huntington s disease ( hd ) is an autosomal dominant , neurodegenerative disorder caused by an extended cag repeat sequence in the hd gene . several mechanisms have been proposed to explain how an expanded cag repeat sequence in the hd gene leads to the symptoms and neuropathology of hd [ 28 ] . we propose an updated review of the mechanisms through which the cholinergic system could be used to modify the functional deficits and neuropathology of hd . the cholinergic hypothesis suggests that dysfunctional acetylcholine ( ach)-containing neurons and dysfunctional cholinergic transmission in the brain significantly contribute to the behavioural symptoms and neuropathology in disease , in this instance hd . the link between the cholinergic hypothesis and neurodegenerative disease was proposed by bartus and colleagues in a seminal review   which referred to findings from previous studies including the cognitive deficits produced by anticholinergic drugs in humans [ 11 , 12 ] and primates , as well as reduced choline acetyltransferase ( chat ) activity , reduced ach release and degeneration of cholinergic neurons in autopsied patients [ 1420 ] . cholinergic neurons in the ventral pallidum and magnocellular cholinergic neurons in other parts of the forebrain , identified by chat staining , displayed a large whole cell conductance , a hyperpolarized resting membrane potential , marked fast inward rectification , a prominent spike afterhyperpolarization ( ahp ) , but did not fire spontaneously . however , these findings were based on a single study in rat brain slices . in addition cholinergic neurons in the basal forebrain of gfp - expressing transgenic mice can be either early - firing or late - firing neurons . the early - firing neurons are more excitable and are more susceptible to depolarization blockade , while displaying prominent spike frequency adaptation . conversely , late firing neurons are less excitable and maintain a tonic discharge at low frequencies . early - firing neurons are thought to be involved in phasic changes in cortical ach release associated with attention , while the late - firing neurons may support general arousal by maintaining tonic ach levels . the distinct electrophysiological properties of cholinergic neurons means that it would be possible to distinguish cholinergic neurons from non - cholinergic neurons in brain slices , which will simplify future physiological and pharmacological studies of these neurons . the existence of two distinct subtypes of basal forebrain cholinergic neurons and their electrophysiological properties suggests that each subtype has a different ach release profile , which is supported by recent studies showing ach release can be measured over seconds or minutes [ 2325 ] . further , it is thought that each subtype is involved in different aspects of synaptic plasticity . the afferent inputs and efferent outputs of cholinergic neurons also vary according to their location . muscarinic cholinoceptive neurons in the neocortex are directly innervated by the magnocellular basal nucleus , while basal forebrain cholinergic neurons receive afferent input from the prefrontal cortex . cholinergic neurons from the basal forebrain and upper brainstem project to several areas including the cerebral cortex , amygdala , hippocampus , olfactory bulb and the thalamic nuclei . in addition cholinergic neurons in and around the pedunculopontine nucleus were shown to send projections to the substantia nigra pars compacta ( snc ) via nicotinic receptors . striatal cholinergic interneurons densely innervate the striatum resulting in the striatum having one of the highest levels of ach in the brain . these neurons , known as tonically active neurons ( tans ) of the striatum , function as pacemaker cells and exhibit single spiking or rhythmic discharges but fire autonomously at rest . they receive dopaminergic input from the snc , glutamatergic input from the thalamus ( from the intralaminar nuclei ) and the cerebral cortex , as well as input from the brain stem . tans modulate other interneurons particularly the fast spiking parvalbumin positive gabaergic interneurons and most importantly the medium spiny neurons ( msns ) through complex synaptic interactions . they project to virtually all msn subtypes , including both d1- and d2-dopamine receptor expressing msns and directly act through m1 receptors located on msns [ 31 , 32 ] . the combined activation of the a2a adenosine and d2 dopamine receptors on cholinergic interneurons decreases the release of ach . this decrease reverses the blockade of l - type calcium channels , which are mediated by