Hippocampal synaptic failure is an early event in experimental parkinsonism with subtle cognitive deficit.

Learning and memory mainly rely on correct synaptic function in the hippocampus and other brain regions. In Parkinson's disease, subtle cognitive deficits may even precede motor signs early in the disease. Hence, we set out to unravel the earliest hippocampal synaptic alterations associated with human α-synuclein overexpression prior to and soon after the appearance of cognitive deficits in a parkinsonism model. We bilaterally injected adeno-associated viral vectors encoding A53T-mutated human α-synuclein into the substantia nigra of rats, and evaluated them 1, 2, 4 and 16 weeks post-inoculation by immunohistochemistry and immunofluorescence to study degeneration and distribution of α-synuclein in the midbrain and hippocampus. The object location test was used to evaluate hippocampal-dependent memory. Sequential window acquisition of all theoretical mass spectrometry-based proteomics and fluorescence analysis of single-synapse long-term potentiation were used to study alterations to protein composition and plasticity in isolated hippocampal synapses. The effect of L-DOPA and pramipexole on long-term potentiation was also tested. Human α-synuclein was found within dopaminergic and glutamatergic neurons of the ventral tegmental area, and in dopaminergic, glutamatergic and GABAergic axon terminals in the hippocampus from 1 week post-inoculation, concomitant with mild dopaminergic degeneration in the ventral tegmental area. In the hippocampus, differential expression of proteins involved in synaptic vesicle cycling, neurotransmitter release and receptor trafficking, together with impaired long-term potentiation were the first events observed (1 week post-inoculation), preceding cognitive deficits (4 weeks post-inoculation). Later on, at 16 weeks post-inoculation, there was a deregulation of proteins involved in synaptic function, particularly those involved in the regulation of membrane potential, ion balance and receptor signalling. Hippocampal long-term potentiation was impaired before and soon after the onset of cognitive deficits, at 1 and 4 weeks post-inoculation, respectively. L-DOPA recovered hippocampal long-term potentiation more efficiently at 4 weeks post-inoculation than pramipexole, which partially rescued it at both time points. Overall, we found impaired synaptic plasticity and proteome dysregulation at hippocampal terminals to be the first events that contribute to the development of cognitive deficits in experimental parkinsonism. Our results not only point to dopaminergic but also to glutamatergic and GABAergic dysfunction, highlighting the relevance of the three neurotransmitter systems in the ventral tegmental area-hippocampus interaction from the earliest stages of parkinsonism. The proteins identified in the current work may constitute potential biomarkers of early synaptic damage in the hippocampus and hence, therapies targeting these could potentially restore early synaptic malfunction and consequently, cognitive deficits in Parkinson's disease.

The role of aging and brain-derived neurotrophic factor signaling in expression of base excision repair genes in the human brain.

DNA damage is a central contributor to the aging process. In the brain, a major threat to the DNA is the considerable amount of reactive oxygen species produced, which can inflict oxidative DNA damage. This type of damage is removed by the base excision repair (BER) pathway, an essential DNA repair mechanism, which contributes to genome stability in the brain. Despite the crucial role of the BER pathway, insights into how this pathway is affected by aging in the human brain and the underlying regulatory mechanisms are very limited. By microarray analysis of four cortical brain regions from humans aged 20-99 years (n = 57), we show that the expression of core BER genes is largely downregulated during aging across brain regions. Moreover, we find that expression of many BER genes correlates positively with the expression of the neurotrophin brain-derived neurotrophic factor (BDNF) in the human brain. In line with this, we identify binding sites for the BDNF-activated transcription factor, cyclic-AMP response element-binding protein (CREB), in the promoter of most BER genes and confirm the ability of BDNF to regulate several BER genes by BDNF treatment of mouse primary hippocampal neurons. Together, these findings uncover the transcriptional landscape of BER genes during aging of the brain and suggest BDNF as an important regulator of BER in the human brain.

Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer's disease.

