Er-Yun Chen

Entorhinal Cortex β-Amyloid Load in Individuals with Mild Cognitive Impairment

Abstract The deposition of β-amyloid within the entorhinal cortex (EC) may play a key role in the development of mild cognitive impairment (MCI) in the elderly. To examine the relationship of β-amyloid deposition to MCI, EC tissue immunostained for this protein was quantitated from a cohort of aged Catholic religious clergy with a clinical diagnosis of MCI and compared to those with no cognitive impairment (NCI) and Alzheimers disease (AD). β-amyloid staining was seen in 12 of the 20 NCI, in 10 of 12 MCI, and in all 12 AD cases within the EC. β-amyloid immunoreactivity displayed two patterns within the EC: (1) a crescent-shaped band within layers 3–4 or (2) bilaminar staining mainly within layers 2–3 and 5–6. Ten cases failed to display any detectable β-amyloid imunoreactivity. Despite the heterogeneity of β-amyloid loads within the clinical groups, decomposing an analysis of variance revealed a significant difference across groups in mean β-amyloid load within the EC based upon a linear trend analysis. Multiple comparisons testing revealed that NCI individuals had a significantly lower mean β-amyloid load (1.32) than AD individuals (4.55). The MCI individuals had a mean intermediate (2.60) load between NCI and AD, but not statistically distinguishable from the mean for either NCI or AD. Spearman rank correlation showed a trend for decreasing MMSE with increasing amyloid load that failed to reach statistical significance. Since many NCI cases displayed β-amyloid loads equal to or greater than that seen in some MCI and some AD cases, it is mostly likely that deposition of this protein is not the sole pathogenic event underlying cognitive impairment in the elderly.

Implants of Encapsulated Human CNTF-Producing Fibroblasts Prevent Behavioral Deficits and Striatal Degeneration in a Rodent Model of Huntington’s Disease

Delivery of neurotrophic molecules to the CNS has gained considerable attention as a potential treatment strategy for neurological disorders. In the present study, a DHFR-based expression vector containing the human ciliary neurotrophic factor (hCNTF) was transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting hCNTF (BHK-hCNTF) were transplanted unilaterally into the rat lateral ventricle. Twelve days later, the same animals received unilateral injections of quinolinic acid (QA; 225 nmol) into the ipsilateral striatum. After surgery, animals were behaviorally tested for apomorphine-induced rotation behavior and for skilled forelimb function using the staircase test. Rats receiving BHK-hCNTF cells rotated significantly less than animals receiving BHK-control cells. No behavioral effects of hCNTF were observed on the staircase test. Nissl-stained sections demonstrated that BHK-hCNTF cells significantly reduced the extent of striatal damage produced by QA. Quantitative analysis of striatal neurons further demonstrated that both choline acetyltransferase- and GAD-immunoreactive neurons were protected by BHK-hCNTF implants. In contrast, a similar loss of NADPH-diaphorase-positive cells was observed in the striatum of both implant groups. Analysis of retrieved capsules revealed numerous viable and mitotically active BHK cells that continued to secrete hCNTF. These results support the concepts that implants of polymer-encapsulated hCNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage and that this strategy may ultimately prove relevant for the treatment of Huntington’s disease.

Distribution of estrogen receptor alpha and beta immunoreactive profiles in the postnatal rat brain

The present study was conducted to identify the localization and possible contribution of the two estrogen receptor (ER) subtypes in the rat brain at postnatal (P) days 3, 7 and 14. Evaluation of the distribution of ERalpha and ERbeta immunoreactive (ir) nuclei did not reveal gender differences at the developmental point times examined. With the exception of the cerebral cortex, the pattern of staining for these ERs was unchanged across the postnatal ages examined. The distribution of ERalpha-ir nuclei was wider than ERbeta-ir during brain development. From P3, ERbeta and ERalpha-ir nuclei were found in different regions of the cerebral cortex, basal forebrain, amygdala, thalamus, hypothalamus, mesencephalon, pons, cerebellum and medulla oblongata. In addition, ERalpha-ir nuclei were exclusively detected in the hippocampal subfields, epithalamus and in several circumventricular organs. ERalpha and ERbeta dual immunofluorescence revealed positive nuclei in the medial part of the bed nucleus of the stria terminalis, periventricular preoptic nucleus and in caudal aspects of the ventrolateral part of the ventromedial hypothalamic nucleus. Although the functional significance of the dual expression of both ERs within the same nuclei remains unknown, it is possible that ERs play different roles in gene regulation within the same cell. The presence of ERs in diverse brain regions through early postnatal periods supports a potential role for estrogens in neural differentiation.

