glycoprotein belonging to the family of olfactomedin domain-containing proteins. A previous study has shown that a mutation in OLFM2 is associated with primary open angle glaucoma in Japanese patients. In the present study, we generated Olfm2 deficient mice by replacing the Olfm2 gene with the LacZ gene. The loss of Olfm2 resulted in no gross abnormalities. However, Olfm2 null mice showed reduced exploration, locomotion, olfactory sensitivity, abnormal motor coordination, and anxiety related behavior. The pattern of the Olfm2 gene expression was studied in the brain and eye using β-galactosidase staining. In the brain, Olfm2 was mainly expressed in the olfactory bulb, cortex, piriform cortex, olfactory trabeculae, and inferior and superior colliculus. In the eye expression was detected mainly in retinal ganglion cells. In Olfm2 null mice, the amplitude of the first negative wave in the visual evoked potential test was significantly reduced as compared with wild-type littermates. Olfm2, similar to Olfm1, interacted with the GluR2 subunit of the AMPAR complexes and Olfm2 co-segregated with the AMPA receptor subunit GluR2 and other synaptic proteins in the synaptosomal membrane fraction upon biochemical fractionation of the adult mice cortex and retina. Immunoprecipitation from the synaptosomal membrane fraction of the Olfm2 null mouse brain cortex using the GluR2 antibody showed reduced levels of several components of the AMPAR complex in the immunoprecipitates including Olfm1, PSD95 and CNIH2. These results suggest that heterodimers of Olfm1 and Olfm2 interact with AMPAR more efficiently than Olfm2 homodimers and that Olfm2 plays a role in the organization of the AMPA receptor complexes.

Intermittent food deprivation (fasting, IF) improves mood and cognition and protects neurons against excitotoxic degeneration in animal models of epilepsy and Alzheimer's disease (AD). The mechanisms by which neuronal networks adapt to IF and how such adaptations impact neuropathological processes are unknown. We show that hippocampal neuronal networks adapt to IF by enhancing GABAergic tone, which is associated with reduced anxiety-like behaviors and improved hippocampus-dependent memory. These neuronal network and behavioral adaptations require the mitochondrial protein deacetylase SIRT3 as they are abolished in SIRT3-deficient mice and wild type mice in which SIRT3 is selectively depleted from hippocampal neurons. In the AppNL-G-F mouse model of AD, IF reduces neuronal network hyperexcitability and ameliorates deficits in hippocampal synaptic plasticity in a SIRT3-dependent manner. These findings demonstrate a role for a mitochondrial protein deacetylase in hippocampal neurons in behavioral and GABAergic synaptic adaptations to IF.

The olfm1a and olfm1b genes in zebrafish encode conserved secreted glycoproteins. These genes are preferentially expressed in the brain and retina starting from 16 h post-fertilization until adulthood. Functions of the Olfm1 gene is still unclear. Here, we produced and analyzed a null zebrafish mutant of both olfm1a and olfm1b genes (olfm1 null). olfm1 null fish were born at a normal Mendelian ratio and showed normal body shape and fertility as well as no visible defects from larval stages to adult. Olfm1 proteins were preferentially localized in the synaptosomes of the adult brain. Olfm1 co-immunoprecipitated with GluR2 and soluble NSF attachment protein receptor complexes indicating participation of Olfm1 in both pre- and post-synaptic events. Phosphorylation of GluR2 was not changed while palmitoylation of GluR2 was decreased in the brain synaptosomal membrane fraction of olfm1 null compared with wt fish. The levels of GluR2, SNAP25, flotillin1, and VAMP2 were markedly reduced in the synaptic microdomain of olfm1 null brain compared with wt. The internalization of GluR2 in retinal cells and the localization of VAMP2 in brain synaptosome were modified by olfm1 null mutation. This indicates that Olfm1 may regulate receptor trafficking from the intracellular compartments to the synaptic membrane microdomain, partly through the alteration of post-translational GluR2 modifications such as palmitoylation. Olfm1 may be considered a novel regulator of the composition and function of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor complex.

The actin-modulating protein Wiskott-Aldrich syndrome protein verprolin homologous-1 (WAVE1) and a novel CNS-specific protein, pancortin, are highly enriched in adult cerebral cortex, but their functions are unknown. Here we show that WAVE1 and pancortin-2 interact in a novel cell death cascade in adult, but not embryonic, cerebral cortical neurons. Focal ischemic stroke induces the formation of a protein complex that includes pancortin-2, WAVE1, and the anti-apoptotic protein Bcl-xL. The three-protein complex is associated with mitochondria resulting in increased association of Bax with mitochondria, cytochrome c release, and neuronal apoptosis. In pancortin null mice generated using a Cre-loxP system, ischemia-induced WAVE1-Bcl-xL interaction is diminished, and cortical neurons in these mice are protected against ischemic injury. Thus, pancortin-2 is a mediator of ischemia-induced apoptosis of neurons in the adult cerebral cortex and functions in a novel mitochondrial/actin-associated protein complex that sequesters Bcl-xL.

Clathrin-coated vesicles are known to play diverse and pivotal roles in cells. The proper formation of clathrin-coated vesicles is dependent on, and highly regulated by, a large number of clathrin assembly proteins. These assembly proteins likely determine the functional specificity of clathrin-coated vesicles, and together they control a multitude of intracellular trafficking pathways, including those involved in embryonic development. In this study, we focus on two closely related clathrin assembly proteins, AP180 and CALM (clathrin assembly lymphoid myeloid leukemia protein), in the developing embryonic rat brain. We find that AP180 begins to be expressed at embryonic day 14 (E14), but only in postmitotic cells that have acquired a neuronal fate. CALM, on the other hand, is expressed as early as E12, by both neural stem cells and postmitotic neurons. In vitro loss-of-function studies using RNA interference (RNAi) indicate that AP180 and CALM are dispensable for some aspects of embryonic neurogenesis but are required for the growth of postmitotic neurons. These results identify the developmental stage of AP180 and CALM expression and suggest that each protein has distinct functions in neural development.