The Tau/A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity.

We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate.

Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer's disease.

Dendritic structure critically determines the electrical properties of neurons and, thereby, defines the fundamental process of input-to-output conversion. The diversity of dendritic architectures enables neurons to fulfill their specialized circuit functions during cognitive processes. It is known that this dendritic integrity is impaired in patients with Alzheimer's disease and in relevant mouse models. It is unknown, however, whether this structural degeneration translates into aberrant neuronal function. Here we use in vivo whole-cell patch-clamp recordings, high-resolution STED imaging, and computational modeling of CA1 pyramidal neurons in a mouse model of Alzheimer's disease to show that structural degeneration and neuronal hyperexcitability are crucially linked. Our results demonstrate that a structure-dependent amplification of synaptic input to action potential output conversion might constitute a novel cellular pathomechanism underlying network dysfunction with potential relevance for other neurodegenerative diseases with abnormal changes of dendritic morphology.

How structure shapes (dys)function: a perspective to understanding brain region-specific degeneration in prion disease.

Structure is a key determinant of function, with the nervous system being no exception. For example, in the nervous system the physiological properties of different synapses may be understood by comparing their structures. However, it is not clear whether specific structural properties of some neurons might play a role in driving their selective removal during chronic neurodegeneration or whether the structural properties might underpin why particular types of synapses or other neuronal compartments are more susceptible to degeneration (i.e., become dysfunctional) in certain brain regions than in others. Our recent study of the ultrastructure of the hippocampus and the cerebellum revealed that early synaptic loss is not a ubiquitous event in a brain undergoing chronic neurodegeneration. The prominent structural differences in proximity of the synaptic environment that are brought about by a degree of synaptic ensheathment by glial cells may help explain why Purkinje cell synapses remain intact, while pyramidal cell synapses progressively degenerate. The intrinsic structural organization of the hippocampal neuropil could contribute to the susceptibility of synapses to extracellular protein misfolding by a relatively higher degree of synaptic exposure to the extracellular environment. We suggest that neuronal structure may determine more than function; it might also predict dysfunction.

Fibronectin aggregation in multiple sclerosis lesions impairs remyelination.

Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.

Brain region specific pre-synaptic and post-synaptic degeneration are early components of neuropathology in prion disease.

Synaptic abnormalities, one of the key features of prion disease pathogenesis, gives rise to functional deficits and contributes to the devastating clinical outcome. The synaptic compartment is the first to succumb in several neurodegenerative diseases linked with protein misfolding but the mechanisms underpinning this are poorly defined. In our current study we document that a focal intrahippocampal injection of the mouse-adapted 22L scrapie strain produces a complex, region-specific pathology in the brain. Our findings reveal that early synaptic changes in the stratum radiatum of the hippocampus, identical to those observed with the ME7 strain, occur when 22L strain is introduced into the hippocampus. The pathology was defined by degenerating Type I pre-synaptic elements progressively enveloped by the post-synaptic density of the dendritic spine. In contrast, the pathology in the cerebellum suggested that dendritic disintegration rather than pre-synaptic abnormalities dominate the early degenerative changes associated with the Purkinje cells. Indeed, both of the major synaptic inputs into the cerebellum, which arise from the parallel and climbing fibers, remained intact even at late stage disease. Immunolabeling with pathway selective antibodies reinforced these findings. These observations demonstrate that neuronal vulnerability to pathological protein misfolding is strongly dependent on the structure and function of the target neurons.

Microglia and synapse: interactions in health and neurodegeneration.

