Vinod Charles

Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA

Huntington disease (HD) is an adult-onset, autosomal dominant inherited human neurodegenerative disorder characterized by hyperkinetic involuntary movements, including motor restlessness and chorea, slowing of voluntary movements and cognitive impairment. Selective regional neuron loss and gliosis in striatum, cerebral cortex, thalamus, subthalamus and hippocampus are well recognized as neuropathological correlates for the clinical manifestations of HD. The underlying genetic mutation is the expansion of CAG trinucleotide repeats (coding for polyglutamines) to 36-121 copies in exon 1 of the HD gene . The HD mRNA and protein product (huntingtin) show widespread distribution, and thus much remains to be understood about the selective and progressive neurodegeneration in HD. To create an experimental animal model for HD, transgenic mice were generated showing widespread expression of full-length human HD cDNA with either 16, 48 or 89 CAG repeats. Only mice with 48 or 89 CAG repeats manifested progressive behavioural and motor dysfunction with neuron loss and gliosis in striatum, cerebral cortex, thalamus and hippocampus. These animals represent clinically relevant models for HD pathogenesis, and may provide insights into the underlying pathophysiological mechanisms of other triplet repeat disorders.

Electroconvulsive Seizures Regulate Gene Expression of Distinct Neurotrophic Signaling Pathways

Electroconvulsive therapy (ECT) remains the treatment of choice for drug-resistant patients with depressive disorders, yet the mechanism for its efficacy remains unknown. Gene transcription changes were measured in the frontal cortex and hippocampus of rats subjected to sham seizures or to 1 or 10 electroconvulsive seizures (ECS), a model of ECT. Among the 3500–4400 RNA sequences detected in each sample, ECS increased by 1.5- to 11-fold or decreased by at least 34% the expression of 120 unique genes. The hippocampus produced more than three times the number of gene changes seen in the cortex, and many hippocampal gene changes persisted with chronic ECS, unlike in the cortex. Among the 120 genes, 77 have not been reported in previous studies of ECS or seizure responses, and 39 were confirmed among 59 studied by quantitative real time PCR. Another 19 genes, 10 previously unreported, changed by <1.5-fold but with very high significance. Multiple genes were identified within distinct pathways, including the BDNF–MAP kinase–cAMP–cAMP response element-binding protein pathway (15 genes), the arachidonic acid pathway (5 genes), and more than 10 genes in each of the immediate-early gene, neurogenesis, and exercise response gene groups. Neurogenesis, neurite outgrowth, and neuronal plasticity associated with BDNF, glutamate, and cAMP–protein kinase A signaling pathways may mediate the antidepressant effects of ECT in humans. These genes, and others that increase only with chronic ECS such as neuropeptide Y and thyrotropin-releasing hormone, may provide novel ways to select drugs for the treatment of depression and mimic the rapid effectiveness of ECT.

Early degenerative changes in transgenic mice expressing mutant huntingtin involve dendritic abnormalities but no impairment of mitochondrial energy production

Mitochondrial defects, which occur in the brain of late-stage Huntingtons disease (HD) patients, have been proposed to underlie the selective neuronal loss in the disease. To shed light on the possible role of mitochondrial energy impairment in the early phases of HD pathophysiology, we carried out Golgi impregnation and quantitative histochemical/biochemical studies in HD full-length cDNA transgenic mice that were symptomatic but had not developed to a stage in which neuronal loss could be documented. Golgi staining showed morphologic abnormalities that included a significant decrease in the number of dendritic spines and a thickening of proximal dendrites in striatal and cortical neurons. In contrast, measurements of mitochondrial electron transport Complexes I-IV did not reveal changes in the striatum and cerebral cortex in these mice. Examination of the neostriatum and cerebral cortex in human presymptomatic and pathological Grade 1 HD cases also showed no change in the activity of mitochondrial Complexes I-IV. These data suggest that dendritic alterations precede irreversible cell loss in HD, and that mitochondrial energy impairment is a consequence, rather than a cause, of early neuropathological changes.

Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: Trophic and tropic effects in a rodent model of Huntington's disease

