Overt Neuronal Loss Research Articles

Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host

How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

Adenosine A2A receptor blockade prevents synaptotoxicity and memory dysfunction caused by beta-amyloid peptides via p38 mitogen-activated protein kinase pathway.

Alzheimer's disease (AD) is characterized by memory impairment, neurochemically by accumulation of beta-amyloid peptide (namely Abeta(1-42)) and morphologically by an initial loss of nerve terminals. Caffeine consumption prevents memory dysfunction in different models, which is mimicked by antagonists of adenosine A(2A) receptors (A(2A)Rs), which are located in synapses. Thus, we now tested whether A(2A)R blockade prevents the early Abeta(1-42)-induced synaptotoxicity and memory dysfunction and what are the underlying signaling pathways. The intracerebral administration of soluble Abeta(1-42) (2 nmol) in rats or mice caused, 2 weeks later, memory impairment (decreased performance in the Y-maze and object recognition tests) and a loss of nerve terminal markers (synaptophysin, SNAP-25) without overt neuronal loss, astrogliosis, or microgliosis. These were prevented by pharmacological blockade [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261); 0.05 mg . kg(-1) . d(-1), i.p.; for 15 d] in rats, and genetic inactivation of A(2A)Rs in mice. Moreover, these were synaptic events since purified nerve terminals acutely exposed to Abeta(1-42) (500 nm) displayed mitochondrial dysfunction, which was prevented by A(2A)R blockade. SCH58261 (50 nm) also prevented the initial synaptotoxicity (loss of MAP-2, synaptophysin, and SNAP-25 immunoreactivity) and subsequent loss of viability of cultured hippocampal neurons exposed to Abeta(1-42) (500 nm). This A(2A)R-mediated control of neurotoxicity involved the control of Abeta(1-42)-induced p38 phosphorylation and was independent from cAMP/PKA (protein kinase A) pathway. Together, these results show that A(2A)Rs play a crucial role in the development of Abeta-induced synaptotoxicity leading to memory dysfunction through a p38 MAPK (mitogen-activated protein kinase)-dependent pathway and provide a molecular basis for the benefits of caffeine consumption in AD.

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ 42) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ 42 were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.

Intraventricular Enzyme Replacement Improves Disease Phenotypes in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive neurodegenerative disease caused by mutations in CLN2, which encodes the lysosomal protease tripeptidyl peptidase 1 (TPP1). LINCL is characterized clinically by progressive motor and cognitive decline, and premature death. Enzyme-replacement therapy (ERT) is currently available for lysosomal storage diseases affecting peripheral tissues, but has not been used in patients with central nervous system (CNS) involvement. Enzyme delivery through the cerebrospinal fluid is a potential alternative route to the CNS, but has not been studied for LINCL. In this study, we identified relevant neuropathological and behavioral hallmarks of disease in a mouse model of LINCL and correlated those findings with tissues from LINCL patients. Subsequently, we tested if intraventricular delivery of TPP1 to the LINCL mouse was efficacious. We found that infusion of recombinant human TPP1 through an intraventricular cannula led to enzyme distribution in several regions of the brain of treated mice. In vitro activity assays confirm increased TPP1 activity throughout the rostral-caudal extent of the brain. Importantly, treated mice showed attenuated neuropathology, and decreased resting tremor relative to vehicle-treated mice. This data demonstrates that intraventricular enzyme delivery to the CNS is feasible and may be of therapeutic value.