The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.

Intra- and extracellular ß-amyloid overexpression via adeno-associated virus-mediated gene transfer impairs memory and synaptic plasticity in the hippocampus.

Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is currently conceptualized as a disease of synaptic failure. Synaptic impairments are robust within the AD brain and better correlate with dementia severity when compared with other pathological features of the disease. Nevertheless, the series of events that promote synaptic failure still remain under debate, as potential triggers such as ß-amyloid (Aß) can vary in size, configuration and cellular location, challenging data interpretation in causation studies. Here we present data obtained using adeno-associated viral (AAV) constructs that drive the expression of oligomeric Aß either intra or extracellularly. We observed that expression of Aß in both cellular compartments affect learning and memory, reduce the number of synapses and the expression of synaptic-related proteins, and disrupt chemical long-term potentiation (cLTP). Together, these findings indicate that during the progression AD the early accumulation of Aß inside neurons is sufficient to promote morphological and functional cellular toxicity, a phenomenon that can be exacerbated by the buildup of Aß in the brain parenchyma. Moreover, our AAV constructs represent a valuable tool in the investigation of the pathological properties of Aß oligomers both in vivo and in vitro.

Hippocampal gene expression patterns linked to late-life physical activity oppose age and AD-related transcriptional decline.

Exercise has emerged as a powerful variable that can improve cognitive function and delay age-associated cognitive decline and Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. To determine if protective mechanisms may occur at the transcriptional level, we used microarrays to investigate the relationship between physical activity levels and gene expression patterns in the cognitively intact aged human hippocampus. In parallel, hippocampal gene expression patterns associated with aging and AD were assessed using publicly available microarray data profiling hippocampus from young (20-59 years), cognitively intact aging (73-95 years) and age-matched AD cases. To identify "anti-aging/AD" transcription patterns associated with physical activity, probesets significantly associated with both physical activity and aging/AD were identified and their directions of expression change in each condition were compared. Remarkably, of the 2210 probesets significant in both data sets, nearly 95% showed opposite transcription patterns with physical activity compared with aging/AD. The majority (>70%) of these anti-aging/AD genes showed increased expression with physical activity and decreased expression in aging/AD. Enrichment analysis of the anti-aging/AD genes showing increased expression in association with physical activity revealed strong overrepresentation of mitochondrial energy production and synaptic function, along with axonal function and myelin integrity. Synaptic genes were notably enriched for synaptic vesicle priming, release and recycling, glutamate and GABA signaling, and spine plasticity. Anti-aging/AD genes showing decreased expression in association with physical activity were enriched for transcription-related function (notably negative regulation of transcription). These data reveal that physical activity is associated with a more youthful profile in the hippocampus across multiple biological processes, providing a potential molecular foundation for how physical activity can delay age- and AD-related decline of hippocampal function.

Inhibition of LTP-Induced Translation by IL-1ß Reduces the Level of Newly Synthesized Proteins in Hippocampal Dendrites.

In rodent hippocampus, the inflammatory cytokine interleukin-1ß (IL-1ß) impairs memory and long-term potentiation (LTP), a major form of plasticity that depends on protein synthesis. A better understanding of the mechanisms by which IL-1ß impairs LTP may help identify targets for preventing cognitive deterioration. We tested whether IL-1ß inhibits protein synthesis in hippocampal neuron cultures following chemically induced LTP (cLTP). Fluorescent-tagging using click-chemistry showed that IL-1ß reduces the level of newly synthesized proteins in proximal dendrites of cLTP stimulated neurons. Relative to controls, in cLTP stimulated neurons, IL-1ß inhibited Akt/mTOR signaling, as well as the upregulation of GluA1, an AMPA receptor subunit, and LIMK1, a kinase that promotes actin polymerization. Notably, a novel TIR domain peptidomimetic (EM163) blocked both the activation of p38 and the suppression of cLTP-dependent protein synthesis by IL-1ß. Our data support a model where IL-1ß suppresses LTP directly in neurons by inhibiting mTOR-dependent translation.