Estrogen receptor immunoreactivity within subregions of the rat forebrain: neuronal distribution and association with perikarya containing choline acetyltransferase.

Administration of the neuroactive steroid hormone estrogen has been shown to effect cholinergic basal forebrain neuronal function. Antibodies directed against the estrogen receptor alpha (ERalpha) revealed dark (type 1) and light (type 2) nuclear positive neurons within the islands of Calleja, endopiriform nucleus, lateral septum, subfields of the cholinergic basal forebrain, bed nucleus of the stria terminalis, striohypothalamic region, medial preoptic region, periventricular, ventromedial, arcuate and tuberal mammillary nuclei of the hypothalamus, reuniens and anterior medial thalamic nuclei, amygdaloid complex, piriform cortex and subfornical organ. In contrast, only a few scattered ERalpha labeled neurons were found in cortex and hippocampus. ERalpha stained cell bodies were not seen in the striatum. Counts of ERalpha labeled neurons in intact female rats revealed significantly more type 2 neurons within the basal forebrain subfields. Quantitation of ERalpha immunoreactive neurons revealed a significant decrease in the relative number of type 1 neurons within the medial septum (MS), horizontal limb of the diagonal band (HDB) and substantia innominata/nucleus basalis (SI/NB) following ovariectomy. Quantitation following choline acetyltransferease (ChAT) immunohistochemistry revealed a significant decrease in the number of ChAT positive neurons within the MS, HDB and SI/NB, but not VDB following ovariectomy. Following ovx, the percentage of double labeled cholinergic basal forebrain neurons also declined significantly within the MS, VDB, HDB and SI/NB. These observations suggest that estrogen effects a subpopulation of cholinergic basal forebrain neurons and may provide insight into the biologic actions of this steroid in Alzheimers disease.

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.

Transgene Expression, Bioactivity, and Safety of CERE-120 (AAV2-Neurturin) Following Delivery to the Monkey Striatum

Neurturin (NTN) is a neurotrophic factor for dopaminergic neurons that may be therapeutic for patients with Parkinsons disease (PD). As a crucial component in a series of nonclinical translational studies aimed at testing whether CERE-120 should advance into clinical trials in PD subjects, we characterized the expression, bioactivity and safety of CERE-120, an adeno-associated virus type-2 (AAV2) vector encoding NTN, following delivery to the striatum of nonhuman primates. Monkeys received bilateral injections of CERE-120 across a tenfold range of doses (6 x 10(10) to 6 x 10(11) vector genomes per animal) or formulation buffer (FB) control. We report here, for the first time, a dose-related: increase in NTN protein expression within the striatum and substantia nigra (SN) pars compacta of nonhuman primates; increase in nigrostriatal tyrosine hydroxylase (TH), (the rate-limited enzyme for dopamine); and activation of phosphorylated signal-regulated kinase (a common neurotrophic signaling event). Additionally, extensive toxicology testing revealed no adverse effects of CERE-120 on in-life measures, neurotoxicity (in any site throughout the brain) or systemic pathology (in any organ or tissue) across the tenfold range of doses. Collectively, these data provide substantial novel evidence for the potential utility of CERE-120 as a novel treatment for PD and support ongoing clinical trials testing CERE-120 in PD patients.

Pramipexole attenuates the dopaminergic cell loss induced by intraventricular 6-hydroxydopamine.

Summary. The D3 preferring dopamine agonist pramipexole has been shown to attenuate the cell loss induced by levodopa in vitro. Pramipexole was herein evaluated in the 6-hydroxydopamine lesion model to determine its in vivo effect. Rats were treated with pramipexole or saline before and after an intracerebroventricular 6-hydroxydopamine injection. In the preliminary study, 6-hydroxydopamine produced a 68% reduction in striatal dopamine and a 62% loss in tyrosine hydroxylase immunoreactive (THir) cell counts in the substantia nigra. Pramipexole treated animals exhibited a 29% and a 27% reduction in striatal dopamine and THir cell counts, respectively. THir cell counts and striatal dopamine were significantly correlated. In the stereological study, 6-hydroxydopamine reduced THir cell counts by 47% in saline treated animals and 26% in pramipexole treated animals. These data demonstrate that pramipexole attenuates the biochemical and THir cell changes normally produced by 6-hydroxydopamine consistent with its neuroprotective actions in vitro.

