Translocation of amyloid precursor protein C-terminal fragment(s) to the nucleus precedes neuronal death due to thiamine deficiency-induced mild impairment of oxidative metabolism.

Thiamine deficiency (TD) is a model of neurodegeneration induced by mild impairment of oxidative metabolism. TD produces time-dependent glial activation, inflammation, oxidative stress, altered metabolism of amyloid precursor protein (APP), exacerbation of plaque formation from APP, and finally, selective neuron death in specific brain regions. The sub-medial thalamic nucleus (SmTN) is the most sensitive region to TD. Alteration in APP metabolism and nuclear translocation of carboxy-terminal fragments (CTF) of APP has been implicated in neuron death in other models of neurodegeneration. These experiments tested whether TD causes translocation of CTF into the nucleus of neurons in the SmTN that are destined to die after 9 days of TD by examining overlapping immunoreactivity (IR) of antibody APP 369 with either Alz90, 6E10 or 4G8 epitopes in the nuclei of the neurons in the SmTN. TD caused the accumulation of the CTF of APP in nuclei of SmTN neurons within 3 days of TD. These changes did not occur in the cortex which is spared in TD. Western blot analysis of nuclear fractions revealed a significant (61%; P < 0.026) increase in CTF 12 levels in TD SmTN (2.08 +/- 0.56) compared to control SmTN (1.29 +/- 0.41). Although TD increased CTF 15 levels in TD SmTN (1.95 +/- 0.73) compared to control SmTN (0.62 +/- 0.52) by 214%; P < 0.665 and decreased the full-length holo-APP levels in TD SmTN (0.32 +/- 0.30) compared to control SmTN (0.47 +/- 0.18) by 34%; P < 0.753, the differences were statistically insignificant. TD did not alter CTF 15 or CTF 12 levels in cortex. These findings demonstrate that changes in APP metabolism occur in early stages of TD, and they may play an important role in TD-induced selective neuronal loss.

Human lupus autoantibodies against NMDA receptors mediate cognitive impairment.

Neuropsychiatric systemic lupus erythematosus, which often entails cognitive disturbances and memory loss, has become a major complication for lupus patients. Previously, we developed a murine model of neuropsychiatric lupus based on Abs that cross-react with dsDNA and the NMDA receptor (NMDAR). We showed that these murine Abs impair cognition when they access the CNS through a breach in the blood-brain barrier (BBB) triggered by lipopolysaccharide. Because studies show that lupus patients possess anti-NMDAR Abs in their serum and cerebrospinal fluid, we decided to investigate whether these human Abs contribute to cognitive dysfunction. Here, we show that serum with reactivity to DNA and NMDAR extracted from lupus patients elicited cognitive impairment in mice receiving the serum intravenously and given lipopolysaccharide to compromise the BBB integrity. Brain histopathology showed hippocampal neuron damage, and behavioral testing revealed hippocampus-dependent memory impairment. To determine whether anti-NMDAR Abs exist in the brains of systemic lupus erythematosus patients, we eluted IgG from a patient's brain. The IgG bound DNA and NMDAR and caused neuronal apoptosis when injected into mouse brains. We examined four more brains of patients with neuropsychiatric lupus and found that they displayed endogenous IgG colocalizing with anti-NMDAR Abs. Our results indicate that lupus patients have circulating anti-NMDAR Abs capable of causing neuronal damage and memory deficit, if they breach the BBB, and that the Abs exist within patients' brains. Which aspects of neuropsychiatric lupus may be mediated by anti-NMDAR Abs, how often, and in which patients are now important clinical questions.

Immunity and acquired alterations in cognition and emotion: lessons from SLE.

Classic immunologic teaching describes the brain as an immunologically privileged site. Studies of neuroimmunology have focused for many years almost exclusively on multiple sclerosis, a disease in which inflammatory cells actually infiltrate brain tissue, and the rodent model of this disease, experimental allergic encephalitis. Over the past decade, however, increasingly, brain-reactive antibodies have been demonstrated in the serum of patients with numerous neurological diseases. The contribution these antibodies make to neuronal dysfunction has, in general, not been determined. Here, we describe recent studies showing that serum antibodies to the N-methyl-D-aspartate receptor occur frequently in patients with systemic lupus erythematosus and can cause alterations in cognition and behavior following a breach in the blood-brain barrier.

