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.

Altered cortical processing of sensory input in Huntington disease mouse models.

Huntington disease (HD), a hereditary neurodegenerative disorder, manifests as progressively impaired movement and cognition. Although early abnormalities of neuronal activity in striatum are well established in HD models, there are fewer in vivo studies of the cortex. Here, we record local field potentials (LFPs) in YAC128 HD model mice versus wild-type mice. In multiple cortical areas, limb sensory stimulation evokes a greater change in LFP power in YAC128 mice. Mesoscopic imaging using voltage-sensitive dyes reveals more extensive spread of evoked sensory signals across the cortical surface in YAC128 mice. YAC128 layer 2/3 sensory cortical neurons ex vivo show increased excitatory events, which could contribute to enhanced sensory responses in vivo. Cortical LFP responses to limb stimulation, visual and auditory input are also significantly increased in zQ175 HD mice. Results presented here extend knowledge of HD beyond ex vivo studies of individual neurons to the intact cortical network.

Endocannabinoid-Specific Impairment in Synaptic Plasticity in Striatum of Huntington's Disease Mouse Model.

Huntington's disease (HD) is an inherited neurodegenerative disease affecting predominantly striatum and cortex that results in motor and cognitive disorders. Before a motor phenotype, animal models of HD show aberrant cortical-striatal glutamate signaling. Here, we tested synaptic plasticity of cortical excitatory synapses onto striatal spiny projection neurons (SPNs) early in the YAC128 mouse model of HD. High-frequency stimulation-induced long-term depression, mediated by the endocannabinoid anandamide and cannabinoid receptor 1 (CB1), was significantly attenuated in male and female YAC128 SPNs. Indirect pathway SPNs, which are more vulnerable in HD, were most affected. Our experiments show metabotropic glutamate receptor and endocannabinoid 2-arachidonoylglycerol-dependent plasticity, as well as direct CB1 activation by agonists, was similar in YAC128 and FVB/N wild-type SPNs suggesting that presynaptic CB1 is functioning normally. These results are consistent with a specific impairment in postsynaptic anandamide synthesis in YAC128 SPN. Strikingly, although suppression of degradation of anandamide was not effective, elevating 2-arachidonoylglycerol levels restored long-term depression in YAC128 striatal neurons. Together, these results have potential implications for neuroprotection and ameliorating early cognitive and motor deficits in HD.SIGNIFICANCE STATEMENT Huntington's disease (HD) is an inherited neurodegenerative disease with no cure. Recent studies find impairment of the endocannabinoid system in animal models but the functional implication for synaptic plasticity in HD remains unclear. Sepers et al. show a selective deficit in synaptic plasticity mediated by the endocannabinoid anandamide, but not 2-arachidonoylglycerol in a mouse model of HD. The deficit is rescued by selectively elevating levels of 2-arachidonoylglycerol produced on-demand. This mechanism could be targeted in the development of future therapeutics for HD.

Alterations in synaptic function and plasticity in Huntington disease.

Huntington disease (HD) is an inherited neurodegenerative disorder caused by an expansion of the CAG repeat region in the first exon of the huntingtin gene. Neurodegeneration, which begins in the striatum and then spreads to other brain areas, is preceded by dysfunction in multiple aspects of neurotransmission across a variety of brain areas. This review will provide an overview of the neurochemical mediators and modulators of synaptic transmission that are disrupted in HD. This includes classical neurotransmitters like glutamate and gamma-aminobutyric acid, modulators such as dopamine, adenosine and endocannabinoids, and molecules like brain-derived neurotrophic factor which affect neurotransmission in a more indirect manner. Alterations in the functioning of these signaling pathways can occur across multiple brain regions such as striatum, cortex and hippocampus, and affect transmission and plasticity at the synapses within these regions, which may ultimately change behaviour and contribute to the pathophysiology of HD. The current state of knowledge in this area has already yielded useful information about the causes of synaptic dysfunction and selective cell death. A full understanding of the mechanisms and consequences of disruptions in synaptic function and plasticity will lend insight into the development of the symptoms of HD, and potential drug targets for ameliorating them.

An enhanced Q175 knock-in mouse model of Huntington disease with higher mutant huntingtin levels and accelerated disease phenotypes.

Huntington disease (HD) model mice with heterozygous knock-in (KI) of an expanded CAG tract in exon 1 of the mouse huntingtin (Htt) gene homolog genetically recapitulate the mutation that causes HD, and might be favoured for preclinical studies. However, historically these mice have failed to phenotypically recapitulate the human disease. Thus, homozygous KI mice, which lack wildtype Htt, and are much less relevant to human HD, have been used. The zQ175 model was the first KI mouse to exhibit significant HD-like phenotypes when heterozygous. In an effort to exacerbate HD-like phenotypes and enhance preclinical utility, we have backcrossed zQ175 mice to FVB/N, a strain highly susceptible to neurodegeneration. These Q175F mice display significant HD-like phenotypes along with sudden early death from fatal seizures. The zQ175 KI allele retains a floxed neomycin resistance cassette upstream of the Htt gene locus and produces dramatically reduced mutant Htt as compared to the endogenous wildtype Htt allele. By intercrossing with mice expressing cre in germ line cells, we have excised the neo cassette from Q175F mice generating a new line, Q175FΔneo (Q175FDN). Removal of the neo cassette resulted in a ∼2 fold increase in mutant Htt and rescue of fatal seizures, indicating that the early death phenotype of Q175F mice is caused by Htt deficiency rather than by mutant Htt. Additionally, Q175FDN mice exhibit earlier onset and a greater variety and severity of HD-like phenotypes than Q175F mice or any previously reported KI HD mouse model, making them valuable for preclinical studies.