Intra-parenchymal injection of tumor necrosis factor-α and interleukin 1-β produces dopamine neuron loss in the rat

Summary.Inflammatory processes are thought to underlie the dopamine (DA) neuron loss seen in Parkinson’s disease (PD). However, it is not known if the inflammation precedes that loss, or is a consequence of it. We injected tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β) into the median forebrain bundle to determine if these pro-inflammatory cytokines could induce DA neuron loss in the substantia nigra (SN) by themselves. The magnitude of the DA cell loss as well as the decreases in striatal DA, were both dose and time to sacrifice dependent. Injecting both cytokines together produced greater cell losses and DA reductions than that seen when the cytokines were injected alone. The DA neuron loss seen was more pronounced in the lateral nigra and its ventral tier and similar to that seen when other toxins are injected. These data suggest that TNFα and IL-1β can induce DA neuron loss by themselves and could produce DA neuron loss independent of other inflammatory events.

Doublecortin-expressing cells persist in the associative cerebral cortex and amygdala in aged nonhuman primates

A novel population of cells that express typical immature neuronal markers including doublecortin (DCX+) has been recently identified throughout the adult cerebral cortex of relatively large mammals (guinea pig, rabbit, cat, monkey and human). These cells are more common in the associative relative to primary cortical areas and appear to develop into interneurons including type II nitrinergic neurons. Here we further describe these cells in the cerebral cortex and amygdala, in comparison with DCX+ cells in the hippocampal dentate gyrus, in three age groups of rhesus monkeys: young adult (12.3 ± 0.2 years, n = 3), mid-age (21.2 ± 1.9 years, n = 3) and aged (31.3 ± 1.8 years, n = 4). DCX+ cells with a heterogeneous morphology persisted in layers II/III primarily over the associative cortex and amygdala in all groups (including in two old animals with cerebral amyloid pathology), showing a parallel decline in cell density with age across regions. In contrast to the cortex and amygdala, DCX+ cells in the subgranular zone diminished in the mid-age and aged groups. DCX+ cortical cells might arrange as long tangential migratory chains in the mid-age and aged animals, with apparently distorted cell clusters seen in the aged group. Cortical DCX+ cells colocalized commonly with polysialylated neural cell adhesion molecule and partially with neuron-specific nuclear protein and γ-aminobutyric acid, suggesting a potential differentiation of these cells into interneuron phenotype. These data suggest a life-long role for immature interneuron-like cells in the associative cerebral cortex and amygdala in nonhuman primates.

Generation and transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice.

EGF-responsive neural stem cells isolated from murine striatum have the capacity to differentiate into both neurons and glia in vitro. Genetic modification of these cells is hindered by a number of problems such as gene stability and transfection efficiency. To circumvent these problems we generated transgenic mice in which the human GFAP promoter directs the expression of human NGF. Neural stem cells isolated from the forebrain of these transgenic animals proliferate and form clusters, which appear identical to stem cells generated from control animals. Upon differentiation in vitro, the transgenic stem cell-derived astrocytes express and secrete bioactive hNGF. Undifferentiated GFAP-hNGF or control stem cells were transplanted into the striatum of adult rats. One and 3 weeks after transplantation, hNGF was detected immunocytochemically in an halo around the transplant sites. In GFAP-hNGF-grafted animals, intrinsic striatal neurons proximal to the graft appear to have taken up hNGF secreted by the grafted cells. Ipsilateral to implants of GFAP-hNGF-secreting cells, choline acetyltransferase-immunoreactive neurons within the striatum were hypertrophied relative to the contralateral side or control-grafted animals. Further, GFAP-hNGF-grafted rats displayed a robust sprouting of p75 neurotrophin receptor-positive fibers emanating from the underlying basal forebrain. These studies indicate that EGF-responsive stem cells which secrete hNGF under the direction of the GFAP promoter display in vitro and in vivo properties similar to that seen following other methods of NGF delivery and this source of cells may provide an excellent avenue for delivery of neurotrophins such as NGF to the central nervous system.