Immunity and behavior: antibodies alter emotion.

Systemic lupus erythematosus is an autoimmune disease in which most patients express Abs that bind double-stranded DNA. Recent work has shown that a subset of lupus Abs can crossreact with the NR2A and NR2B subunits of the NMDA receptor. This receptor is expressed in neurons throughout the brain but is at highest density within cells of the hippocampus, amygdala, and hypothalamus. The neurons in the CNS are normally protected from brain-reactive Abs by the blood-brain barrier (BBB); however, a breach in the barrier's integrity exposes neurons to potentially pathogenic Abs. Previously, we have shown that mice that are immunized with a peptide mimetope of DNA produce lupus-like Abs that crossreact with DNA and the NMDA receptor. Moreover, after abrogation of the BBB by treatment with lipopolysaccharide, the immunized mice display hippocampal neuron damage with ensuing memory impairment. Given that rises in epinephrine can increase cerebral blood flow and can cause leaks in the BBB, we decided to investigate whether epinephrine could act as a permissive agent for Ab-mediated neurotoxicity. Here, we show that peptide-immunized mice, given epinephrine to open the BBB, lose neurons in the lateral amygdala and develop a behavioral disorder characterized by a deficient response to fear-conditioning paradigms. Thus, the agent used to open the BBB determines which brain region is made vulnerable to neurotoxic Abs, and Abs that penetrate brain tissue can cause changes not only in cognitive competence, but also in emotional behavior.

Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons.

Septin 5, a parkin substrate, is a vesicle- and membrane-associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinson's disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron-specific toxicity in SN-DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well-characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin-1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co-transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN-DAergic neurons. These mice acquired a progressive l-DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l-DOPA-responsive motor deficits can occur even in the presence of elevated DA.

CD40-CD40L interactions promote neuronal death in a model of neurodegeneration due to mild impairment of oxidative metabolism.

Abnormalities in oxidative processes, region-selective neuron loss, inflammation and diminished activity of thiamine-dependent enzymes characterize age-related neurodegenerative diseases. Thiamine deficiency (TD) models the selective neurodegeneration that accompanies mild impairment of oxidative metabolism. As in human neurodegenerative diseases, alterations in multiple cell types accompany the TD-induced neurodegeneration. The current studies demonstrate that CD40 and CD40 ligand (CD40L), two co-stimulatory immune molecules, are involved in TD-induced selective neuronal death. TD induced CD40 immunoreactivity in microglia and CD40L immunoreactivity in astrocytes. Both CD40-positive microglia and CD40L-positive astrocytes increased during the progressive TD-induced neuronal death. In early stages of TD, targeted deletion of CD40 diminished the number of CD40L-positive astrocytes and reduced neuronal death by 35%. The number of CD40L-positive astrocytes increased whenever the number of NeuN-positive neurons decreased. In early stages of TD, deletion of CD40L diminished CD40-positive microglia and reduced the neuronal death by 64%. In advanced phases of TD, neither CD40 nor CD40L deletion protected against neuronal death. The data show for the first time that TD induces expression of CD40 by the microglia and CD40L by astrocytes. The results indicate that CD40-CD40L interactions promote neuronal death in early stages of TD, but that at later phases the protective effects of the diminished CD40 or CD40L are over-ridden by other mechanisms.

CD40L deletion delays neuronal death in a model of neurodegeneration due to mild impairment of oxidative metabolism.

Inflammatory/immune processes are important in the pathogenesis of neurodegenerative diseases. Thiamine deficiency (TD) models the region selective neuronal loss in brain that accompanies mild impairment of oxidative metabolism. TD induces well-defined alterations in neurons, microglia, astrocytes, and endothelial cells. To test the role of inflammatory/immune mechanisms in TD-induced neurodegeneration, the temporal profile of neurodegeneration was compared to the activation of CD68-positive microglia and ICAM-1-positive endothelial cells during TD in wild type mice and in CD40L-/- mice. CD40L-/- delayed the onset of TD-induced neuronal death as well as the activation of microglia and endothelial cells. The current results suggest that CD40L-mediated immune and inflammatory responses have a role in TD-induced neuronal death.

