The increasing awareness of the susceptibility of olfactory acuity in people with prodromal neurodegenerative disease, relative to other sensory modalities, and our deeper understanding of the physiology underlying olfactory perception32, 33
have made the olfactory neural network in animals a more widely used model to investigate mechanisms of neurodegenerative disease34, 35
. The amenability of the mouse peripheral olfactory neural circuit to genetic, imaging, and behavioral characterization renders it a powerful model system to elucidate the actions of disease proteins16, 27
. One of the unique aspects of this neural circuit is the continuous renewal of OSNs in adults36
, necessitating precise axon targeting throughout its lifetime to maintain the integrity of the circuit. We exploit this physiologic structural plasticity to visualize dysfunction caused by the expression of genes that cause neurologic disease. Here, we report that expression of the Swedish mutation of human APP in mice in vivo
alters the connectivity and function of the peripheral olfactory neural circuit in the absence of plaques.
Our initial observation of this axon guidance phenotype occurred in well characterized lines that overexpress hAPPsw throughout the brain. Taking advantage of methodology to specifically express genes in the peripheral olfactory neural circuit37
, we generated and examined two lines of mice that overexpress human APP alleles exclusively in the presynaptic neurons of this circuit. Aβ production is facilitated in one line that expresses hAPPsw and impeded in the other line that expresses hAPPmv12
. Overproduction of hAPPsw in the presynaptic neurons was sufficient to perturb the structural connectivity of the peripheral olfactory neural circuit. The absence of a phenotype in the hAPPmv line corroborated our findings in the mouse line overexpressing the wild type form of hAPP. Together with preserved fidelity of axon targeting in the I5 line overexpressing wild type hAPP throughout the brain, these important controls indicate that overexpression of human APP is not sufficient to alter OSN axonal connectivity. Rather, our findings suggest that products of BACE1-mediated cleavage of APP critically modulate neuronal function — in congruence with results from studies in hippocampal slices13, 15, 38
, in induced human neurons derived from skin fibroblasts39
and studies of BACE1 heterozygous and null mice40
We postulate that Aβ is the BACE1 cleavage product of APP involved in mediating the connectivity phenotype. The presence of the connectivity phenotype in mice expressing hAPPsw, but not in the I5 line expressing hAPPwt and not in the CORMAC line expressing hAPPmv, supports this hypothesis. Moreover, expression of human Aβ40 or human Aβ42 in the olfactory epithelium using a viral vector delivered intranasally phenocopies the alteration in the projection map of OSN axons. Since we observe these axon targeting deficits in young mice prior to the onset of plaques in the broadly expressing lines and since we do not observe amyloid plaques in lines that overexpress hAPP isoforms exclusively in OSNs or in the virally-infected mice, this novel axonal phenotype is independent of amyloid plaque deposition. This phenotype is unlikely to be caused by an in utero anomaly since we can induce it in adult control mice by intranasal expression of Aβ40 or Aβ42. Together, these data are consistent with a model that soluble Aβ triggers axonal dysfunction in the absence of amyloid plaques in vivo, although we cannot disprove that insoluble Aβ could also cause similar changes.
In addition to altering structural connectivity, expression of hAPPsw exclusively in OSNs has functional consequences as indicated by two lines of evidence. First, relative to control littermates or CORMAC mice, CORMAP mice exhibit significant reductions in olfactory acuity in two distinct behavioral paradigms, one paradigm employing an odor that evokes an aversive response while the other paradigm employs an odor that evokes an appetitive response. We interpret these olfactory-mediated behavioral responses as reflecting the function of the peripheral olfactory neural circuit in CORMAP and CORMAC lines — an interpretation afforded by the restricted expression pattern of hAPPsw and hAPPmv, respectively. Deficits in an odor habituation paradigm have been reported in Tg2576 mice at ages 6 - 7 months that correlated with amyloid plaque deposition34
. By contrast, the behavioral phenotype of CORMAP mice is detectable at 3 to 5 months of age and is independent of amyloid plaques. Secondly, the expression of two independent activity-dependent markers in postsynaptic neurons in the olfactory bulb were significantly reduced in CORMAP mice relative to littermate controls. The magnitude of reduction of these activity-dependent markers (approx. 25%) exceeds the frequency of expression of hAPPsw in mature OSNs (approx. 12%). This disproportional response in the functional data raises the possibility that hAPPsw may be acting in a non-cell autonomous manner, as seen in studies in hippocampal slices by Malinow's laboratory13, 38
. Our attempts to determine whether a non-cell autonomous mechanism underlies the structural connectivity phenotype in the CORMAP line have not been conclusive to date. Future studies employing expression of hAPP isoforms by specific OR promotors are aimed to address this question directly.
Our data illustrate a detrimental effect of heightened Aβ levels on a neural circuit in the absence of plaques. The CORMAP line offers a quantifiable outcome of the actions of hAPPsw, and likely Aβ, which is distinct from amyloid plaque production and that could be utilized to assess therapies targeting aberrant neural plasticity due to Aβ. While olfactory deficits have been shown in patients with prodromal and mild Alzheimer's disease, demonstration of alterations in the map of OSN projections in the human olfactory bulb in AD is necessary to postulate that the CORMAP mouse is a model for the disease. Increasing evidence indicates that Aβ levels rise after neuronal injury by increased expression of APP and/or BACE141
. Established risk factors for late onset AD, e.g. head trauma and vascular insults, result in elevated Aβ production42
. Further delineation of the mechanism of action of human Aβ in this model system may provide insight into neural circuit and network dysfunction in the long, preclinical stage of Alzheimer's disease.