Véronique M. André

Full-Length Human Mutant Huntingtin with a Stable Polyglutamine Repeat Can Elicit Progressive and Selective Neuropathogenesis in BACHD Mice

To elucidate the pathogenic mechanisms in Huntingtons disease (HD) elicited by expression of full-length human mutant huntingtin (fl-mhtt), a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) was developed expressing fl-mhtt with 97 glutamine repeats under the control of endogenous htt regulatory machinery on the BAC. BACHD mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuron degeneration. Power analyses reveal the robustness of the behavioral and neuropathological phenotypes, suggesting BACHD as a suitable fl-mhtt mouse model for preclinical studies. Additional analyses of BACHD mice provide novel insights into how mhtt may elicit neuropathogenesis. First, unlike previous fl-mhtt mouse models, BACHD mice reveal that the slowly progressive and selective pathogenic process in HD mouse brains can occur without early and diffuse nuclear accumulation of aggregated mhtt (i.e., as detected by immunostaining with the EM48 antibody). Instead, a relatively steady-state level of predominantly full-length mhtt and a small amount of mhtt N-terminal fragments are sufficient to elicit the disease process. Second, the polyglutamine repeat within fl-mhtt in BACHD mice is encoded by a mixed CAA-CAG repeat, which is stable in both the germline and somatic tissues including the cortex and striatum at the onset of neuropathology. Therefore, our results suggest that somatic repeat instability does not play a necessary role in selective neuropathogenesis in BACHD mice. In summary, the BACHD model constitutes a novel and robust in vivo paradigm for the investigation of HD pathogenesis and treatment.

Pathophysiology of Huntington's disease: time-dependent alterations in synaptic and receptor function

Huntingtons disease (HD) is a progressive, fatal neurological condition caused by an expansion of CAG (glutamine) repeats in the coding region of the Huntington gene. To date, there is no cure but great strides have been made to understand pathophysiological mechanisms. In particular, genetic animal models of HD have been instrumental in elucidating the progression of behavioral and physiological alterations, which had not been possible using classic neurotoxin models. Our groups have pioneered the use of transgenic HD mice to examine the excitotoxicity hypothesis of striatal neuronal dysfunction and degeneration, as well as alterations in excitation and inhibition in striatum and cerebral cortex. In this review, we focus on synaptic and receptor alterations of striatal medium-sized spiny (MSNs) and cortical pyramidal neurons in genetic HD mouse models. We demonstrate a complex series of alterations that are region-specific and time-dependent. In particular, many changes are bidirectional depending on the degree of disease progression, that is, early vs. late, and also on the region examined. Early synaptic dysfunction is manifested by dysregulated glutamate release in striatum followed by progressive disconnection between cortex and striatum. The differential effects of altered glutamate release on MSNs originating the direct and indirect pathways is also elucidated, with the unexpected finding that cells of the direct striatal pathway are involved early in the course of the disease. In addition, we review evidence for early N-methyl-D-aspartate receptor (NMDAR) dysfunction leading to enhanced sensitivity of extrasynaptic receptors and a critical role of GluN2B subunits. Some of the alterations in late HD could be compensatory mechanisms designed to cope with early synaptic and receptor dysfunctions. The main findings indicate that HD treatments need to be designed according to the stage of disease progression and should consider regional differences.

Morphological and electrophysiological characterization of abnormal cell types in pediatric cortical dysplasia.

The mechanisms responsible for seizure generation in cortical dysplasia (CD) are unknown, but morphologically abnormal cells could contribute. We examined the passive and active membrane properties of cells from pediatric CD in vitro. Normal‐ and abnormal‐appearing cells were identified morphologically by using infrared videomicroscopy and biocytin in slices from children with mild to severe CD. Electrophysiological properties were assessed with patch clamp recordings. Four groups of abnormal‐appearing cells were observed. The first consisted of large, pyramidal cells probably corresponding to cytomegalic neurons. Under conditions that reduced the contribution of K+ conductances, these cells generated large Ca2+ currents and influx when depolarized. When these cells were acutely dissociated, peak Ca2+ currents and densities were greater in cytomegalic compared with normal‐appearing pyramidal neurons. The second group included large, nonpyramidal cells with atypical somatodendritic morphology that could correspond to “balloon” cells. These cells did not display active voltage‐ or ligand‐gated currents and did not appear to receive synaptic inputs. The third group included misoriented and dysmorphic pyramidal neurons, and the fourth group consisted of immature‐looking pyramidal neurons. Electrophysiologically, neurons in these latter two groups did not display significant abnormalities when compared with normal‐appearing pyramidal neurons. We conclude that there are cells with abnormal intrinsic membrane properties in pediatric CD. Among the four groups of cells, the most abnormal electrophysiological properties were displayed by cytomegalic neurons and large cells with atypical morphology. Cytomegalic neurons could play an important role in the generation of epileptic activity.

Epileptogenesis in pediatric cortical dysplasia: The dysmature cerebral developmental hypothesis

Cortical dysplasia (CD) is the most frequent pathology found in pediatric epilepsy surgery patients with a nearly 80% incidence in children younger than 3 years of age. Younger cases are more likely to have multilobar and severe forms of CD compared with older patients with focal and mild CD. Using clinico-pathologic techniques, we have initiated studies that unravel the timing of CD pathogenesis that in turn suggest mechanisms of epileptogenesis. Morphological comparisons provided the first clue when we observed that cytomegalic neurons have similarities with human subplate cells, and balloon cells have features analogous to radial glia. This suggested that failure of prenatal cell degeneration before birth could explain the presence of postnatal dysmorphic cells in CD tissue. Neuronal density and MRI volumes indicate that there were more neurons than expected in CD tissue, and they were probably produced in later neurogenesis cell cycles. Together these findings imply that there is partial failure in later phases of cortical development that might explain the distinctive histopathology of CD. If correct, epileptogenesis should be the consequence of incomplete cellular maturation in CD tissue. In vitro electrophysiological findings are consistent with this notion. They show that balloon cells have glial features, cytomegalic neurons and recently discovered cytomegalic interneurons reveal atypical hyperexcitable intrinsic membrane properties, there are more GABA than glutamate spontaneous synaptic inputs onto neurons, and in a subset of cells NMDA and GABA(A) receptor-mediated responses and subunit expression are similar to those of immature neurons. Our studies support the hypothesis that there are retained prenatal cells and neurons with immature cellular and synaptic properties in pediatric CD tissue. We propose that local interactions of dysmature cells with normal postnatal neurons produce seizures. This hypothesis will drive future studies aimed at elucidating mechanisms of epileptogenesis in pediatric CD tissue.

Differential electrophysiological properties of dopamine D1 and D2 receptor-containing striatal medium-sized spiny neurons

The electrophysiological properties of distinct subpopulations of striatal medium‐sized spiny neurons (MSSNs) were compared using enhanced green fluorescent protein as a reporter gene for identification of neurons expressing dopamine D1 and D2 receptor subtypes in mice. Whole‐cell patch‐clamp recordings in slices revealed that passive membrane properties were similar in D1 and D2 cells. All MSSNs displayed hyperpolarized resting membrane potentials but the threshold for firing action potentials was lower in D2 than in D1 neurons. In voltage clamp, the frequency of spontaneous excitatory postsynaptic currents was higher in D2 than in D1 cells and large‐amplitude inward currents (> 100 pA) were observed only in D2 cells. After tetrodotoxin this difference was reduced, suggesting that sodium conductances contribute to the increased frequencies in D2 cells. After pharmacological blockade of GABAA receptors, a subset of D2 cells also displayed large spontaneous membrane depolarizations and complex responses to stimulation of the corticostriatal pathway. To further characterize ionotropic glutamate receptor function, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) was applied onto dissociated MSSNs. Application of AMPA alone or in the presence of cyclothiazide (an AMPA receptor desensitization blocker) evoked larger currents in D1 than in D2 cells. Together, these data demonstrate significant differences in electrophysiological properties of subpopulations of MSSNs defined by selective expression of D1 and D2 receptors. D2 cells display increased excitability and reflect ongoing cortical activity more faithfully than D1 cells, an effect that is independent of postsynaptic AMPA receptors and probably results from stronger synaptic coupling. This could help to explain the increased vulnerability of D2 MSSNs in neurodegenerative disorders.

Age-Dependent Alterations of Corticostriatal Activity in the YAC128 Mouse Model of Huntington Disease

Huntington disease is a genetic neurodegenerative disorder that produces motor, neuropsychiatric, and cognitive deficits and is caused by an abnormal expansion of the CAG tract in the huntingtin (htt) gene. In humans, mutated htt induces a preferential loss of medium spiny neurons in the striatum and, to a lesser extent, a loss of cortical neurons as the disease progresses. The mechanisms causing these degenerative changes remain unclear, but they may involve synaptic dysregulation. We examined the activity of the corticostriatal pathway using a combination of electrophysiological and optical imaging approaches in brain slices and acutely dissociated neurons from the YAC128 mouse model of Huntington disease. The results demonstrated biphasic age-dependent changes in corticostriatal function. At 1 month, before the behavioral phenotype develops, synaptic currents and glutamate release were increased. At 7 and 12 months, after the development of the behavioral phenotype, evoked synaptic currents were reduced. Glutamate release was decreased by 7 months and was markedly reduced by 12 months. These age-dependent alterations in corticostriatal activity were paralleled by a decrease in dopamine D2 receptor modulation of the presynaptic terminal. Together, these findings point to dynamic alterations at the corticostriatal pathway and emphasize that therapies directed toward preventing or alleviating symptoms need to be specifically designed depending on the stage of disease progression.

Increased GABAergic function in mouse models of Huntington's disease: reversal by BDNF.

Huntingtons disease (HD) is characterized by loss of striatal γ‐aminobutyric acid (GABA)ergic medium‐sized spiny projection neurons (MSSNs), whereas some classes of striatal interneurons are relatively spared. Striatal interneurons provide most of the inhibitory synaptic input to MSSNs and use GABA as their neurotransmitter. We reported previously alterations in glutamatergic synaptic activity in the R6/2 and R6/1 mouse models of HD. In the present study, we used whole‐cell voltage clamp recordings to examine GABAergic synaptic currents in MSSNs from striatal slices in these two mouse models compared to those in age‐matched control littermates. The frequency of spontaneous GABAergic synaptic currents was increased significantly in MSSNs from R6/2 transgenics starting around 5–7 weeks (when the overt behavioral phenotype begins) and continuing in 9–14‐week‐old mice. A similar increase was observed in 12–15‐month‐old R6/1 transgenics. Bath application of brain‐derived neurotrophic factor, which is downregulated in HD, significantly reduced the frequency of spontaneous GABAergic synaptic currents in MSSNs from R6/2 but not control mice at 9–14 weeks. Increased GABA current densities also occurred in acutely isolated MSSNs from R6/2 animals. Immunofluorescence demonstrated increased expression of the ubiquitous α1 subunit of GABAA receptors in MSSNs from R6/2 animals. These results indicate that increases in spontaneous GABAergic synaptic currents and postsynaptic receptor function occur in parallel to progressive decreases in glutamatergic inputs to MSSNs. In conjunction, both changes will severely alter striatal outputs to target areas involved in the control of movement.

