Heterogenous effect of early adulthood stress on cognitive aging and synaptic function in the dentate gyrus.

Cognitive aging widely varies among individuals due to different stress experiences throughout the lifespan and vulnerability of neurocognitive mechanisms. To understand the heterogeneity of cognitive aging, we investigated the effect of early adulthood stress (EAS) on three different hippocampus-dependent memory tasks: the novel object recognition test (assessing recognition memory: RM), the paired association test (assessing episodic-like memory: EM), and trace fear conditioning (assessing trace memory: TM). Two-month-old rats were exposed to chronic mild stress for 6 weeks and underwent behavioral testing either 2 weeks or 20 months later. The results show that stress and aging impaired different types of memory tasks to varying degrees. RM is affected by combined effect of stress and aging. EM became less precise in EAS animals. TM, especially the contextual memory, showed impairment in aging although EAS attenuated the aging effect, perhaps due to its engagement in emotional memory systems. To further explore the neural underpinnings of these multi-faceted effects, we measured long-term potentiation (LTP), neural density, and synaptic density in the dentate gyrus (DG). Both stress and aging reduced LTP. Additionally, the synaptic density per neuron showed a further reduction in the stress aged group. In summary, EAS modulates different forms of memory functions perhaps due to their substantial or partial dependence on the functional integrity of the hippocampus. The current results suggest that lasting alterations in hippocampal circuits following EAS could potentially generate remote effects on individual variability in cognitive aging, as demonstrated by performance in multiple types of memory.

PTK2 regulates tau-induced neurotoxicity via phosphorylation of p62 at Ser403.

Tau is a microtubule-associated protein that forms insoluble filaments that accumulate as neurofibrillary tangles in neurodegenerative diseases such as Alzheimer's disease and other related tauopathies. A relationship between abnormal Tau accumulation and ubiquitin-proteasome system impairment has been reported. However, the molecular mechanism linking Tau accumulation and ubiquitin proteasome system (UPS) dysfunction remains unclear. Here, we show that overexpression of wild-type or mutant (P301L) Tau increases the abundance of polyubiquitinated proteins and activates the autophagy-lysosome pathway in mammalian neuronal cells. Previous studies found that PTK2 inhibition mitigates toxicity induced by UPS impairment. Thus, we investigated whether PTK2 inhibition can attenuate Tau-induced UPS impairment and cell toxicity. We found that PTK2 inhibition significantly reduces Tau-induced death in mammalian neuronal cells. Moreover, overexpression of WT or mutant Tau increased the phosphorylation levels of PTK2 and p62. We also confirmed that PTK2 inhibition suppresses Tau-induced phosphorylation of PTK2 and p62. Furthermore, PTK2 inhibition significantly attenuated the climbing defect and shortened the lifespan in the Drosophila model of tauopathy. In addition, we observed that phosphorylation of p62 is markedly increased in Alzheimer's disease patients with tauopathies. Taken together, our results indicate that the UPS dysfunction induced by Tau accumulation might contribute directly to neurodegeneration in tauopathies and that PTK2 could be a promising therapeutic target for tauopathies.

Automated Synapse Detection Method for Cerebellar Connectomics.

The connectomic analyses of large-scale volumetric electron microscope (EM) images enable the discovery of hidden neural connectivity. While the technologies for neuronal reconstruction of EM images are under rapid progress, the technologies for synapse detection are lagging behind. Here, we propose a method that automatically detects the synapses in the 3D EM images, specifically for the mouse cerebellar molecular layer (CML). The method aims to accurately detect the synapses between the reconstructed neuronal fragments whose types can be identified. It extracts the contacts between the reconstructed neuronal fragments and classifies them as synaptic or non-synaptic with the help of type information and two deep learning artificial intelligences (AIs). The method can also assign the pre- and postsynaptic sides of a synapse and determine excitatory and inhibitory synapse types. The accuracy of this method is estimated to be 0.955 in F1-score for a test volume of CML containing 508 synapses. To demonstrate the usability, we measured the size and number of the synapses in the volume and investigated the subcellular connectivity between the CML neuronal fragments. The basic idea of the method to exploit tissue-specific properties can be extended to other brain regions.

IQSEC3 Deletion Impairs Fear Memory Through Upregulation of Ribosomal S6K1 Signaling in the Hippocampus.

