Phosphodiesterase 9A Inhibition Facilitates Corticostriatal Transmission in Wild-Type and Transgenic Rats That Model Huntington's Disease.

Huntington's disease (HD) results from abnormal expansion in CAG trinucleotide repeats within the HD gene, a mutation which leads to degeneration of striatal medium-sized spiny neurons (MSNs), deficits in corticostriatal transmission, and loss of motor control. Recent studies also indicate that metabolism of cyclic nucleotides by phosphodiesterases (PDEs) is dysregulated in striatal networks in a manner linked to deficits in corticostriatal transmission. The current study assessed cortically-evoked firing in electrophysiologically-identified MSNs and fast-spiking interneurons (FSIs) in aged (9-11 months old) wild-type (WT) and BACHD transgenic rats (TG5) treated with vehicle or the selective PDE9A inhibitor PF-04447943. WT and TG5 rats were anesthetized with urethane and single-unit activity was isolated during low frequency electrical stimulation of the ipsilateral motor cortex. Compared to WT controls, MSNs recorded in TG5 animals exhibited decreased spike probability during cortical stimulation delivered at low to moderate stimulation intensities. Moreover, large increases in onset latency of cortically-evoked spikes and decreases in spike probability were observed in FSIs recorded in TG5 animals. Acute systemic administration of the PDE9A inhibitor PF-04447943 significantly decreased the onset latency of cortically-evoked spikes in MSNs recorded in WT and TG5 rats. PDE9A inhibition also increased the proportion of MSNs responding to cortical stimulation and reversed deficits in spike probability observed in TG5 rats. As PDE9A is a cGMP specific enzyme, drugs such as PF-04447943 which act to facilitate striatal cGMP signaling and glutamatergic corticostriatal transmission could be useful therapeutic agents for restoring striatal function and alleviating motor and cognitive symptoms associated with HD.

Reduced presynaptic vesicle stores mediate cellular and network plasticity defects in an early-stage mouse model of Alzheimer's disease.

BACKGROUND: Identifying effective strategies to prevent memory loss in AD has eluded researchers to date, and likely reflects insufficient understanding of early pathogenic mechanisms directly affecting memory encoding. As synaptic loss best correlates with memory loss in AD, refocusing efforts to identify factors driving synaptic impairments may provide the critical insight needed to advance the field. In this study, we reveal a previously undescribed cascade of events underlying pre and postsynaptic hippocampal signaling deficits linked to cognitive decline in AD. These profound alterations in synaptic plasticity, intracellular Ca2+ signaling, and network propagation are observed in 3-4 month old 3xTg-AD mice, an age which does not yet show overt histopathology or major behavioral deficits. METHODS: In this study, we examined hippocampal synaptic structure and function from the ultrastructural level to the network level using a range of techniques including electron microscopy (EM), patch clamp and field potential electrophysiology, synaptic immunolabeling, spine morphology analyses, 2-photon Ca2+ imaging, and voltage-sensitive dye-based imaging of hippocampal network function in 3-4 month old 3xTg-AD and age/background strain control mice. RESULTS: In 3xTg-AD mice, short-term plasticity at the CA1-CA3 Schaffer collateral synapse is profoundly impaired; this has broader implications for setting long-term plasticity thresholds. Alterations in spontaneous vesicle release and paired-pulse facilitation implicated presynaptic signaling abnormalities, and EM analysis revealed a reduction in the ready-releasable and reserve pools of presynaptic vesicles in CA3 terminals; this is an entirely new finding in the field. Concurrently, increased synaptically-evoked Ca2+ in CA1 spines triggered by LTP-inducing tetani is further enhanced during PTP and E-LTP epochs, and is accompanied by impaired synaptic structure and spine morphology. Notably, vesicle stores, synaptic structure and short-term plasticity are restored by normalizing intracellular Ca2+ signaling in the AD mice. CONCLUSIONS: These findings suggest the Ca2+ dyshomeostasis within synaptic compartments has an early and fundamental role in driving synaptic pathophysiology in early stages of AD, and may thus reflect a foundational disease feature driving later cognitive impairment. The overall significance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic vesicle stores, synaptic plasticity, and network propagation, which directly impact memory encoding.

Impact of Vortioxetine on Synaptic Integration in Prefrontal-Subcortical Circuits: Comparisons with Escitalopram.

