We generated tissue specific Atg7 conditional knockout mice using the Cre/lox technique. Atg7flox/flox mice were bred with mice expressing Cre recombinase downstream of either the dopamine transporter (DAT) or Engrailed-1 (En) promoters. Engrailed-1 expresses Cre widely in the hindbrain, cerebellum, and midbrain, including the dopaminergic cells of the SNpc and ventral tegmental area (VTA) whereas the DAT promoter drives Cre exclusively in dopaminergic cells (). A reduction in the levels of Atg7 protein in Atg7flox/flox; En-Cre mice verified appropriate gene targeting and inactivation in the model system (). Although Engrailed-1 is expressed widely, there are cells and cell processes in those regions where the En promoter is not active. As a result, there is residual Atg7 immunoreactivity in those protein lysates.
Figure 1 Generation of mice with the conditional deletion of Atg7. A, Cre recombinase activity (blue coloration) in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), hindbrain (HB), and cerebellum (CB) of mice expressing Cre downstream of (more ...)
Mice with the conditional Atg7 deletion are born healthy and indistinguishable from control littermates. Although the survival rate of Atg7flox/flox; DAT-Cre mice is comparable to the Atg7flox/flox controls, Atg7flox/flox; En-Cre mice displayed a 76% reduction in survival by one year of age (). The more widespread loss of autophagic function in the Atg7flox/flox; En-Cre mice compared to the isolated effect on dopamine neurons in the Atg7flox/flox; DAT-Cre mice is the likely cause of increased mortality.
In addition, we performed a locomotor activity analysis of these mice as a measure of bradykinesia. Paradoxically the Atg7flox/flox; DAT-Cre mice were found to be more active than control mice during the wake cycle (). This trend continued in older mice as well, but was no longer statistically significant when compared to controls. Interestingly, the Atg7flox/flox; En-Cre mice displayed decreased locomotor activity, tremulousness, weight loss and a wide-based and ataxic gait presumably due to the additional brainstem, midbrain, spinal cord, and cerebellar pathology (data not shown).
The effect of Atg7 deficiency on dopaminergic neuron survival was investigated using immunohistochemical techniques and stereological analysis. We observed an age-dependent loss of Tyrosine hydroxylase (TH) immunoreactive cells in the SNpc of mice with the conditional deletion of Atg7
(). Adult Atg7flox/flox
; DAT-Cre and Atg7flox/flox
; En-Cre mice displayed a 40% and 60% loss of SNpc dopamine neurons, respectively, compared to adult Atg7
control mice (). The corresponding decrease in Nissl positive neuron count and lack of significant difference in age and genotype-matched Nissl and TH counts further support the loss of neurons rather than loss of cellular TH immunoreactivity (). Despite a trend toward fewer cells, Atg7-deficient juvenile mice did not show a significant loss of TH neurons compared to controls (). Consistent with these data, HPLC analysis confirmed an age-dependent decrease in striatal dopamine content in the affected adult brain (). We detected a 55.5% and a 64.5% reduction in striatal dopamine in adult Atg7flox/flox
; DAT-Cre and Atg7flox/flox
; En-Cre mice, respectively, whereas a significant decrease in striatal dopamine was not observed in juvenile mice. These results suggest that dopamine neurons are sensitive to impaired autophagy in vivo
and are associated with a progressive age-dependent cell loss. Autophagy impairment in the SNpc also resulted in a modest increase in reactive astrocytes as indicated by GFAP staining (). Additionally, as expected from previous work, we observed Purkinje cell loss in the Atg7flox/flox
; En-Cre mice (Komatsu et al., 2006
Figure 2 Progressive loss of TH neurons. A, Impaired autophagy induced TH positive neuronal cell loss in adult Atg7 flox/flox; DAT-Cre mice compared to an age-matched Atg7 flox/flox control. Scale bar, 500 μm. B, Stereological analysis of TH positive cell (more ...)
Given the PD-like neuronal loss in Atg7 deficient mice, we examined the tissue for the presence of inclusions using hematoxylin and eosin (H&E) staining of paraffin-embedded SNpc sections. Neuronal inclusions are present in the SNpc of Atg7-deficient mice, but not Atg7flox/flox
controls (). The inclusions were often perinuclear, but also appeared in the neuropil. They stained intensely for ubiquitin and for the ubiquitin-binding protein and autophagy substrate p62 (Komatsu et al., 2007
). Interestingly, although we did not observe significant cell loss in the SNpc of juvenile Atg7flox/flox
; DAT-Cre mice, inclusions were present and were significantly smaller (1.1 ± 0.23 vs. 1.7± 0.43 μm average diameter, P=0.0013) than those seen at older ages (). Similar inclusions were detected in Atg7flox/flox
; En-Cre mice (). We verified the presence of ubiquitin using immunoblots and demonstrated an increase in polyubiquitinated proteins in the SDS-soluble fractions of midbrain/hindbrain lysates of Atg7flox/flox
; En-Cre mice compared to control mice while ubiquitin levels remained unchanged in the cortex of the Atg7 conditional knockout mice ().
Figure 3 Detection of inclusions in Atg7 conditional knockout mice. A, Inclusions (arrowheads) are present in the Atg7 conditional knockout sections of 10-month-old mice. Ubiquitin (Ub) and p62 positive inclusions are present in the cytoplasm of neuronal cells. (more ...)
As polyubiquitin is known to exist in various forms depending on the lysine residue involved in the linkage, we sought to determine whether a particular ubiquitin linkage serves as a specific autophagy signal using linkage-specific antibodies (Newton et al., 2008
). The anti-K48-linked polyubiquitin antibody stains inclusions within dopamine neurons in the SNpc of Atg7flox/flox
; DAT-Cre mice and in Atg7flox/flox
; En-Cre mice (). The accumulation of this form of ubiquitin was confirmed in midbrain/hindbrain lysates of Atg7flox/flox
; En-Cre mice using immunoblots (). Based on the assessment of two animals, there was a 54% increase in total K48 ubiquitin and a 45% increase in high molecular weight K48 ubiquitin in Atg7flox/flox
; En-Cre mice compared to Atg7flox/flox
controls. Staining for K63-linked ubiquitin in autophagy deficient mice suggests little co-localization of p62 and K63-linked polyubiquitin in aggregates of dopamine neurons in the SNpc of the conditional knockouts (data not shown). Western blot analysis of K63-linked polyubiquitin in Atg7flox/flox and Atg7flox/flox
; En-Cre midbrain/hindbrain lysates indicates the K63 protein is not increased in Atg7 deficient animals compared to control.
Figure 4 Ubiquitin-linked protein changes and α-synuclein accumulation resulting from autophagy impairment. A, Co-localization of p62 and K48-linked polyubiquitin in aggregates within dopamine neurons of the SNpc in both Atg7 flox/flox; DAT-Cre and Atg7 (more ...)
Inclusions containing p62 were observed in the dopaminergic cells of Atg7-deficient mice and in addition were found to co-localize with K48-linked polyubiquitin (). The accumulation of this protein was verified using immunoblots showing a dramatic increase in p62 levels in midbrain/hindbrain lysates taken from autophagy deficient mice (). Although α-synuclein inclusions were not present (data not shown), using immunoblot analysis, we demonstrated elevated levels of low molecular weight (14kDa) α-synuclein in the brains of mice with impaired autophagy (). This provides in vivo evidence that α-synuclein levels are influenced by the process of autophagy.