Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease

Abstract

Current research on Parkinson’s disease (PD) pathogenesis requires relevant animal models that mimic the gradual and progressive development of neuronal dysfunction and degeneration that characterizes the disease. Polymorphisms in engrailed 1 (En1), a homeobox transcription factor that is crucial for both the development and survival of mesencephalic dopaminergic neurons, are associated with sporadic PD. This suggests En1 mutant mice might be a promising candidate PD model. Indeed, a mouse that lacks one En1 allele exhibits decreased mitochondrial complex I activity and progressive midbrain dopamine neuron degeneration in adulthood, both features associated with PD. We aimed to further characterize the disease-like phenotype of these En1+/– mice with focus on early neurodegenerative changes that can be utilized to score efficacy of future disease modifying studies. We observed early terminal defects in the dopaminergic nigrostriatal pathway in En1+/– mice. Several weeks before a significant loss of dopaminergic neurons in the substantia nigra (SN) could be detected, we found that striatal terminals expressing high levels of dopaminergic neuron markers TH, VMAT2, and DAT were dystrophic and swollen. Using transmission electron microscopy, we identified electron dense bodies consistent with abnormal autophagic vacuoles in these terminal swellings. In line with these findings, we detected an up-regulation of the mTOR pathway, concurrent with a downregulation of the autophagic marker LC3B, in ventral midbrain and nigral dopaminergic neurons of En1+/– mice. This supports the notion that autophagic protein degradation is reduced in the absence of one En1 allele. We imaged the nigrostriatal pathway using the CLARITY technique and observed many fragmented axons in the medial forebrain bundle of En1+/ – mice, consistent with axonal maintenance failure. Using in vivo electrochemistry, we found that nigrostriatal terminals in the dorsal striatum were severely deficient in dopamine release and reuptake. Our findings support a progressive retrograde degeneration of En1+/– nigrostriatal neurons, akin to what is suggested to occur in PD. We suggest that using En1+/– mice as a model will provide further key insights into PD pathogenesis, and propose that axon terminal integrity and function can be utilized to estimate dopaminergic neuron health and efficacy of experimental PD therapies.

Is Axonal Degeneration a Key Early Event in Parkinson’s Disease?

“…All of them present some form of axonal abnormalities in TH-positive nigrostriatal fibers before the degenerative processes are detected in the cell bodies. Interestingly, very similar manifestation of early axonopathy is present in all of them: 'swollen' structures called 'spheroids' or 'axonal swellings' appearing in the terminal parts [10,11,13,15,17] or medial forebrain bundle portions of nigrostriatal axons [12,14,15]. Similar axonal spheroids were described in the post-mortem brains of PD [18] and Diffuse Lewy Body Disease patients [19], and they are also seen in other neurodegenerative diseases such as multiple sclerosis (MS).…”

“…This research was elegantly summarized by Dr. Robert Burke in his talk and recent review [9]. The models that were discussed include: AAV/2 human a-syn-A53T in rat [10], AAV/6 human a-syn-WT in rat [11], hBAC-LRRK2 in mouse [12,13], mBAC-Nurr1(fl/fl)XmBACDAT-CreT2 in mouse [14] and Engrailed1 +/-mouse [15,16], and it is likely that there are other models where such changes are yet to be reported. All of them present some form of axonal abnormalities in TH-positive nigrostriatal fibers before the degenerative processes are detected in the cell bodies.…”

“…Similar axonal spheroids were described in the post-mortem brains of PD [18] and Diffuse Lewy Body Disease patients [19], and they are also seen in other neurodegenerative diseases such as multiple sclerosis (MS). In aforementioned rodent models, the axo-degenerative processes are accompanied by other axonal abnormalities that mimic findings made in PD, such as loss of dopaminergic phenotype [11,14,15], changes in levels of axonal transport proteins [10], and autophagy markers [15]. 'It is important to emphasize that although this evidence suggests that axons are the first site of degenerative change, this does not necessarily mean that the primary disturbance at the molecular level first occurs in the axons.…”

“…The lack of one Engrailed-1 allele causes slow progressive neurodegeneration of nigrostriatal pathway. The axonal 'swellings' stained by TH, dopamine transporter (DAT) and VMAT appear in the striatum in small numbers as early as 8 days after birth and their size and number increases with age [15,20]. A reduction in tissue levels of dopamine in the striatum is not detected until 8 weeks, and it is followed by degeneration of dopaminergic neurons in substantia nigra [15,16].…”

“…The nigral neurons that project to the most dorsal striatum also exhibit profound reductions in evoked dopamine release and reuptake. Changes in autophagy pathways have been defined in Engrailed1-deficient mice [15], as well as increased sensitivity to oxidative stress [21], abnormalities in mitochondrial function [22] and axonal guidance [23]. All of these features might contribute to the axonal degeneration (see Fig.…”