Expression Analyses of POGZ, A Responsible Gene for Neurodevelopmental Disorders, during Mouse Brain Development.

POGZ is a heterochromatin protein 1 α-binding protein and regulates gene expression. On the other hand, accumulating pieces of evidence indicate that the POGZ gene abnormalities are involved in various neurodevelopmental disorders. In this study, we prepared a specific antibody against POGZ, anti-POGZ, and carried out biochemical and morphological characterization with mouse brain tissues. Western blotting analyses revealed that POGZ is expressed strongly at embryonic day 13 and then gradually decreased throughout the brain development process. In immunohistochemical analyses, POGZ was found to be enriched in cerebrocortical and hippocampal neurons in the early developmental stage. The nuclear expression was also detected in Purkinje cells in cerebellum at postnatal day (P)7 and P15 but disappeared at P30. In primary cultured hippocampal neurons, while POGZ was distributed mainly in the nucleus, it was also visualized in axon and dendrites with partial localization at synapses in consistency with the results obtained in biochemical fractionation analyses. The obtained results suggest that POGZ takes part in the regulation of synaptic function as well as gene expression during brain development.

Distinct brain pathologies associated with Alzheimer's disease biomarker-related phospho-tau 181 and phospho-tau 217 in App knock-in mouse models of amyloid-ß amyloidosis.

Phospho-tau 217, phospho-tau 231 and phospho-tau 181 in cerebrospinal fluid and plasma are promising biomarkers for the diagnosis of Alzheimer's disease. All these p-tau proteins are detected in neurofibrillary tangles in brains obtained post-mortem from Alzheimer's disease patients. However, increases in p-tau levels in cerebrospinal fluid and plasma during the preclinical stage of Alzheimer's disease correlate with amyloid-ß burden and precede neurofibrillary tangles in brains, suggesting that these p-tau proteins are indicative of amyloid-ß-mediated brain pathology. In addition, phospho-tau 217 has greater sensitivity than phospho-tau 181, though it is unclear whether each of these p-tau variants contributes to the same or a different type of neuropathology prior to neurofibrillary tangle formation. In this study, we evaluated the intracerebral localization of p-tau in App knock-in mice with amyloid-ß plaques without neurofibrillary tangle pathology (AppNLGF ), in App knock-in mice with increased amyloid-ß levels without amyloid-ß plaques (AppNL ) and in wild-type mice. Immunohistochemical analysis showed that phospho-tau 217 and phospho-tau 231 were detected only in AppNLGF mice as punctate structures around amyloid-ß plaques, overlapping with the tau pathology marker, AT8 epitope phospho-tau 202/205/208. Moreover, phospho-tau 217 and phospho-tau 202/205/208 colocalized with the postsynaptic marker PSD95 and with a major tau kinase active, GSK3ß. In contrast and similar to total tau, phospho-tau 181 signals were readily detectable as fibre structures in wild-type and AppNL mice and colocalized with an axonal marker neurofilament light chain. In AppNLGF mice, these phospho-tau 181-positive structures were disrupted around amyloid-ß plaques and only partially overlapped with phospho-tau 217. These results indicate that phospho-tau 217, phospho-tau 231 and a part of phospho-tau 181 signals are markers of postsynaptic pathology around amyloid-ß plaques, with phospho-tau 181 also being a marker of axonal abnormality caused by amyloid-ß burden in brains.

Widespread Reduced Density of Noradrenergic Locus Coeruleus Axons in the App Knock-In Mouse Model of Amyloid-ß Amyloidosis.

BACKGROUND: The locus coeruleus (LC), a brainstem nucleus comprising noradrenergic neurons, is one of the earliest regions affected by Alzheimer's disease (AD). Amyloid-ß (Aß) pathology in the cortex in AD is thought to exacerbate the age-related loss of LC neurons, which may lead to cortical tau pathology. However, mechanisms underlying LC neurodegeneration remain elusive. OBJECTIVE: Here, we aimed to examine how noradrenergic neurons are affected by cortical Aß pathology in AppNL-G-F/NL-G-F knock-in mice. METHODS: The density of noradrenergic axons in LC-innervated regions and the LC neuron number were analyzed by an immunohistochemical method. To explore the potential mechanisms for LC degeneration, we also examined the occurrence of tau pathology in LC neurons, the association of reactive gliosis with LC neurons, and impaired trophic support in the brains of AppNL-G-F/NL-G-F mice. RESULTS: We observed a significant reduction in the density of noradrenergic axons from the LC in aged AppNL-G-F/NL-G-F mice without neuron loss or tau pathology, which was not limited to areas near Aß plaques. However, none of the factors known to be related to the maintenance of LC neurons (i.e., somatostatin/somatostatin receptor 2, brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) were significantly reduced in AppNL-G-F/NL-G-F mice. CONCLUSION: This study demonstrates that cortical Aß pathology induces noradrenergic neurodegeneration, and further elucidation of the underlying mechanisms will reveal effective therapeutics to halt AD progression.

Biochemical and Morphological Characterization of a Guanine Nucleotide Exchange Factor ARHGEF9 in Mouse Tissues.

