Calcium dysregulation and compensation in cortical pyramidal neurons of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that predominantly affects striatal medium-sized spiny neurons and cortical pyramidal neurons (CPNs). It has been proposed that perturbations in Ca2+ homeostasis could play a role in CPN alterations. To test this hypothesis, we used the R6/2 mouse model of juvenile HD at different stages of disease progression; presymptomatic, early symptomatic, and late symptomatic. We combined whole-cell patch-clamp recordings of layer 2/3 CPNs with two-photon laser scanning microscopy to image somatic and dendritic Ca2+ transients associated with evoked action potentials (APs). We found that the amplitude of AP-induced Ca2+ transients recorded at the somata of CPNs was significantly reduced in presymptomatic and late symptomatic R6/2 mice compared with wild-type (WT) littermates. However, reduced amplitudes were compensated by increases in decay times, so that Ca2+ transient areas were similar between genotypes. AP-induced Ca2+ transients in CPN proximal dendrites were variable and differences did not reach statistical significance, except for reduced areas in the late symptomatic group. In late symptomatic mice, a specific store-operated Ca2+ channel antagonist, EVP4593, reduced somatic Ca2+ transient amplitude similarly in WT and R6/2 CPNs. In contrast, dantrolene, a ryanodine receptor (RyR) antagonist, and nifedipine, an L-type Ca2+ channel blocker, significantly reduced both somatic Ca2+ transient amplitude and area in R6/2 but not WT CPNs. These findings demonstrate that perturbations of Ca2+ homeostasis and compensation occur in CPNs before and after the onset of overt symptoms, and suggest RyRs and L-type Ca2+ channels as potential targets for therapeutic intervention.NEW & NOTEWORTHY We used two-photon microscopy to examine calcium influx induced by action potentials in cortical pyramidal neurons from a mouse model of Huntington's disease (HD), the R6/2. The amplitude of somatic calcium transients was reduced in R6/2 mice compared with controls. This reduction was compensated by increased decay times, which could lead to reduced calcium buffering capacity. L-type calcium channel and ryanodine receptor blockers reduced calcium transient area in HD neurons, suggesting new therapeutic avenues.

Early impairment of thalamocortical circuit activity and coherence in a mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive, fatal neurodegenerative disorder characterized by motor, cognitive, and psychiatric disturbances. There is no known cure for HD, but its progressive nature allows for early therapeutic intervention. Currently, much of the research has focused on the striatum, however, there is evidence suggesting that disruption of thalamocortical circuits could underlie some of the early symptoms of HD. Loss of both cortical pyramidal neurons (CPNs) and thalamic neurons occurs in HD patients, and cognitive, somatosensory, and attention deficits precede motor abnormalities. However, the role of thalamocortical pathways in HD progression has been understudied. Here, we measured single unit activity and local field potentials (LFPs) from electrode arrays implanted in the thalamus and primary motor cortex of 4-5 month-old male and female Q175 mice. We assessed neuronal activity under baseline conditions as well as during presentation of rewards delivered via actuation of an audible solenoid valve. HD mice showed a significantly delayed licking response to the reward stimulus. At the same time, neuronal activation to the reward was delayed in thalamic neurons, CPNs and fast-spiking cortical interneurons (FSIs) of HD mice. In addition, thalamocortical coherence increased at lower frequencies in HD relative to wildtype mice. Together, these data provide evidence that impaired cortical and thalamic responses to reward stimuli, and impaired thalamocortical coherence, may play an important early role in motor, cognitive, and learning deficits in HD patients.

Neuronal Network Topology Indicates Distinct Recovery Processes after Stroke.

Despite substantial recent progress in network neuroscience, the impact of stroke on the distinct features of reorganizing neuronal networks during recovery has not been defined. Using a functional connections-based approach through 2-photon in vivo calcium imaging at the level of single neurons, we demonstrate for the first time the functional connectivity maps during motion and nonmotion states, connection length distribution in functional connectome maps and a pattern of high clustering in motor and premotor cortical networks that is disturbed in stroke and reconstitutes partially in recovery. Stroke disrupts the network topology of connected inhibitory and excitatory neurons with distinct patterns in these 2 cell types and in different cortical areas. These data indicate that premotor cortex displays a distinguished neuron-specific recovery profile after stroke.

Cortical Network Dynamics Is Altered in Mouse Models of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric disturbances. Although evidence indicates that projections from motor cortical areas play a key role in the development of dysfunctional striatal activity and motor phenotype, little is known about the changes in cortical microcircuits and their role in the development of the HD phenotype. Here we used two-photon laser-scanning microscopy to evaluate network dynamics of motor cortical neurons in layers II/III in behaving transgenic R6/2 and knock-in Q175+/- mice. Symptomatic R6/2 mice displayed increased motion manifested by a significantly greater number of motion epochs, whereas symptomatic Q175 mice displayed decreased motion. In both models, calcium transients in symptomatic mice displayed reduced amplitude, suggesting decreased bursting activity. Changes in frequency were genotype- and time-dependent; for R6/2 mice, the frequency was reduced during both motion and nonmotion, whereas in symptomatic Q175 mice, the reduction only occurred during nonmotion. In presymptomatic Q175 mice, frequency was increased during both behavioral states. Interneuronal correlation coefficients were generally decreased in both models, suggesting disrupted interneuronal communication in HD cerebral cortex. These results indicate similar and contrasting effects of the HD mutation on cortical ensemble activity depending on mouse model and disease stage.

Neurophysiological Assessment of Huntington's Disease Model Mice.