BACKGROUND: IQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. METHODS: We generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were used to address our question. RESULTS: We found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6K1-mediated signaling in the hippocampus but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density but not deficits in long-term potentiation maintenance. Finally, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF (adenosine diphosphate ribosylation factor-guanine nucleotide exchange factor) inactive IQSEC3 mutant. CONCLUSIONS: Our results suggest that IQSEC3-mediated balanced synaptic inhibition in hippocampal CA1 is critical for the proper formation of hippocampus-dependent fear memory.

LRRTM3 regulates activity-dependent synchronization of synapse properties in topographically connected hippocampal neural circuits.

Synaptic cell-adhesion molecules (CAMs) organize the architecture and properties of neural circuits. However, whether synaptic CAMs are involved in activity-dependent remodeling of specific neural circuits is incompletely understood. Leucine-rich repeat transmembrane protein 3 (LRRTM3) is required for the excitatory synapse development of hippocampal dentate gyrus (DG) granule neurons. Here, we report that Lrrtm3-deficient mice exhibit selective reductions in excitatory synapse density and synaptic strength in projections involving the medial entorhinal cortex (MEC) and DG granule neurons, accompanied by increased neurotransmitter release and decreased excitability of granule neurons. LRRTM3 deletion significantly reduced excitatory synaptic innervation of hippocampal mossy fibers (Mf) of DG granule neurons onto thorny excrescences in hippocampal CA3 neurons. Moreover, LRRTM3 loss in DG neurons significantly decreased mossy fiber long-term potentiation (Mf-LTP). Remarkably, silencing MEC-DG circuits protected against the decrease in the excitatory synaptic inputs onto DG and CA3 neurons, excitability of DG granule neurons, and Mf-LTP in Lrrtm3-deficient mice. These results suggest that LRRTM3 may be a critical factor in activity-dependent synchronization of the topography of MEC-DG-CA3 excitatory synaptic connections. Collectively, our data propose that LRRTM3 shapes the target-specific structural and functional properties of specific hippocampal circuits.

Selective Regional Loss of Cortical Synapses Lacking Presynaptic Mitochondria in the 5xFAD Mouse Model.

Synaptic loss in Alzheimer's disease (AD) is strongly correlated with cognitive impairment. Accumulating evidence indicates that amyloid pathology leads to synaptic degeneration and mitochondrial damage in AD. However, it remains unclear whether synapses and presynaptic mitochondria are differentially affected in various cortical regions of the AD brain at the ultrastructural level. Using serial block-face scanning electron microscopy, we assessed synaptic structures in the medial prefrontal cortex (mPFC) and primary visual cortex (V1) of the 5xFAD mouse model of AD. At 6 months of age, 5xFAD mice exhibited significantly elevated levels of amyloid deposition in layer 2/3 of the mPFC but not V1. Accordingly, three-dimensional reconstruction of synaptic connectivity revealed a significant reduction in excitatory synaptic density in layer 2 of the mPFC, but not V1, of male transgenic mice. Notably, the density of synapses lacking presynaptic mitochondria was selectively decreased in the mPFC of 5xFAD mice, with no change in the density of mitochondria-containing synapses. Further classification of spines into shape categories confirmed a preferential loss of thin spines whose presynaptic boutons were largely devoid of mitochondria in the 5xFAD mPFC. Furthermore, the number of mitochondria per bouton in spared mitochondria-containing boutons was reduced in the mPFC, but not V1, of 5xFAD mice. Collectively, these results highlight region-specific vulnerability of cortical synapses to amyloid deposition and suggest that the presence of presynaptic mitochondria may affect synaptic degeneration in AD.

Overexpression of pigment epithelium-derived factor in placenta-derived mesenchymal stem cells promotes mitochondrial biogenesis in retinal cells.