Cognition and immunity; antibody impairs memory.

Patients with lupus (SLE) experience progressive cognitive loss without evidence of CNS vascular disease or inflammation. SLE patients produce anti-DNA antibodies that crossreact with NMDA receptors and are capable of mediating excitotoxic death. We now show that mice induced by antigen to express these antibodies have no neuronal damage until breakdown of the blood-brain barrier occurs. Following administration of lipopolysaccharide (LPS) to immunized mice, antibodies gain access to the brain. They bind preferentially to hippocampal neurons and cause neuronal death with resulting cognitive dysfunction and altered hippocampal metabolism on magnetic resonance spectroscopy. Memantine, an NMDA receptor antagonist, given prior to LPS administration, prevents neuronal damage. Thus, systemic immune responses can cause cognitive impairment in the absence of an inflammatory cascade, implicating the immune system in yet another arena of human pathobiology. Furthermore, NMDA receptor antagonists prevent antibody-mediated damage and may constitute a new approach to therapy in SLE.

Cross-linking cellular prion protein triggers neuronal apoptosis in vivo.

Neuronal death is a prominent, but poorly understood, pathological hallmark of prion disease. Notably, in the absence of the cellular prion protein (PrPC), the disease-associated isoform, PrPSc, appears not to be intrinsically neurotoxic, suggesting that PrPC itself may participate directly in the prion neurodegenerative cascade. Here, cross-linking PrPC in vivo with specific monoclonal antibodies was found to trigger rapid and extensive apoptosis in hippocampal and cerebellar neurons. These findings suggest that PrPC functions in the control of neuronal survival and provides a model to explore whether cross-linking of PrPC by oligomeric PrPSc can promote neuronal loss during prion infection.

Microglial phagocytosis of dopamine neurons at early phases of apoptosis.

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.

Reversal of thiamine deficiency-induced neurodegeneration.

Neurodegenerative diseases are characterized by abnormalities in oxidative processes, region-selective neuron loss, and diminished thiamine-dependent enzymes. Thiamine deficiency (TD) diminishes thiamine dependent enzymes, alters mitochondrial function, impairs oxidative metabolism, and causes selective neuronal death. In mice, the time course of TD-induced changes in neurons and microglia were determined in the brain region most sensitive to TD. Significant neuron loss (29%) occurred after 8 or 9 days of TD (TD8-9) and increased to 90% neuron loss by TD10-11. The number of microglia increased 16% by TD8 and by nearly 400% on TD11. Hemeoxygenase-1 (HO-1)-positive microglia were not detectable at TD8, yet increased dramatically coincident with neuron loss. To test the duration of TD critical for irrevocable changes, mice received thiamine after various durations of TD. Thiamine administration on TD8 blocked further neuronal loss and induction of HO-1-positive microglia, whereas other microglial changes persisted. Thiamine only partially reversed effects on TD9, and was ineffective on TD10-11. These studies indicate that irreversible steps leading to neuronal death and induction of HO-1-positive microglia occur on TD9. The results indicate that TD induces alterations in neurons. endothelial cells, and microglia contemporaneously. This model provides a unique paradigm for elucidating the molecular mechanisms involved in neuronal commitment to neuronal death cascades and contributory microglial activity.

Oxidative stress regulated genes in nigral dopaminergic neuronal cells: correlation with the known pathology in Parkinson's disease.

