John R. O'Kusky

TARGETED DISRUPTION OF THE HUNTINGTON'S DISEASE GENE RESULTS IN EMBRYONIC LETHALITY AND BEHAVIORAL AND MORPHOLOGICAL CHANGES IN HETEROZYGOTES

Huntingtons disease (HD) is an incurable neuropsychiatric disease associated with CAG repeat expansion within a widely expressed gene that causes selective neuronal death. To understand its normal function, we have created a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homolog of the HD gene. Homozygotes die before embryonic day 8.5, initiate gastrulation, but do not proceed to the formation of somites or to organogenesis. Mice heterozygous for the Hdhex5 mutation display increased motor activity and cognitive deficits. Neuropathological assessment of two heterozygous mice shows significant neuronal loss in the subthalamic nucleus. These studies show that the HD gene is essential for postimplantation development and that it may play an important role in normal functioning of the basal ganglia.

Mutant mouse models of insulin-like growth factor actions in the central nervous system

Insulin-like growth factor-I (IGF-I) and its cognate receptor, the type 1 IGF receptor (IGF1R), as well as high-affinity IGF binding proteins (IGFBP) that modulate IGF-I actions, are expressed throughout the course of brain development. These observations, taken together with studies in cultured neural cells demonstrating a variety of IGF-I growth-promoting activities, provide a strong argument for IGF-I having a central role in the growth and development of the CNS. This report reviews studies of brain development in mutant mice with alterations of IGF-I expression or action. Transgenic (Tg) mice overexpressing IGF-I postnatally exhibit brain overgrowth characterized by increased neuron and oligodendrocyte number, as well as marked increases in myelination. Mutant mice with ablated IGF-I and IGF1R expression, as well as those with overexpression of IGFBPs capable of inhibiting IGF actions, exhibit brain growth retardation with a variety of growth deficits. These studies confirm a role for IGF-I in neural development, and indicate that IGF-I stimulates neurogenesis and synaptogenesis, facilitates oligodendrocyte development, promotes neuron and oligodendrocyte survival, and stimulates myelination. Evidence from experiments in these mouse models also indicates that IGF-I has a role in recovery from neural injury.

In vivo effects of insulin‐like growth factor‐I (IGF‐I) on prenatal and early postnatal development of the central nervous system

The in vivo actions of insulin‐like growth factor‐I (IGF‐I) on prenatal and early postnatal brain development were investigated in transgenic (Tg) mice that overexpress IGF‐I prenatally under the control of regulatory sequences from the nestin gene. Tg mice demonstrated increases in brain weight of 6% by embryonic day (E) 18 and 27% by postnatal day (P) 12. In Tg embryos at E16, the volume of the cortical plate was significantly increased by 52% and total cell number was increased by 54%. S‐phase labeling with 5‐bromo‐2′‐deoxyuridine revealed a 13–15% increase in the proportion of labeled neuroepithelial cells in Tg embryos at E14. In Tg mice at P12, significant increases in regional tissue volumes were detected in the cerebral cortex (29%), subcortical white matter (52%), caudate‐putamen (37%), hippocampus (49%), dentate gyrus (71%) and habenular complex (48%). Tg mice exhibited significant increases in the total number of neurons in the cerebral cortex (27%), caudate‐putamen (27%), dentate gyrus (69%), medial habenular nucleus (61%) and lateral habenular nucleus (36%). In the cerebral cortex and subcortical white matter of Tg mice, the total numbers of glial cells were significantly increased by 37% and 42%, respectively. The numerical density of apoptotic cells in the cerebral cortex, labeled by antibodies against active caspase‐3, was reduced by 26% in Tg mice at P7. Our results demonstrate that IGF‐I can both promote proliferation of neural cells in the embryonic central nervous system in vivo and inhibit their apoptosis during postnatal life.

Insulin-Like Growth Factor-I Accelerates the Cell Cycle by Decreasing G1 Phase Length and Increases Cell Cycle Reentry in the Embryonic Cerebral Cortex