The present study examined whether implants of epidermal growth factor (EGF)‐responsive stems cells derived from transgenic mice in which the glial fibrillary acid protein (GFAP) promoter directs the expression of human nerve growth factor (hNGF) could prevent the degeneration of striatal neurons in a rodent model of Huntingtons disease (HD). Rats received intrastriatal transplants of GFAP‐hNGF stem cells or control stem cells followed 9 days later by an intrastriatal injection of quinolinic acid (QA). Nissl stains revealed large striatal lesions in rats receiving control grafts, which, on average, encompassed 12.78 mm3. The size of the lesion was significantly reduced (1.92 mm3) in rats receiving lesions and GFAP‐hNGF transplants. Rats receiving QA lesions and GFAP‐hNGF‐secreting grafts stem cell grafts displayed a sparing of striatal neurons immunoreactive (ir) for glutamic acid decarboxylase, choline acetyltransferase, and neurons histochemically positive for nicotinamide adenosine diphosphate. Intrastriatal GFAP‐hNGF‐secreting implants also induced a robust sprouting of cholinergic fibers from subjacent basal forebrain neurons. The lesioned striatum in control‐grafted animals displayed numerous p75 neurotrophin‐ir (p75NTR) astrocytes, which enveloped host vasculature. In rats receiving GFAP‐hNGF‐secreting stem cell grafts, the astroglial staining pattern was absent. By using a mouse‐specific probe, stem cells were identified in all animals. These data indicate that cellular delivery of hNGF by genetic modification of stem cells can prevent the degeneration of vulnerable striatal neural populations, including those destined to die in a rodent model of HD, and supports the emerging concept that this technology may be a valuable therapeutic strategy for patients suffering from this disease. J. Comp. Neurol. 387:96–113, 1997.

Overexpression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase in a transgenic mouse model of Huntington’s disease

Huntingtons disease is due to an expansion of CAG repeats in the huntingtin gene. Huntingtin interacts with several proteins including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We performed immunohistochemical analysis of GAPDH expression in the brains of transgenic mice carrying the huntingtin gene with 89 CAG repeats. In all wild-type animals examined, GAPDH was evenly distributed among the different cell types throughout the brain. In contrast, the majority of transgenic mice showed GAPDH overexpression, with the most prominent GAPDH changes observed in the caudate putamen, globus pallidus, neocortex, and hippocampal formation. Double staining for NeuN and GFAP revealed that GAPDH overexpression occurred exclusively in neurons. Nissl staining analysis of the neocortex and caudate putamen indicated 24 and 27% of cell loss in transgenic mice, respectively. Subcellular fluorescence analysis revealed a predominant increase in GAPDH immunostaining in the nucleus. Thus, we conclude that mutation of huntingtin is associated with GAPDH overexpression and nuclear translocation in discrete populations of brain neurons.

Atrophy of cholinergic basal forebrain neurons following excitotoxic cortical lesions is reversed by intravenous administration of an NGF conjugate

Nerve growth factor (NGF) has been shown to sustain the viability and modulate the function of cholinergic basal forebrain neurons. However, under normal circumstances, NGF does not cross the blood-brain barrier (BBB) following systemic administration making this neurotrophin unavailable to NGF-responsive neurons within the central nervous system (CNS). Recently, a non-invasive method for delivering NGF to the brain was established in which NGF was conjugated to an antibody directed against the transferrin receptor (OX-26) [15, 16]. This conjugation facilitates the transfer of NGF from the systemic circulation to the CNS via the transferrin transport system. In the present study, we tested whether intravenous administration of an OX-26-NGF conjugate could reverse the atrophy of cholinergic basal forebrain neurons following removal of the target sites. Lesions of the left cerebral cortex were created by epidural application of N-methyl-D-aspartic acid (NMDA). Seventy-five days later, cholinergic nucleus basalis neurons were atrophic ipsilateral to the lesion relative to the contralateral side in control rats receiving intravenous injections of vehicle or a non-conjugated mixture of OX-26 and NGF. In contrast, intravenous injections of the OX-26-NGF conjugate restored the size of nucleus basalis perikarya to within normal limits relative to the unlesioned contralateral side. Immunohistochemical studies using rat serum albumen antisera indicated that the BBB was closed at the time of treatment indicating that this trophic effect did not result from NGF crossing through a compromised BBB at the site of the lesion. These data demonstrate that systemic administration of a neurotrophic factor-antibody conjugate, intended to circumvent the BBB, can provide trophic influences to degenerating cholinergic basal forebrain neurons. These data support the emerging concept that the conjugate method can facilitate the transfer of impermeable therapeutic compounds across the BBB.

Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models.

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntingtons disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntingtons disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.

Excitotoxic and metabolic damage to the rodent striatum: role of the P75 neurotrophin receptor and glial progenitors.