Prefrontal-subcortical circuits support executive functions which often become dysfunctional in psychiatric disorders. Vortioxetine is a multimodal antidepressant that is currently used in the clinic to treat major depressive disorder. Mechanisms of action of vortioxetine include serotonin (5-HT) transporter blockade, 5-HT1A receptor agonism, 5-HT1B receptor partial agonism, and 5-HT1D, 5-HT3, and 5-HT7 receptor antagonism. Vortioxetine facilitates 5-HT transmission in the medial prefrontal cortex (mPFC), however, the impact of this compound on related prefrontal-subcortical circuits is less clear. Thus, the current study examined the impact of systemic vortioxetine administration (0.8 mg/kg, i.v.) on spontaneous spiking and spikes evoked by electrical stimulation of the mPFC in the anterior cingulate cortex (ACC), medial shell of the nucleus accumbens (msNAc), and lateral septal nucleus (LSN) in urethane-anesthetized rats. We also examined whether vortioxetine modulated afferent drive in the msNAc from hippocampal fimbria (HF) inputs. Similar studies were performed using the selective 5-HT reuptake inhibitor [selective serotonin reuptake inhibitors (SSRI)] escitalopram (1.6 mg/kg, i.v.) to enable comparisons between the multimodal actions of vortioxetine and SSRI-mediated effects. No significant differences in spontaneous activity were observed in the ACC, msNAc, and LSN across treatment groups. No significant impact of treatment on mPFC-evoked responses was observed in the ACC. In contrast, vortioxetine decreased mPFC-evoked activity recorded in the msNAc as compared to parallel studies in control and escitalopram treated groups. Thus, vortioxetine may reduce mPFC-msNAc afferent drive via a mechanism that, in addition to an SSRI-like effect, requires 5-HT receptor modulation. Recordings in the LSN revealed a significant increase in mPFC-evoked activity following escitalopram administration as compared to control and vortioxetine treated groups, indicating that complex modulation of 5-HT receptors by vortioxetine may offset SSRI-like effects in this region. Lastly, neurons in the msNAc were more responsive to stimulation of the HF following both vortioxetine and escitalopram administration, indicating that elevation of 5-HT tone and 5-HT receptor modulation may facilitate excitatory hippocampal synaptic drive in this region. The above findings point to complex 5-HT receptor-dependent effects of vortioxetine which may contribute to its unique impact on the function of prefrontal-subcortical circuits and the development of novel strategies for treating mood disorders.

Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Emerging pathways driving early synaptic pathology in Alzheimer's disease.

The current state of the AD research field is highly dynamic is some respects, while seemingly stagnant in others. Regarding the former, our current lack of understanding of initiating disease mechanisms, the absence of effective treatment options, and the looming escalation of AD patients is energizing new research directions including a much-needed re-focusing on early pathogenic mechanisms, validating novel targets, and investigating relevant biomarkers, among other exciting new efforts to curb disease progression and foremost, preserve memory function. With regard to the latter, the recent disappointing series of failed Phase III clinical trials targeting Aß and APP processing, in concert with poor association between brain Aß levels and cognitive function, have led many to call for a re-evaluation of the primacy of the amyloid cascade hypothesis. In this review, we integrate new insights into one of the earliest described signaling abnormalities in AD pathogenesis, namely intracellular Ca2+ signaling disruptions, and focus on its role in driving synaptic deficits - which is the feature that does correlate with AD-associated memory loss. Excess Ca2+release from intracellular stores such as the endoplasmic reticulum (ER) has been well-described in cellular and animal models of AD, as well as human patients, and here we expand upon recent developments in ER-localized release channels such as the IP3R and RyR, and the recent emphasis on RyR2. Consistent with ER Ca2+ mishandling in AD are recent findings implicating aspects of SOCE, such as STIM2 function, and TRPC3 and TRPC6 levels. Other Ca2+-regulated organelles important in signaling and protein handling are brought into the discussion, with new perspectives on lysosomal regulation. These early signaling abnormalities are discussed in the context of synaptic pathophysiology and disruptions in synaptic plasticity with a particular emphasis on short-term plasticity deficits. Overall, we aim to update and expand the list of early neuronal signaling abnormalities implicated in AD pathogenesis, identify specific channels and organelles involved, and link these to proximal synaptic impairments driving the memory loss in AD. This is all within the broader goal of identifying novel therapeutic targets to preserve cognitive function in AD.

Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington's Disease Models.

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.

Nitric oxide signaling is recruited as a compensatory mechanism for sustaining synaptic plasticity in Alzheimer's disease mice.

Synaptic plasticity deficits are increasingly recognized as causing the memory impairments which define Alzheimer's disease (AD). In AD mouse models, evidence of abnormal synaptic function is present before the onset of cognitive deficits, and presents as increased synaptic depression revealed only when synaptic homeostasis is challenged, such as with suppression of ryanodine receptor (RyR)-evoked calcium signaling. Otherwise, at early disease stages, the synaptic physiology phenotype appears normal. This suggests compensatory mechanisms are recruited to maintain a functionally normal net output of the hippocampal circuit. A candidate calcium-regulated synaptic modulator is nitric oxide (NO), which acts presynaptically to boost vesicle release and glutamatergic transmission. Here we tested whether there is a feedforward cycle between the increased RyR calcium release seen in presymptomatic AD mice and aberrant NO signaling which augments synaptic plasticity. Using a combination of electrophysiological approaches, two-photon calcium imaging, and protein biochemistry in hippocampal tissue from presymptomatic 3xTg-AD and NonTg mice, we show that blocking NO synthesis results in markedly augmented synaptic depression mediated through presynaptic mechanisms in 3xTg-AD mice. Additionally, blocking NO reduces the augmented synaptically evoked dendritic calcium release mediated by enhanced RyR calcium release. This is accompanied by increased nNOS levels in the AD mice and is reversed upon normalization of RyR-evoked calcium release with chronic dantrolene treatment. Thus, recruitment of NO is serving a compensatory role to boost synaptic transmission and plasticity during early AD stages. However, NO's dual role in neuroprotection and neurodegeneration may convert to maladaptive functions as the disease progresses.

Facilitation of corticostriatal transmission following pharmacological inhibition of striatal phosphodiesterase 10A: role of nitric oxide-soluble guanylyl cyclase-cGMP signaling pathways.

The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 µm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade.

Calcium channelopathies and Alzheimer's disease: insight into therapeutic success and failures.

Calcium ions are versatile and universal biological signaling factors that regulate numerous cellular processes ranging from cell fertilization, to neuronal plasticity that underlies learning and memory, to cell death. For these functions to be properly executed, calcium signaling requires precise regulation, and failure of this regulation may tip the scales from a signal for life to a signal for death. Disruptions in calcium channel function can generate complex multi-system disorders collectively referred to as "calciumopathies" that can target essentially any cell type or organ. In this review, we focus on the multifaceted involvement of calcium signaling in the pathophysiology of Alzheimer's disease (AD), and summarize the various therapeutic options currently available to combat this disease. Detailing the series of disappointing AD clinical trial results on cognitive outcomes, we emphasize the urgency to design alternative therapeutic strategies if synaptic and memory functions are to be preserved. One such approach is to target early calcium channelopathies centrally linked to AD pathogenesis.

Early presynaptic and postsynaptic calcium signaling abnormalities mask underlying synaptic depression in presymptomatic Alzheimer's disease mice.

Alzheimer's disease (AD)-linked presenilin (PS) mutations result in pronounced endoplasmic reticulum calcium disruptions that occur before detectable histopathology and cognitive deficits. More subtly, these early AD-linked calcium alterations also reset neurophysiological homeostasis, such that calcium-dependent presynaptic and postsynaptic signaling appear functionally normal yet are actually operating under aberrant calcium signaling systems. In these 3xTg-AD mouse brains, upregulated ryanodine receptor (RyR) activity is associated with a shift toward synaptic depression, likely through a reduction in presynaptic vesicle stores and increased postsynaptic outward currents through small-conductance calcium-activated potassium SK2 channels. The deviant RyR-calcium involvement in the 3xTg-AD mice also compensates for an intrinsic predisposition for hippocampal long-term depression (LTD) and reduced long-term potentiation (LTP). In this study, we detail the impact of disrupted RyR-mediated calcium stores on synaptic transmission properties, LTD, and calcium-activated membrane channels of hippocampal CA1 pyramidal neurons in presymptomatic 3xTg-AD mice. Using electrophysiological recordings in young 3xTg-AD and nontransgenic (NonTg) hippocampal slices, we show that increased RyR-evoked calcium release in 3xTg-AD mice "normalizes" an altered synaptic transmission system operating under a shifted homeostatic state that is not present in NonTg mice. In the process, we uncover compensatory signaling mechanisms recruited early in the disease process that counterbalance the disrupted RyR-calcium dynamics, namely increases in presynaptic spontaneous vesicle release, altered probability of vesicle release, and upregulated postsynaptic SK channel activity. Because AD is increasingly recognized as a "synaptic disease," calcium-mediated signaling alterations may serve as a proximal trigger for the synaptic degradation driving the cognitive loss in AD.

