Mechanisms of synapse-to-nucleus calcium signalling in striatal neurons and impairments in Huntington's disease.

Huntington's disease (HD) is a monogenic disorder with autosomal dominant inheritance. In HD patients, neurons in the striatum and cortex degenerate, leading to motor, psychiatric and cognitive disorders. Dysregulated synaptic function and calcium handling are common in many neurodegenerative diseases, including HD. N-methyl-D-aspartate (NMDA) receptor function is enhanced in HD at extrasynaptic sites, altering the balance of calcium-dependent neuronal survival versus death signalling pathways. Endoplasmic reticulum (ER) calcium handling is also abnormal in HD. The ER, which is continuous with the nuclear envelope, is purportedly involved in nuclear calcium signalling; based on this, we hypothesised that nuclear calcium signalling is altered in HD. We explored this hypothesis with calcium imaging techniques, including simultaneous epifluorescent imaging of cytosolic and nuclear calcium using jRCaMP1b and GCaMP3 sensors, respectively, in striatal spiny projection neurons in cortical-striatal co-cultures from the YAC128 mouse model of HD. Our data show contributions from a variety of calcium channels to nuclear calcium signalling. NMDA receptors (NMDARs) play an essential role in initiating action potential-dependent calcium signalling to the nucleus, and ryanodine receptors (RyR) contribute to both cytosolic and nuclear calcium signals. Unlike previous reports in glutamatergic hippocampal and cortical neurons, we found that in GABAergic striatal neurons, L-type voltage-gated calcium channels (CaV) contribute to cytosolic, but not nuclear calcium signalling. Calcium imaging also suggests impairments in nuclear calcium signalling in HD striatal neurons, where spontaneous action potential-dependent calcium transients in the nucleus were smaller in YAC128 striatal neurons compared to those of wild-type (WT). Our results elucidate mechanisms involved in action potential-dependent nuclear calcium signalling in GABAergic striatal neurons, and have revealed a clear deficit in this signalling in HD.

Activin A targets extrasynaptic NMDA receptors to ameliorate neuronal and behavioral deficits in a mouse model of Huntington disease.

Cortical-striatal synaptic dysfunction, including enhanced toxic signaling by extrasynaptic N-methyl-d-aspartate receptors (eNMDARs), precedes neurodegeneration in Huntington disease (HD). A previous study showed Activin A, whose transcription is upregulated by calcium influx via synaptic NMDARs, suppresses eNMDAR signaling. Therefore, we examined the role of Activin A in the YAC128 HD mouse model, comparing it to wild-type controls. We found decreased Activin A secretion in YAC128 cortical-striatal co-cultures, while Activin A overexpression in this model rescued altered eNMDAR expression. Striatal overexpression of Activin A in vivo improved motor learning on the rotarod task, and normalized striatal neuronal eNMDAR-mediated currents, membrane capacitance and spontaneous excitatory postsynaptic current frequency in the YAC128 mice. These results support the therapeutic potential of Activin A signaling and targeting eNMDARs to restore striatal neuronal health and ameliorate behavioral deficits in HD.

Axonal ER Ca2+ Release Selectively Enhances Activity-Independent Glutamate Release in a Huntington Disease Model.

Action potential (AP)-independent (miniature) neurotransmission occurs at all chemical synapses but remains poorly understood, particularly in pathologic contexts. Axonal endoplasmic reticulum (ER) Ca2+ stores are thought to influence miniature neurotransmission, and aberrant ER Ca2+ handling is implicated in progression of Huntington disease (HD). Here, we report elevated mEPSC frequencies in recordings from YAC128 mouse (HD-model) neurons (from cortical cultures and striatum-containing brain slices, both from male and female animals). Pharmacological experiments suggest that this is mediated indirectly by enhanced tonic ER Ca2+ release. Calcium imaging, using an axon-localized sensor, revealed slow AP-independent ER Ca2+ release waves in both YAC128 and WT cultures. These Ca2+ waves occurred at similar frequencies in both genotypes but spread less extensively and were of lower amplitude in YAC128 axons, consistent with axonal ER Ca2+ store depletion. Surprisingly, basal cytosolic Ca2+ levels were lower in YAC128 boutons and YAC128 mEPSCs were less sensitive to intracellular Ca2+ chelation. Together, these data suggest that elevated miniature glutamate release in YAC128 cultures is associated with axonal ER Ca2+ depletion but not directly mediated by ER Ca2+ release into the cytoplasm. In contrast to increased mEPSC frequencies, cultured YAC128 cortical neurons showed less frequent AP-dependent (spontaneous) Ca2+ events in soma and axons, although evoked glutamate release detected by an intensity-based glutamate-sensing fluorescence reporter in brain slices was similar between genotypes. Our results indicate that axonal ER dysfunction selectively elevates miniature glutamate release from cortical terminals in HD. This, together with reduced spontaneous cortical neuron firing, may cause a shift from activity-dependent to -independent glutamate release in HD, with potential implications for fidelity and plasticity of cortical excitatory signaling.SIGNIFICANCE STATEMENT Miniature neurotransmitter release persists at all chemical neuronal synapses in the absence of action potential firing but remains poorly understood, particularly in disease states. We show enhanced miniature glutamate release from cortical neurons in the YAC128 mouse Huntington disease model. This effect is mediated by axonal ER Ca2+ store depletion, but is not obviously due to elevated ER-to-cytosol Ca2+ release. Conversely, YAC128 cortical pyramidal neurons fired fewer action potentials and evoked cortical glutamate release was similar between WT an YAC128 preparations, indicating axonal ER depletion selectively enhances miniature glutamate release in YAC128 mice. These results extend our understanding of action potential independent neurotransmission and highlight a potential involvement of elevated miniature glutamate release in Huntington disease pathology.

Impairment and Restoration of Homeostatic Plasticity in Cultured Cortical Neurons From a Mouse Model of Huntington Disease.

Huntington disease (HD) is an inherited neurodegenerative disorder caused by a mutation in the huntingtin gene. The onset of symptoms is preceded by synaptic dysfunction. Homeostatic synaptic plasticity (HSP) refers to processes that maintain the stability of networks of neurons, thought to be required to enable new learning and cognitive flexibility. One type of HSP is synaptic scaling, in which the strength of all of the synapses onto a cell increases or decreases following changes in the cell's level of activity. Several pathways implicated in synaptic scaling are dysregulated in HD, including brain-derived neurotrophic factor (BDNF) and calcium signaling. Here, we investigated whether HSP is disrupted in cortical neurons from an HD mouse model. We treated cultured cortical neurons from wild-type (WT) FVB/N or YAC128 HD mice with tetrodotoxin (TTX) for 48 h to silence action potentials and then recorded miniature excitatory postsynaptic currents. In WT cultures, these increased in both amplitude and frequency after TTX treatment, and further experiments showed that this was a result of insertion of AMPA receptors and formation of new synapses, respectively. Manipulation of BDNF concentration in the culture medium revealed that BDNF signaling contributed to these changes. In contrast to WT cortical neurons, YAC128 cultures showed no response to action potential silencing. Strikingly, we were able to restore the TTX-induced changes in YAC128 cultures by treating them with pridopidine, a drug which enhances BDNF signaling through stimulation of the sigma-1 receptor (S1R), and with the S1R agonist 3-PPP. These data provide evidence for disruption of HSP in cortical neurons from an HD mouse model that is restored by stimulation of S1R. Our results suggest a potential new direction for developing therapy to mitigate cognitive deficits in HD.

Cause or compensation?-Altered neuronal Ca2+ handling in Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder of typically middle-aged onset for which there is no disease-modifying treatment. Caudate and putamen medium-sized spiny projection neurons (SPNs) most severely degenerate in HD. However, it is unclear why mutant huntingtin protein (mHTT) is preferentially toxic to these neurons or why symptoms manifest only relatively late in life. mHTT interacts with numerous neuronal proteins. Likewise, multiple SPN cellular processes have been described as altered in various HD models. Among these, altered neuronal Ca2+ influx and intracellular Ca2+ handling feature prominently and are addressed here. Specifically, we focus on extrasynaptic NMDA-type glutamate receptors, endoplasmic reticulum IP3 receptors, and mitochondria. As mHTT is expressed throughout development, compensatory processes will likely be mounted to mitigate any deleterious effects. Although some compensations can lessen mHTT's disruptive effects, others-such as upregulation of the ER-refilling store-operated Ca2+ channel response-contribute to pathogenesis. A causation-based approach is therefore necessary to decipher the complex sequence of events linking mHTT to neurodegeneration, and to design rational therapeutic interventions. With this in mind, we highlight evidence, or lack thereof, that the above alterations in Ca2+ handling occur early in the disease process, clearly interact with mHTT, and show disease-modifying potential when reversed in animals.