Neurogenesis in the developing cerebral cortex of mice occurs in the dorsal telencephalon between embryonic day 11 (E11) and E17, during which time the majority of cortical projection neurons and some glia are produced from proliferating neuroepithelial cells in the ventricular zone. The number of cells produced by this process is governed by several factors, including cell cycle kinetics and the proportion of daughter cells exiting the cell cycle after a given round of cell division. The in vivo effects of IGF-I on cell cycle kinetics were investigated in nestin/IGF-I transgenic (Tg) embryos, in which IGF-I is overexpressed in the cerebral cortex and dorsal telencephalon. These Tg mice have been shown to exhibit increased cell number in the cortical plate by E16 and increased numbers of neurons and glia in the cerebral cortex during postnatal development. Cumulative S phase labeling with 5-bromo-2′-deoxyuridine revealed a decrease in total cell cycle length (TC) in Tg embryos on E14. This decrease in TC was found to result entirely from a reduction in the length of the G1 phase of the cell cycle from 10.66 to 8.81 hr, with no significant changes in the lengths of the S, G2, and M phases. Additionally, the proportion of daughter cells reentering the cell cycle was significantly increased by 15% in Tg embryos on E14-E15 compared with littermate controls. These data demonstrate that IGF-I regulates progenitor cell division in the ventricular zone by reducing G1 phase length and decreasing TC but increases cell cycle reentry.

Increased insulin-like growth factor-I (IGF-I) expression during early postnatal development differentially increases neuron number and growth in medullary nuclei of the mouse.

Morphometric analyses of the medulla were performed in transgenic mice that overexpress insulin-like growth factor-I (IGF-I) postnatally and in non-transgenic littermates. The total volume of the medulla was increased in transgenic mice at all postnatal ages studied: 14 days (18%), 21 days (23%), 28 days (23%), and 35 days (27%). By 35 days of age, the volumes of individual medullary nuclei were also increased: nucleus of the tractus solitarius (NTS, 59%), dorsal motor nucleus of the vagus (DMV, 84%), hypoglossal nucleus (HN, 29%) and facial nucleus (FN, 21%). Neuron number in transgenic mice was significantly greater in NTS (50%) and DMV (53%), but not in the HN or the FN. Motor neurons in DMV, HN and FN of transgenic mice exhibited increases in mean profile areas of the soma and decreased numerical densities, suggesting increases in neuritic outgrowth. These results point to IGF-I actions in promoting neuron survival and growth, and suggest that IGF-I has differential effects on distinct neuron populations, possibly depending upon its time of expression.

Toward Understanding the Molecular Pathology of Huntington's Disease

Huntingtons Disease (HD) is caused by expansion of a CAG trinucleotide beyond 35 repeats within the coding region of a novel gene. Recently, new insights into the relationship between CAG expansion in the HD gene and pathological mechanisms have emerged. Survival analysis of a large cohort of affected and at‐risk individuals with CAG sizes between 39 and 50 repeats have yielded probability curves of developing HD symptoms and dying of HD by a certain age. Animals transgenic for the first exon of huntingtin with large CAG repeats lengths have been reported to have a complex neurological phenotype that bears interesting similarities and differences to HD. The repertoire of huntingtin‐inter‐acting proteins continues to expand with the identification of HIP1, a protein whose yeast homologues have known functions in regulating events associated with the cytoskeleton. The ability of huntingtin to interact with two of its four known protein partners appears to be influenced by CAG length. Caspase 3 (apopain), a key cysteine protease known to play a seminal role in neural apoptosis, has also been demonstrated to specifically cleave huntingtin in a CAG length‐dependent manner. Many of these features are combined in a model suggesting mechanisms by whi h the pathogenesis of HD may be initiated. The development of appropriate in vitro and animal models for HD will allow the validity of these models to be tested.

Neuronal degeneration in the basal ganglia and loss of pallido-subthalamic synapses in mice with targeted disruption of the Huntington's disease gene

Huntingtons disease (HD) is a progressive neurodegenerative disorder associated with CAG repeat expansion within a novel gene (IT15). We have previously created a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homologue of the HD gene. Homozygotes for the Hdhex5 mutation exhibit embryolethality before embryonic day 8.5, while heterozygotes survive to adulthood and display increased motor activity and cognitive deficits. Detailed morphometric and stereological analyses of the basal ganglia in adult heterozygous mice were performed by light and electron microscopy. Morphometric analyses demonstrated a significant loss of neurons from both the globus pallidus (29%) and the subthalamic nucleus (51%), with a normal complement of neurons in the caudate-putamen and substantia nigra. The ultrastructural appearance of sporadic degenerating neurons in these regions indicated apoptosis. The highest frequency of apoptotic neurons was observed in the globus pallidus and subthalamic nucleus. Stereological analyses in the subthalamic nucleus revealed a significant decrease in the numerical density of symmetric synapses (43%), suggesting a relatively selective loss of inhibitory pallido-subthalamic afferents. Immunohistochemistry using antibodies against enkephalin and substance-P was unremarkable in heterozygotes, indicating a normal complement of enkephalin-immunoreactive striatopallidal afferents and substance-P-immunoreactive striatopeduncular and striatonigral afferents in these animals. These findings show that loss of an intact huntingtin protein is associated with significant morphological alterations in the basal ganglia of adult mice, indicating an important role for this protein during development of the central nervous system.

