Enhanced striatopallidal gamma-aminobutyric acid (GABA)A receptor transmission in mouse models of huntington's disease.

BACKGROUND: Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin gene. This mutation leads to progressive dysfunction that is largely attributable to dysfunction of the striatum. The earliest signs of striatal pathology in HD are found in indirect pathway gamma-Aminobutyric acid (GABA)-ergic spiny projection neurons that innervate the external segment of the globus pallidus (GPe). What is less clear is whether the synaptic coupling of spiny projection neurons with GPe neurons changes in HD. OBJECTIVES: The principal goal of this study was to determine whether striatopallidal synaptic transmission was altered in 2 mouse models of HD. METHODS: Striatopallidal synaptic transmission was studied using electrophysiological and optogenetic approaches in ex vivo brain slices from 2 HD models: Q175 heterozygous (het) and R6/2 mice. RESULTS: Striatopallidal synaptic transmission increased in strength with the progression of behavioral deficits in Q175 and R6/2 mice. The alteration in synaptic transmission was evident in both prototypical and arkypallidal GPe neurons. This change did not appear attributable to an increase in the probability of GABA release but, rather, to an enhancement in the postsynaptic response to GABA released at synaptic sites. This alteration significantly increased the ability of striatopallidal axon terminals to pause ongoing GPe activity. CONCLUSIONS: In 2 mouse models of HD, striatopallidal synaptic transmission increased in parallel with the progression of behavioral deficits. This adaptation could compensate in part for the concomitant deficit in the ability of corticostriatal signals to activate spiny projection neurons and pause GPe activity. © 2019 International Parkinson and Movement Disorder Society.

Age- and sex-related changes in cortical and striatal nitric oxide synthase in the Q175 mouse model of Huntington's disease.

In Huntington's disease (HD), corticostriatal and striatopallidal projection neurons preferentially degenerate as a result of mutant huntingtin expression. Pathological deficits in nitric oxide (NO) signaling have also been reported in corticostriatal circuits in HD, however, the impact of age and sex on nitrergic transmission is not well characterized. Thus, we utilized NADPH-diaphorase (NADPH-d) histochemistry and qPCR assays to assess neuronal NO synthase (nNOS) activity/expression in aged male and female Q175 heterozygous mice. Compared to age-matched controls, male Q175 mice exhibited reductions in NADPH-d staining in the motor cortex at 21, but not, 16 months of age. Comparisons across genotypes showed that striatal NADPH-d staining was significantly decreased at both 16 and 21 months of age. Comparisons within sexes in 21 month old mice revealed a decrease in striatal NADPH-d staining in males, but no changes were detected in females. Significant correlations between cortical and striatal NADPH-d staining deficits were also observed in males and females at both ages. To directly assess the role of constitutively active NOS isoforms in these changes, nNOS and endothelial NOS (eNOS) mRNA expression levels were examined in R6/2 (3 month old) and Q175 (11.5 month old) mice using qPCR assays. nNOS transcript expression was decreased in the cortex (40%) and striatum (54%) in R6/2 mice. nNOS mRNA down-regulation in striatum of Q175 animals was more modest (19%), and no changes were detected in cortex. eNOS expression was not changed in the cortex or striatum of Q175 mice. The current findings point to age-dependent deficits in nNOS activity in the HD cortex and striatum which appear first in the striatum and are more pronounced in males. Together, these observations and previous studies indicate that decreases in nitrergic transmission progress with age and are likely to contribute to corticostriatal circuit pathophysiology particularly in male patients with HD.

Development and characterization of a CNS-penetrant benzhydryl hydroxamic acid class IIa histone deacetylase inhibitor.

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ∼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for ∼8 h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.

HDAC4 reduction: a novel therapeutic strategy to target cytoplasmic huntingtin and ameliorate neurodegeneration.

Histone deacetylase (HDAC) 4 is a transcriptional repressor that contains a glutamine-rich domain. We hypothesised that it may be involved in the molecular pathogenesis of Huntington's disease (HD), a protein-folding neurodegenerative disorder caused by an aggregation-prone polyglutamine expansion in the huntingtin protein. We found that HDAC4 associates with huntingtin in a polyglutamine-length-dependent manner and co-localises with cytoplasmic inclusions. We show that HDAC4 reduction delayed cytoplasmic aggregate formation, restored Bdnf transcript levels, and rescued neuronal and cortico-striatal synaptic function in HD mouse models. This was accompanied by an improvement in motor coordination, neurological phenotypes, and increased lifespan. Surprisingly, HDAC4 reduction had no effect on global transcriptional dysfunction and did not modulate nuclear huntingtin aggregation. Our results define a crucial role for the cytoplasmic aggregation process in the molecular pathology of HD. HDAC4 reduction presents a novel strategy for targeting huntingtin aggregation, which may be amenable to small-molecule therapeutics.