Oxidative stress (OS) is a primary pathogenic mechanism of nigral dopaminergic (DA) cell death in Parkinson's disease (PD). Oxidative damage, Lewy body formation and decreased mitochondrial complex I activity are the consistent pathological findings in PD. In nigral DA neurons, however, it is unknown whether any gene expressional changes induced by OS contribute to the typical PD pathology. Here, using microarray analysis, we identified several groups of genes in the nigral DA cell line, SN4741 [J. Neurosci. 19 (1999) 10; J. Neurochem. 76 (2001) 1010], that were regulated by OS. Approximately 36 significantly regulated genes that encode functional molecules of nuclear subunits of mitochondrial complex I, exocytosis and membrane trafficking proteins, markers for OS and oxidoreductases, regulatory molecules of apoptosis and unidentified EST clones were further analysed. OS modulated the expression of specific genes, of which physiological dysfunctions have been implicated in PD. For instance, the expression of the nuclear-encoded subunits of mitochondrial complex I, B8 and B17, were significantly down-regulated by OS, possibly contributing to selective defect in mitochondrial complex I activity in PD. Furthermore, syntaxin 8 and heme oxygenase-1 (HO-1) are most dramatically up-regulated by OS in DA cells. Syntaxin 8 is a SNARE protein, regulating lipid vesicle docking and fusion as well as early endosome membrane recycling. Lipid membranes are significantly oxidative-damaged in PD. HO-1 is an important cytoplasmic constituent of Lewy bodies, a pathological hallmark of idiopathic PD. Thus, our findings provide novel molecular probes that may be useful in unraveling the molecular mechanism(s) of OS-induced pathogenesis in PD. Further functional characterization of the affected genes including ESTs can help elucidate the underlying molecular pathology as well as develop biomarkers for monitoring degenerating DA neurons in PD.

Age-related microglial activation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in C57BL/6 mice.

Microglial activation was investigated in the brains of young (3 months old) and older (9-12 months old) mice following administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tyrosine hydroxylase (TH)-positive neuronal loss differed significantly between young and older mice. Importantly, the two groups clearly demonstrated a distinct microglial activation pattern. In young mice which showed TH neuronal loss at 1 day (33.4%), 3 days (45.1%), 7 days (47.1%) and 14 days (46.9%), microglial activation was first observed at 1 day, with lesser activation at 3 days and none shown later than 7 days. In contrast, in older mice which showed TH neuronal loss at 1 day (49.6%), 3 days (56.1%), 7 days (71.7%) and 14 days (72.1%), microglial activation occurred at 1 day, further intensified at 3-7 days, and was largely abated by 14 days. The double immunohistochemistry further demonstrated that the activated microglia surrounded dopaminergic neurons in older mice at 7 days, which was sharply in contrast to the young mice which were devoid of massive microglial activation in the SN later than 3 days after MPTP treatment. The present study suggests that age-related microglial activation in the SN may be relevant to the higher susceptibility to MPTP neurotoxicity in older mice.

17 beta-estradiol treatment retards excitotoxic delayed degeneration in substantia nigra reticulata neurons.

Estrogen treatment offers neuro-protection in animal experiments in which excitotoxic mechanisms destroy neurons. In a model of delayed neuronal degeneration that depends on excitotoxicity, we tested whether females had an altered susceptibility, and whether physiologic doses of estrogen administered after the brain insult would protect susceptible neurons. Females were ovariectomized, exposed to striatal-pallidal ibotenic acid injury that caused delayed degeneration of substantia nigra neurons, and treated with 17 beta -estradiol (30 microg, subcutaneously every other day, beginning 2 days after the striatal injury) or vehicle. At 6 and 8 days post lesion, the 17beta-estradiol treatment group maintained over 87 and 70% of control nigral neuron number, respectively. Physiologic levels of estrogen delivered days after the excitotoxic stress completely protected neurons in the substantia nigra reticulata 6 days post lesion and slowed degeneration 8 days post lesion.

Amyloid precursor protein gene disruption attenuates degeneration of substantia nigra compacta neurons following axotomy.

Our past work has shown that the C-terminal fragment of amyloid precursor protein (APP) translocated to the nucleus in neurons destined for delayed excitotoxic degeneration. To test whether nuclear APP fragments also play a role in the progressive loss of dopaminergic (DA) substantia nigra compacta (SNc) neurons, we performed unilateral medial forebrain bundle (MFB) transection on APP wild type (WT) and on mice with disruption of the APP gene (KO). In WT mice immunoreactivity for APP C-terminal, beta-amyloid and Alz90 epitopes appeared in the nuclei of axotomized DA neurons at 3 days post-lesion (dpl), persisted at 7 dpl and was absent in 14 dpl mice. APP N-terminal immunoreactivity was restricted to the cytosol at all time points, precluding the possibility of full length APP in the nucleus. Nuclear localization of APP epitopes was absent in neurons of the contralateral SNc or in neurons of the ipsilateral ventral tegmental area and SN reticulata. The presence of APP C-terminal and Alz90 domains was confirmed by Western blotting performed on the nuclear fraction of the SN ipsilateral to the axotomy. Quantitative morphometric analysis revealed that WT mice demonstrated earlier and more profound loss of tyrosine hydroxylase+SNc neurons than did KO mice. These data showed that a novel nuclear C-terminal fragment appeared coincident with SNc neuron degeneration, and that APP deficiency correlated with significant neuroprotection in vivo.