Early glial dysfunction in epilepsy

Early glial dysfunction in epilepsy

Early Axonopathy Preceding Neurofibrillary Tangles in Mutant Tau Transgenic Mice

Neurodegenerative diseases characterized by brain and spinal cord involvement often show widespread accumulations of tau aggregates. We have generated a transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). These mice developed age-dependent brain and hippocampal atrophy, central and peripheral axonopathy, progressive motor impairment with neurogenic muscle atrophy, and neurofibrillary tangles and had decreased survival. Axonal spheroids and axonal atrophy developed early before neurofibrillary tangles. Neurofibrillary inclusions developed in neurons at 3 months and were of two types, suggestive of a selective vulnerability of neurons to form different types of fibrillary aggregates. A first type of tau-positive neurofibrillary tangles, more abundant in the forebrain, were composed of ribbon-like 19-nm-wide filaments and twisted paired helical filaments. A second type of tau and neurofilament-positive neurofibrillary tangles, more abundant in the spinal cord and the brainstem, were composed of 10-nm-wide neurofilaments and straight 19-nm filaments. Unbiased stereological analysis indicated that total number of pyramidal neurons and density of neurons in the lumbar spinal cord were not reduced up to 12 months in Tg30tau mice. This Tg30tau model thus provides evidence that axonopathy precedes tangle formation and that both lesions can be dissociated from overt neuronal loss in selected brain areas but not from neuronal dysfunction.

MAPK-activated Protein Kinase 2 Deficiency in Microglia Inhibits Pro-inflammatory Mediator Release and Resultant Neurotoxicity: RELEVANCE TO NEUROINFLAMMATION IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER DISEASE

MAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon gamma-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2-/-) mice demonstrated significant inhibition in release of tumor necrosis factor alpha, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1alpha on stimulation with LPS + interferon gamma or amyloid-beta peptide (1-42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon gamma-stimulated or amyloid-beta peptide (1-42)-stimulated MK2-/- microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with beta-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor alpha, macrophage inflammatory protein 1alpha, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented.

Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice

Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of beta-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimer's disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between beta-amyloid and tau alterations may take place in vivo.

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.

Alterations in dopamine and benzodiazepine receptor binding precede overt neuronal pathology in mice modelling early Huntington disease pathogenesis

Huntington disease (HD) is an inherited, late onset, progressive neurodegenerative disorder. Primary degeneration appears to selectively occur in striatal medium spiny neurones but this is most likely preceded by a period of neuronal dysfunction. Altered levels of neurotransmitter receptors may disrupt neuronal function and contribute to a toxic environment within the brain. In the present study, a knock-in HD mouse modelling early stages of the disease was used to determine whether alterations in neurotransmitter receptor densities occurred before overt neuronal loss. Receptor autoradiography demonstrated reduced dopamine D 2 and increased benzodiazepine receptor binding in the striatum of HD animals compared to wild-type littermates. The density of benzodiazepine receptor binding was also increased in the cerebral cortex of the HD mice. Changes in opioid and dopamine D 1 receptor densities were more subtle and influenced by the genetic background of the mice. Our findings are consistent with the hypothesis that alterations in neurotransmitter receptor density precede cell loss and may be an active cellular response to the initial stages of HD pathogenesis.

Building a More Perfect Beast: APP Transgenic Mice with Neuronal Loss

While there have been a number of successful attempts to develop transgenic mice that express mutant amyloid precursor protein (APP) and develop amyloid deposits that have features similar to those in aging and Alzheimer’s disease (AD),1 until now there has been either no neuronal loss or less than convincing evidence in favor of neuronal loss. In this issue of The American Journal of Pathology, Schmitz and colleagues2 provide compelling evidence for age-dependent neuronal loss in the hippocampus of bigenic transgenic mice expressing a double mutation in APP (Swedish APP mutation APPK670N, M671L (APPsw) and APPV717I, under the mouse Thy1 promoter) and mutant presenilin-1 (PS-1 M146L under the pHMG promoter). The neuronal loss was ascertained by unbiased stereologic methods on Nissl-stained sections. The animals showed significant (30 to 35%) neuronal loss in the hippocampal pyramidal layer compared to controls, which was disproportionate to the degree of amyloid deposition. No neuronal loss was detected in the dentate fascia of the hippocampus, the only other region assessed for neuronal loss. While the regions analyzed are of interest, if the model is to have relevance to AD, it will be important in future studies to extend neuronal counts to limbic and association cortical regions, which are especially vulnerable to amyloid deposition in humans. The behavioral consequences, if any, of neuronal loss in this model are not described in this report or other descriptions of this model3 and are left to future work.

