Noriaki Ohkawa

Neuronal Stimulation Induces Autophagy in Hippocampal Neurons That Is Involved in AMPA Receptor Degradation after Chemical Long-Term Depression

Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP–lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K–Akt–mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.

N‐acetyltransferase ARD1‐NAT1 regulates neuronal dendritic development

ARD1 and NAT1 constitute an N‐acetyltransferase complex where ARD1 holds the enzymatic activity of the complex. The ARD1–NAT1 complex mediates N‐terminal acetylation of nascent polypeptides that emerge from ribosomes after translation. ARD1 may also acetylate the internal lysine residues of proteins. Although ARD1 and NAT1 have been found in the brain, the physiological role and substrates of the ARD1–NAT1 complex in neurons remain unclear. Here we investigated role of N‐acetyltransferase activity in the process of neuronal development. Expression of ARD1 and NAT1 increased during dendritic development, and both proteins colocalized with microtubules in dendrites. The ARD1–NAT1 complex displayed acetyltransferase activity against a purified microtubule fraction in vitro. Inhibition of the complex limited the dendritic extension of cultured neurons. These findings suggest that the ARD1–NAT1 complex has acetyltransferase activity against microtubules in dendrites. Regulation by acetyltransferase activity is a novel mechanism that is required for dendritic arborization during neuronal development.

The microtubule destabilizer stathmin mediates the development of dendritic arbors in neuronal cells

The regulation of microtubule dynamics is important for the appropriate arborization of neuronal dendrites during development, which in turn is critical for the formation of functional neural networks. Here we show that stathmin, a microtubule destabilizing factor, is downregulated at both the expression and activity levels during cerebellar development, and this down-regulation contributes to dendritic arborization. Stathmin overexpression drastically limited the dendritic growth of cultured Purkinje cells. The stathmin activity was suppressed by neural activity and CaMKII-dependent phosphorylation at Ser16, which led to dendritic arborization. Stathmin phosphorylation at Ser16 was mediated by the activation of voltage-gated calcium channels and metabotropic glutamate receptor 1. Although overexpression of SCG10, a member of the stathmin family, also limited the dendritic arborization, SCG10 did not mediate the CaMKII regulation of dendritic development. These results suggest that calcium elevation activates CaMKII, which in turn phosphorylates stathmin at Ser16 to stabilize dendritic microtubules. siRNA knockdown of endogenous stathmin significantly reduced dendritic growth in Purkinje cells. Thus, these data suggest that proper regulation of stathmin activity is a key factor for controlling the dendritic microtubule dynamics that are important for neuronal development.

Molecular cloning and characterization of neural activity-related RING finger protein (NARF): a new member of the RBCC family is a candidate for the partner of myosin V

Activity‐dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long‐term potentiation (L‐LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP‐related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)‐ and kainate (KA)‐mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR‐based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ‐treated mice. One of these genes, neural activity‐related RING finger protein (NARF), encodes a new protein containing a RING finger, B‐box zinc finger, coiled‐coil (RBCC domain) and β‐propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up‐regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP‐fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein‐fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C‐terminal β‐propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ‐induced seizure. These observations indicated that NARF expression is enhanced by seizure‐related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.

Distribution of AP-2 subtypes in the adult mouse brain

In the mammalian central nervous system (CNS), transcription factor activator protein 2 (AP-2) is one of the critical regulatory factors for neural gene expression and neural development. As AP-2 has diverged into several subtypes, i.e. AP-2alpha, -2beta, and 2.2, we investigated the distribution of the AP-2 subtypes in the adult mouse brain by in situ hybridization using subtype-specific probes. Though AP-2 was essentially expressed in most regions of the brain, the hippocampus and cerebellum Purkinje cells exhibited a relatively high concentration of transcripts of any of the AP-2 subtypes. Among AP-2alpha variants, the expression of variant 1 was considerably lower than that of variant 3. Hence, the expression pattern of AP-2alpha variant 3 is suggested to represent the major gene expression of AP-2alpha. On the other hand, the expression of AP-2beta messenger RNA (mRNA) was higher than that of AP-2alpha in many regions. Especially, the olfactory bulb, hippocampus, cerebellum, and cerebral cortex contained an abundance of these mRNAs. Different from those of AP-2alpha, AP-2beta mRNAs were detected in considerable amounts in the glanular cells as well as in Purkinje cells. AP-2.2 gene expression was weak throughout the brain. Consequently, we found that various AP-2 subtypes and variants were expressed in a similar distribution pattern with each having its own specific intensity but that their precise distribution profiles were not exactly the same. In the mature brain, AP-2 is thought to regulate neural gene expression through specific and redundant association with a target gene.