m1 muscarinic receptors located on msns , which triggers the entry of calcium into the msn . this might in turn trigger endocannabinoid release at the postsynaptic sites of both d1- and d2r - expressing msns and thus depress the corticostriatal and thalamostriatal glutamatergic synaptic transmission of both the direct and indirect pathways [ 31 , 32 ] . mouse models of hd have shown anatomical changes of striatal cholinergic neurons and reduced thalamic input to striatal cholinergic interneurons . since cholinergic interneurons have different effects on the striatal spiny projection neurons of the direct and indirect pathways , the reduction of glutamatergic transmission from corticostriatal and thalamostriatal projections may be responsible for early motor impairments observed in hd . studies in post - mortem human hd brains and animal models have shown that dysfunctional cholinergic neurotransmission and cholinergic neurons may play a significant role in hd pathogenesis . post - mortem brains of patients with hd have shown a reduction in the levels of chat , vesicular acetylcholine transporter ( vacht ) , vesicular monoamine transporter type-2 ( vmat2 ) and the pyruvate dehydrogenase complex , an essential enzyme for ach synthesis [ 3439 ] . conversely , choline levels were down in the csf of hd patients but there was no reduction in the levels of acetylcholinesterase ( ache ) , an enzyme which degrades ach . young r6/1 transgenic mice exhibited reductions in ache activity and chat and vacht messenger ribonucleic acid ( mrna ) and protein levels [ 41 , 15 ] . transgenic r6/2 mice also showed a significant reduction in the electrically - evoked release of ach in striatal tissue , as well as a decrease in chat activity and synaptosomal high - affinity choline uptake with age [ 42 , 43 ] . in hd , striatal gabaergic msns are preferentially degraded , while large aspiny cholinergic interneurons are relatively spared [ 44 , 45 ] . however , the surviving striatal cholinergic neurons showed decreased ach release , vacht binding and significant reductions in chat activity , but persistent ache activity [ 34 , 4547 ] . during the early and mid - stages of hd , these cholinergic neurons demonstrate neuronal dysfunction without cell death , which may be involved in the early symptomatology of hd [ 41 , 48 ] . the dysfunction observed in striatal cholinergic neurons may impact ach - dependent processes such as the induction of synaptic plasticity [ 43 , 47 ] , and may significantly affect the function of other neuronal populations , including msns [ 4850 ] . however , in the later stages of hd , the loss of aspiny cholinergic neurons has been observed [ 5153 ] . some have shown a reduction in muscarinic acetylcholine receptor ( machr ) binding , and a decrease in the number of m2 machrs and total machrs [ 35 , 37 ] . conversely , early studies reported no change in the levels of nachrs in post - mortem human hd brains [ 54 , 55 ] . however , these studies also reported no alterations in the level of chat in human hd brains , which has since been refuted by subsequent studies , and further research is therefore needed to clarify the levels of nachrs in the hd brain [ 3437 ] . decreased levels of ngf , an essential regulator of cholinergic neuronal survival and cholinergic neurotransmission , were observed in hd patients and r6/1 mice [ 48 , 57 ] . altered levels of ngf receptors have also been reported in the hippocampus of hd patients and in r6/1 and hdh mice [ 58 , 59 ] . the first procholinergic treatment for hd was the ach precursor , choline , which significantly reduced chorea in three out of eight patients after they were administered choline ( table  1 ) . however , choline showed no beneficial effects in other clinical studies [ 61 , 62 ] . another therapeutic strategy was to increase ach levels through the use of acetylcholinesterase inhibitors ( acheis ) which inhibited the breakdown of ach by the enzyme ache ( fig .  