Pigment epithelium-derived factor (PEDF) plays a role in protecting retinal pigment epithelial (RPE) cells from oxidative stress (OS), a causative factor of RPE cell death. Genetically modified mesenchymal stem cells (MSCs) can be used to treat critical and incurable retinal diseases. Here, we overexpressed PEDF in placenta-derived MSCs (PD-MSCsPEDF, PEDF+) using a nonviral gene delivery system and evaluated the characteristics of PD-MSCsPEDF and their potential regenerative effects on RPE cells damaged by H2O2-induced OS. PD-MSCsPEDF maintained their stemness, cell surface marker, and differentiation potential characteristics. Compared to naive cells, PD-MSCsPEDF promoted mitochondrial respiration by enhancing biogenesis regulators (e.g., NRF1, PPARGC1A, and TFAM) as well as antioxidant enzymes (e.g., HMOXs, SODs, and GPX1). Compared to OS-damaged RPE cells cocultured with naive cells, OS-damaged RPE cells cocultured with PD-MSCsPEDF showed PEDF upregulation and VEGF downregulation. The expression levels of antioxidant genes and RPE-specific genes, such as RPE65, RGR, and RRH, were significantly increased in RPE cells cocultured with PD-MSCsPEDF. Furthermore, OS-damaged RPE cells cocultured with PD-MSCsPEDF had dramatically enhanced mitochondrial functions, and antiapoptotic effects improved due to cell survival signaling pathways. In the H2O2-induced retinal degeneration rat model, compared to administration of the naive counterpart, intravitreal administration of PD-MSCsPEDF alleviated proinflammatory cytokines and restored retinal structure and function by increasing PEDF expression and decreasing VEGF expression. Intravitreal administration of PD-MSCsPEDF also protected retinal degeneration against OS by increasing antioxidant gene expression and regulating the mitochondrial ROS levels and biogenesis. Taken together, PEDF overexpression in PD-MSCs improved the mitochondrial activities and induced OS-damaged RPE cell regeneration by regulating the oxidative status and mitochondrial biogenesis in vitro and in vivo. These data suggest that genetic modification of PEDF in PD-MSCs might be a new cell therapy for the treatment of retinal degenerative diseases.

RAPGEF2 mediates oligomeric Aß-induced synaptic loss and cognitive dysfunction in the 3xTg-AD mouse model of Alzheimer's disease.

AIMS: Amyloid-ß (Aß) oligomers trigger synaptic degeneration that precedes plaque and tangle pathology. However, the signalling molecules that link Aß oligomers to synaptic pathology remain unclear. Here, we addressed the potential role of RAPGEF2 as a novel signalling molecule in Aß oligomer-induced synaptic and cognitive impairments in human-mutant amyloid precursor protein (APP) mouse models of Alzheimer's disease (AD). METHODS: To investigate the role of RAPGEF2 in Aß oligomer-induced synaptic and cognitive impairments, we utilised a combination of approaches including biochemistry, molecular cell biology, light and electron microscopy, behavioural tests with primary neuron cultures, multiple AD mouse models and post-mortem human AD brain tissue. RESULTS: We found significantly elevated RAPGEF2 levels in the post-mortem human AD hippocampus. RAPGEF2 levels also increased in the transgenic AD mouse models, generating high levels of Aß oligomers before exhibiting synaptic and cognitive impairment. RAPGEF2 upregulation activated the downstream effectors Rap2 and JNK. In cultured hippocampal neurons, oligomeric Aß treatment increased the fluorescence intensity of RAPGEF2 and reduced the number of dendritic spines and the intensities of synaptic marker proteins, while silencing RAPGEF2 expression blocked Aß oligomer-induced synapse loss. Additionally, the in vivo knockdown of RAPGEF2 expression in the AD hippocampus prevented cognitive deficits and the loss of excitatory synapses. CONCLUSIONS: These findings demonstrate that the upregulation of RAPGEF2 levels mediates Aß oligomer-induced synaptic and cognitive disturbances in the AD hippocampus. We propose that an early intervention regarding RAPGEF2 expression may have beneficial effects on early synaptic pathology and memory loss in AD.

Altered presynaptic function and number of mitochondria in the medial prefrontal cortex of adult Cyfip2 heterozygous mice.

