Striatonigral distribution of a fluorescent reporter following intracerebral delivery of genome editors.

Introduction: Targeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) project to the striatum, and the striatum contains neurons that project to the substantia nigra pars reticulata (SNpr). Methods: Here, we report the effect of injecting genome editors into the striatum of Ai14 reporter mice, which have a LoxP-flanked stop cassette that prevents expression of the red fluorescent protein tdTomato. Two weeks following intracerebral delivery of either synthetic nanocapsules (NCs) containing CRISPR ribonucleoprotein targeting the tdTomato stop cassette or adeno-associated virus (AAV) vectors expressing Cre recombinase, the brains were collected, and the presence of tdTomato was assessed in both the striatum and SN. Results: TdTomato expression was observed at the injection site in both the NC- and AAV-treated groups and typically colocalized with the neuronal marker NeuN. In the SN, tdTomato-positive fibers were present in the pars reticulata, and SNpr area expressing tdTomato correlated with the size of the striatal genome edited area. Conclusion: These results demonstrate in vivo anterograde axonal transport of reporter gene protein products to the SNpr following neuronal genome editing in the striatum.

Expression of tau and phosphorylated tau in the brain of normal and hemiparkinsonian rhesus macaques.

Tau is a neuronal protein involved in microtubule stabilization and intracellular vesicle transport in axons. In neurodegenerative disorders termed "tauopathies," like Alzheimer's and Parkinson's disease, tau becomes hyperphosphorylated and forms intracellular inclusions. Rhesus macaques are widely used for studying ageing processes and modeling neurodegenerative disorders, yet little is known about endogenous tau expression in their brains. In this study, immunohistochemical methods were used to map and characterize total tau, 3R- and 4R-tau isoforms, and phosphorylated tau (pThr231-tau and pSer202/Thr205-tau/AT8) expression bilaterally in 16 brain regions of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced hemiparkinsonian adult rhesus macaques. Tau-immunoreactivity (-ir), including both 3R and 4R isoforms, was observed throughout the brain, with varying regional intensities. The anterior cingulate cortex, entorhinal cortex, and hippocampus displayed the most robust tau-ir, while the subthalamic nucleus and white matter regions had minimal expression. Tau was present in neurons of gray matter regions; it was preferentially observed in fibers of the globus pallidus and substantia nigra and in cell bodies of the thalamus and subthalamic nucleus. In white matter regions, tau was abundantly present in oligodendrocytes. Additionally, neuronal pThr231-tau-ir was abundant in all brain regions, but not AT8-ir. Differences in regional and intracellular protein expression were not detected between control subjects and both brain hemispheres of MPTP-treated animals. Specifically, tau-ir in the substantia nigra of all subjects colocalized with GABAergic neurons. Overall, this report provides an in-depth characterization of tau expression in the rhesus macaque brain to facilitate future investigations for understanding and modeling tau pathology in this species.

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.

Alpha-synuclein and tau are abundantly expressed in the ENS of the human appendix and monkey cecum.

α-Synuclein (α-syn) proteinopathy in the neurons of the Enteric Nervous System (ENS) is proposed to have a critical role in Parkinson's disease (PD) onset and progression. Interestingly, the ENS of the human appendix harbors abundant α-syn and appendectomy has been linked to a decreased risk and delayed onset of PD, suggesting that the appendix may influence PD pathology. Common marmosets and rhesus macaques lack a distinct appendix (a narrow closed-end appendage with a distinct change in diameter at the junction with the cecum), yet the cecal microanatomy of these monkeys is similar to the human appendix. Sections of human appendix (n = 3) and ceca from common marmosets (n = 4) and rhesus macaques (n = 3) were evaluated to shed light on the microanatomy and the expression of PD-related proteins. Analysis confirmed that the human appendix and marmoset and rhesus ceca present thick walls comprised of serosa, muscularis externa, submucosa, and mucosa plus abundant lymphoid tissue. Across all three species, the myenteric plexus of the ENS was located within the muscularis externa with nerve fibers innervating all layers of the appendix/ceca. Expression of α-syn and tau in the appendix/cecum was present within myenteric ganglia and along nerve fibers of the muscularis externa and mucosa in all species. In the myenteric ganglia α-syn, p-α-syn, tau and p-tau immunoreactivities (ir) were not significantly different across species. The percent area above threshold of α-syn-ir and tau-ir in the nerve fibers of the muscularis externa and mucosa were greater in the human appendix than in the NHP ceca (α-syn-ir p<0.05; tau-ir p<0.05). Overall, this study provides critical translational evidence that the common marmoset and rhesus macaque ceca are remarkably similar to the human appendix and, thus, that these NHP species are suitable for studying the development of PD linked to α-syn and tau pathological changes in the ENS.

