Characterization of Age-Dependent and Progressive Cortical Neuronal Degeneration in Presenilin Conditional Mutant Mice

Presenilins are the major causative genes of familial Alzheimer''s disease (AD). Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO) mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold) and apoptotic (TUNEL+, 7.4-fold) neurons, which represent ∼0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (∼9%) and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen cortical neurons. Together, our findings show that loss of presenilins in cortical neurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss of cortical neurons in the aging brain. The low occurrence and significant delay of apoptosis among cortical neurons lacking presenilins suggest that loss of presenilins may induce apoptotic neuronal death through disruption of cellular homeostasis rather than direct activation of apoptosis pathways.     

Conditional deletion of Notch1 and Notch2 genes in excitatory neurons of postnatal forebrain does not cause neurodegeneration or reduction of Notch mRNAs and proteins

Activation of Notch signaling requires intramembranous cleavage by γ-secretase to release the intracellular domain. We previously demonstrated that presenilin and nicastrin, components of the γ-secretase complex, are required for neuronal survival in the adult cerebral cortex. Here we investigate whether Notch1 and/or Notch2 are functional targets of presenilin/γ-secretase in promoting survival of excitatory neurons in the adult cerebral cortex by generating Notch1, Notch2, and Notch1/Notch2 conditional knock-out (cKO) mice. Unexpectedly, we did not detect any neuronal degeneration in the adult cerebral cortex of these Notch cKO mice up to ～2 years of age, whereas conditional inactivation of presenilin or nicastrin using the same αCaMKII-Cre transgenic mouse caused progressive, striking neuronal loss beginning at 4 months of age. More surprisingly, we failed to detect any reduction of Notch1 and Notch2 mRNAs and proteins in the cerebral cortex of Notch1 and Notch2 cKO mice, respectively, even though Cre-mediated genomic deletion of the floxed Notch1 and Notch2 exons clearly took place in the cerebral cortex of these cKO mice. Furthermore, introduction of Cre recombinase into primary cortical cultures prepared from postnatal floxed Notch1/Notch2 pups, where Notch1 and Notch2 are highly expressed, completely eliminated their expression, indicating that the floxed Notch1 and Notch2 alleles can be efficiently inactivated in the presence of Cre. Together, these results demonstrate that Notch1 and Notch2 are not involved in the age-related neurodegeneration caused by loss of presenilin or γ-secretase and suggest that there is no detectable expression of Notch1 and Notch2 in pyramidal neurons of the adult cerebral cortex.     

Shp2 in Forebrain Neurons Regulates Synaptic Plasticity,Locomotion, and Memory Formation in Mice

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K⁺-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.     

Cdk5 is required for the positioning and survival of GABAergic neurons in developing mouse striatum

Cyclin‐dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron‐specific activating subunit. It was previously reported that Cdk5^−/− mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5^−/− embryos. To further study this abnormal development, we generated and analyzed Dlx5/6‐Cre p35 conditional KO (cKO); p39^−/− mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6‐Cre p35cKO; p39^−/− mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6‐Cre p35cKO; p39^−/− mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell‐autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Counting statistics of 1/f fluctuations in neuronal spike trains

The mesencephalic reticular formation (MRF) neurons are regarded as contributing to the activation of the celebral cortex. In this paper, the statistical features of single neuronal activities in MRF of cat during dream sleep are investigated; the neuronal spike train exhibits 1/f fluctuations. Counting statistics is applied to the neuronal spike train giving rise to a variance/mean curve which follows at -law. For an interpretation of these findings, the clustering Poisson process is applied which not only gives rise to at -law but also suggests a generation mechanism. The MRF neuronal activities are closely fitted by the clustering Poisson process and the underlying statistical parameters can be estimated. These findings strongly suggest that neuronal activities can be interpreted as superposition of randomly occuring clusters ( = bursts of spikes).     

TrkB受体依赖的PV神经元调控小鼠视觉方位辨别能力的机制

神经营养因子-酪氨酸受体激酶B （tyrosine receptor kinase B,TrkB）信号通路在调控初级视皮层（primary visual cortex,V1）兴奋与抑制平衡上发挥着重要的作用,以往的研究揭示了其通过增加兴奋性传递效率来调控皮层兴奋性水平的机制,却并未阐明TrkB受体如何通过抑制系统来调控兴奋与抑制平衡,进而影响视觉皮层功能。为了探讨TrkB信号通路如何特异性地调控最主要的抑制性神经元——PV神经元进而对小鼠视觉皮层功能产生影响,本研究通过病毒特异性地降低V1区的PV神经元上TrkB受体的表达水平,并通过在体多通道电生理手段记录初级视皮层抑制性与兴奋性神经元功能变化,通过行为学实验测试小鼠的方位辨别能力改变。结果表明,初级视觉皮层中的PV抑制性神经元上的TrkB受体表达减少会显著增加兴奋性神经元的反应强度,减弱抑制性神经元与兴奋性神经元的方位辨别能力,增加二者的信噪比,但是小鼠个体水平的方位辨别能力出现下降。这些结果说明,TrkB信号通路并非单纯通过增加靶向PV神经元的兴奋性传递来调控PV神经元的功能,其对神经元信噪比的影响也并非由于抑制系统的增强所致。     