ARHGEF9, also known as Collybistin, a guanine nucleotide exchange factor for Rho family GTPases, is thought to play an essential role in the mammalian brain. In this study, we prepared a specific polyclonal antibody against ARHGEF9, anti-ARHGEF9, and carried out expression analyses with mouse tissues especially brain. Western blotting analyses demonstrated tissue-dependent expression profiles of ARHGEF9 in the young adult mouse, and strongly suggested a role during brain development. Immunohistochemical analyses revealed developmental stage-dependent expression profiles of ARHGEF9 in cerebral cortex, hippocampus and cerebellum. ARHGEF9 exhibited partial localization at dendritic spines in cultured hippocampal neurons. From the obtained results, anti-ARHGEF9 was found to be a useful tool for biochemical and cell biological analyses of ARHGEF9.

It's green outside: tracking cell surface proteins with pH-sensitive GFP.

Green fluorescent protein (GFP) and mutated GFP variants have proved to be immensely powerful tools that have had a profound impact on research in biological sciences. This review considers the development, use and future implications of pH-dependent GFP variants (e.g. pHluorins). These proteins hold considerable promise for the relatively non-invasive monitoring of events such as exocytosis, endocytosis and protein surface expression in living neurons with high spatial and temporal resolution.

Intravital oxygen radical imaging in normal and ischemic rat cortex.

OBJECTIVE: We examined reactive oxygen species (ROS) generation on cerebral ischemia/reperfusion by intravital fluorescence imaging. METHODS: In anesthetized adult rats, a fluorescent dye (5 microL), MitoSOX (5 micromol/L) for superoxide radical (.O2-), and hydroxyphenyl fluorescein (20 micromol/L) for hydroxyl radical (.OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope on 10-minute forebrain ischemia. Because hemoglobin absorbs excitation and emission lights, ischemia may affect the change in fluorescence intensity (FI) inside the brain. To examine the effects of ischemia on the FI change, fluoromicrospheres (0.2-microm diameter) were used to mimic a dye and FI was analyzed in the same manner as when using ROS indicators. Their FI increased to 129% during ischemia (n=3/mimicking each dye), and based on the results, FI of ROS indicators was corrected. RESULTS: After correcting the FI of MitoSOX and hydroxyphenyl fluorescein, they showed no change during ischemia, whereas the raw data showed the increase. In the early period of reperfusion, FI significantly (n=5/each, P<.01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. To test the feasibility of our imaging, edaravone (3.0 mg/kg) was used. The treatment completely scavenged .OH, but did not do so in .O2- generation. CONCLUSION: ROS production increased in the early period of reperfusion but not during ischemia, which was location selective, being significant in the areas adjacent to the arteries. Our method was useful for investigating intracellular in situ ROS production.

GIF-0173 protects against cerebral infarction through DP1 receptor activation.

The neuroprotective effects and mechanism of action of GIF-0173, a Delta12-prostaglandin J analogue, were investigated in the early phase of cerebral ischemia. GIF-0173 was administered intravenously immediately following middle cerebral artery occlusion (MCAO) in photochemically induced thrombosis model of rat. Neurological scores and infarct sizes were examined at 24 h after MCAO. Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry for 1 h after MCAO. In cultured cortical neurons obtained from 1-day-old rats, the effects of GIF-0173 on the excitotoxicity induced by glutamate were examined. Morphological changes, neuronal death, and changes in intracellular calcium concentration ([Ca(2+)](i)) were also examined. GIF-0173 improved neurological scores and reduced the infarct size in a dose-dependent manner following MCAO. But GIF-0173 did not improve CBF after MCAO. GIF-0173 also prevented glutamate-induced neuronal death and acute cellular swelling in primary cultures in a dose-dependent manner, indicating that it inhibited neuronal necrosis. GIF-0173 dose-dependently suppressed the glutamate-induced increase in [Ca(2+)](i), but could not inhibit NMDA-induced calcium influx. The effects of GIF-0173 against glutamate-induced [Ca(2+)](i) increase were reversed by addition of non-specific prostaglandin D (PGD(2)) receptor antagonist and were comparable to the effects of PGD(2) DP1 receptor agonist, which prevented [Ca(2+)](i) increase and neuronal death. We conclude that GIF-0173 reduces cerebral infarction and protects cultured neurons against glutamate-induced excitotoxicity by inhibiting [Ca(2+)](i) increase through DP1 receptor activation.

Brain-derived neurotrophic factor signal enhances and maintains the expression of AMPA receptor-associated PDZ proteins in developing cortical neurons.

Postsynaptic molecules with PDZ domains (PDZ proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. In rat neocortical development, the protein expression of AMPA-type glutamate receptor GluR1 lagged behind its mRNA expression and rather paralleled an increase in PDZ protein levels. One of the neurotrophins, brain-derived neurotrophic factor (BDNF), appeared to contribute to this process, regulating the PDZ protein expression. In neocortical cultures, BDNF treatment upregulated SAP97, GRIP1, and Pick1 PDZ proteins. Conversely, BDNF gene targeting downregulated these same PDZ molecules. The BDNF-triggered increases in PDZ proteins resulted in the elevation of their total association with the AMPA receptors GluR1 and GluR2/3, which led to the increase in AMPA receptor proteins. When Sindbis viruses carrying GluR1 or GluR2 C-terminal decoys disrupted their interactions, GluR2 C-terminal decoys inhibited both BDNF-triggered GluR1 and GluR2/3 increases, whereas GluR1 C-terminal decoys blocked only the BDNF-triggered GluR1 increase. In agreement, coexpression of SAP97 and GluR1 in nonneuronal HEK293 cells increased both proteins compared with their single transfection, implying mutual stabilization. This work reveals a novel function of BDNF in postsynaptic development by regulating the PDZ protein expression.