Electrophysiological and cell imaging techniques are powerful tools for understanding alterations in neuronal activity in Huntington's disease (HD), a fatal neurological disorder caused by an expansion of CAG repeats in the HTT gene. Changes in neuronal activity often precede the behavioral manifestations of HD, therefore, understanding the electrophysiology of HD is critical for identifying potential prodromal markers and therapeutic targets. This chapter outlines the basic methodology behind four major electrophysiological and imaging techniques used in HD mouse models: patch clamp recordings, optogenetics, in vivo electrophysiology, and Ca2+ imaging, as well as some of the advancements in HD research using each of these techniques.

Quantitative Electroencephalographic Biomarkers in Preclinical and Human Studies of Huntington's Disease: Are They Fit-for-Purpose for Treatment Development?

A major focus in development of novel therapies for Huntington's disease (HD) is identification of treatments that reduce the burden of mutant huntingtin (mHTT) protein in the brain. In order to identify and test the efficacy of such therapies, it is essential to have biomarkers that are sensitive to the effects of mHTT on brain function to determine whether the intervention has been effective at preventing toxicity in target brain systems before onset of clinical symptoms. Ideally, such biomarkers should have a plausible physiologic basis for detecting the effects of mHTT, be measureable both in preclinical models and human studies, be practical to measure serially in clinical trials, and be reliably measurable in HD gene expansion carriers (HDGECs), among other features. Quantitative electroencephalography (qEEG) fulfills many of these basic criteria of a "fit-for-purpose" biomarker. qEEG measures brain oscillatory activity that is regulated by the brain structures that are affected by mHTT in premanifest and early symptom individuals. The technology is practical to implement in the laboratory and is well tolerated by humans in clinical trials. The biomarkers are measureable across animal models and humans, with findings that appear to be detectable in HDGECs and translate across species. We review here the literature on recent developments in both preclinical and human studies of the use of qEEG biomarkers in HD, and the evidence for their usefulness as biomarkers to help guide development of novel mHTT lowering treatments.

Modulation of learning and memory by cytokines: signaling mechanisms and long term consequences.

This review describes the role of cytokines and their downstream signaling cascades on the modulation of learning and memory. Immune proteins are required for many key neural processes and dysregulation of these functions by systemic inflammation can result in impairments of memory that persist long after the resolution of inflammation. Recent research has demonstrated that manipulations of individual cytokines can modulate learning, memory, and synaptic plasticity. The many conflicting findings, however, have prevented a clear understanding of the precise role of cytokines in memory. Given the complexity of inflammatory signaling, understanding its modulatory role requires a shift in focus from single cytokines to a network of cytokine interactions and elucidation of the cytokine-dependent intracellular signaling cascades. Finally, we propose that whereas signal transduction and transcription may mediate short-term modulation of memory, long-lasting cellular and molecular mechanisms such as epigenetic modifications and altered neurogenesis may be required for the long lasting impact of inflammation on memory and cognition.

Akt-induced tamoxifen resistance is associated with altered FKHR regulation.

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses, is associated with a poor pathophenotype in breast carcinoma, and is involved in hormone and chemotherapy resistance, including resistance to the antiestrogen, tamoxifen. Akt promotes cell survival by phosphorylating and inactivating proapoptotic proteins and increasing the transcription of survival genes. To explore the role that specific components of the Akt kinase pathway play in the cellular response to tamoxifen, we transfected MCF-7 cells with an expression plasmid for a constitutively active Akt. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Initial analysis of the molecular responses in the Akt/MCF-7 xenografts to tamoxifen suggests that high Akt activity alters apoptotic responses to tamoxifen. Control MCF-7 xenografts demonstrated activation of the proapoptotic forkhead (FKHR) transcription factor in response to tamoxifen treatment, while the xenografts expressing the constitutively active Akt transgene demonstrated no alterations in FKHR expression. In addition, TUNEL analysis demonstrated higher levels of apoptosis in the control xenografts in response to tamoxifen treatment compared to the Akt xenografts. Inhibition of Akt activity in vitro restored apoptotic responses to tamoxifen in the Akt/MCF-7 cells to those observed in the control cells. These data suggest that alteration of survival responses is an important mechanism by which Akt confers resistance to tamoxifen.

Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity.

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses. Studies show that high Akt activity in breast carcinoma is associated with a poor pathophenotype, as well as hormone and chemotherapy resistance. Additionally, high Akt activity is associated with other features of poor prognosis. Thus, a chemotherapeutic agent directed specifically toward tumors with high Akt activity could prove extremely potent in treating those breast tumors with the most aggressive phenotypes. Several studies have demonstrated that rapamycin, which inhibits mammalian target of rapamycin (mTOR), a downstream target of Akt, sensitizes certain resistant cancer cells to chemotherapeutic agents. This study evaluated the efficacy of mTOR inhibition in the treatment of tamoxifen-resistant breast carcinoma characterized by high Akt activity. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Cotreatment with the mTOR inhibitor rapamycin in vitro, or the ester of rapamycin, CCI-779 (Wyeth) in vivo, inhibited mTOR activity and restored sensitivity to tamoxifen, suggesting that Akt-induced tamoxifen resistance is mediated in part by signaling through the mTOR pathway. Although the mechanism underlying the synergism remains to be understood, the results were associated with rapamycin's ability to block transcriptional activity mediated by estrogen receptor alpha, as assessed by reporter gene assays with estrogen-responsive element luciferase. These data corroborate prior findings indicating that Akt activation induces resistance to tamoxifen in breast cancer cells. Importantly, these data indicate a novel mechanism for tamoxifen resistance and suggest that blockage of the phosphatidylinositol 3'-kinase/Akt signaling pathway by mTOR inhibition effectively restores the susceptibility of these cells to tamoxifen. These data may have implication for future clinical studies of mTOR inhibition in breast carcinoma.