Transient appearance of amyloid precursor protein plaques in the brain of thymectomized rats after human leptomeningeal cell grafts.

Cells cultured from Alzheimer disease leptomeninges or skin were grafted into the cortex of adult thymectomized rats. At 3 days post-implant, plaque-like aggregates were found in the cortex, corpus callosum, septum and caudate nucleus. These structures were immunopositive for human amyloid precursor protein (APP), human amyloid beta peptide (Abeta), cathepsin D, apolipoprotein E and ubiquitin. Aberrant tau+ neurites, reactive astrocytes and microglia were associated with many aggregates. Although birefringent amyloid occupied the central area of most aggregates, these structures had disappeared by l month post-implant. Abeta and APP produced by grafted non-neural human cells can penetrate rat brain and form plaque-like structures, which can be effectively cleared by the rat.

APP knockout attenuates microglial activation and enhances neuron survival in substantia nigra compacta after axotomy.

Focal microglial activation and progressive dopaminergic neurodegeneration in substantia nigra compacta (SNc) have characterized Parkinson's disease (PD). We have hypothesized that the microglial response may be provoked by molecular signals from chronically stressed SNc neurons. To test whether amyloid precursor protein (APP) could serve as such a signal, we evaluated microglial activation in SN after unilateral transection of the medial forebrain bundle (MFB) in mice either wild-type (WT) or null (KO) for APP. WT and KO mice displayed comparable microglial response at the MFB transection site. In WT mice microglial activation was first apparent in the ipsilateral SN at 3 days postlesion (dpl), marked by morphological change and increased isolectin immunoreactivity. The microglial response intensified at 7 dpl and persisted in the medial nigra through 14 dpl. In contrast, in KO mice activated microglia appeared predominantly at 7 dpl, with little activation at 3 dpl and none at 14 dpl. Neuron number in affected WT SNc at 14 dpl was significantly reduced compared with loss in affected KO SNc. The delayed and limited local microglial activation and increased neuron survival in response to distal axotomy of SNc neurons in APP KO mice are consistent with the important role APP in neuronal stress responses in vivo.

Marked dopaminergic cell loss subsequent to developmental, intranigral expression of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) shows potent neuroprotective as well as neurorestorative actions on the adult neurons impacted in animal models of Parkinson's disease (PD). Long-term pharmaco-physiological effects of GDNF on developing dopaminergic (DA) neurons have not yet been explored because of technical difficulties in producing prolonged cell type-specific delivery of this neurotrophic factor in mammalian embryonic brain. The current studies used our previously characterized 9.0-kb tyrosine hydroxylase promoter to produce transgenic mice with neuronal cell type-specific expression of GDNF in substantia nigra pars compacta (SNc) and locus coeruleus (LC). These mice were used to test the parsimonious hypothesis that increased developmental expression of GDNF in SNc and LC would significantly enhance the number of postmitotic adult neurons. To our surprise, adult transgenic mice carrying the TH9.0kb-GDNF hybrid gene showed dramatic reductions in both the numbers and the volumes of SNc-DA and LC-noradrenergic (NA) neurons by quantitative morphometric analysis. The decrease in the number of DA neurons was apparent as early as postnatal day 2, the period before the major naturally occurring apoptotic cell death in midbrain. Aged transgenic mice exhibited no further significant deficits in motor behaviors. These data suggest that continuous, early developmental GDNF expression exerts physiological effects on newly differentiated, immature dopamine neurons that differ from those observed on more mature and adult DA neurons. Further elucidation of the mechanisms underlying differential GDNF actions will greatly improve the pharmacological efficacy of GDNF in fetal neural transplantation as well as adult neuronal gene therapy in PD patients.