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice.

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

Identification of a Potent, Selective, and Brain-Penetrant Rho Kinase Inhibitor and its Activity in a Mouse Model of Huntington's Disease.

The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.

Developing HDAC4-Selective Protein Degraders To Investigate the Role of HDAC4 in Huntington's Disease Pathology.

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).

HDinHD: A Rich Data Portal for Huntington's Disease Research.

HDinHD (Huntington's Disease in High Definition; HDinHD.org) is an open online portal for the HD research community that presents a synthesized view of HD-related scientific data. Here, we present a broad overview of HDinHD and highlight the newly launched HDinHD Explorer tool that enables researchers to discover and explore a wide range of diverse yet interconnected HD-related data. We demonstrate the utility of HDinHD Explorer through data mining of a single collection of newly released in vivo therapeutic intervention study reports alongside previously published reports.

Evaluation of 5-(Trifluoromethyl)-1,2,4-oxadiazole-Based Class IIa HDAC Inhibitors for Huntington's Disease.

Using an iterative structure-activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in vivo. Given the lack of understanding of endogenous class IIa HDAC substrates, we developed a surrogate readout to measure compound effects in vivo, by exploiting the >100-fold selectivity compound 12 exhibits over class I/IIb HDACs. We achieved adequate brain exposure with compound 12 in mice to estimate a class I/IIb deacetylation EC50, using class I substrate H4K12 acetylation and global acetylation levels as a pharmacodynamic readout. We observed excellent correlation between the compound 12 in vivo pharmacodynamic response and in vitro class I/IIb cellular activity. Applying the same relationship to class IIa HDAC inhibition, we estimated the compound 12 dose required to inhibit class IIa HDAC activity, for use in preclinical models of Huntington's disease.

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.

In vitro phosphodiesterase 10A (PDE10A) binding in whole hemisphere human brain using the PET radioligand [18F]MNI-659.

Highly specific and sensitive biomarkers for pathologies related to dysfunctions in the basal ganglia circuit are of great value to assess therapeutic efficacy not only clinically to establish an early diagnosis, but also in terms of monitoring the efficacy of therapeutic interventions and decelerated neurodegeneration. The phosphodiesterase 10A (PDE10A) enzyme plays a central role in striatal signaling and is implicated in several neuropsychiatric disorders involving striatal pathology, such as Huntingtons disease (HD) and schizophrenia. Inhibition of PDE10A activates the neurons in the striatum and consequently leads to alteration of behavioral aspects modulated by the striatal circuit. [18F]MNI-659, (2-(2-(3-(4-(2-[18F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione), is a newly developed PET radioligand that shows a high binding to PDE10A in the human brain in vivo. In the present study, we examined the in vitro binding of [18F]MNI-659 in human postmortem brain to gain a better understanding of the presence, density, disease-related alterations and therapy related to changes in PDE10A expression. The results show high specific binding of [18F]MNI-659 in the caudate nucleus, putamen and the hippocampal formation. Low specific [18F]MNI-659 binding was detected in nucleus accumbens in comparison to the caudate nucleus and putamen. In vitro binding studies with [18F]MNI-659 will facilitate in elucidating better understanding of the role of PDE10A activity in health and disease that may lead to new diagnostic opportunities in HD.

Real-time measurement of exocytosis and endocytosis using interference of light.

We describe a new approach for making real-time measurements of exocytosis and endocytosis in neurons and neuroendocrine cells. The method utilizes interference reflection microscopy (IRM) to image surface membrane in close contact with a glass coverslip (the "footprint"). At the synaptic terminal of retinal bipolar cells, the footprint expands during exocytosis and retracts during endocytosis, paralleling changes in total surface area measured by capacitance. In chromaffin cells, IRM detects the fusion of individual granules as the appearance of bright spots within the footprint with spatial and temporal resolution similar to total internal reflection fluorescence microscopy. Advantages of IRM over capacitance are that it can monitor changes in surface area while cells are electrically active and it can be applied to mammalian neurons with relatively small synaptic terminals. IRM reveals that vesicles at the synapse of bipolar cells rapidly collapse into the surface membrane while secretory granules in chromaffin cells do not.