After injury, the striatum displays several morphologic responses that may play a role in both regenerative and degenerative events. One such response is the de novo expression of the low‐affinity p75 neurotrophin receptor (p75NTR), a gene that plays critical roles in central nervous system (CNS) cell death pathways. The present series of experiments sought to elucidate the cellular origins of this p75NTR response, to define the conditions under which p75NTR is expressed after striatal injury, and how this receptor expression is associated with neuronal plasticity. After chemical lesions, by using either the excitotoxin quinolinic acid (QA) or the complex II mitochondria inhibitor 3‐nitropropionic acid (3‐NP), we compared the expression of the p75NTR receptor within the rat striatum at different survival times. Intrastriatal administration of QA between 7 days and 21 days postlesion induced p75NTR expression in astrocytes that was preferentially distributed throughout the lesion core. P75NTR immunoreactivity within astrocytes was seen at high (100–220 nmol) but not low (50 nmol) QA doses. Seven and 21 days after 3‐NP lesions, p75NTR expression was present in astrocytes at all doses tested (100–1,000 nmol). However, in contrast to QA, these cells were located primarily around the periphery of the lesion and not within the lesion core. At the light microscopic level p75NTR immunoreactive elements resembled vasculature: but did not colocalize with the pan endothelium cell marker RecA‐1. In contrast, p75NTR‐containing astrocytes colocalized with nestin, vimentin, and 5‐bromo‐2‐deoxyuridine, indicating that these cells are newly born astrocytes. Additionally, striatal cholinergic neurons were distributed around the lesion core expressed p75NTR 3–5 days after lesion in both QA and 3‐NP lesions. These cells did not coexpress the pro‐apoptotic degradation enzyme caspase‐3. Taken together, these data indicate that striatal lesions created by means of excitotoxic or metabolic mechanisms trigger the expression of p75NTR in structures related to progenitor cells. The expression of the p75NTR receptor after these chemical lesions support the concept that this receptor plays a role in the initiation of endogenous cellular events associated with CNS injury. J. Comp. Neurol. 444:291–305, 2002.

Altered expression of hippocampal dentate granule neuron genes in a mouse model of human 22q11 deletion syndrome

Hemizygous deletion of a 3 Mb region of 22q11.2 is found in 1/4000 humans and produces 22q11 deletion syndrome (22q11DS). Up to 35% of 22q11DS patients develop schizophrenia, making it the second highest risk factor for schizophrenia. A mouse model for 22q11DS, the Df1/+ mouse, carries a hemizygous deletion in a region syntenic with the human deletion. Df1/+ mice are mostly viable but display deficits in prepulse inhibition and learning and memory, two common traits of schizophrenia thought to result, at least in part, from defects in hippocampal neurons. We used oligonucleotide microarrays and QRT-PCR to evaluate gene expression changes in hippocampal dentate granule neurons of Df1/+ mice versus wild-type littermates (n=12/group). The expression of only 287 genes changed with p value significance below 0.05 by microarray, yet 12 of the 21 Df1 region genes represented on the array showed highly significantly reduced expression compared to wild-type controls (33% on average, p values from 10(-3) to 10(-7)). Variants in two of these genes, COMT and PRODH, have been linked with schizophrenia. Overlap of the 287 genes with the reportedly reduced expression of mitochondrial, ubiquitin/proteasome, and synaptic plasticity genes in schizophrenia dentate granule neurons, was not significant. However, modest increases in expression of mitochondrial electron transport genes were observed in the Df1/+ mice. This perhaps indicates a compensation for mitochondrial dysfunction caused by the strongly reduced expression of the Df1 region-encoded mitochondrial enzymes proline dehydrogenase (Prodh) and thioredoxin reductase 2 (Txnrd2).

Dissociation of p75 receptors and nerve growth factor neurotrophic effects: lack of p75 immunoreactivity in striatum following physical trauma, excitotoxicity and NGF administration

While the function and regulation of the low affinity (p75) nerve growth factor (NGF) receptor in the central nervous system (CNS) remains a mystery, one of the more intriguing observations involves its response to injury in the adult rat striatum. Following mechanical injury to the striatum, a re-expression of striatal p75 receptors and mRNA purportedly occurs (apparently mediated by elevations in NGF), thus reversing the natural loss of these phenotypic markers that is known to occur during development. This observation has important implications for understanding both the regulation of NGF neurotrophic activity and the role of the p75 receptor, for it implies that the presence of this receptor may be required for NGF trophic activity in the CNS. In an effort to gain a greater understanding of the function and regulation of the low affinity p75 NGF receptor, we performed a series of experiments to study the injury-induced, re-expression phenomenon in the striatum. In the first experiment, we duplicated the mechanical, cannula-induced injury used in the original study. In a follow-up study, we exacerbated that injury by infusing quinolinic acid directly into the striatum. In a third study, the mechanical injury was complemented with chronic striatal infusions of NGF. In a final study, we examined striatal tissue from rats who had been protected from striatal quinolinic acid neurotoxicity by administration of NGF. In no instance was the re-expression of p75 striatal receptors observed, despite positive controls for (a) effective neural trauma, confirmed by histologic and immunocytochemical methods, (b) effective antibody staining, confirmed by appropriate basal forebrain p75 immunoreactivity, and (c) effective biological activity of exogenous NGF, confirmed by hypertrophy of choline acetyltransferase (ChAT)-positive striatal neurons and protection of ChAT-positive striatal neurons against excitotoxicity. At least two important conclusions can be drawn from these studies: (1) the presence or induction of low affinity p75 receptors is not necessary, while the presence of constitutive high affinity tropomyosin related kinase (trk) NGF receptors seem sufficient for NGF trophic activity in the CNS, and (2) the variables necessary to induce re-expression of p75 striatal receptors in adult rats have not yet been elucidated and are apparently complex.