Myelin Basic Protein and Cardiac Sympathetic Neurodegeneration in Nonhuman Primates.

Minimal myelination is proposed to be a contributing factor to the preferential nigral neuronal loss in Parkinson's disease (PD). Similar to nigral dopaminergic neurons, sympathetic neurons innervating the heart have long, thin axons which are unmyelinated or minimally myelinated. Interestingly, cardiac sympathetic loss in PD is heterogeneous across the heart, yet the spatial relationship between myelination and neurodegeneration is unknown. Here, we report the mapping of myelin basic protein (MBP) expression across the left ventricle of normal rhesus macaques (n = 5) and animals intoxicated with systemic 6-OHDA (50 mg/kg iv) to model parkinsonian cardiac neurodegeneration (n = 10). A subset of 6-OHDA-treated rhesus received daily dosing of pioglitazone (5 mg/kg po; n = 5), a PPARγ agonist with neuroprotective properties. In normal animals, MBP-immunoreactivity (-ir) was identified surrounding approximately 14% of axonal fibers within nerve bundles of the left ventricle, with more myelinated nerve fibers at the base level of the left ventricle than the apex (p < 0.014). Greater MBP-ir at the base was related to a greater number of nerve bundles at that level relative to the apex (p < 0.05), as the percent of myelinated nerve fibers in bundles was not significantly different between levels of the heart. Cardiac sympathetic loss following 6-OHDA was associated with decreased MBP-ir in cardiac nerve bundles, with the percent decrease of MBP-ir greater in the apex (84.5%) than the base (52.0%). Interestingly, cardiac regions and levels with more MBP-ir in normal animals showed attenuated sympathetic loss relative to areas with less MBP-ir in 6-OHDA + placebo (r = -0.7, p < 0.014), but not in 6-OHDA + pioglitazone (r = -0.1) subjects. Our results demonstrate that myelination is present around a minority of left ventricle nerve bundle fibers, is heterogeneously distributed in the heart of rhesus macaques, and has a complex relationship with cardiac sympathetic neurodegeneration and neuroprotection.

Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys.

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD1. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues1,2. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.

Acute Exposure to the Food-Borne Pathogen Listeria monocytogenes Does Not Induce α-Synuclein Pathology in the Colonic ENS of Nonhuman Primates.

INTRODUCTION: Gastrointestinal (GI) inflammation elicited by environmental factors is proposed to trigger Parkinson's disease (PD) by stimulating accumulation of pathological α-synuclein (α-syn) in the enteric nervous system (ENS), which then propagates to the central nervous system via the vagus nerve. The goal of this study was to model, in nonhuman primates, an acute exposure to a common food-borne pathogen in order to assess whether the related acute GI inflammation could initiate persistent α-syn pathology in the ENS, ultimately leading to PD. METHODS: Adult female cynomolgus macaques were inoculated by oral gavage with 1×108 colony-forming units (CFUs) Listeria monocytogenes (LM, n=10) or vehicle (mock, n=3) and euthanized 2 weeks later. Evaluations included clinical monitoring, blood and fecal shedding of LM, and postmortem pathological analysis of colonic and cecal tissues. RESULTS: LM inoculation of healthy adult cynomolgus macaques induced minimal to mild clinical signs of infection; LM shedding in feces was not seen in any of the animals nor was bacteremia detected. Colitis varied from none to moderate in LM-treated subjects and none to minimal in mock-treated subjects. Expression of inflammatory markers (HLA-DR, CD3, CD20), oxidative stress (8-OHDG), α-syn, and phosphorylated-α-syn in the enteric ganglia was not significantly different between treatment groups. DISCUSSION: Our results demonstrate that cynomolgus macaques orally inoculated with LM present with a clinical response that resembles human LM exposure. They also suggest that acute exposure to food-borne pathogens is not sufficient to induce significant and persistent α-syn changes in healthy adult female subjects. Based on the results of this limited experimental setting, we propose that, if LM has a role in PD pathology, other underlying factors or conditions, such as male sex, inflammatory bowel disease, exposure to toxins, dysbiosis, and/or aging, are needed to be present.

Post mortem evaluation of inflammation, oxidative stress, and PPARγ activation in a nonhuman primate model of cardiac sympathetic neurodegeneration.