A series of discoveries spanning for the last few years has challenged our view of microglial function, the main form of immune defense in the brain. The surveillance of neuronal circuits executed by each microglial cell overseeing its territory occurs in the form of regular, dynamic interactions. Microglial contacts with individual neuronal compartments, such as dendritic spines and axonal terminals, ensure that redundant or dysfunctional elements are recognized and eliminated from the brain. Microglia take on a new shape that is large and amoeboid when a threat to brain integrity is detected. In this defensive form, they migrate to the endangered sites, where they help to minimize the extent of the brain insult. However, in neurodegenerative diseases that are associated with misfolding and aggregation of synaptic proteins, these vital defensive functions appear to be compromised. Many microglial functions, such as phagocytosis, might be overwhelmed during exposure to the abnormal levels of misfolded proteins in their proximity. This might prevent them from attending to their normal duties, such as the stripping of degenerating synaptic terminals, before neuronal function is irreparably impaired. In these conditions microglia become chronically activated and appear to take on new, destructive roles by direct or indirect inflammatory attack.

The role of activity in synaptic degeneration in a protein misfolding disease, prion disease.

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration.

Morphological and functional abnormalities in mitochondria associated with synaptic degeneration in prion disease.

Synaptic and dendritic pathology is a well-documented component of prion disease. In common with other neurodegenerative diseases that contain an element of protein misfolding, little is known about the underlying mechanisms of synaptic degeneration. In particular, in prion disease the relationship between synaptic malfunction, degeneration, and mitochondria has been neglected. We investigated a wide range of mitochondrial parameters, including changes in mitochondrial density, inner membrane ultrastructure, functional properties and nature of mitochondrial DNA from hippocampal tissue of mice with prion disease, which have ongoing synaptic pathology. Our results indicate that despite a lack of detectable changes in either mitochondrial density or expression of the mitochondrial proteins, mitochondrial function was impaired when compared with age-matched control animals. We observed changes in mitochondrial inner membrane morphology and a reduction in the cytochrome c oxidase activity relative to a sustained level of mitochondrial proteins such as porin and individual, functionally important subunits of complex II and complex IV. These data support the idea that mitochondrial dysfunction appears to occur due to inhibition or modification of respiratory complex rather than deletions of mitochondrial DNA. Indeed, these changes were seen in the stratum radiatum where synaptic pathology is readily detected, indicating that mitochondrial function is impaired and could potentially contribute to or even initiate the synaptic pathology in prion disease.

Reactive hypertrophy of synaptic varicosities within the hippocampus of prion-infected mice.

Prion diseases are characteristically accompanied by extensive synaptic pathology that can occur during the preclinical phase of the disease and, in animal models, correlates with the first decline of hippocampus-dependent cognitive functions. This pathology is defined by abnormally shaped synapses in which the postsynaptic membrane modifies its curvature and potentially engulfs the juxtaposed presynaptic membrane. Using the intrahippocampally injected ME7 prion model, we further detailed the structural alterations of the population of ostensibly intact synaptic compartments within the hippocampus during this period of extensive synaptic loss. A disease stage-dependent increase in the average PSD (postsynaptic density) area, the average length of the active zone and the average number of synaptic vesicles indicated that the synapses that were visualized as the animal progressed to end-stage disease were undergoing hypertrophy. Similar findings in samples from AD (Alzheimer's disease) patients, aged and senile individuals, and animal models of neurodegenerative diseases suggest synaptic swelling as synaptic loss is initiated and/or compensatory reaction to counteract the synaptic loss.

Degenerating synaptic boutons in prion disease: microglia activation without synaptic stripping.

A growing body of evidence suggests that the loss of synapses is an early and major component of a number of neurodegenerative diseases. Murine prion disease offers a tractable preparation in which to study synaptic loss in a chronic neurodegenerative disease and to explore the underlying mechanisms. We have previously shown that synaptic loss in the hippocampus underpins the first behavioral changes and that there is a selective loss of presynaptic elements. The microglia have an activated morphology at this stage but they have an anti-inflammatory phenotype. We reasoned that the microglia might be involved in synaptic stripping, removing synapses undergoing a degenerative process, and that this gives rise to the anti-inflammatory phenotype. Analysis of synaptic density revealed a progressive loss from 12 weeks post disease initiation. The loss of synapses was not associated with microglia processes; instead, we found that the postsynaptic density of the dendritic spine was progressively wrapped around the degenerating presynaptic element with loss of subcellular components. Three-dimensional reconstructions of these structures from Dual Beam electron microscopy support the conclusion that the synaptic loss in prion disease is a neuron autonomous event facilitated without direct involvement of glial cells. Previous studies described synapse engulfment by developing and injured neurons, and we suggest that this mechanism may contribute to developmental and pathological changes in synapse numbers.

