HD is a progressive disorder and new evidence indicates that the onset of the characteristic neurological symptoms is preceded by a protracted premanifest phase of disease [34
]. Because carriers can be readily identified with genetic testing, early implementation of neuroprotective treatments will eventually be possible once such treatments are identified. At this time, however, no cure or methods to delay or prevent the onset of the devastating symptoms of HD are available, and the development of such treatments is predicated on preclinical testing in mouse models of the disease. Of the many models available, the CAG140 mice present many advantages both because they express a full length mutated protein and because they express robust behavioral deficits that are suitable for drug testing, from an early age. Previous characterization of the pathological features of this model has been limited to a description of huntingtin pathology [32
] and the discovery of late stage striatal atrophy and neuronal loss [15
In the present study, we show that this striatal atrophy develops over time as with HD. We observed no deficit in striatal volume at 4 months of age but by 12 months there was decreased volume in both male and female homozygote KI mice. In addition, we show that atrophy of the corpus callosum is present at 20–26 months of age and that surviving neurons in the striatum show significant morphological alterations at that age. These data further establish the validity of this mouse model for studies of neurodegeneration and its prevention, and provide time course information on some of the major endpoint measures and robust differences that can be used in preclinical drug testing of neuroprotective therapies for HD.
It is noteworthy that the decrease in striatal volume we have observed in CAG140 mice (repeat length of ~119 CAGs in this study) does not occur until many months after the onset of behavioral deficits that have been detected as early as 1 month of age in this model. Carriers of the HD mutation also show subtle behavioral deficits many years before the onset of clear neurological symptoms [34
]. Similarly, CAG94 KI mice show ~14% loss in striatal volume, but no loss in neuronal number, at approximately 2 years of age whereas behavioral deficits begin at 2 months [32
]. Nevertheless, as in HD [34
], striatal atrophy was detected in our model many months before the onset of overt behavioral deficits (approximately 20–26 months of age [15
]). Notably, the striatal atrophy detected at this late age is similar to the “one third to one half loss” demonstrated in HD patients at the time of diagnosis [2
Alterations in white matter tracts have been described to occur very early in carriers of the HD mutation [48
]. Neither the volumes of the cerebral cortex nor the corpus callosum, measured in the same sections as the striatum, were altered at 12 months, when striatal atrophy was already evident. This result in cortex however, should be interpreted with caution because it reflects only a small portion, and a global measure of cortex can miss specific atrophy of defined cortical layers, as has been observed in patient brain [40
]. Similarly, our corpus callosum measurements were global, which could obscure subtle changes, as have been found in HD [3
]. Nevertheless, a significant decrease in the volume of the corpus callosum was detected at 20–26 months of age, confirming that white matter tracts are affected in this model, as they are at early stages of HD [41
The existence of morphological anomalies of surviving striatal neurons in post-mortem tissue from HD patients has been well documented but these studies are usually performed at relatively advanced stages of disease [9
]. We found that at a time when about 40% of striatal neurons are lost and overt behavioral deficits just become apparent, remaining striatal neurons in our mouse model present significant morphological alterations suggesting the presence of a slow, ongoing neurodegenerative process. Specifically, the neurons showed a significant reduction in spine density, preferentially lost in the distal branches of the dendrite, as well as a reduced dendritic arborization, and a trend toward a reduction in total dendritic branch length. Thus, the CAG140 KI mice offers an opportunity to analyze the mechanisms that lead to cellular pathology characteristic of HD, in addition to allowing the testing of potential neuroprotective agents.