Variants of the cytoplasmic FMR1-interacting protein (CYFIP) gene family, CYFIP1 and CYFIP2, are associated with numerous neurodevelopmental and neuropsychiatric disorders. According to several studies, CYFIP1 regulates the development and function of both pre- and post-synapses in neurons. Furthermore, various studies have evaluated CYFIP2 functions in the postsynaptic compartment, such as regulating dendritic spine morphology; however, no study has evaluated whether and how CYFIP2 affects presynaptic functions. To address this issue, in this study, we have focused on the presynapses of layer 5 neurons of the medial prefrontal cortex (mPFC) in adult Cyfip2 heterozygous (Cyfip2+/-) mice. Electrophysiological analyses revealed an enhancement in the presynaptic short-term plasticity induced by high-frequency stimuli in Cyfip2+/- neurons compared with wild-type neurons. Since presynaptic mitochondria play an important role in buffering presynaptic Ca2+, which is directly associated with the short-term plasticity, we analyzed presynaptic mitochondria using electron microscopic images of the mPFC. Compared with wild-type mice, the number, but not the volume or cristae density, of mitochondria in both presynaptic boutons and axonal processes in the mPFC layer 5 of Cyfip2+/- mice was reduced. Consistent with an identification of mitochondrial proteins in a previously established CYFIP2 interactome, CYFIP2 was detected in a biochemically enriched mitochondrial fraction of the mouse mPFC. Collectively, these results suggest roles for CYFIP2 in regulating presynaptic functions, which may involve presynaptic mitochondrial changes.

Haploinsufficiency of Cyfip2 Causes Lithium-Responsive Prefrontal Dysfunction.

OBJECTIVE: Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments for CYFIP2-associated brain disorders remain largely unknown. Here, we characterized Cyfip2 heterozygous (Cyfip2+/- ) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of the Cyfip2+/- mice and specified a neuronal function mediating its efficacy. METHODS: We performed behavioral analyses of Cyfip2+/- mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses of Cyfip2+/- prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections. RESULTS: Adult Cyfip2+/- mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adult Cyfip2+/- PFC that was restricted to layer 5 (L5) neurons. Consistently, adult Cyfip2+/- mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability of Cyfip2+/- L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adult Cyfip2+/- PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior. INTERPRETATION: These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adult Cyfip2+/- mice, which can be implicated in CYFIP2-associated brain disorders. ANN NEUROL 2020;88:526-543.

Elevated protein synthesis in microglia causes autism-like synaptic and behavioral aberrations.

Mutations that inactivate negative translation regulators cause autism spectrum disorders (ASD), which predominantly affect males and exhibit social interaction and communication deficits and repetitive behaviors. However, the cells that cause ASD through elevated protein synthesis resulting from these mutations remain unknown. Here we employ conditional overexpression of translation initiation factor eIF4E to increase protein synthesis in specific brain cells. We show that exaggerated translation in microglia, but not neurons or astrocytes, leads to autism-like behaviors in male mice. Although microglial eIF4E overexpression elevates translation in both sexes, it only increases microglial density and size in males, accompanied by microglial shift from homeostatic to a functional state with enhanced phagocytic capacity but reduced motility and synapse engulfment. Consequently, cortical neurons in the mice have higher synapse density, neuroligins, and excitation-to-inhibition ratio compared to control mice. We propose that functional perturbation of male microglia is an important cause for sex-biased ASD.

Microtubule-associated protein 2 mediates induction of long-term potentiation in hippocampal neurons.

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Neurotrophic interactions between neurons and astrocytes following AAV1-Rheb(S16H) transduction in the hippocampus in vivo.

BACKGROUND AND PURPOSE: We recently reported that AAV1-Rheb(S16H) transduction could protect hippocampal neurons through the induction of brain-derived neurotrophic factor (BDNF) in the rat hippocampus in vivo. It is still unclear how neuronal BDNF produced by AAV1-Rheb(S16H) transduction induces neuroprotective effects in the hippocampus and whether its up-regulation contributes to the enhance of a neuroprotective system in the adult brain. EXPERIMENTAL APPROACH: To determine the presence of a neuroprotective system in the hippocampus of patients with Alzheimer's disease (AD), we examined the levels of glial fibrillary acidic protein, BDNF and ciliary neurotrophic factor (CNTF) and their receptors, tropomyocin receptor kinase B (TrkB) and CNTF receptor α(CNTFRα), in the hippocampus of AD patients. We also determined whether AAV1-Rheb(S16H) transduction stimulates astroglial activation and whether reactive astrocytes contribute to neuroprotection in models of hippocampal neurotoxicity in vivo and in vitro. KEY RESULTS: AD patients may have a potential neuroprotective system, demonstrated by increased levels of full-length TrkB and CNTFRα in the hippocampus. Further AAV1-Rheb(S16H) transduction induced sustained increases in the levels of full-length TrkB and CNTFRα in reactive astrocytes and hippocampal neurons. Moreover, neuronal BDNF produced by Rheb(S16H) transduction of hippocampal neurons induced reactive astrocytes, resulting in CNTF production through the activation of astrocytic TrkB and the up-regulation of neuronal BDNF and astrocytic CNTF which had synergistic effects on the survival of hippocampal neurons in vivo. CONCLUSIONS AND IMPLICATIONS: The results demonstrated that Rheb(S16H) transduction of hippocampal neurons could strengthen the neuroprotective system and this intensified system may have a therapeutic value against neurodegeneration in the adult brain.

PTK2/FAK regulates UPS impairment via SQSTM1/p62 phosphorylation in TARDBP/TDP-43 proteinopathies.

TARDBP/TDP-43 (TAR DNA binding protein) proteinopathies are a common feature in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer disease (AD). However, the molecular mechanisms underlying TARDBP-induced neurotoxicity are largely unknown. In this study, we demonstrated that TARDBP proteinopathies induce impairment in the ubiquitin proteasome system (UPS), as evidenced by an accumulation of ubiquitinated proteins and a reduction in proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2/FAK (PTK2 protein tyrosine kinase 2) as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduced ubiquitin aggregates and attenuated TARDBP-induced cytotoxicity in a Drosophila model of TARDBP proteinopathies. We further identified that phosphorylation of SQSTM1/p62 (sequestosome 1) at S403 (p-SQSTM1 [S403]), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TARDBP overexpression and is dependent on the activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of SQSTM1 (SQSTM1S403A) significantly repressed the accumulation of insoluble poly-ubiquitinated proteins and neurotoxicity induced by TARDBP overexpression in neuronal cells. In addition, TBK1 (TANK binding kinase 1), a kinase that phosphorylates S403 of SQSTM1, was found to be involved in the PTK2-mediated phosphorylation of SQSTM1. Taken together, our data suggest that the PTK2-TBK1-SQSTM1 axis plays a critical role in the pathogenesis of TARDBP by regulating neurotoxicity induced by UPS impairment. Therefore, targeting the PTK2-TBK1-SQSTM1 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TARDBP proteinopathies.Abbreviations: ALP: macroautophagy/autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; ATXN2: ataxin 2; BafA1: bafilomycin A1; cCASP3: cleaved caspase 3; CSNK2: casein kinase 2; FTLD: frontotemporal lobar degeneration; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; OPTN: optineurin; PTK2/FAK: PTK2 protein tyrosine kinase 2; SQSTM1/p62: sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system.

Therapeutic Potential of AAV1-Rheb(S16H) Transduction Against Alzheimer's Disease.

We recently reported that adeno-associated virus serotype 1-constitutively active Ras homolog enriched in brain [AAV1-Rheb(S16H)] transduction of hippocampal neurons could induce neuron-astroglia interactions in the rat hippocampus in vivo, resulting in neuroprotection. However, it remains uncertain whether AAV1-Rheb(S16H) transduction induces neurotrophic effects and preserves the cognitive memory in an animal model of Alzheimer's disease (AD) with characteristic phenotypic features, such as ß-amyloid (Aß) accumulation and cognitive impairments. To assess the therapeutic potential of Rheb(S16H) in AD, we have examined the beneficial effects of AAV1-Rheb(S16H) administration in the 5XFAD mouse model. Rheb(S16H) transduction of hippocampal neurons in the 5XFAD mice increased the levels of neurotrophic signaling molecules, including brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and their corresponding receptors, tropomyosin receptor kinase B (TrkB) and CNTF receptor α subunit (CNTFRα), respectively. In addition, Rheb(S16H) transduction inhibited Aß production and accumulation in the hippocampus of 5XFAD mice and protected the decline of long-term potentiation (LTP), resulting in the prevention of cognitive impairments, which was demonstrated using novel object recognition testing. These results indicate that Rheb(S16H) transduction of hippocampal neurons may have therapeutic potential in AD by inhibiting Aß accumulation and preserving LTP associated with cognitive memory.