Cardiac dysautonomia is a common nonmotor symptom of Parkinson's disease (PD) associated with loss of sympathetic innervation to the heart and decreased plasma catecholamines. Disease-modifying strategies for PD cardiac neurodegeneration are not available, and biomarkers of target engagement are lacking. Systemic administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) recapitulates PD cardiac dysautonomia pathology. We recently used positron emission tomography (PET) to visualize and quantify cardiac sympathetic innervation, oxidative stress, and inflammation in adult male rhesus macaques (Macaca mulatta; n = 10) challenged with 6-OHDA (50mg/kg; i.v.). Twenty-four hours post-intoxication, the animals were blindly and randomly assigned to receive daily doses of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n = 5; 5mg/kg p.o.) or placebo (n = 5). Quantification of PET radioligand uptake showed increased oxidative stress and inflammation one week after 6-OHDA which resolved to baseline levels by twelve weeks, at which time pioglitazone-treated animals showed regionally preserved sympathetic innervation. Here we report post mortem characterization of heart and adrenal tissue in these animals compared to age and sex matched normal controls (n = 5). In the heart, 6-OHDA-treated animals showed a significant loss of sympathetic nerve fibers density (tyrosine hydroxylase (TH)-positive fibers). The anatomical distribution of markers of sympathetic innervation (TH) and inflammation (HLA-DR) significantly correlated with respective in vivo PET findings across left ventricle levels and regions. No changes were found in alpha-synuclein immunoreactivity. Additionally, CD36 protein expression was increased at the cardiomyocyte intercalated discs following PPARγ-activation compared to placebo and control groups. Systemic 6-OHDA decreased adrenal medulla expression of catecholamine producing enzymes (TH and aromatic L-amino acid decarboxylase) and circulating levels of norepinephrine, which were attenuated by PPARγ-activation. Overall, these results validate in vivo PET findings of cardiac sympathetic innervation, oxidative stress, and inflammation and illustrate cardiomyocyte CD36 upregulation as a marker of PPARγ target engagement.

Colonic inflammation affects myenteric alpha-synuclein in nonhuman primates.

Background: Parkinson's disease (PD) patients frequently present gastrointestinal (GI) dysfunction that, in many cases, predates the onset of motor symptoms. In PD, the presynaptic protein alpha-synuclein (α-syn) undergoes pathological changes, including phosphorylation and aggregation leading to the formation of Lewy bodies, which can be found in neurons of the enteric nervous system (ENS). Inflammation has been proposed as a possible trigger of α-syn pathology. Interestingly, patients with inflammatory bowel disease and irritable bowel syndrome, conditions associated with GI inflammation, are at higher risk of developing PD. Captive common marmosets (Callithrix jacchus) develop colitis, providing a natural platform to assess the relationship between α-syn pathology and GI inflammation. Materials and Methods: Sections of proximal colon from marmosets with colitis (n=5; 5.3±2.3 years old; 4 male) and normal controls (n=5; 4.1±1.6 years old; 1 male) were immunostained against protein gene product 9.5 (PGP9.5), human leukocyte antigen DR (HLA-DR), cluster of differentiation 3 (CD3), cluster of differentiation 20 (CD20), glial fibrillary acidic protein (GFAP), 8-hydroxy-2'-deoxyguanosine (8-OHdG), α-syn, and serine 129 phosphorylated α-syn (p-α-syn). Immunoreactivity of each staining in the myenteric plexus was quantified using NIH ImageJ software. Results: Marmosets with colitis had significantly increased expression of inflammatory markers (HLA-DR, p<0.02; CD3, p<0.008), oxidative stress (8-OHdG, p<0.05), and p-α-syn (p<0.02) and decreased expression of α-syn (p<0.04) in the colonic myenteric ganglia compared to normal, healthy controls. Conclusion: Colonic inflammation is associated with changes in α-syn expression and phosphorylation in the myenteric plexus of common marmosets. Future evaluation of the vagus nerve and brain of animals with colitis will be key to assess the contribution of colitis-induced ENS α-syn pathology to PD-like pathology in the brain.

In vivo imaging of inflammation and oxidative stress in a nonhuman primate model of cardiac sympathetic neurodegeneration.