Neuronal injury and loss after traumatic brain injury: time course and regional variability

We have examined regional neuronal injury after traumatic brain injury using Fluoro-Jade, an acidic dye that exhibits a marked affinity for both the degenerating neuronal cell body and its processes and have determined the extent to which early injury corresponds to regional patterns of neuronal loss. Rats ( n=45) were subjected to lateral fluid percussion brain injury and euthanized at 3 h to 28 days post injury. Complementary Fluoro-Jade, silver impregnation methods and TUNEL were used to assess neuronal injury. Neuronal loss was evaluated in sections immunostained for NeuN, a neuronal specific nuclear protein. Overt neuronal cell loss was evident by 7 days post injury in the cortex, hippocampus and thalamus. Injured neurons were apparent in the ipsilateral cortex bordering the impact site, hippocampus (CA1 and dentate), thalamus, and vermis of the cerebellum as early as 3 h post injury. Degenerating neurons were maximal by 1 and 3 days in the cortex and hippocampus, by 3 and 7 days in the cerebellum, and by 7 days in the thalamus. The regional distribution of Fluoro-Jade-labeled neurons corresponded to a similar pattern of silver and TUNEL staining. Together, these findings demonstrate a regionally specific temporal pattern of neuronal injury that results in overt neuronal cell loss within both cortical and subcortical regions.

Learning deficits in mice with persistent Borna disease virus infection of the CNS associated with elevated chemokine expression

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes a CD8 + T cell-mediated neurological disease in certain mouse strains. We established asymptomatic persistent central nervous system (CNS) infections in mutant C57BL/10J mice that lack functional CD8 + T cells. When analyzed at adult age for spatial learning abilities in a water maze, BDV-infected mice showed slightly impaired escape performance while their exploratory behavior in an openfield test was indistinguishable from uninfected control mice. Histological and molecular biological analysis revealed extensive viral spread throughout the CNS of infected animals. Most neurons of the hippocampus contained viral antigen, but there was no overt loss of neurons from this structure. We found almost unchanged levels of the proinflammatory cytokines IL-1β and TNF-α, but clearly increased levels of the chemokines IP-10 and RANTES in brains of infected mice. Re-examination of water maze data revealed that only infected mice with IP-10 transcript levels above a certain threshold showed impaired performance, whereas the performance of infected mice with lower IP-10 levels was indistinguishable from uninfected controls. This suggests that BDV infection can disturb the function of the mammalian CNS without causing overt neuronal loss, and that the magnitude of virus-induced chemokine production in the CNS correlates with the degree of impairment.

NNOS Expression in Reactive Astrocytes Correlates with Increased Cell Death Related DNA Damage in the Hippocampus and Entorhinal Cortex in Alzheimer's Disease

The immunocytochemical distribution of the neuronal form of nitric oxide synthase (nNOS) was compared with neuropathological changes and with cell death related DNA damage (as revealed by in situ end labeling, ISEL) in the hippocampal formation and entorhinal cortex of 12 age-matched control subjects and 12 Alzheimer's disease (AD) patients. Unlike controls, numerous nNOS-positive reactive astrocytes were found in AD patients around β-amyloid plaques in CA1 and subiculum and at the places of clear and overt neuron loss, particularly in the entorhinal cortex layer II and CA4. This is the first evidence of nNOS-like immunoreactivity in reactive astrocytes in AD. In contrast to controls, in all but one AD subject, large numbers of ISEL-positive neuronal nuclei and microglial cells were found in the CA1 and CA4 regions and subiculum. Semiquantitative analysis showed that neuronal DNA fragmentation in AD match with the distribution of nNOS-expressing reactive astroglial cells in CA1 (r = 0.74, P < 0.01) and CA4 (r = 0.58, P < 0.05). A portion of the nNOS-positive CA2/CA3 pyramidal neurons was found to be spared even in the most affected hippocampi. A significant inverse correlation between nNOS expression and immunoreactivity to abnormally phosphorylated tau proteins (as revealed by AT8 monoclonal antibody) in perikarya of these CA2/3 neurons (r = −0.85, P < 0.01) suggests that nNOS expression may provide selective resistance to neuronal degeneration in AD. In conclusion, our results imply that an upregulated production of NO by reactive astrocytes may play a key role in the pathogenesis of AD.