NeuroD regulates neuronal migration

NeuroD is required for the survival of many subtypes of developing neurons in the vertebrate central nervous system. Because NeuroD-deficient neurons in the hippocampus, cerebellum, and inner ear die prematurely in the early stage of neurogenesis, the role of NeuroD during the later stages of neurogenesis of these cell subtypes is not well understood. In addition, the mechanism of NeuroD-deficient neuronal death has not been investigated. It was hypothesized that NeuroD-dependent neuronal death occurs through a Bax-dependent apoptotic pathway. Based on this hypothesis, this study attempted to rescue neuronal cell death by deleting the Bax gene in NeuroD null mice to investigate the role of NeuroD in surviving neurons. The NeuroD and Bax double null mice displayed a decrease in the number of apoptotic cells in the hippocampus and the cerebellum and the rescue of vestibulocochlear ganglion (VCG) neurons that failed to migrate and innervate. In addition, at E13.5, the NeuroD^−/−Bax^−/− VCG neurons failed to express TrkB and TrkC, which are known to be essential for the survival of those neurons. These data suggest that neuronal death in NeuroD null mice is mediated by Bax-dependent apoptosis and that NeuroD is required for the migration of VCG neurons. Finally, these data show that TrkB and TrkC expression in E13.5 VCG neurons requires NeuroD and that TrkB and TrkC expression may be necessary for the normal migration and innervations of those neurons.     

Corticofugal effects on the neuronal activity in the emotiogenic/motivational zones of the hypothalamus

Neuronal responses to stimulation of the proreal (field 8) and cingular (field 24) cortices, pyriform lobe (periamygdalar cortex), and hippocampus (CA3) were studied in the lateral (HL) and ventromedial (Hvm) hypothalamus, dorsal hypothalamic region (aHd), and projection region of the medial forelimb bundle (MFB); single and repeated (series of a 6–300 sec⁻¹ frequency) stimuli were used. At single stimulations, the minimum proportion of inhibitory responses with respect to excitatory effects was observed when the neocortex (the proreal gyrus) was stimulated; this proportion became successively greater at stimulations of the intermediate cortex (the cingular gyrus) and paleocortex (the pyriform cortex), while stimulation of the archicortex (the hippocampus) evoked mostly inhibitory responses. At repeated stimulation of the cortical structures, inhibitory responses prevailed in the neurons under study: their total number was nearly four times larger than that of excitatory reactions. The response patterns to single and serial stimulations of the cortical structures allowed us to demonstrate: (i) significant diversity of the influences received by hypothalamic neurons from the cortical structures and (ii) the dependence of the pattern of these influences on the phylogenetic specificity of the above structures.     

Inhibition of excitatory neuronal cell death by cell-permeable calcineurin autoinhibitory peptide

In glutamate-mediated excitatory neuronal cell death, immunosuppressants (FK506, Cys-A) are powerful agents that protect neurons from apoptosis. Immunosuppressants inhibit two types of enzyme, calcium/calmodulin-dependent protein phosphatase (calcineurin: CaN), and peptidyl-prolyl cis-trans-isomerase (PPIase) activity such as the FKBP family. In this study, we used a protein transduction approach to determine the functional role of CaN and to produce a potential therapeutic agent for glutamate-mediated neuronal cell death. We created a novel cell-permeable CaN autoinhibitory peptide using the 11 arginine protein transduction domain. This peptide was highly efficient at transducing into primary culture neurons, potently inhibited CaN phosphatase activities, and inhibited glutamate-mediated neuronal cell death. These results showed that CaN plays an important role in excitatory neuronal cell death and cell-permeable CaN autoinhibitory peptide could be a new drug to protect neurons from excitatory neuronal death.     

Role for NMDA receptors in visceral nociceptive transmission in the anterior cingulate cortex of viscerally hypersensitive rats

We have identified colorectal distension (CRD)-responsive neurons in the anterior cingulate cortex (ACC) and demonstrated that persistence of a heightened visceral afferent nociceptive input to the ACC induces ACC sensitization. In the present study, we confirmed that rostral ACC neurons of sensitized rats [induced by chicken egg albumin (EA)] exhibit enhanced spike responses to CRD. Simultaneous in vivo recording and reverse microdialysis of single ACC neurons showed that a low dose of glutamate (50 microM) did not change basal ACC neuronal firing in normal rats but increased ACC neuronal firing in EA rats from 18 +/- 2 to 32 +/- 3.8 impulses/10 s. A high dose of glutamate (500 microM) produced 1.95-fold and a 4.27-fold increases of ACC neuronal firing in sham-treated rats and in EA rats, respectively, suggesting enhanced glutamatergic transmission in the ACC neurons of EA rats. Reverse microdialysis of the 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainite receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) reduced basal and abolished CRD-induced ACC neuronal firing in normal rats. In contrast, microdialysis of N-methyl-d-aspartate (NMDA) receptor antagonist AP5 had no effect on ACC neuronal firing in normal rats. However, AP5 produced 86% inhibition of ACC neuronal firing evoked by 50 mmHg CRD in the EA rats. In conclusion, ACC nociceptive transmissions are mediated by glutamate AMPA receptors in the control rats. ACC responses to CRD are enhanced in viscerally hypersensitive rats. The enhancement of excitatory glutamatergic transmission in the ACC appears to mediate this response. Furthermore, NMDA receptors mediate ACC synaptic responses after the induction of visceral hypersensitivity.     

Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death

Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a^−/− mice exhibit significantly enhanced Ca²⁺ retention capacity and accelerated Ca²⁺ uptake rate. When challenged with hydrogen peroxide (H₂O₂), ASIC1a^−/− neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H₂O₂-induced neuronal death, which is MPT dependent, is reduced in ASIC1a^−/− neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a^−/− mouse brain, which also displays marked changes (>2-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death.     

Inhibitory effects of actinomycin D and cycloheximide on neuronal death in adult Manduca sexta

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomysin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response. 1994 John Wiley & Sons, Inc.