Impaired development of cortico-striatal synaptic connectivity in a cell culture model of Huntington's disease.

Huntington's disease (HD) is a genetically inherited neurodegenerative disease caused by a mutation in the gene encoding the huntingtin protein. This mutation results in progressive cell death that is particularly striking in the striatum. Recent evidence indicates that early HD is initially a disease of the synapse, in which subtle alterations in synaptic neurotransmission, particularly at the cortico-striatal (C-S) synapse, can be detected well in advance of cell death. Here, we used a cell culture model in which striatal neurons are co-cultured with cortical neurons, and monitored the development of C-S connectivity up to 21days in vitro (DIV) in cells cultured from either the YAC128 mouse model of HD or the background strain, FVB/N (wild-type; WT) mice. Our data demonstrate that while C-S connectivity in WT co-cultures develops rapidly and continuously from DIV 7 to 21, YAC128 C-S connectivity shows no significant growth from DIV 14 onward. Morphological and electrophysiological data suggest that a combination of pre- and postsynaptic mechanisms contribute to this effect, including a reduction in both the postsynaptic dendritic arborization and the size and replenishment rate of the presynaptic readily releasable pool of excitatory vesicles. Moreover, a chimeric culture strategy confirmed that the most robust impairment in C-S connectivity was only observed when mutant huntingtin was expressed both pre- and postsynaptically. In all, our data demonstrate a progressive HD synaptic phenotype in this co-culture system that may be exploited as a platform for identifying promising therapeutic strategies to prevent early HD-associated synaptopathy.

Purkinje cell axonal swellings enhance action potential fidelity and cerebellar function.

Axonal plasticity allows neurons to control their output, which critically determines the flow of information in the brain. Axon diameter can be regulated by activity, yet how morphological changes in an axon impact its function remains poorly understood. Axonal swellings have been found on Purkinje cell axons in the cerebellum both in healthy development and in neurodegenerative diseases, and computational models predicts that axonal swellings impair axonal function. Here we report that in young Purkinje cells, axons with swellings propagated action potentials with higher fidelity than those without, and that axonal swellings form when axonal failures are high. Furthermore, we observed that healthy young adult mice with more axonal swellings learn better on cerebellar-related tasks than mice with fewer swellings. Our findings suggest that axonal swellings underlie a form of axonal plasticity that optimizes the fidelity of action potential propagation in axons, resulting in enhanced learning.

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.

Region-specific pro-survival signaling and global neuronal protection by wild-type Huntingtin.

BACKGROUND: Huntington's disease (HD), caused by polyglutamine expansion in huntingtin (Htt), results in severe neurodegeneration in the striatum, and to a lesser extent, cortex and hippocampus. In contrast, non-expanded huntingtin (wildtype, wtHtt) enhances pro-survival trophic factor BDNF expression and protects striatal neurons from excitotoxicity, a mechanism thought to contribute to HD pathophysiology; however, it is unknown whether these effects of wtHtt extend to other brain areas. OBJECTIVE: Test wtHtt's role in pro-survival signaling and neuroprotection in striatum, cortex and hippocampus. METHODS: Levels of nuclear phosphorylated cAMP response element-binding protein (pCREB), a regulator of pro-survival gene transcription, and resistance to NMDA-induced apoptosis in primary neuronal cultures - hippocampal and corticostriatal co-culture -were assessed using immunocytochemistry and excitotoxicity assays, respectively. Cultures from wild-type FVB/N (WT) mice were compared with those from YAC18 mice on an FVB/N background, expressing both human, full-length wtHtt and normal levels of murine Htt. RESULTS: Basal pCREB was higher in YAC18 striatal but not cortical or hippocampal neurons; however, all three types showed decreased apoptosis in YAC18 vs. WT cultures. Increased striatal neuronal pCREB required wtHtt overexpression in both cortical and striatal neurons. Reduced response to exogenous BDNF, or its soluble scavenger TrkB-Fc, suggested enhanced BDNF signaling contributes to increased YAC18 striatal pCREB. CONCLUSION: Basal pro-survival signaling does not predict neuronal vulnerability to apoptosis in our culture system, since wtHtt overexpression elevates basal pCREB selectively in striatal neurons but is more globally neuroprotective. These results extend knowledge of the physiological roles of huntingtin, facilitating development of HD therapeutics.