Our study used the first α
-syn transgenic rat bearing the human A30P and A53T mutations under the control of the TH promoter. As previously stated [19
] transgenic animals displayed some long-lasting olfactory deficits and the human-mutated α
-syn protein was observed in the OB, the SNpc, and the LC. It was colocalized with TH immunostaining (as shown for the OB in the present paper) which is consistent with the fact that TH was the transgene promoter. Olfactory deficits appeared long before the motor alterations as 18-month old animal did not present yet any deficit in movement initiation. Deficits in motor coordination appeared at 19 months of age (not shown). Twenty-five- month-old transgenic rats were used in this study as clinical and pathological manifestations of the α
-syn mutations appear in advanced age in PD, generally [22
]. Tyrosine hydroxylase was used as the promoter in order to obtain the human α
-syn synthesis only in catecholaminergic structures. Indeed, we were able to observe transgene expression in the 3 main catecholaminergic brain areas involved in the course of PD: the OB, the SNpc, and the locus coeruleus. To date, there is a growing evidence of a prion-like transmission of α
-syn contained in aggregates from donor cell to recipient cell [30
]. However, it did not seem to be the case in our transgenic rat as non-TH positive brain structures did not contain any human α
-syn molecule. However, we cannot rule out that this mechanism did not happen within catecholaminergic structures, thus potentiating the effect of the transgene.
The OB is a brain region of particular interest because Lewy neurites and bodies are present in this area in the very early stages of the PD [31
]. These inclusions consist of aggregated form of α
-syn with other components such as phosphorylated neurofilaments and ubiquitin [31
]. As in PD patients, we have shown that our mutant human A53T and A30P α
-syn expressing rat presented protein aggregates in the glomerular layer suggesting an implication of the human-mutated α
-syn in the cellular processing of aggregates, which could in turn alter local OB proliferation.
Our data showed an increased number of proliferative cells in the glomerular layer but not in the granular cell layer. It is worth mentioning here that the BrdU protocol used in the present study rather revealed local OB proliferation than migrated cells from the SVZ to the OB as the animals were given BrdU for 5
days and sacrificed 5
hours after the last injection.
Interestingly, Winner et al. [32
] showed in 2-month-old female Wistar rats that the local dividing cells represented less than 5% of the total number of new cells. Their total number of BrdU positive cells in the granular (8,200 cells) and glomerular layer (250) are lower than ours (16,600 cells and 2,350 cells, respectively). This important difference in numbers can be related to the concentration of BrdU used in Winner's study being half of the one we used, to the time of the sacrifice after the last injection (2
h versus 5
h in our study), to the age of the animal (2
months versus 25
months) and could point out for a few cells an increase in granular and glomerular layer local proliferation due to aging.
No variation in the SVZ proliferation was induced by the double α
-syn mutation. This later result can be explained by the absence of any transgene expression within the SVZ. Our observation in the SVZ is in agreement with the findings of Maxreiter et al. [21
] using a mouse expressing the human A30P mutant form of the α
-syn, who did not find any change in SVZ proliferation. Using mice expressing the A30P mutant form of α
-syn under the control of the calcium/calmodulin-dependent protein kinase II alpha (CaMK) promoter [21
] or expressing the A53T mutant form of α
-syn under the control of the PDGF-promoter [22
], two groups studying OB neurogenesis found a decrease in newly generated neurons in the glomerular and granular layers. Taken together, the data on OB neurogenesis and from our own investigation suggest that human α
-syn A30P/A53T mutations impacts newly generated neuroblasts during OB integration/differentiation as well as local OB proliferation. In contrast to our observations and certainly due to the promoter they used, Winner et al. [22
] and Maxreiter et al. [21
] also observed some transgene expression in noncatecholaminergic structures. Some data suggest that the mutated α
-syn could spread using a prion-like transmission from cell to cell [30
]. As a result, it is possible that a more “α
-syn toxic brain environment” was created in the A30P and the A53T transgenic mouse brains than in our rat brain. The increased local proliferation that we noticed in the glomerular layer is in agreement with data from the glomerular layer of PD patients [23
]. This later finding suggests that our rat model is a suitable tool concerning the effects of the α
-syn mutations in the OB.
As proliferative cells within the glomerular layer are known to differentiate in DA neurons [24
], we investigated the TH innervation within the glomerular layer. We observed an increase in width of the TH positive area in the glomerular layer without an increase in the size of the glomeruli (except for the smaller interval in transgenic animals) suggesting that this increased TH innervation was not induced by an increased glomerular layer areas (as an increased glomeruli size would have increased the size of the glomerular layer which in turn could have enlarged the pattern of TH innervation). This result is agreement with the 100% increase in DA cell number in the glomerular layer from PD patients [23
]. Although the mechanisms underlying the enhanced DA innervation in the OB glomerular layer remains to be determined, various growth factors which play an important role in OB proliferation and DA differentiation could be involved such as BDNF, GDNF and CNTF [33
]. Interestingly, this increase in DA innervation observed in our transgenic rat and in PD patients could explain, at least in part, the olfactory deficit observed both in our rat and in patients as DA in the OB has an inhibitory action. Hyposmia can be detected in PD patients in early stage of disease. Our transgenic rats have been tested for olfactory function at different ages (from one week to 25
months of age) and at 6 months of age they presented an alteration of olfaction. Dopamine has an important role in mediating olfactory information into the brain [34
]. TH innervation is found exclusively in glomerular layer of the OB [25
]. In our rat, the increase in TH innervation in this area might suggest an increase in DA release. DA is known to induce an inhibition between olfactory receptor cells and mitral cells within glomerular layer [23
]. D2 receptors are the most abundant subtype of DA receptors in the glomerular layer [35
] and are involved in the decrease in synaptic transmission [34
]. The increase of DA neurons caused by the A30P and the A53T mutant forms of α
-syn could induce a depression in synaptic transmission and therefore compromises the threshold for olfaction. This circuit is the first step in the process of final consciousness of smell and therefore is essential for the proper function of olfactory circuits. Data from biopsies of patients diagnosed with PD support the idea that olfactory impairment in PD do not result from damage to the olfactory epithelium but is the consequence of central-nervous alterations [36
]. Thus our rat could be a good model to investigate the role of human mutated α
-syn in the development of olfactory deficits.
In conclusion, we generated a human double mutated α-syn (A30P and A53T) transgenic rat presenting an alteration of the local proliferation in the glomerular layer but neither in the granular cell layer of the OB nor in the SVZ. In addition, an increase in DA glomerular layer innervation was noticed, which might be related to the increased proliferation observed in this layer. Further investigation should examine the time course of the changes in the olfactory function in regards to alterations in OB local proliferation as well as elucidate the role of the increased DA function in the olfactory deficits we observed in our transgenic rat.