Gait Ignition Failure in JNPL3 Human Tau-mutant Mice.

Cognitive impairments and motor dysfunction are commonly observed behavioral phenotypes in genetic animal models of neurodegenerative diseases. JNPL3 transgenic mice expressing human P301L-mutant tau display motor disturbances with age- and gene dose-dependent development of neurofibrillary tangles, suggesting that tau pathology causes neurodegeneration associated with motor behavioral abnormalities. Although gait ignition failure (GIF), a syndrome marked by difficulty in initiating locomotion, has been described in patients with certain forms of tauopathies, transgenic mouse models mirroring human GIF syndrome have yet to be reported. Using the open field and balance beam tests, here we discovered that JNPL3 homozygous mice exhibit a marked delay of movement initiation. The elevated plus maze excluded the possibility that hesitation to start in JNPL3 mice was caused by enhanced levels of anxiety. Considering the normal gait ignition in rTg4510 mice expressing the same mutant tau in the forebrain, GIF in JNPL3 mice seems to arise from abnormal tau deposition in the hindbrain areas involved in locomotor initiation. Accordingly, immunohistochemistry revealed highly phosphorylated paired helical filament tau in JNPL3 brainstem areas associated with gait initiation. Together, these findings demonstrate a novel behavioral phenotype of impaired gait initiation in JNPL3 mice and underscore the value of this mouse line as a tool to study the neural mechanisms and potential treatments for human GIF syndrome.

Inhibition of Polo-like kinase 2 ameliorates pathogenesis in Alzheimer's disease model mice.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by pathological hallmarks of neurofibrillary tangles and amyloid plaques. The plaques are formed by aggregation and accumulation of amyloid ß (Aß), a cleavage fragment of amyloid precursor protein (APP). Enhanced neuronal activity and seizure events are frequently observed in AD, and elevated synaptic activity promotes Aß production. However, the mechanisms that link synaptic hyperactivity to APP processing and AD pathogenesis are not well understood. We previously found that Polo-like kinase 2 (Plk2), a homeostatic repressor of neuronal overexcitation, promotes APP ß-processing in vitro. Here, we report that Plk2 stimulates Aß production in vivo, and that Plk2 levels are elevated in a spatiotemporally regulated manner in brains of AD mouse models and human AD patients. Genetic disruption of Plk2 kinase function reduces plaque deposits and activity-dependent Aß production. Furthermore, pharmacological Plk2 inhibition hinders Aß formation, synapse loss, and memory decline in an AD mouse model. Thus, Plk2 links synaptic overactivity to APP ß-processing, Aß production, and disease-relevant phenotypes in vivo, suggesting that Plk2 may be a potential target for AD therapeutics.

Different types of multiple-synapse boutons in the cerebellar cortex between physically enriched and ataxic mutant mice.

Experience-dependent synapse remodeling is associated with information storage in the nervous system. Neuronal synapses show alteration in various neurological and cognitive disorders in their structure and function. At the ultrastructural level, parallel fiber boutons contacting multiple spines of Purkinje cells in the cerebellar cortex are commonly observed in physiologically enriched animals as well as pathological ataxic mutants. However, the dendritic origin of those spines on parallel fiber multiple-synapse boutons (MSBs) has been poorly understood. Here, we investigated this issue by 3-dimensional ultrastructural analysis to determine synaptic connectivity of MSBs in both mice housed in physically enriched environment and cerebellar ataxic mutants. Our results demonstrated that environmental enrichment selectively induced MSBs to contact spines from the same parent dendrite, indicating focal strengthening of synapse through the simultaneous activation of two adjacent spines. In contrast, ataxic mutants displaying impaired motor coordination had significantly more MSBs involving spines originating from different neighboring dendrites compared to both wild-type and environmentally enriched animals, suggesting that compromising multiple synapse formation may lead to abnormal motor behavior in the mutant mice. These findings propose that environmental stimulation in normal animals mainly involves the refinement of preexisting synaptic networks, whereas pathological ataxic conditions may results from less-selective but compromising multiple synaptic formation. This study underscores that different types of multiple synapse boutons may have disparate effects on cerebellar synaptic transmission.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo.

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson's disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.