It is clear that both intrinsic and extrinsic mechanisms contribute to the demise of striatal neurons in HD [11
]. A role for cortical inputs has been documented in mouse models [12
], however another intriguing possibility is the potential role of dysfunction in the other main striatal input, the dopaminergic neurons from the Substantia nigra pars compacta that are more commonly thought of as the primary deficit in Parkinson’s disease. In humans, a reduction in dopamine receptors has been demonstrated in both symptomatic and asymptomatic HD patients [1
]. Early loss of dopamine receptors, abnormal transcriptional levels of dopamine-regulated proteins, reduced dopamine release, reduced dopamine levels, have all been observed in the striatum of HD mice [5
]. In CAG 140 KI mice, we have shown reduction in D1 and D2 dopamine receptor mRNAs as early as 4 months of age [14
]. On the presynaptic side, some studies have detected a loss of striatal dopamine in post-mortem brains of HD patients [26
] although the data have been inconsistent [30
]. In the present study, we have measured immunoreactivity of TH, the rate-limiting enzyme of catecholamine synthesis and a marker for dopamine terminals in the striatum (which contains very few noradrenergic terminals). Interestingly, several studies have reported an association between striatal dopamine deficiency and spine loss in medium sized spiny neurons [4
]. In fact, changes in dopamine function influencing striatal spines in HD could either be pre- or post-synaptic. Importantly, our measurements here were made using a fluorescently-labeled secondary antibody and measured with a microarray scanner. This method provides enhanced correlation of signal intensity to antigen concentration because, contrary to diaminobenzidine based immunodetection techniques, it does not rely on an enzymatic reaction and it uses the expanded dynamic range of fluorescence. TH immunofluorescence was markedly reduced, and although no non-perfused tissue from these very old mice was available to perform neurochemical studies, previous data in R6/2 mice, a severe model of HD, suggests that striatal dopamine is reduced in old mutant mice [16
It is tempting to interpret these data to suggest that dopamine therapy could be beneficial in HD. Unfortunately, although L-dopa administration was found to improve short-term symptoms in R6/2 mouse models, its long-term administration was deleterious to behavior and survival [16
]. Moreover, high-dose L-dopa treatment has not been shown to restore spine density back to normal levels in either PD models or patients, demonstrating that an increase in dopamine does not recover the pathology [8
]. Perhaps dopamine needs to be administered earlier, or other factors present in dopaminergic or other terminals contribute to the morphological deficits in striatal neurons.
Indeed, another factor that could lead to spine loss is excess glutamate. Studies in mouse models of HD have revealed increases in corticostriatal glutamate currents that occur early in the course of the disease [24
]. In our study, we found that distal branches of the dendrites suffered greater spine loss than those of the proximal dendritic branches. As the distal dendrites are associated with growth and with relatively greater spine densities, they are more susceptible to excessive activity of glutamatergic inputs and the resulting increase in calcium levels that could lead to their degeneration [9
]. At later disease stage, some mouse models exhibit a disconnection of the corticostriatal synapse [7
]. Although it is not known whether a cortical disconnection results in spine degeneration, or whether the spine degeneration is itself the cause of the cortical disconnection, it is clear that at the late stage examined in our study, neuronal function is severely compromised.
In conclusion, we report loss in striatal volume from 12 months of age in both male and female mice, with loss in corpus callosum volume by 20–26 months, and show that surviving neurons in the striatum of 20–26 month old mice exhibit degenerative changes in morphology, in particular loss of both immature and mature spines in distal branches. This was accompanied by a decrease in striatal TH immunohistochemical staining, suggesting a loss of dopaminergic input to the striatum at this age. Together, these data reveal additional endpoint measures for neuroprotective studies. Our data clearly indicate that therapies should target earlier stages of pathophysiology, prior to the occurrence of irreversible cellular impairments that occur during disease progression. This is because the striatal atrophy, and the morphological anomalies, present in striatal neurons in our mouse model at 20–26 months are likely to correspond to early manifest HD. The CAG140 KI mice provides a useful model to analyze the effects of potential neuroprotective treatments on both short- and long-term effects of the HD mutation, correlating to different stages of disease. Furthermore, the protracted period of time between the appearance of subtle but robust behavioral deficits at 1 month of age and evidence of frank neurodegeneration allows for the testing of early and sustained neuroprotective interventions in this model, and provides a long therapeutic time window in this model of HD, as occurs in HD itself.
Here, we show striatal atrophy is already present at 12 months in CAG140 KI mice
We also show late-disease-stage (20–26m) atrophy in white matter in CAG140 KI mice
Late-disease-stage KI striatal neurons display extensive morphological degeneration
DA input may be altered in late-disease-stage KI striatum, as striatal TH is reduced
CAG140 KI mice are highly suited for testing neuroprotective therapies