Temporal synaptic tagging by I(h) activation and actin: involvement in long-term facilitation and cAMP-induced synaptic enhancement.

Presynaptic I(h) channels become activated during a tetanus through membrane hyperpolarization resulting from Na(+) accumulation and electrogenic Na(+)/K(+) exchange. I(h) activation is obligatory for inducing long-term facilitation (LTF), a long-lasting synaptic strengthening. cAMP-induced synaptic enhancement also requires I(h) activation, and both processes are sensitive to actin depolymerization. Other mechanisms are responsible for expression of the responses. Once initiated, continued response to cAMP is I(h) and actin independent. Moreover, LTF-induced activation of I(h) renders subsequent cAMP enhancement insensitive to both I(h) blockers and actin depolymerization. This actin-stabilized "temporal synaptic tagging" set by I(h) activation is prolonged when I(h) is activated concurrent with an elevation in presynaptic calcium concentration ([Ca(2+)]i), permitting the further strengthening of synapses given appropriate additional stimuli.

Behavioral and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest therapeutic potential for psychiatric indications.

The receptor tyrosine kinase product of the anaplastic lymphoma kinase (ALK) gene has been implicated in oncogenesis as a product of several chromosomal translocations, although its endogeneous role in the hematopoietic and neural systems has remained poorly understood. We describe that the generation of animals homozygous for a deletion of the ALK tyrosine kinase domain leads to alterations in adult brain function. Evaluation of adult ALK homozygotes (HOs) revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain. ALK HO animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel object-recognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. Altogether, these results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia and depression, with an underlying deregulated monoaminergic signalling.

Unravelling and Exploiting Astrocyte Dysfunction in Huntington's Disease.

Astrocytes are abundant within mature neural circuits and are involved in brain disorders. Here, we summarize our current understanding of astrocytes and Huntington's disease (HD), with a focus on correlative and causative dysfunctions of ion homeostasis, calcium signaling, and neurotransmitter clearance, as well as on the use of transplanted astrocytes to produce therapeutic benefit in mouse models of HD. Overall, the data suggest that astrocyte dysfunction is an important contributor to the onset and progression of some HD symptoms in mice. Additional exploration of astrocytes in HD mouse models and humans is needed and may provide new therapeutic opportunities to explore in conjunction with neuronal rescue and repair strategies.

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease.

Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntington's disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.

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.

Efficacy of selective PDE4D negative allosteric modulators in the object retrieval task in female cynomolgus monkeys (Macaca fascicularis).

Cyclic adenosine monophosphate (cAMP) signalling plays an important role in synaptic plasticity and information processing in the hippocampal and basal ganglia systems. The augmentation of cAMP signalling through the selective inhibition of phosphodiesterases represents a viable strategy to treat disorders associated with dysfunction of these circuits. The phosphodiesterase (PDE) type 4 inhibitor rolipram has shown significant pro-cognitive effects in neurological disease models, both in rodents and primates. However, competitive non-isoform selective PDE4 inhibitors have a low therapeutic index which has stalled their clinical development. Here, we demonstrate the pro-cognitive effects of selective negative allosteric modulators (NAMs) of PDE4D, D159687 and D159797 in female Cynomolgous macaques, in the object retrieval detour task. The efficacy displayed by these NAMs in a primate cognitive task which engages the corticostriatal circuitry, together with their suitable pharmacokinetic properties and safety profiles, suggests that clinical development of these allosteric modulators should be considered for the treatment of a variety of brain disorders associated with cognitive decline.

The PDE1/5 Inhibitor SCH-51866 Does Not Modify Disease Progression in the R6/2 Mouse Model of Huntington's Disease.

Huntington's disease is a neurodegenerative disorder caused by mutations in the CAG tract of huntingtin. Several studies in HD cellular and rodent systems have identified disturbances in cyclic nucleotide signaling, which might be relevant to pathogenesis and therapeutic intervention. To investigate whether selective phosphodiesterase (PDE) inhibitors can improve some aspects of disease pathogenesis in HD models, we have systematically evaluated the effects of a variety of cAMP and cGMP selective PDE inhibitors in various HD models. Here we present the lack of effect in a variety of endpoints of the PDE subtype selective inhibitor SCH-51866, a PDE1/5 inhibitor, in the R6/2 mouse model of HD, after chronic oral dosing.

HDAC4 does not act as a protein deacetylase in the postnatal murine brain in vivo.

Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation we employed the immunoaffinity-based AcetylScan proteomic method. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only Calml4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain.