A few neuropsychiatric diseases like autism and attention deficit hyperactive disorder etc. are thought to be neurodevelopmental disorders while others like Huntington’s, Alzheimer’s, Parkinson’s disease are designated as adult or elderly onset disorders [37–41]. It is being increasingly recognized that deregulation of developmental neuronal apoptosis or glial death may result in functional deficits and associated diseases in the later years of life [42]. In the absence of human tissues for analysis, animal models are valuable resources for mechanistic explanations of a disease. Ours is the first study using unbiased stereology on developing nigra that compares the developmental trajectories of nigral DA neurons in two distinct mice strains and the F1 progeny of their crossbreds. The studies were conducted in MPTP-sensitive C57BL/6J, MPTP resistant CD-1 mice and the F1 generation of their reciprocal crossbreds. Our model offers the advantage of “no use of neurotoxins”, since most other studies on PCD relied on chemical lesions to investigate target dependence in development.
The first two postnatal weeks are crucial for the rodent midbrain DA system, for being associated with culmination of neuronal migration, maturation of perikarya, extension of axons and their terminal differentiation, which occurs alongside the apoptotic death of neurons that fail to mature [43, 25, 44, 26]. Stressors during this period prime the DA system to subsequent insults like prenatal stress, maternal separation, ischemia, hypoxia and excitotoxicity etc. resulting in the delayed attainment of DA phenotype [45], reduction in the number of DA neurons, exaggerated response to neurotoxins [46, 47] and affect the DA neurotransmission at adulthood [48]. In fetal non-human primates [24] peak PCD of DA neurons occurs at E80 and approximately at mid-gestation in humans, when approximately 50% neurons are pruned. The time period P2 in rats equates with E79/80 in monkeys [24], thus P2 in mice is a critical window of vulnerability and protective modalities could be developed if the relevant molecular underpinnings are understood.
The clusters of TH-ir neurons found along the midline during early postnatal development i.e. P2 and P6 probably represent the migrating neurons or other dopaminergic clusters. The gradual increase in numbers till P14 point at the culmination of neuronal migration [49–51], or addition of TH-ir neurons by postnatal neurogenesis [52–54, 49]. The third likelihood is that progressively more neurons show detectable levels of TH expression i.e. they attain DA phenotype [55, 56, 45]. These factors reduce the likelihood of extensive cell loss during this phase, thus maintaining the steady increase in numbers. In an earlier study using unbiased stereology, Jackson-Lewis et al. (2000) also reported a gradual increase in nigral dopaminergic neurons till P14. However, the small decline in the numbers after P14 observed in our study was contrasted by absence of cell loss in their study. Our observation is corroborated by the findings of Oo and Burke [25], who showed a smaller second peak of dopaminergic neuronal loss around P14. The data of Jackson-Lewis et al. [23] represent cumulative results of two different mice strains i.e. C57BL/6J and CD-1, which may underlie the discrepancy. The delay in attainment of adult nigral architecture in C57BL/6J i.e. at P18, may be attributed to the ongoing cell loss vis-à-vis the early maturation at P14 in CD-1 and the crossbreds. Our observations on C57BL/6J, match those of Oo and Burke [25], who showed two peaks of loss i.e. at P2 and P14 in rats, proposing a biphasic nature of programmed cell death. We propose an additional minor peak at P22 in C57BL/6J, complemented by TUNEL reaction, caspase-3 expression and reduction in TH-ir dopaminergic neurons. Both caspase-mediated and caspase-independent cell death pathways, associated with mitochondria and endoplasmic reticulum respectively, work in unison to cause neurodegeneration[57]. The enhancement of AIF corroborates with the role of caspase independent cell death in developing human midbrain [58]. Elevated AIF expression at P14 in addition to high caspase-3 expression at P22, corroborated by TUNEL-expression, further illustrate the alliance between the two cell death pathways in the instance of DA neurons.
Classical studies suggest that GDNF protects nigral neuron from apoptosis [59, 60]. Recent studies consider it as a potent therapeutic for PD [61, 62]. It is expressed in the striatum and reaches SNpc through retrograde transport[63]. Therefore, its mRNA synthesis and protein expression during striatal development are critical[64]. The upregulation of GDNF in the striatum from day 2 to day 14, in contrast to reduced expression in the SNpc at analogous stages, compares well with earlier studies[64, 65] and it may be associated with the refinement of the nigrostriatal connections. GDNF expression at P2 may be paracrine/nigral in origin, since the nigro-striatal connections are yet not formed. At later stages, the source may switch to striatum. The higher levels in the crossbreds suggest better neuroprotection in them, which could be conjectured during nigral development in the Anglo-Indians, scaling up their dopaminergic reserve. It might be worthwhile to examine the early embryonic periods and develop preventative strategies.
Cellular hypertrophy following second peak of apoptosis noted exclusively in C57BL/6J may be a physiological process corresponding to the expansion of nerve terminals, also seen in other amine systems [49, 66–68]. Alternatively it may be a predisposing factor; since larger neurons are vulnerable to degeneration [69]. Hypertrophy of DA neurons in aging human nigra was considered as a compensation for sub-threshold neurodegeneration [10] and its absence as a marker for resilience [12]. The gradual decrease in cellular TH over development alongside an increase in soma area, suggests that overall dopamine synthesis remains stable through development. Amongst the strains, F1X2 showed best preservation of TH levels.
A correlation between the nigral volume and the neuronal numbers through development and across strains indicates that volume can be an indirect measure of DA neuronal number. Nigral volumetry by MRI is a diagnostic tool for PD, based on a similar premise. The striatal volume varies in healthy individuals [70, 71] and imparts differential susceptibility to psychiatric disorders [72–74]. Anthropometric studies suggest that children with smaller head circumference and cerebral volume may develop Huntington’s disease [37] reflecting the developmental origin of the disease. Cognitive capabilities were better protected in Alzheimer’s disease patients with relatively larger head circumference [75]. Therefore, nigral volumetry may be a valuable tool to identify the predisposed individuals/populations.
A hypothetical division of the postnatal period in three phases reveals that the second phase that defines the neuronal numbers is more critical. Such differences may be envisioned in the Caucasian, Asian-Indians, and their admixed population imparting them varying degrees of susceptibility to develop PD [9, 12, 11, 6, 15, 34, 35].
Finally, the findings of fewer neurons throughout development in C57BL/6J nigra; posit the presence of excessive endogenous toxins or pro-apoptotic factors at these stages. This possibility was strengthened by corresponding high levels of caspase-3 and AIF. The higher cellular packing density at P14 in C57BL/6J may represent increased competition for trophic factors. Whereas, minimal loss of DA cells, higher GNDF and controlled expression of pro-apoptotic factors in CD-1 and the crossbreds, hint at better endogenous milieu. Developmental apoptosis sculpts aberrant neural connections[76, 77]to regulate the final neural numbers in adult multicellular organisms [78, 79]. Since both the mice strains are non-transgenic and genetically distinct, we hypothesize that genetic constitution may predispose individuals or populations even in absence of major mutations. In late eighties, Fahn [5] hypothesised that the brain wiring during development may influence the number of neurons at birth and other cellular features which may be unique to the individuals, making them either vulnerable or resilient to PD. He also proposed that the metabolic processes in the brain may govern vulnerability to develop PD [5]. Both these hypotheses hereby stand validated, hinting at the developmental origin of vulnerability to MPTP. It may therefore be possible that susceptibility to PD originates during nigral development in humans.
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