TNFα and IL-1ß but not IL-18 Suppresses Hippocampal Long-Term Potentiation Directly at the Synapse.

CNS inflammatory responses are linked to cognitive impairment in humans. Research in animal models supports this connection by showing that inflammatory cytokines suppress long-term potentiation (LTP), the best-known cellular correlate of memory. Cytokine-induced modulation of LTP has been previously studied in vivo or in brain slices, two experimental approaches containing multiple cell populations responsive to cytokines. In their target cells, cytokines commonly increase the expression of multiple cytokines, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Whether cytokines suppress LTP by direct effects on neurons or by indirect mechanisms is still an open question. Here, we evaluated the effect of a major set of inflammatory cytokines including tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) on chemically-induced LTP (cLTP) in isolated hippocampal synaptosomes of mice, using fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). We found that TNFα and IL-1ß suppress synaptosomal cLTP. In contrast, cLTP was not affected by IL-18, at a concentration previously shown to block LTP in hippocampal slices. We also found that IL-18 does not impair cLTP or brain-derived neurotrophic factor (BDNF) signaling in primary hippocampal neuronal cultures. Thus, using both synaptosomes and neuron cultures, our data suggest that IL-18 impairs LTP by indirect mechanisms, which may depend on non-neuronal cells, such as glia. Notably, our results demonstrate that TNFα and IL-1ß directly suppress hippocampal plasticity via neuron-specific mechanisms. A better understanding of the brain's cytokine networks and their final molecular effectors is crucial to identify specific targets for intervention.

IL-1ß suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation.

BACKGROUND: Brain inflammation including increases in inflammatory cytokines such as IL-1ß is widely believed to contribute to the pathophysiology of Alzheimer's disease. Although IL-1ß-induced impairments in long-term potentiation (LTP) in acute hippocampal slices and memory functions in vivo have been well documented, the neuron-specific molecular mechanisms of IL-1ß-mediated impairments of LTP and memory remain unclear. METHODS: This study uses an in vitro approach in primary hippocampal neurons to evaluate the effect of IL-1ß on chemical LTP (cLTP)-induced structural plasticity and signaling. RESULTS: We found that IL-1ß reduces both the surface expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 and the spine growth following cLTP. These effects of IL-1ß were mediated by impairing actin polymerization during cLTP, as IL-1ß decreased the cLTP-induced formation of F-actin, and the effect of IL-1ß on cLTP-induced surface expression of GluA1 can be mimicked by latrunculin, a toxin that disrupts dynamics of actin filaments, and can be prevented by jasplakinolide, a cell-permeable peptide that stabilizes F-actin. Moreover, live-cell imaging demonstrated that IL-1ß decreased the stability of the actin cytoskeleton in spines, which is required for LTP consolidation. We further examined the role of sphingolipid signaling in the IL-1ß-mediated impairment of spine plasticity and found that both the neutral sphingomyelinase inhibitor GW4869 and the inhibitor of Src kinase PP2 attenuated the IL-1ß-mediated suppression of cLTP-induced surface expression of GluA1 and actin polymerization. CONCLUSIONS: These findings support a mechanism by which IL-1ß, via the sphingomyelinase/ceramide/Src pathway, impairs structural spine remodeling essential for LTP consolidation and memory.

On the road towards the global analysis of human synapses.