Correction for Irizarry et al., Aβ Deposition Is Associated with Neuropil Changes, but not with Overt Neuronal Loss in the Human Amyloid Precursor Protein V717F (PDAPP) Transgenic Mouse

Correction: In the article “Aβ Deposition Is Associated with Neuropil Changes, but not with Overt Neuronal Loss in the Human Amyloid Precursor Protein V717F (PDAPP) Transgenic Mouse,” by Michael C. Irizarry, Ferdie Soriano, Megan McNamara, Keith J. Page, Dale Schenk, Dora Games, and Bradley T.

Aβ Deposition Is Associated with Neuropil Changes, but not with Overt Neuronal Loss in the Human Amyloid Precursor Protein V717F (PDAPP) Transgenic Mouse

The PDAPP transgenic mouse overexpresses human amyloid precursor protein V717F (PDAPP minigene) and develops age-related cerebral amyloid-beta protein (Abeta) deposits similar to senile plaques in Alzheimer's disease. We find age-related cortical and limbic Abeta deposition that begins at 8 months and progresses to cover 20-50% of the neuropil in cingulate cortex, entorhinal cortex, and hippocampus of 18-month-old heterozygotic animals. The regional patterns of transgene expression and amyloid deposition suggest that Abeta deposits occur at the terminals of overexpressing neurons. Amyloid deposition is associated with dystrophic neurites and extensive gliosis. However, stereological analysis shows that there is no overt neuronal loss in entorhinal cortex, CA1 hippocampal subfield, or cingulate cortex through 18 months of age. In addition, there is no apparent loss of mRNA encoding neuronal synaptic, cytoskeletal, or metabolic proteins. Thus, widespread Abeta deposition in 18-month-old heterozygotic mice produces neuritic alterations and gliosis without widespread neuronal death.

Increased vulnerability of the midly traumatized rat brain to cerebral ischemia: the use of controlled secondary ischemia as a research tool to identify common or different mechanisms contributing to mechanical and ischemic brain injury

Fasted Wistar rats were subjected to either a mild mechanical injury, 6 min of transient forebrain ischemia, or a mild mechanical injury followed 1 h later by 6 min of forebrain ischemia. EEG and evoked potentials were assessed intermittently and morphological analyses were performed after 7 das postinjury survival. In all groups complete qualitative recovery of electrical activity and general behavior was observed with 7-day survival. However, rats subjected to combined concussion and ischemia displayed EEG spike activity and a delayed return of EEG and evoked potentials during acute recovery not evident in other groups. No overt neuronal cells loss was seen in trauma alone and was minimal or absent in ischemia alone. However, extensive bilateral CA1 and subicular pyramidal cell loss was found in the septal and mid-dorsal hippocampi in the combined trauma and ischemia group. In contrast, no overt axonal injury was found in any group. We conclude that even mild mechanical injury can potentiate selective ischemic hippocampal neuronal necrosis in the absence of overt axonal injury. This potentiation also occurs in conjunction with more generalized electrophysiological disturbances such as EEG evidence of postischemic neuronal hyperactivity suggesting that mild concussion may also decrease the threshold for post-ischemic neuronal excitation. These results suggest the potential of this model for examining common or different injury mechanisms in mechanical and ischemic brain injury.