1 ) . the achei , rivastigmine , enhanced cognitive performance and slowed motor deterioration in an open - label clinical trial of 21 hd patients . in a follow - up study , patients treated with rivastigmine showed increased motor performance , reduced chorea , increased cognitive function and reduced functional disability , relative to controls , suggesting that rivastigmine may have long - term benefits as a potential treatment for hd . a smaller clinical study on four hd patients reported that rivastigmine improved cognitive and behavioural function , but failed to improve motor function or functional performance ( table  1 ) . the achei , donepezil , improved the cognitive function of r6/2 mice in a simple visual discrimination task ( table  2 ) . however , the beneficial effects of donepezil did not translate to the clinic [ 67 , 68 ] . galantamine , another achei , attenuated neurological deficits , reduced striatal lesion volumes and decreased the number of apoptotic cells in the 3-nitropropionic acid rat model of hd . further , a 35 year old male patient treated with galantamine displayed an improvement of psychotic symptoms and chorea , but showed no change in cognitive function . interestingly , the beneficial effects of galantamine were attenuated by mecamylamine , a nachr antagonist , which suggests that the beneficial effects of galantamine may be mediated through nachr activation . acheis can also activate the cholinergic anti - inflammatory pathway , which reduces the neuroinflammation associated with hd pathology [ 71 , 72 ] . these anti - inflammatory effects were inhibited by the 7  nachr antagonist mla , but not by the 42  nachr antagonist , dihydro--erythroidine , which suggests that the anti - inflammatory effects of the cholinergic anti - inflammatory pathway are mediated through 7  nachrs [ 7376 ] . physostigmine , an achei which stimulates both nachrs and machrs , was shown to reduce chorea in two studies [ 77 , 78 ] . muscarinic receptor activation was also shown to reverse impairments in spatial memory , enhance hippocampal cell proliferation in the dentate gyrus and the ca1 region of the hippocampus and reverse deficits in hippocampal neurogenesis ( fig .  1 ) . an alternative therapeutic strategy was to use anticholinergic agents , such as muscarinic receptor antagonists , to reduce chorea since anticholinergics cause sedation . the machr antagonist , scopolamine , decreased chorea but increased lack of coordination , sedation and confusion . another machr antagonist , benztropine , produced similar but milder effects , while the peripheral anticholinergic agent , glycopyrrolate , had no effect on chorea . conversely , the m1  machr antagonist , olanzapine , improved motor function   and psychiatric function   in human hd patients . other studies reported that olanzapine improved both motor and psychiatric function when administered alone   or together with valproate . the m4  machr positive allosteric modulator vu0467154 was reported to improve motor function and reduce synaptic deficits in transgenic yac128 mice . further , the depotentiation of spiny neurons was blocked by either scopolamine or hemicholinium in mouse and rat models , which suggests that the reversal of ltp requires the activation of muscarinic receptors . the administration of ngf and ngf - promoting drugs , cells or microspheres , increased motor coordination , reduced clasping , increased spatial working memory [ 48 , 88 ] , showed neuroprotective effects [ 8994 ] , reversed cholinergic neuron atrophy , promoted hippocampal neurogenesis , and increased the expression of chat and vacht [ 48 , 95 ] in rodent models of hd ( table  2 ) . there are therefore several potential targets for therapeutic intervention in the cholinergic system including ( 1 ) increasing the amount of the cholinergic survival factor ngf ( 2 ) increasing the amount of the ach precursor choline ( 3 ) using acheis which inhibit aches from breaking down ach and ( 4 ) using machr / nachr ligands to activate machrs and nachrs . of these , the most promising therapeutic intervention appears to be increasing ngf , because if ngf can restore cholinergic neurons to optimal function and enhance ach release to enable proper msn function , there is no need to artificially add choline , acheis or machr / nachr ligands to the system . studies showing the dysfunction of cholinergic transmission , cholinergic receptors , cholinergic neurons and their associated neurotrophic factors in human hd patients and animal models of hd , together with the close link between cholinergic neurons and msns , suggest that the dysfunctional cholinergic system plays a vital role in the symptoms and neuropathology of hd . this provides researchers with a rationale to develop potential therapies for hd by targeting the cholinergic system . cholinergic treatments have shown beneficial effects in both pre - clinical and clinical studies , and have improved motor , cognitive and psychiatric function , as well as reducing striatal lesions , synaptic deficits , excitotoxicity , cell atrophy and cell death [ 63 , 64 , 69 , 85 , 88 ] . however , different studies often reported different efficacies for the same therapy , possibly due to differences in the model or criteria used to determine efficacy , dose administration technique and dosing regimen used . in order to achieve optimal results , these factors need to be optimised . in addition , a potential cholinergic therapeutic should be administered during the stage of the disease where it can have the greatest effect , for example , a machr agonist should be administered before significant loss of machrs occurs . nonetheless , we believe that the beneficial effects of cholinergic treatments in pre - clinical and clinical hd studies demonstrates that this is a promising avenue of hd research which requires further investigation , and that enhancing cholinergic function may lead to the development of a potential therapy for hd . the interdependent nature of the cholinergic dysfunction and its associated neurotrophic factors means that a potential treatment targeted towards increasing cholinergic neurotrophic factors may also have beneficial effects on cholinergic neuronal survival and neurotransmission . in addition , the widespread effects of the cholinergic system on brain function suggests that treatments targeted towards the normalization of cholinergic transmission in hd may have other unseen beneficial effects , such as improving the function of other neuronal populations , such as msns [ 48 , 50 , 96 ] . based on the available data ( see table  1 ) , one of the most - promising therapeutic avenues appears to be increasing the levels of ngf , either through direct administration , the administration of ngf - promoting drugs , such as riluzole , or ngf - secreting cells or microspheres . ngf is an important survival factor for cholinergic neurons , and thus mediates cholinergic transmission as well as the function of neurons innervated by cholinergic neurons , such as msns . to investigate the pre - clinical efficacy of ngf , an animal model that replicates the human condition as closely as possible such as the yac128 transgenic mouse model of hd , this mouse contains the full - length human hd gene and exhibits the motor , cognitive and psychiatric symptoms of hd , as well as the neuropathological characteristics , such as striatal cell loss , huntingtin aggregates and altered synaptic currents [ 97 , 98 ] . ngf does not cross the blood - brain - barrier , thus , we would deliver ngf by surgically implanting microspheres in the striatum of the mice to achieve a controlled and sustained delivery of ngf . the mice would undergo behavioural testing , and various neuropathological endpoints including msn and cholinergic cell survival as well as synaptic function [ 48 , 85 , 100 , 101 ] . since therapies geared towards a single target have failed to show beneficial effects in the clinic , a therapy , such as ngf , which can affect multiple targets , may have a higher chance of exhibiting beneficial effects in a clinical trial . recent studies have focused on specific cholinergic receptor subtype agonists and antagonists , in particular those targeting 42 and 7 nicotinic acetylcholine receptors ( nachrs ) . the selective 42 agonists zy-1 and tc-6683 increased cognitive function in preclinical animal models of ad , with tc-6683 having progressed to phase 2 clinical trials for ad [ 104 , 105 ] . the selective 42 agonist altinicline , and partial 42 agonists cytisine and ispronicline displayed antidepressant - like effects [ 106 , 107 , 108 ] . further , administration of evp-6124 , an 7  nachr partial agonist , and tc-5619 , a full 7  nachr agonist produced improvements in the memory of rats [ 110 , 111 ] . given the beneficial effects of 42 and 7  nachr agonists on motor , cognitive and psychiatric function , it may be beneficial to pursue a potential therapy which activates both 42 and 7  nachrs . one such drug is varenicline , a potent , partial agonist at 42  nachrs , and a potent , full agonist at 7  nachrs [ 112 , 113 ] . varenicline also enhanced the cognitive function of mice [ 115 , 116 ] and rats . the potentially advantageous therapies described above which directly or indirectly target the cholinergic system may benefit hd patients , and should therefore be further investigated .
in this review , we outline the role of the cholinergic system in huntington s disease , and briefly describe the dysfunction of cholinergic transmission , cholinergic neurons , cholinergic receptors and cholinergic survival factors observed in post - mortem human brains and animal models of huntington s disease . 
 we postulate how the dysfunctional cholinergic system can be targeted to develop novel therapies for huntington s disease , and discuss the beneficial effects of cholinergic therapies in pre - clinical and clinical studies .