Synapses are essential units for the flow of information in the brain. Over the last 70 years, synapses have been widely studied in multiple animal models including worms, fruit flies, and rodents. In comparison, the study of human synapses has evolved significantly slower, mainly because of technical limitations. However, three novel methods allowing the analysis of molecular, morphological, and functional properties of human synapses may expand our knowledge of the human brain. Here, we briefly describe these methods, and evaluate how the information provided by each unique approach may contribute to the functional and anatomical analysis of the synaptic component of human brain circuitries. In particular, using tissue from cryopreserved human brains, synaptic plasticity can be studied in isolated synaptosomes by fluorescence analysis of single-synapse long-term potentiation (FASS-LTP), and subpopulations of synapses can be thoroughly assessed in the ribbons of brain tissue by array tomography (AT). Currently, it is also possible to quantify synaptic density in the living human brain by positron emission tomography (PET), using a novel synaptic radio-ligand. Overall, data provided by FASS-LTP, AT, and PET may significantly contribute to the global understanding of synaptic structure and function in both healthy and diseased human brains, thus directly impacting translational research.

Abnormalities of Dopamine D3 Receptor Signaling in the Diseased Brain.

Dopamine D3 receptors (D3R) modulate neuronal activity in several brain regions including cortex, striatum, cerebellum, and hippocampus. A growing body of evidence suggests that aberrant D3R signaling contributes to multiple brain diseases, such as Parkinson's disease, essential tremor, schizophrenia, and addiction. In line with these findings, D3R has emerged as a potential target in the treatment of neurological disorders. However, the mechanisms underlying neuronal D3R signaling are poorly understood, either in healthy or diseased brain. Here, I review the molecular mechanisms involved in D3R signaling via monomeric D3R and heteromeric receptor complexes (e.g., D3R-D1R, D3R-D2R, D3R-A2aR, and D3R-D3nf). I focus on D3R signaling pathways that, according to recent reports, contribute to pathological brain states. In particular, I describe evidence on both quantitative (e.g., increased number or affinity) and qualitative (e.g., switched signaling) changes in D3R that has been associated with brain dysfunction. I conclude with a description of basic mechanisms that modulate D3R signaling such as desensitization, as disruption of these mechanisms may underlie pathological changes in D3R signaling. Because several lines of evidence support the idea that imbalances in D3R signaling alter neural function, a better understanding of downstream D3R pathways is likely to reveal novel therapeutic strategies toward dopamine-related brain disorders.

Cytokines and cytokine networks target neurons to modulate long-term potentiation.

Cytokines play crucial roles in the communication between brain cells including neurons and glia, as well as in the brain-periphery interactions. In the brain, cytokines modulate long-term potentiation (LTP), a cellular correlate of memory. Whether cytokines regulate LTP by direct effects on neurons or by indirect mechanisms mediated by non-neuronal cells is poorly understood. Elucidating neuron-specific effects of cytokines has been challenging because most brain cells express cytokine receptors. Moreover, cytokines commonly increase the expression of multiple cytokines in their target cells, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Here, we review evidence on both direct and indirect-mediated modulation of LTP by cytokines. We also describe novel approaches based on neuron- and synaptosome-enriched systems to identify cytokines able to directly modulate LTP, by targeting neurons and synapses. These approaches can test multiple samples in parallel, thus allowing the study of multiple cytokines simultaneously. Hence, a cytokine networks perspective coupled with neuron-specific analysis may contribute to delineation of maps of the modulation of LTP by cytokines.

IL-1ß impairs retrograde flow of BDNF signaling by attenuating endosome trafficking.