Loss of cardiac postganglionic sympathetic innervation is a characteristic pathology of Parkinson's disease (PD). It progresses over time independently of motor symptoms and is not responsive to typical anti-parkinsonian therapies. Cardiac sympathetic neurodegeneration can be mimicked in animals using systemic dosing of the neurotoxin 6-hydroxydopamine (6-OHDA). As in PD, 6-OHDA-induced neuronal loss is associated with increased inflammation and oxidative stress. To assess the feasibility of detecting changes over time in cardiac catecholaminergic innervation, inflammation, and oxidative stress, myocardial positron emission tomography with the radioligands [11C]meta-hydroxyephedrine (MHED), [11C]PBR28 (PBR28), and [61Cu]diacetyl-bis(N(4))-methylthiosemicarbazone (ATSM) was performed in 6-OHDA-intoxicated adult, male rhesus macaques (n = 10; 50 mg/kg i.v.). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone, which is known to have anti-inflammatory and anti-oxidative stress properties, was administered to five animals (5 mg/kg, PO); the other five were placebo-treated. One week after 6-OHDA, cardiac MHED uptake was significantly reduced in both groups (placebo, 86% decrease; pioglitazone, 82%); PBR28 and ATSM uptake increased in both groups but were attenuated in pioglitazone-treated animals (PBR28 Treatment × Level ANOVA p < 0.002; ATSM Mann-Whitney p = 0.032). At 12 weeks, partial recovery of MHED uptake was significantly greater in the pioglitazone-treated group, dependent on left ventricle circumferential region and axial level (Treatment × Region × Level ANOVA p = 0.034); 12-week MHED uptake significantly correlated with tyrosine hydroxylase immunoreactivity across cardiac anatomy (p < 0.000002). PBR28 and ATSM uptake returned to baseline levels by 12 weeks. These radioligands thus hold potential as in vivo biomarkers of mechanisms of cardiac neurodegeneration and neuroprotection.

A monoclonal antibody-GDNF fusion protein is not neuroprotective and is associated with proliferative pancreatic lesions in parkinsonian monkeys.

Glial cell line derived neurotrophic factor (GDNF) is a neurotrophic factor that has neuroprotective effects in animal models of Parkinson's disease (PD) and has been proposed as a PD therapy. GDNF does not cross the blood brain barrier (BBB), and requires direct intracerebral delivery to be effective. Trojan horse technology, in which GDNF is coupled to a monoclonal antibody (mAb) against the human insulin receptor (HIR), has been proposed to allow GDNF BBB transport (ArmaGen Technologies Inc.). In this study we tested the feasibility of HIRMAb-GDNF to induce neuroprotection in parkinsonian monkeys, as well as its tolerability and safety. Adult rhesus macaques were assessed throughout the study with a clinical rating scale, a computerized fine motor skills task and general health evaluations. Following baseline measurements, the animals received a unilateral intracarotid artery MPTP injection. Seven days later the animals were evaluated, matched according to disability and blindly assigned to receive twice a week i.v. treatments (vehicle, 1 or 5 mg/kg HIRmAb-GDNF) for a period of three months. HIRmAb-GDNF did not improve parkinsonian motor symptoms and induced a dose-dependent hypersensitivity reaction. Quantification of dopaminergic striatal optical density and stereological nigral cell counts did not demonstrate differences between treatment groups. Focal pancreatic acinar to ductular metaplasia (ADM) was noted in four of seven animals treated with 1 mg/kg HIRmAb-GDNF; two of four with ADM also had focal pancreatic intraepithelial neoplasia 1B (PanIN-1B) lesions. Minimal to mild, focal to multifocal, nonsuppurative myocarditis was noted in all animals in the 5 mg/kg treatment group. Our results demonstrate that HIRmAb-GDNF dosing in a monkey model of PD is not an effective neuroprotective strategy and may present serious health risks that should be considered when planning future use of the IR antibody as a carrier, or of any systemic treatment of a GDNF-containing molecule.

The PPAR-γ agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys.

BACKGROUND: Activation of the peroxisome proliferator-activated receptor gamma (PPAR-γ) has been proposed as a possible neuroprotective strategy to slow down the progression of early Parkinson's disease (PD). Here we report preclinical data on the use of the PPAR-γ agonist pioglitazone (Actos®; Takeda Pharmaceuticals Ltd.) in a paradigm resembling early PD in nonhuman primates. METHODS: Rhesus monkeys that were trained to perform a battery of behavioral tests received a single intracarotid arterial injection of 20 ml of saline containing 3 mg of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Twenty-four hours later the monkeys were assessed using a clinical rating scale, matched accordingly to disability, randomly assigned to one of three groups [placebo (n = 5), 2.5 (n = 6) or 5 (n = 5) mg/kg of pioglitazone] and their treatments started. