HIV and FIV glycoproteins increase cellular tau pathology via cGMP-dependent kinase II activation

As the development of combination antiretroviral therapy (cART) against human immunodeficiency virus (HIV) drastically improves the lifespan of individuals with HIV, many are now entering the prime age when Alzheimer's disease (AD)-like symptoms begin to manifest. Hyperphosphorylated tau, a known AD pathological characteristic, has been prematurely increased in the brains of HIV-infected patients as early as in their 30s and is increased with age. This thus suggests that HIV infection may lead to accelerated AD phenotypes. However, whether HIV infection causes AD to develop more quickly in the brain is not yet fully determined. Interestingly, we have previously revealed that viral glycoproteins, HIV gp120 and feline immunodeficiency virus (FIV) gp95, induce neuronal hyperexcitation via cGMP-dependent kinase II (cGKII) activation in cultured hippocampal neurons. Here, we use cultured mouse cortical neurons to demonstrate that HIV gp120 and FIV gp95 are sufficient to increase cellular tau pathology, including intracellular tau hyperphosphorylation and tau release to the extracellular space. We further reveal that viral glycoprotein-induced cellular tau pathology requires cGKII activation. Together, HIV infection likely accelerates AD-related tau pathology via cGKII activation.

Calcium-/Calmodulin-Dependent Protein Kinase II (CaMKII) Inhibition Induces Learning and Memory Impairment and Apoptosis

Objectives. Inhibition of calcium-/calmodulin- (CaM-) dependent kinase II (CaMKII) is correlated with epilepsy. However, the specific mechanism that underlies learning and memory impairment and neuronal death by CaMKII inhibition remains unclear. Materials and Methods. In this study, KN93, a CaMKII inhibitor, was used to investigate the role of CaMKII during epileptogenesis. We first identified differentially expressed genes (DEGs) in primary cultured hippocampal neurons with or without KN93 treatment using RNA-sequencing. Then, the impairment of learning and memory by KN93-induced CaMKII inhibition was assessed using the Morris water maze test. In addition, Western blotting, immunohistochemistry, and TUNEL staining were performed to determine neuronal death, apoptosis, and the relative signaling pathway. Results. KN93-induced CaMKII inhibition decreased cAMP response element-binding (CREB) protein activity and impaired learning and memory in Wistar and tremor (TRM) rats, an animal model of genetic epilepsy. CaMKII inhibition also induced neuronal death and reactive astrocyte activation in both the Wistar and TRM hippocampi, deregulating mitogen-activated protein kinases. Meanwhile, neuronal death and neuron apoptosis were observed in PC12 and primary cultured hippocampal neurons after exposure to KN93, which was reversed by SP600125, an inhibitor of c-Jun N-terminal kinase (JNK). Conclusions. CaMKII inhibition caused learning and memory impairment and apoptosis, which might be related to dysregulated JNK signaling.

A Novel Effect of PDLIM5 In α7 Nicotinic Acetylcholine Receptors Up-Regulation And Surface Expression

α7 neuronal nicotinic acetylcholine receptors (α7nAChRs) are expressed widely in the brain, where they contribute to a variety of behaviors including arousal and cognition, participate in a number of neurodegenerative disorders including Alzheimer’s and Parkinson’s disease, and is responsible for nicotine addiction. Although recent studies indicate that the PDZ-containing proteins comprising PSD-95 family co-localize with nicotinic acetylcholine receptors and mediate downstream signaling in the neurons, the mechanisms by which α7nAChRs are regulated are still less well understood. Here we show that the regulation of the α7nAChRs is controlled by PDLIM5 in the endogenous PDZ domain proteins family. We find that chronic exposure to 1 μM nicotine up-regulated both α7, β2-contained nAChRs and PDLIM5 in primary cultured hippocampal neurons, and the up-regulation of α7nAChRs and PDLIM5 is increased more on the cell membrane than the cytoplasm. Interestingly, the α7nAChRs and β2nAChRs display distinct patterns of expression, with α7 co-localized more with PDLIM5. Meanwhile, PDLIM5 interacts with native brain α7 but not β2 nAChRs in neurons. Moreover, knocking down of PDLIM5 in heterologous cells abolishes nicotine-induced up-regulation of α7nAChRs. In cultured hippocampal neurons, shRNA against PDLIM5 decreased both surface clustering of α7nAChRs and α7nAChRs mediated currents. Proteomics analysis shows PDLIM5 interacts with α7nAChRs through the PDZ domain and the interaction between PDLIM5 and α7nAChRs can be promoted by nicotine. Collectively, our data suggest a novel cellular role of PDLIM5 in regulating α7nAChRs, which may be relevant to plastic changes in the nervous system.

Overexpression of Tropomyosin Isoform Tpm3.1 Does Not Alter Synaptic Function in Hippocampal Neurons

Tropomyosin (Tpm) has been regarded as the master regulator of actin dynamics. Tpms regulate the binding of the various proteins involved in restructuring actin. The actin cytoskeleton is the predominant cytoskeletal structure in dendritic spines. Its regulation is critical for spine formation and long-term activity-dependent changes in synaptic strength. The Tpm isoform Tpm3.1 is enriched in dendritic spines, but its role in regulating the synapse structure and function is not known. To determine the role of Tpm3.1, we studied the synapse structure and function of cultured hippocampal neurons from transgenic mice overexpressing Tpm3.1. We recorded hippocampal field excitatory postsynaptic potentials (fEPSPs) from brain slices to examine if Tpm3.1 overexpression alters long-term synaptic plasticity. Tpm3.1-overexpressing cultured neurons did not show a significantly altered dendritic spine morphology or synaptic activity. Similarly, we did not observe altered synaptic transmission or plasticity in brain slices. Furthermore, expression of Tpm3.1 at the postsynaptic compartment does not increase the local F-actin levels. The results suggest that although Tpm3.1 localises to dendritic spines in cultured hippocampal neurons, it does not have any apparent impact on dendritic spine morphology or function. This is contrary to the functional role of Tpm3.1 previously observed at the tip of growing neurites, where it increases the F-actin levels and impacts growth cone dynamics.

Deficiency of Intellectual Disability-Related Gene Brpf1 Attenuated Hippocampal Excitatory Synaptic Transmission and Impaired Spatial Learning and Memory Ability

Patients with monoallelic bromodomain and PHD finger-containing protein 1 (BRPF1) mutations showed intellectual disability. The hippocampus has essential roles in learning and memory. Our previous work indicated that Brpf1 was specifically and strongly expressed in the hippocampus from the perinatal period to adulthood. We hypothesized that mouse Brpf1 plays critical roles in the morphology and function of hippocampal neurons, and its deficiency leads to learning and memory deficits. To test this, we performed immunofluorescence, whole-cell patch clamp, and mRNA-Seq on shBrpf1-infected primary cultured hippocampal neurons to study the effect of Brpf1 knockdown on neuronal morphology, electrophysiological characteristics, and gene regulation. In addition, we performed stereotactic injection into adult mouse hippocampus to knock down Brpf1 in vivo and examined the learning and memory ability by Morris water maze. We found that mild knockdown of Brpf1 reduced mEPSC frequency of cultured hippocampal neurons, before any significant changes of dendritic morphology showed. We also found that Brpf1 mild knockdown in the hippocampus showed a decreasing trend on the spatial learning and memory ability of mice. Finally, mRNA-Seq analyses showed that genes related to learning, memory, and synaptic transmission (such as C1ql1, Gpr17, Htr1d, Glra1, Cxcl10, and Grin2a) were dysregulated upon Brpf1 knockdown. Our results showed that Brpf1 mild knockdown attenuated hippocampal excitatory synaptic transmission and reduced spatial learning and memory ability, which helps explain the symptoms of patients with BRPF1 mutations.

EFFECTS OF ARTIFICIAL HYPOINSULINEMIA ON SYNAPTIC ACTIVITY AND PLASTICITY OF GLUTAMATERGIC NEUROTRANSMISSION IN CULTURE OF HIPPOCAMPAL NEURONS

We investigated the effect of chronic hypoinsulinemia on the level of synaptic activity and short-term plasticity in cultured hippocampal neurons. Hypoinsulinemia was induced by culturing mature (16-20 days in vitro) rat’s hippocampal neurons without insulin for 1, 2, and 4 days. The control insulin concentration was 100 nM. Spontaneous and evoked glutamatergic excitatory postsynaptic currents (sEPSC and eEPSC, respectively) in these neurons were analyzed using the whole-cell patch-clamp method and the method of local electrical stimulation of individual axon. Hypoinsulinemia during the 1st, 2nd and 4th days led to significantly reduction of the mean sEPSC’s frequency to 49.9 ± 15.8% (n = 6), 8.5 ± 7.7% (n = 6) and 16.6 ± 5.2% (n = 8) respectively, relative to control. Also, there was a decrease of the average sEPSC’s amplitudes to 52.6 ± 5.5% (n = 6), 36.6 ± 5.8% (n = 6) and 43.9 ± 8.4% (n = 8), respectively, relative to control. Quantal analysis of the sEPSC’s amplitudes showed a decrease of multivesicular glutamate release at the synapses under such conditions. Hypoinsulinemia caused a shift in the direction of short-term plasticity in glutamatergic hippocampal synapses from potentiation to depression. The paired-pulse ratio decreased from 1.83 ± 0.25 in the control to 0.59 ± 0.07, 0.77 ± 0.07, and 0.80 ± 0.06 after the 1st, 2nd, and 4th days under cultivation without insulin. Accordingly, the ratio of the coefficients of variation of eEPSC’s amplitudes (CV2/ CV1) increased from 0.82 ± 0.07 to 1.30 ± 0.28, 1.52 ± 0.27, and 1.61 ± 0.24. The presented results indicate a significant reduction of synaptic activity and decrease in the probability of multivesicular release of glutamate at the synapses of cultured hippocampal neurons under hypoinsulinemia.

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.