BACKGROUND: Pro-inflammatory cytokines accumulate in the brain with age and Alzheimer's disease and can impair neuron health and cognitive function. Brain-derived neurotrophic factor (BDNF) is a key neurotrophin that supports neuron health, function, and synaptic plasticity. The pro-inflammatory cytokine interleukin-1ß (IL-1ß) impairs BDNF signaling but whether it affects BDNF signaling endosome trafficking has not been studied. METHODS: This study uses an in vitro approach in primary hippocampal neurons to evaluate the effect of IL-1ß on BDNF signaling endosome trafficking. Neurons were cultured in microfluidic chambers that separate the environments of the cell body and its axon terminal, enabling us to specifically treat in axon compartments and trace vesicle trafficking in real-time. RESULTS: We found that IL-1ß attenuates BDNF signaling endosomes throughout networks in cultures. In IL-1ß-treated cells, overall BDNF endosomal density was decreased, and the colocalization of BDNF endosomes with presynaptic terminals was found to be more than two times higher than in control cultures. Selective IL-1ß treatment to the presynaptic compartment in microfluidic chamber attenuated BDNF endosome flux, as measured by reduced BDNF-GFP endosome counts in the somal compartment. Further, IL-1ß decreased the BDNF-induced phosphorylation of Erk5, a known BDNF retrograde trafficking target. Mechanistically, the deficiency in trafficking was not due to impaired endocytosis of the BDNF-TrkB complex, or impaired transport rate, since BDNF endosomes traveled at the same rate in both control and IL-1ß treatment groups. Among the regulators of presynaptic endosome sorting is the post-translational modification, ubiquitination. In support of this possibility, the IL-1ß-mediated suppression of BDNF-induced Erk5 phosphorylation can be rescued by exogenous ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that regulates ubiquitin and endosomal trafficking. CONCLUSIONS: We observed a state of neurotrophic resistance whereby, in the prolonged presence of IL-1ß, BDNF is not effective in delivering long-distance signaling via the retrograde transport of signaling endosomes. Since IL-1ß accumulation is an invariant feature across many neurodegenerative diseases, our study suggest that compromised BDNF retrograde transport-dependent signaling may have important implications in neurodegenerative diseases.

Pharmacological Rescue of Long-Term Potentiation in Alzheimer Diseased Synapses.

Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.

H3K9me3 Inhibition Improves Memory, Promotes Spine Formation, and Increases BDNF Levels in the Aged Hippocampus.

An increasing number of studies show that an altered epigenetic landscape may cause impairments in regulation of learning and memory-related genes within the aged hippocampus, eventually resulting in cognitive deficits in the aged brain. One such epigenetic repressive mark is trimethylation of H3K9 (H3K9me3), which is typically implicated in gene silencing. Here, we identify, for the first time, an essential role for H3K9me3 and its histone methyl transferase (SUV39H1) in mediating hippocampal memory functions. Pharmacological inhibition of SUV39H1 using a novel and selective inhibitor decreased levels of H3K9me3 in the hippocampus of aged mice, and improved performance in the objection location memory and fear conditioning tasks and in a complex spatial environment learning task. The inhibition of SUV39H1 induced an increase in spine density of thin and stubby but not mushroom spines in the hippocampus of aged animals and increased surface GluR1 levels in hippocampal synaptosomes, a key index of spine plasticity. Furthermore, there were changes at BDNF exon I gene promoter, in concert with overall BDNF levels in the hippocampus of drug-treated animals compared with control animals. Together, these data demonstrate that SUV39H1 inhibition and the concomitant H3K9me3 downregulation mediate gene transcription in the hippocampus and reverse age-dependent deficits in hippocampal memory. SIGNIFICANCE STATEMENT: Cognitive decline is a debilitating condition associated with not only neurodegenerative diseases but also aging in general. However, effective treatments have been slow to emerge so far. In this study, we demonstrate that epigenetic regulation of key synaptic proteins may be an underlying, yet reversible, cause of this decline. Our findings suggest that histone 3 trimethylation is a probable target for pharmacological intervention that can counteract cognitive decline in the aging brain. Finally, we provide support to the hypothesis that, by manipulating the enzyme that regulates H3K9me3 (using a newly developed specific inhibitor of SUV39H1), it is possible to alter the chromatin state of subjects and restore memory and synaptic function in the aging brain.

Synapse-specific IL-1 receptor subunit reconfiguration augments vulnerability to IL-1ß in the aged hippocampus.

