Early Parkinson's disease symptoms in α-synuclein transgenic monkeys

Abstract

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that can be caused by genetic mutations in α-synuclein (α-syn) or duplication of wild-type α-syn; PD is characterized by the deposition of α-syn aggregates, indicating a gain of toxicity from accumulation of α-syn. Although the major neuropathologic feature of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra, non-motor symptoms including anxiety, cognitive defect and sleep disorder precede the onset of motor impairment, and many clinical symptoms of PD are not caused by degeneration of DA neurons. Non-human primate models of PD are important for revealing the early pathology in PD and identifying effective treatments. We established transgenic PD rhesus monkeys that express mutant α-syn (A53T). Six transgenic A53T monkeys were produced via lentiviral vector expressing A53T in fertilized monkey eggs and subsequent embryo transfer to surrogates. Transgenic A53T is expressed in the monkey brain and causes age-dependent non-motor symptoms, including cognitive defects and anxiety phenotype, without detectable sleeping disorders. The transgenic α-syn monkeys demonstrate the specific early symptoms caused by mutant α-syn and provide insight into treatment of early PD.

Figure 1.

Expression of lentiviral A53T vector in rodent neuronal cells. (A) A53T is linked with ECFP via F2A and expressed under the human ubiquitin (hUBC) promoter in the lentiviral vector. (B) Cultured mouse striatal neurons transduced by lentiviral A53T show positive staining by anti-α-syn. Scale bar: 10 µm.

Figure 2.

Generation of transgenic A53T monkeys. (A) The numbers of embryos and surrogates used and live transgenic monkeys generated. (B) Picture of monkey 110217, which was the first newborn transgenic A53T monkey. (C) PCR verified the integration of the transgenic A53T DNA into the monkey DNAs in different tissues of a monkey aborted on gestation day 170.

Figure 3.

PCR genotyping and copy numbers of transgenic A53T monkeys. (A) The PCR products from seven live transgenic A53T monkeys and one wild-type monkey were obtained with two sets of primers specific to transgenic A53T-ECFP construct. β-Actin DNA served as an internal control for normalization and the endogenous monkey α-syn gene was normalized as two copies. (B) The general information of transgenic monkeys and assessment of copy numbers of transgenic A53T in monkeys via quantitative PCR.

Figure 4.

Expression of transgenic A53T in monkey tissues. (A) Western blotting of different tissues from WT and the transgenic A53T monkey that was aborted on gestation day 170. The blots were probed with antibodies to ECFP, α-syn and GAPDH. ECFP-A53T fusion protein, A53T, and ECFPN are indicated on the blots. Note that A53T (arrow) above a nonspecific band (arrowhead) and ECFP are more abundant than the non-cleaved A53T-ECFP. The control is an aborted wild-type monkey. (B) Anti-α-syn immunostaining of the substantia nigra of wild-type monkey and the A53T transgenic monkey that died from a difficult birth. Hematoxylin staining (blue) was used for nuclear counterstaining. The brain from a stillborn wild-type monkey served as a control. Low magnification micrographs (10×) are presented. (C) Anti-α-syn immunostaining (40×) of the substantia nigra, striatum and cortex of transgenic A53T monkey. Neuronal cells expressing transgenic A53T were intensively labeled by anti-α-syn in their cell bodies and neurites. Scale bars: 100 µm (B), 10 µm (C).

Figure 5.

Soluble transgenic A53T in the brain of a newborn transgenic A53T monkey. (A) Double immunostaining of the monkey striatum showing that the neuron expressing transgenic A53T is also labeled by anti-NeuN, a neuronal marker. Scale bar: 10 µm. (B) The substantia nigra of a stillborn transgenic A53T monkey was stained by anti-α-syn (left panel) and anti-S129 (right panel). Note that only soluble A53T was seen in neuronal cells and anti-S129 (1:100 dilution) did not show noticeable staining signals. (C) Quantitation of the numbers of neurons per image (400×) that were labeled by anti-α-syn. Control is the result from a non-transgenic wild-type stillborn monkey. The staining result with anti-S129 was also included. ***P < 0.001 compared with the control.

Figure 6.

Behavioral examination of control and transgenic A53T monkeys. (A and B) Photos of a male transgenic A53T monkey (110217), which was examined for finding food and cognitive function (A) and picking up candy in a spinning plate (B). (C and D) Two A53T transgenic female monkeys (120666 and 120676) at 1.5 year of age and their age-matched wild-type controls (120660, 120671 and 120696), the oldest A53T transgenic monkey (110217, 2.5 year old) and three 2.5 year-old wild-type male monkeys (110215, 110225, and 11058), and GFP transgenic monkey (08431) at 5 years of age were examined. Times (s) for the control and A53T monkeys to pick up food in the red box among five different colored boxes are shown in (C). The numbers of failures to immediately pick up candies in a rotating plate per test are presented in (D). The GFP transgenic monkey was not included in this test since his fingers were too large to fit into the holes in the spinning plate. The results (mean ± SEM) were obtained from repeated experiments (n = 8–12) after training.

Figure 7.

Depressive and anxiety-like phenotype of A53T transgenic monkeys. (A) Depressive and anxiety-related stereotypic phenotype of the oldest A53T monkey (110217). The frequency and duration (mean ± SEM) of circling behavior over the course of an hour were obtained by video recording. The monkeys were examined for 7 consecutive days. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control monkeys. (B) Lack of sleep disorders in transgenic A53T monkeys. Quantitative analysis of the frequency and duration of wake-up of monkeys from late night to early morning (11:00 pm–6:00 am). The oldest A53T monkey (110217) and its age-matched control (110215) were observed for 5–6 consecutive days for each experiment. The results from three experiments are presented.

Figure 8.

Magnetic resonance imaging (MRI) of the brain regions in 32-month-old wild type and A53T transgenic (110217) monkeys. Three scanned coronal images in each monkey are presented. The region containing the substantia nigra is indicated by arrows. No significant difference is seen between these two monkeys.