Fibronectin attenuates process outgrowth in oligodendrocytes by mislocalizing MMP-9 activity.

The extension of multiple oligodendroglial branched processes towards axons is an important event during the early stages of myelination that likely requires remodeling of the extracellular matrix (ECM) microenvironment via matrix metalloproteinases (MMPs). Here we investigated whether fibronectin-mediated inhibition of myelin sheet formation in oligodendrocytes correlated with an altered MMP activity. Our data reveal that fibronectin enhanced, in a PKC-dependent manner, the net activity of MMP-9, but not its expression, in conditioned medium of oligodendrocytes. Residual cellular MMP-9 activity on fibronectin was confined to the cell body, whereas MMP-9 activity on laminin-2 was localized along extending processes of oligodendrocytes. The mislocalization of MMP-9 activity on fibronectin correlated with a perturbed outgrowth of oligodendroglial processes. In conclusion, our findings suggest that ECM molecules influence both the net activity of secreted MMP and the spatial distribution of cell-associated MMP activity, and thereby morphological oligodendrocyte differentiation.

Selective presynaptic degeneration in the synaptopathy associated with ME7-induced hippocampal pathology.

Intrahippocampal injection of the murine modified scrapie (ME7) induces a model of prion disease in vivo. Animals inoculated with ME7 brain homogenate were compared to controls at 8, 12 and 21 weeks. The data show that the accumulation of misfolded prion (PrP(Sc)) coincided with selective reduction in presynaptic protein expression early in disease. This loss is independent of a change in the number of cell bodies in CA3 that provide the major presynaptic input to the stratum radiatum. Electron microscopy of the stratum radiatum independently evidenced a progressive decrease in the number of synapses during disease. Further, the number of postsynaptic specializations lacking an intact presynaptic specialization increased from 12 to 21 weeks. This suggests that the presynaptic compartment is selectively disrupted when the previously reported first behavioural deficits are observed in this model. This synaptic pathology or "synaptopathy" may represent the earliest neuronal dysfunction in this and other protein misfolding induced neurodegenerative diseases.

Fibronectin impedes "myelin" sheet-directed flow in oligodendrocytes: a role for a beta 1 integrin-mediated PKC signaling pathway in vesicular trafficking.

Differentiation of oligodendrocytes results in the formation of the myelin sheath, a dramatic morphological alteration that accompanies cell specialization. Here, we demonstrate that changes in the extracellular microenvironment may regulate these morphological changes by altering intracellular vesicular trafficking of myelin sheet-directed proteins. The data reveal that fibronectin, in contrast to laminin-2, decreased membrane-directed transport of endogenous NCAM 140 and the model viral protein VSV G, both proteins normally residing in the myelin membrane. The underlying mechanism relies on an integrin-mediated activation of PKC, which causes stable phosphorylation of MARCKS. As a result, dynamic reorganization of the cortical actin cytoskeleton necessary for the targeting of vesicular trafficking to the myelin sheet is precluded, a prerequisite for morphological differentiation. These data are discussed in the context of the demyelinating disease multiple sclerosis, i.e., that leakage of fibronectin across the blood-brain barrier may impede myelination by interference with intracellular myelin sheet-directed membrane transport.

CRF and urocortin differentially modulate GluRdelta2 expression and distribution in parallel fiber-Purkinje cell synapses.

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluRdelta2 mRNA. CRF, in contrast, reduces the number of delta2-positive dendritic shafts per cell, implying that the increase of GluRdelta2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluRdelta2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRF treatment results in a clustered distribution of GluRdelta2 along the postsynaptic density, whereas UCN treatment provides a linear distribution.