The fornix provides multiple biomarkers to characterize circuit disruption in a mouse model of Alzheimer's disease

Abstract

Multivariate biomarkers are needed for detecting Alzheimer's disease (AD), understanding its etiology, and quantifying the effect of therapies. Mouse models provide opportunities to study characteristics of AD in well-controlled environments that can help facilitate development of early interventions. The CVN-AD mouse model replicates multiple AD hallmark pathologies, and we identified multivariate biomarkers characterizing a brain circuit disruption predictive of cognitive decline. In vivo and ex vivo magnetic resonance imaging (MRI) revealed that CVN-AD mice replicate the hippocampal atrophy (6%), characteristic of humans with AD, and also present changes in subcortical areas. The largest effect was in the fornix (23% smaller), which connects the septum, hippocampus, and hypothalamus. In characterizing the fornix with diffusion tensor imaging, fractional anisotropy was most sensitive (20% reduction), followed by radial (15%) and axial diffusivity (2%), in detecting pathological changes. These findings were strengthened by optical microscopy and ultrastructural analyses. Ultrastructual analysis provided estimates of axonal density, diameters, and myelination-through the g-ratio, defined as the ratio between the axonal diameter, and the diameter of the axon plus the myelin sheath. The fornix had reduced axonal density (47% fewer), axonal degeneration (13% larger axons), and abnormal myelination (1.5% smaller g-ratios). CD68 staining showed that white matter pathology could be secondary to neuronal degeneration, or due to direct microglial attack. In conclusion, these findings strengthen the hypothesis that the fornix plays a role in AD, and can be used as a disease biomarker and as a target for therapy.

Keywords: Alzheimer's disease; Diffusion tensor imaging; Electron microscopy; Fornix; Mouse models.

Figure 1. In vivo MRI identified focal volume changes, including reduced fornix and septohippocampal areas (t maps of the log Jacobian for deformation fields, thresholded at FDR=10%, unitless)

Regions of the neocortex (S1/S2) and cerebellum were enlarged, whereas olfactory regions, basal ganglia, thalamus, ventral tegmentum, and superior colliculus were reduced. Lateral ventricles appeared enlarged. Abbreviations: AO: accessory olfactory; CPu: caudate putamen; Cblm: cerebellum; Hc: hippocampus; Olf: olfactory; Re: reuniens thalamic nucleus; RRF: retrorubral field; SFi: septofimbrial nucleus; SC: superior colliculus; SN: substantia nigra; VTA: ventral tegmental area; VThal: ventral thalamus; f: fornix; ic: internal capsule; cc: corpus callosum.

Figure 2. Ex vivo DTI identified significant local volume differences in CVN-AD mice relative to controls (t maps, thresholded at FDR= 5%, unitless)

Atrophy was observed in the olfactory areas, preoptic, hypothalamus, lateral amygdala, hippocampal commissure, septofimbrial nucleus, triangular septum, hippocampus, subiculum, ventral thalamic nuclei, superior colliculus, and pons, as well as the fornix and internal capsule. Areas of hypertrophy included the somatosensory cortex, corpus callosum (medially, and periventricular), dorsal thalamus, baso-medial amygdala, cerebellum (lobule 10), and the lateral reticular nucleus. (B) Volumes for selected regions of interest in both genotypes. Data are presented as medians (line), mean (circle), quantile (box, 25–75% of data range), range (whiskers). Abbreviations: AD: anterodorsal thalamic nuclei; AO: anterior olfactory; DEnt: dorsolateral entorhinal cortex; DS: dorsal subiculum; Ect: ectorhinal cortex; HcDG: dentate gyrus of hippocampus; LDDM: latero-dorsal thalamic nucleus (dorsomedial); LA: lateral amygdala; LRt: lateral reticular nucleus; LSI: lateral septal nucleus, intermediate part; LMol: lacunosum moleculare layer of the hippocampus; Olf: olfactory; Pn: pons; SC: superior colliculus; SFi: septofimbrial nucleus; cc: corpus callosum; f: fornix; ic: internal capsule.

Figure 3. Fractional anisotropy (FA) differences between CVN-AD and mNos2^−/− controls (t statistics, thresholded at FDR=5%, unitless)

(A) For a mask including the whole brain we observed a strong effect for the fornix, and within the thalamus only. (B) Restricting the mask to white matter (FA > 0.3), we found significant reductions in the fornix, corpus callosum, and internal capsule, as well as in cerebral peduncle, and middle cerebellar peduncle. (C) ROI based differences in FA. Abbreviations: cc: corpus callosum; cp: cerebral peduncle; dhc (vhc): dorsal (ventral) hippocampal commissure; ic: internal capsule; f: fornix; mcp: middle cerebellar peduncle.

Figure 4. DTI multiparameter changes in CVN-AD mice (t statistics thresholded at p<0.05)

A) Axial diffusivity changes (E1, associated with axonal density) were significantly decreased in the optic tract, cerebral peduncle, vestibulocochlear nerve (n8), ventral spinocerebellar tract, and middle cerebellar peduncle (~7%), and increased in stria terminalis (which did not survive permutation corrections). B) Radial diffusivity (RD) associated with myelination) was significantly increased in several white mater tracts and pontine areas (p<0.05). The fornix, and hippocampal commissure were >10% increased. The difference for fimbria, corpus callosum, anterior commissure and brachium of the superior colliculus showed a trend (p<0.1). c) Secondary eigenvalues changes and ADC point to the ventral hippocampal commissure and fimbria.

Figure 5. Decreased myelin content in the fimbria of CVN-AD mice was detected through luxol fast blue image contrast relative to the cortex for three regions of interest (fi: fimbria, cc: corpus callosum, ac: anterior commissure) indicated (d=-3.6, 8% contrast difference, p<0.01).

Data are presented in contrast to similar sections through the MR, where FA reductions are shown in blue; and as mean ± standard error of the mean. N = 4 animals/group.

Figure 6. Representative transmission electron microscopy images through the fornix are indicative of abnormal white matter in CVN-AD mice (7100 x magnification)

Lower density of myelinated axons, increased variability in axonal size and shape, as well as enlarged G ratios, were found in CVN-AD mice compared to mNos2^−/− control mice. Abnormalities of myelination were more frequently seen in CVN mice, and included ballooning, abnormal lamellation, and myelin figures in degenerated axons.

Figure 7. Several vulnerable areas identified by morphometric and/or DTI changes also stained for reactive microglia in CVN-AD mice, but not in controls

In particular areas of the hippocampus (CA1, CA3), as well as its connections - fimbria and alveus (A) stained for CD 68 (phagocytic marker) in CVN-AD mice (A), but not in controls (B). GFAP staining also revealed higher density and darker staining for reactive astrocytes in CVN mice (C) than in controls (D). In contrast Abeta staining was mostly found in gray matter areas, and not in the fimbria white matter, and only in CVN mice (E), but not on controls. The structures involved are part of a circuitry known to be involved in human AD (fx: fornix, fi: fimbria, Hc: hippocampus; Hyp: hypothalamus) (F).