The role of axonal transport in neurodegenerative diseases remains poorly understood. While there are indications that axonal transport deficits contribute to neurodegeneration, in vivo detection has been very difficult and invasive. The technique defined by this paper extends the use of MEMRI to quantify and compare in vivo axonal transport in normal and transgenic mouse models. This novel methodology opens up the possibility for in vivo, longitudinal studies that could contribute unique information in mouse models of neurodegeneration. Signal intensity data from thirty minutes of recording in the ONL allows for extrapolation of the rate (ΔSI/Time) at which Mn2+ is transported from the olfactory epithelium to the ONL. The difference in signal intensity produced by Mn2+ is also visible through qualitative assessment, as seen in . The data confirm that the nasal lavage and data acquisition are reliable and reproducible and that without Mn2+ the SI does not change.
It has been noted previously that physiological temperatures (36.9ºC in mice) are important for normal axonal transport (Cosens, Thacker et al. 1976
; Van der Linden, Van Meir et al. 2004
). To determine the effect of temperature on Mn2+
transport, baseline transport rates at 37.0ºC were compared to transport rates at 30.3ºC (). The drop in temperature corresponded with a reduction in rate of Mn2+
transport indicating that Mn2+
is transported in a temperature dependent manner. Temperature dependence was further established by finding that Mn2+
transport rates returned to normal when the body temperature of the animal was reestablished at 37.0ºC.
Another component known to be necessary for normal axonal transport is the microtubule network. Although the role of microtubules in Mn2+
transport in anatomical MEMRI data has already been demonstrated (Sloot and Gramsbergen 1994
; Pautler, Silva et al. 1998
), we evaluated the dependence of dynamic Mn2+
transport upon microtubule integrity. The microtubule disruptor colchicine was utilized because it is known to bind tightly to tubulin and prevent the polymerization of microtubules (Hastie 1991
; Han, Malak et al. 1998
). By preventing assembly of microtubules, the ability of motor proteins to bind and transport cargo is also prevented. Colchicine has the added benefit of having been applied through nasal lavage and intracerebral injections in previous studies (Sloot and Gramsbergen 1994
; Pautler, Silva et al. 1998
). Here, the dose 1 mg/kg, a dose less than one-half that of previous studies, was used. Colchicine and vehicle controls (0.9% saline) were tested and results show that the colchicine significantly and dramatically blocked the rate of Mn2+
transport in the ONL. Data acquired using MEMRI confirmed Mn2+
enhancement in the olfactory turbinates at the base of the olfactory bulb, indicating continued Mn2+
influx into the cells and axonal areas following colchicine treatment. We paid particular attention to this because microtubule alterations are capable of changing calcium influx into the cell. The data in indicate that Mn2+
influx was not hampered by the colchicine. The data shown here verify that Mn2+
transport is dependent upon microtubule-based axonal transport and that the MEMRI measurement is a quantifiable indicator of axonal transport.
Having established that Mn2+ transport measured by MEMRI is reflective of axonal transport, the axonal transport rates of Tg2576 animals and wildtype littermate animals were evaluated at ages 3–4, 7–8, and 11–14 months of age utilizing MEMRI. Significant differences in axonal transport between wildtype and Tg2576 mice were apparent in the 7–8 months aged group, at which point the axonal transport rate for APP overexpressing mice was 48% less than controls. When older animals, 11–14 months, were analyzed they were found to have an even more pronounced deficit, 82% less. We verified on scanned 7–8 month old animals that their olfactory bulbs were indeed negative for amyloid plaques compared to the olfactory of 12 month old Tg2576 animals with positive amyloid plaque accumulation in the olfactory bulb.
Studies analyzing Aβ accumulation, the cleavage product of APP, in these animals demonstrate that insoluble Aβ appears at 6–9 months (Kawarabayashi, Younkin et al. 2001
). The definite biochemical changes take place by the 10th
month leaving minimal Aβ deposition followed by diffuse plaques that are visible by 12 months (Kawarabayashi, Younkin et al. 2001
). Combined with the present study, this evidence suggests that the axonal transport deficits are occurring in conjunction with the accumulation of insoluble Aβ and prior to Aβ plaque formation in this particular AD model.
The cause of the differences in axonal transport rates of the Tg2576 mouse model at different ages is unclear. One possibility could be that the calcium influx is decreased resulting in less Mn2+
available to be transported down the axon. However, in this mouse model the calcium influx increases with age instead of decreasing suggesting that Mn2+
entry into the cell is equally unimpeded as our images show (Xie 2004
). It has also been reported that the cytoskeletal proteins α and β tubulin are genetically up-regulated in the Tg2576 animal (Reddy, McWeeney et al. 2004
). This could result in abnormal microtubule function and further strengthens our data that axonal transport decreases abnormally compared to controls.
Magnetic resonance imaging (MRI) is one of the best imaging methodologies for studying soft tissues and monitoring biological processes as they occur in vivo
. There are currently many attempts to identify and study AD animal models through MRI imaging. Numerous MRI studies have targeted the assessment of plaque formation with and without the use of endogenously applied contrast agents (Redwine, Kosofsky et al. 2003
; Helpern, Jensen et al. 2004
; Helpern, Lee et al. 2004
; Jack, Garwood et al. 2004
; Lee, Falangola et al. 2004
; Song, Kim et al. 2004
; Rohner, Staab et al. 2005
; Sun, Song et al. 2005
; Sykova, Vorisek et al. 2005
; Vanhoutte, Dewachter et al. 2005
). For example, MRI has been used to identify individual plaques in the Tg2576/Presinilin1 double mutant, a mouse model of AD (Jack, Garwood et al. 2004
). Many studies have evaluated changes in anatomy, such as the high resolution, in vitro
MRI study on fixed brains of transgenic mice containing the PDAPP mutation. The data from this in vitro
study showed dentate gyrus shrinkage prior to the appearance of plaques (Redwine, Kosofsky et al. 2003
). Other MRI studies include the assessment of alteration in the diffusion of water in the gray and white matter of mouse models of AD. This work demonstrated alterations in the diffusion of water in the Tg2576 mouse brain after plaque formation (Sun, Song et al. 2005
). With the exception of the in vitro
studies by Redwine et al, all MRI studies to date on AD mouse models have demonstrated alterations post plaque formation. Here we extend the use Manganese Enhanced MRI (MEMRI) to assess in vivo
axonal transport, and therefore neuronal function, in the Tg2576 AD mouse model.
The data reported here establishes that functional deficits in axons begin prior to the appearance of Aβ plaques. This is supported by an in vitro
study that found axonal swellings starting at four months in axons from mice also carrying the Swedish AD mutation (Stokin, Lillo et al. 2005
). This finding implicates that axonal swellings do not form in response to amyloid deposition, but that the swellings may instigate the accumulation of Aβ similar to that seen in acute neuronal injury.
The evidence presented here indicates that MEMRI is a very promising tool for detecting early signs of abnormal physiological deficits. Using MEMRI we were able to identify and quantify the difference in axonal transport rates in vivo in the Tg2576 mouse model of AD during the progression of the AD phenotype. Data from the Tg2576 mouse indicates that deficits coincide with the timing of previously reported biochemical changes, but prior to histologically visible plaques. The ability to measure axonal transport rates in vivo opens up many new and exciting opportunities for the characterization of disease states as well as assessing the efficacy of diagnosis and therapeutic intervention.