Stabilizing ER Ca2+ channel function as an early preventative strategy for Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca(2+) homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca(2+) signaling, predominantly through the ER-localized inositol triphosphate (IP(3)) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca(2+) upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca(2+) imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca(2+) signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aß deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca(2+) aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.

Early calcium dysregulation in Alzheimer's disease: setting the stage for synaptic dysfunction.

Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disorder with no known cure or clear understanding of the mechanisms involved in the disease process. Amyloid plaques, neurofibrillary tangles and neuronal loss, though characteristic of AD, are late stage markers whose impact on the most devastating aspect of AD, namely memory loss and cognitive deficits, are still unclear. Recent studies demonstrate that structural and functional breakdown of synapses may be the underlying factor in AD-linked cognitive decline. One common element that presents with several features of AD is disrupted neuronal calcium signaling. Increased intracellular calcium levels are functionally linked to presenilin mutations, ApoE4 expression, amyloid plaques, tau tangles and synaptic dysfunction. In this review, we discuss the role of AD-linked calcium signaling alterations in neurons and how this may be linked to synaptic dysfunctions at both early and late stages of the disease.

Generation of dendritic Ca2+ oscillations as a consequence of altered ryanodine receptor function in AD neurons.

Ryanodine receptor (RyR)-mediated Ca(2+) dysregulation is associated with Alzheimer's disease (AD) neuropathology. Using 2-photon Ca(2+) imaging and patch clamp recordings in brain slice preparations from young 3xTg-AD and NonTg control mice, we recently demonstrated that RyR-mediated Ca(2+) -induced Ca(2+) release (CICR) is substantially increased within dendrites from AD neurons, such that synaptic stimulation alone is sufficient to generate aberrant CICR. We also observed supra-additive Ca(2+) release upon coincident RyR activation with synaptic stimulation in 3xTg-AD mice. Here, we describe an additional observed phenomenon: generation of patterned Ca(2+) oscillations in the spines and dendrites from AD neurons upon coincident RyR and synaptic stimulation. As the temporal entrainment of Ca(2+) signals influences many downstream cellular and synaptic functions, these abnormal oscillatory patterns may be associated with the structural and functional breakdown of synapses in AD.

Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice.

Presenilin mutations result in exaggerated endoplasmic reticulum (ER) calcium release in cellular and animal models of Alzheimer's disease (AD). In this study, we examined whether dysregulated ER calcium release in young 3xTg-AD neurons alters synaptic transmission and plasticity mechanisms before the onset of histopathology and cognitive deficits. Using electrophysiological recordings and two-photon calcium imaging in young (6-8 weeks old) 3xTg-AD and non-transgenic (NonTg) hippocampal slices, we show a marked increase in ryanodine receptor (RyR)-evoked calcium release within synapse-dense regions of CA1 pyramidal neurons. In addition, we uncovered a deviant contribution of presynaptic and postsynaptic ryanodine receptor-sensitive calcium stores to synaptic transmission and plasticity in 3xTg-AD mice that is not present in NonTg mice. As a possible underlying mechanism, the RyR2 isoform was found to be selectively increased more than fivefold in the hippocampus of 3xTg-AD mice relative to the NonTg controls. These novel findings demonstrate that 3xTg-AD CA1 neurons at presymptomatic ages operate under an aberrant, yet seemingly functional, calcium signaling and synaptic transmission system long before AD histopathology onset. These early signaling alterations may underlie the later synaptic breakdown and cognitive deficits characteristic of later stage AD.