Type 1 insulin-like growth factor receptor signaling is essential for the development of the hippocampal formation and dentate gyrus.

Type 1 insulin‐like growth factor receptor (IGF1R) signaling in neuronal development was studied in mutant mice with blunted igf1r gene expression in nestin‐expressing neuronal precursors. At birth [postnatal (P) day 0] brain weights were reduced to 37% and 56% of controls in mice homozygous (nes‐igf1r−/−) and heterozygous (nes‐igf1r−/Wt) for the null mutation, respectively, and this brain growth retardation persisted postnatally. Stereological analysis demonstrated that the volumes of the hippocampal formation, CA fields 1–3, dentate gyrus (DG), and DG granule cell layer (GCL) were decreased by 44–54% at P0 and further by 65–69% at P90 in nes‐igf1r−/Wt mice. In nes‐igf1r−/− mice, volumes were 29–31% of controls at P0 and, in the two mice that survived to P90, 6–19% of controls, although the hilus could not be identified. Neuron density did not differ among the mice at any age studied; therefore, decreased volumes were due to reduced cell number. In postnatal nes‐igf1r−/Wt mice, the percentage of apoptotic cells, as judged by activated caspase‐3 immunostaining, was increased by 3.5–5.3‐fold. The total number of proliferating DG progenitors (labeled by BrdU incorporation and Ki67 staining) was reduced by ∼50%, but the percentage of these cells was similar to the percentages in littermate controls. These findings suggest that 1) the postnatal reduction in DG size is due predominantly to cell death, pointing to the importance of the IGF1R in regulating postnatal apoptosis, 2) surviving DG progenitors remain capable of proliferation despite reduced IGF1R expression, and 3) IGF1R signaling is necessary for normal embryonic brain development.

Methylmercury-induced movement and postural disorders in developing rat: regional analysis of brain catecholamines and indoleamines

Subcutaneous administration of methylmercury (MeHg) to rats during early postnatal development resulted in movement and postural disorders by day 22-24. Tissue concentrations of norepinephrine (NE), serotonin (5-HT), dopamine (DA) and selected metabolites were measured in the cerebral cortex, spinal cord and caudate-putamen at the onset of neurological impairment and at two subclinical stages of toxicity. In the cerebral cortex there was a significant increase in tissue concentrations of 5-HT (54-81%) and 5-hydroxyindoleacetic acid (HIAA, 133-178%) at the onset of neurological impairment. Similar increases were detected in the spinal cord for 5-HT (19-43%) and HIAA (98-123%) as well as an increase in the concentration of NE (42-51%). In the caudate-putamen there were significant increases in the concentrations of NE (98-116%), HIAA (108-124%) and DA (28-29%) with a significant decrease in the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC, 20-27%); however, tissue levels of homovanillic acid (HVA) did not change significantly. Many of these changes were detected at subclinical stages of MeHg toxicity. The ratio of HIAA/5-HT, which is frequently used as an estimate of turnover for 5-HT, was significantly increased in all 3 tissues at the onset of neurological impairment (38-94%) and at one subclinical stage (47-114%). The ratio of (DOPAC + HVA)/DA was significantly decreased in caudate-putamen at all 3 stages of toxicity (18-40%). These changes indicate altered metabolism in aromatic amine systems in the developing central nervous system during the pathogenesis of MeHg-induced movement and postural disorder.

Acetylcholine and aromatic amine systems in postmortem brain of an infant with Down's syndrome

Adult cases of Downs syndrome often show histologic and biochemical changes comparable to those seen in severe Alzheimers disease, but it is not known whether these are congenital or acquired defects. Cell counts of the basal forebrain cholinergic system innervating the cortex in a 5.5-month-old male infant with Downs indicated about 50% of the number of cells expected at birth but this is in the range of cell numbers found in healthy middle-aged normals. The noradrenergic system of the locus ceruleus has the expected complement of cells for normal newborns. The activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), glutamate decarboxylase, and tyrosine hydroxylase in a number of brain regions are reported for this infant, two cases of crib death, and a group of normal adults. The regional distributions of the enzymes in the infants were generally as expected from adult control data except for that of ChAT in one of the two cases of crib death; the AChE activities seemed extraordinarily high, especially in the case of Downs syndrome. Data on the concentrations of the catecholamines, serotonin, and their metabolites are also given but, like the enzyme data, are difficult to interpret in the absence of controls for the neonatal period.