Alterations in Cortical Excitation and Inhibition in Genetic Mouse Models of Huntington's Disease

Previously, we identified progressive alterations in spontaneous EPSCs and IPSCs in the striatum of the R6/2 mouse model of Huntingtons disease (HD). Medium-sized spiny neurons from these mice displayed a lower frequency of EPSCs, and a population of cells exhibited an increased frequency of IPSCs beginning at ∼40 d, a time point when the overt behavioral phenotype begins. The cortex provides the major excitatory drive to the striatum and is affected during disease progression. We examined spontaneous EPSCs and IPSCs of somatosensory cortical pyramidal neurons in layers II/III in slices from three different mouse models of HD: the R6/2, the YAC128, and the CAG140 knock-in. Results revealed that spontaneous EPSCs occurred at a higher frequency, and evoked EPSCs were larger in behaviorally phenotypic mice whereas spontaneous IPSCs were initially increased in frequency in all models and subsequently decreased in R6/2 mice after they displayed the typical R6/2 overt behavioral phenotype. Changes in miniature IPSCs and evoked IPSC paired-pulse ratios suggested altered probability of GABA release. Also, in R6/2 mice, blockade of GABAA receptors induced complex discharges in slices and seizures in vivo at all ages. In conclusion, altered excitatory and inhibitory inputs to pyramidal neurons in the cortex in HD appear to be a prevailing deficit throughout the development of the disease. Furthermore, the differences between synaptic phenotypes in cortex and striatum are important for the development of future therapeutic approaches, which may need to be targeted early in the development of the phenotype.

Differential susceptibility to excitotoxic stress in YAC128 mouse models of Huntington disease between initiation and progression of disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG tract in the HD gene. Polyglutamine expansion of huntingtin (htt) results in early, progressive loss of medium spiny striatal neurons, as well as cortical neurons that project to the striatum. Excitotoxicity has been postulated to play a key role in the selective vulnerability of striatal neurons in HD. Early excitotoxic neuropathological changes observed in human HD brain include increased quinolinate (QUIN) concurrent with proliferative changes such as increased spine density and dendritic length. In later stages of the disease, degenerative-type changes are apparent, such as loss of dendritic arborization, a reduction in spine density and reduced levels of 3-hydroxykynurenine and QUIN. It is currently unknown whether sensitivity to excitotoxic stress varies between initiation and progression of disease. Here, we have assessed the excitotoxic phenotype in the YAC128 mouse model of HD by examining the response to excitotoxic stress at different stages of disease. Our results demonstrate that YAC128 mice display enhanced sensitivity to NMDA ex vivo and QUIN in vivo before obvious phenotypic changes. In contrast, 10-month-old symptomatic YAC128 mice are resistant to QUIN-induced neurotoxicity. These findings are paralleled by a significant increase in NMDAR-mediated membrane currents in presymptomatic YAC128 dissociated medium spiny neurons progressing to reduced NMDAR-mediated membrane currents with disease progression. These data highlight the dynamic nature of the mutant htt-mediated excitotoxic phenotype and suggests that therapeutic approaches to HD may need to be altered, depending on the stage and development of the disease.

Dopamine and glutamate in Huntington's disease: A balancing act.

Huntingtons disease (HD) is caused by a CAG repeat expansion in exon 1 of the HD gene resulting in a long polyglutamine tract in the N‐terminus of the protein huntingtin. Patients carrying the mutation display chorea in early stages followed by akinesia and sometimes dystonia in late stages. Other major symptoms include depression, anxiety, irritability or aggressive behavior, and apathy. Although many neuronal systems are affected, dysfunction and subsequent neurodegeneration in the basal ganglia and cortex are the most apparent pathologies. In HD, the primary hypothesis has been that there is an initial overactivity of glutamate neurotransmission that produces excitotoxicity followed by a series of complex changes that are different in the striatum and in the cortex. This review will focus on evidence for alterations in dopamine (DA)–glutamate interactions in HD, concentrating on the striatum and cortex. The most recent evidence points to decreases in DA and glutamate neurotransmission as the HD phenotype develops. However, there is some evidence for increased DA and glutamate functions that could be responsible for some of the early HD phenotype. Significant evidence indicates that glutamate and dopamine neurotransmission is affected in HD, compromising the fine balance in which DA modulates glutamate‐induced excitation in the basal ganglia and cortex. Restoring the balance between glutamate and dopamine could be helpful to treat HD symptoms.