Overlapping memory trace indispensable for linking, but not recalling, individual memories

Unrelated memories get blurred together If one retrieves two memories around the same time, a small number of neurons will become involved in both memories. Yokose et al. investigated the cellular ensemble mechanisms underlying the association between two such memories. In mice, a small population of neurons mediates the association. Memory traces for two independent emotional memories in the brain partially overlapped when the two memories were retrieved synchronously to create a linkage. Suppressing the activity of the overlapping memory trace interrupted the linkage without damaging the original memories. Science, this issue p. 398 In mice, repeated simultaneous reactivation of two initially separated memory traces links them together. Memories are not stored in isolation from other memories but are integrated into associative networks. However, the mechanisms underlying memory association remain elusive. Using two amygdala-dependent behavioral paradigms—conditioned taste aversion (CTA) and auditory-cued fear conditioning (AFC)—in mice, we found that presenting the conditioned stimulus used for the CTA task triggered the conditioned response of the AFC task after natural coreactivation of the memories. This was accompanied through an increase in the overlapping neuronal ensemble in the basolateral amygdala. Silencing of the overlapping ensemble suppressed CTA retrieval-induced freezing. However, retrieval of the original CTA or AFC memory was not affected. A small population of coshared neurons thus mediates the link between memories. They are not necessary for recalling individual memories.

Cellular tagging as a neural network mechanism for behavioural tagging

Behavioural tagging is the transformation of a short-term memory, induced by a weak experience, into a long-term memory (LTM) due to the temporal association with a novel experience. The mechanism by which neuronal ensembles, each carrying a memory engram of one of the experiences, interact to achieve behavioural tagging is unknown. Here we show that retrieval of a LTM formed by behavioural tagging of a weak experience depends on the degree of overlap with the neuronal ensemble corresponding to a novel experience. The numbers of neurons activated by weak training in a novel object recognition (NOR) task and by a novel context exploration (NCE) task, denoted as overlapping neurons, increases in the hippocampal CA1 when behavioural tagging is successfully achieved. Optical silencing of an NCE-related ensemble suppresses NOR–LTM retrieval. Thus, a population of cells recruited by NOR is tagged and then preferentially incorporated into the memory trace for NCE to achieve behavioural tagging.

Transcriptional regulation of mouse type 1 inositol 1,4,5-trisphosphate receptor gene by NeuroD-related factor.

Abstract: The type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box‐I element, which is located —334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E‐box consensus sequence, is involved in the up‐regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS‐related basic helix‐loop‐helix (bHLH) factors bind to the box‐I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS‐related bHLH factors, neuronal differentiation factor (NeuroD)‐related factor (NDRF), specifically bound to the box‐I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co‐expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue‐specific regulation of IP3R1 gene expression in the CNS.

Spine Formation Pattern of Adult-Born Neurons Is Differentially Modulated by the Induction Timing and Location of Hippocampal Plasticity

In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn’t respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP–induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.

Motor discoordination of transgenic mice overexpressing a microtubule destabilizer, stathmin, specifically in Purkinje cells

The proper regulation of microtubule (MT) structure is important for dendritic and neural circuit development. However, the relationship between the regulation of the MTs in dendrites and the formation of neural function is still unclear. Stathmin is a MT destabilizer, and we have previously reported that the expression and the activity of stathmin is downregulated during cerebellar Purkinje cell (PC) development. In this study, we generated transgenic mice that specifically overexpress the constitutively active form of stathmin in the PCs. These mutant mice did not show any obvious morphological or excitatory transmission abnormalities in the cerebellum. In contrast, we observed a decline in the expression of MAP2 and KIF5 signal in the PC dendrites and a discoordination of motor function in the mutant mice, although they displayed normal general behavior. These data indicate that the overexpression of stathmin disrupts dendritic MT organization, motor protein distribution, and neural function in PCs.