Abstract
Postnatal Disruption of the Disintegrin/Metalloproteinase ADAM10 in Brain Causes Epileptic Seizures, Learning Deficits, Altered Spine Morphology, and Defective Synaptic Functions
Prox J, Bernreuther C, Altmeppen H, Grendel J, Glatzel M, D'Hooge R, Stroobants S, Ahmed T, Balschun D, Willem M, Lammich S, Isbrandt D, Schweizer M, Horré K, De Strooper B, Saftig P. J Neurosci 2013;33:12915–12928.
The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid β (Aβ) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPβ and increased levels of sAPPβ and endogenous Aβ peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.
Commentary
Epilepsy is a comorbidity in patients with Alzheimer's disease (AD). The cleavage of amyloid precursor protein (APP) by β- and γ-secretases produces amyloid β peptides, which are the main component of amyloid plaques. Alternatively, APP may be cleaved by an α-secretase to form the soluble fragment sAPPa, which promotes neuronal survival. Shifting the proteolysis of APP from the amyloidogenic pathway to α-secretase generation is a potential therapeutic strategy for AD and associated epilepsy.
Molecules that remove extracellular domains from trans-membrane proteins are collectively known as “sheddases,” and the largest family is the ADAM (a disintegrin and metalloprotease) family. Based on expression patterns and transgenic mouse studies, ADAM10 protein (also known as CD156c) has been proposed to be the main α-secretase in the generation of sAPPa. In overexpression studies, ADAM10 is neuroprotective in mice that overexpress APP in the kainate seizure model (1) and reduces β-amyloid deposition and cognitive dysfunction. However, total loss of the Adam10 gene in the mouse is embryonic lethal (2), and conditional deletions targeted to forebrain neuroprogenitors severely disrupted brain development, terminating in late embryonic mortality and intracranial hemorrhages (3). Thus, the role of ADAM10 as the α-secretase in the processing of APP in adult animal models could not be ascertained.
The postnatal function of ADAM10 is revealed by using a neuronal specific Cre-loxP strategy with the Adam10 floxed mouse bred with the CamKIIa-Cre driver mouse strain (4). The expression of CamKIIa commences at postnatal day 5 (P5), circumventing the embryonic and perinatal lethality of the previous mouse models. Based on CamKIIa expression patterns, ADAM10 was expected to be eliminated from the cerebral cortex, hippocampus, olfactory bulb, and amygdala, and to a lesser extent in the striatum, thalamus, and hypothalamus. Many of the Adam10/CamKIIa conditional mutant mice died around P18–P27, during the time of synaptogenesis, while some mice lived to 2 months, but they were smaller in size and weight than their control littermates. Repetitive behavioral seizures were observed as early as P14, with some cases being tonic seizures that ended with respiratory distress and death. Depth electrodes implanted into the hippocampus recorded electrographic seizures in 20 percent of the Adam10/CamKIIa conditional mutant mice and in none of the control mice. In summary, genetic deletion of Adam10 retarded growth and initiated a seizure phenotype.
During normal ontogeny, ADAM10 levels increase with postnatal age, in particular throughout synaptogenesis. The levels of ADAM10 in the targeted genetic mutant mice were measured by Western blot analysis. Decreased levels of ADAM10 were observed as early as P5, at the beginning of the CamKIIa–mediated deletion, and continued to barely detectable levels at P17. The sAPPa levels were nearly absent in targeted regions, confirming the role of ADAM10 as the main α-secretase for APP. The balance of the soluble APP fragments was shifted to the Aß species, which may ultimately lead to increased amyloid deposition and plaques in very young mice. In addition, the lack of sAPPa in its role in promoting neuronal survival may account for the seizure phenotype in juvenile animals. Dysregulation of APP processing is also observed in patients with Down syndrome, some of whom experience seizures during childhood. The DS phenotype is not really similar. Most childhood DS is infantile spasms and otherwise epilepsy tends to start in adulthood.
At P20, the hippocampus displayed no obvious abnormalities or neuronal loss. In older mice (20 and 30 weeks old), gliosis and activation of microglial were observed. Behavioral testing showed normal circadian cycles and open field motor activity, but decreased motor performance was reported for the rotarod test. The major deficits were observed during spatial memory testing in the Morris water maze, when the Adam10/CamKIIa conditional mutant mice were not able to learn the task during the acquisition phase and did not show preference for the target area during the memory probe test, reflecting the spatial memory deficits observed in other mouse models of AD.
The circuitry of the hippocampus was analyzed on multiple levels. Recordings of the Schaffer collateral pathway in hippocampal slices revealed no differences in basal synaptic transmission as measured by input–output curves. A significant difference in paired-pulse ratios (a measure of short-term plasticity) was noted at one of the six interstimulus intervals that were tested. However, long-term potentiation (LTP), a major mechanism for long-term memory formation, was severely attenuated in the Adam10 /CamKIIa conditional mutant mice. While control mice exhibited LTP for more than 2 h, the Adam10/CamKIIa conditional mutant mice only displayed short-term potentiation for only a few minutes. The LTP deficit can be partially rescued with the addition of sAPPa to the recording solution, supporting the role of Adam10 as critical for the generation of sAPPa for normal function. In vivo hippocampal recordings under urethane anesthesia were divided into sleep-like (rapid eye movement [REM]) and non-REM sleep-like episodes. The numbers of ripples and sharp waveforms observed in non-REM sleep-like episodes were similar between control and Adam10/CamKIIa mutant mice. Analysis of the local field potentials (LFPs) in the REM episodes indicated reduced power spectra in the theta and gamma frequency ranges and a lower cross-frequency coupling between theta and gamma oscillations in the Adam10/CamKIIa mutant mice.
The altered electrophysiological function of the Adam10/CamKIIa mice suggested postsynaptic defects. Combined biochemical and immunohistochemical analysis demonstrated decreased levels of PSD-95 (post-synaptic density −95) scaffold molecule, and NMDA (N-methyl-D-aspartate) receptor subunits 2A, 2B, and NR1. Anatomically, the Adam10/CamKIIa conditional mutant mice had a reduction in the apical dendrites in the stratum radiatum and altered distribution of shapes of the spines. The Adam10/CamKIIa conditional mutant mice had more stubby and enlarged spines compared to the controls’ small and round spines, similar to morphologies reported in aged APP null mice and APP-deficient neurons. However, electron micrographs indicated that the synapses were regularly formed. The changes in spine morphology may be correlated with abnormal cleavage and shedding of cell adhesion molecules that serve as substrates for ADAM10, and Western blots confirmed decreased C-terminal fragments of N-cadherin and nectin-1, but normal full length N-cadherin and nectin-1 supporting loss of α-secretase activity.
The recent data support an independent role for ADAM10 in AD and epilepsy. Impaired processing of APP in the absence of Adam10 or γ-secretase may lead to increased network hyperexcitability (4, 5). Recent reports have identified two rare human mutations in the human gene ADAM10, that correspond with late-onset AD (6). A promoter haplotype that increases ADAM10 expression was associated with lower plaque load and higher cognitive scores in patients without AD, implying that greater ADAM10 expression produced more sAPPa that served to protect the hippocampus from degeneration (7). The combination of human and animal research supports the potential for therapeutic agents that modulate ADAM10 to prevent or delay AD and the epilepsy comorbidity.