In the aged brain, synaptic plasticity and memory show increased vulnerability to impairment by the inflammatory cytokine interleukin 1ß (IL-1ß). In this study, we evaluated the possibility that synapses may directly undergo maladaptive changes with age that augment sensitivity to IL-1ß impairment. In hippocampal neuronal cultures, IL-1ß increased the expression of the IL-1 receptor type 1 and the accessory coreceptor AcP (proinflammatory), but not of the AcPb (prosurvival) subunit, a reconfiguration that potentiates the responsiveness of neurons to IL-1ß. To evaluate whether synapses develop a similar heightened sensitivity to IL-1ß with age, we used an assay to track long-term potentiation (LTP) in synaptosomes. We found that IL-1ß impairs LTP directly at the synapse and that sensitivity to IL-1ß is augmented in aged hippocampal synapses. The increased synaptic sensitivity to IL-1ß was due to IL-1 receptor subunit reconfiguration, characterized by a shift in the AcP/AcPb ratio, paralleling our culture data. We suggest that the age-related increase in brain IL-1ß levels drives a shift in IL-1 receptor configuration, thus heightening the sensitivity to IL-1ß. Accordingly, selective blocking of AcP-dependent signaling with Toll-IL-1 receptor domain peptidomimetics prevented IL-1ß-mediated LTP suppression and blocked the memory impairment induced in aged mice by peripheral immune challenge (bacterial lipopolysaccharide). Overall, this study demonstrates that increased AcP signaling, specifically at the synapse, underlies the augmented vulnerability to cognitive impairment by IL-1ß that occurs with age.

Modulation of direct pathway striatal projection neurons by muscarinic M4-type receptors.

Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D1-type DA, M1-and M4-type ACh receptors, while iSPNs express D2-type DA and M1-type ACh receptors. Actions of M1-, D1-, and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools such as mamba-toxins, and BAC D(1 or 2)-eGFP transgenic mice to show that activation of M4-type receptors with bath applied muscarine enhances Ca(2+)-currents through CaV1-channels in dSPNs and not in iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically driven stimulation. The increases in Ca(2+)-current and excitability were blocked specifically by mamba toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradoxically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed.

Rapamycin and interleukin-1ß impair brain-derived neurotrophic factor-dependent neuron survival by modulating autophagy.

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF's support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF's suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1ß impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.

Brain-derived neurotrophic factor-dependent synaptic plasticity is suppressed by interleukin-1ß via p38 mitogen-activated protein kinase.

Evolving evidence suggests that brain inflammation and the buildup of proinflammatory cytokine increases the risk for cognitive decline and cognitive dysfunction. Interleukin-1ß (IL-1ß), acting via poorly understood mechanisms, appears to be a key cytokine in causing these deleterious effects along with a presumably related loss of long-term potentiation (LTP)-type synaptic plasticity. We hypothesized that IL-1ß disrupts brain-derived neurotrophic factor (BDNF) signaling cascades and thereby impairs the formation of filamentous actin (F-actin) in dendritic spines, an event that is essential for the stabilization of LTP. Actin polymerization in spines requires phosphorylation of the filament severing protein cofilin and is modulated by expression of the immediate early gene product Arc. Using rat organotypic hippocampal cultures, we found that IL-1ß suppressed BDNF-dependent regulation of Arc and phosphorylation of cofilin and cAMP response element-binding protein (CREB), a transcription factor regulating Arc expression. IL-1ß appears to act on BDNF signal transduction by impairing the phosphorylation of insulin receptor substrate 1, a protein that couples activation of the BDNF receptor TrkB to downstream signaling pathways regulating CREB, Arc, and cofilin. IL-1ß upregulated p38 mitogen-activated protein kinase (MAPK), and inhibiting p38 MAPK prevented IL-1ß from disrupting BDNF signaling. IL-1ß also prevented the formation of F-actin in spines and impaired the consolidation, but not the induction, of BDNF-dependent LTP in acute hippocampal slices. The suppressive effect of IL-1ß on F-actin and LTP was prevented by inhibiting p38 MAPK. These findings define a new mechanism for the action of IL-1ß on LTP and point to a potential therapeutic target to restore synaptic plasticity.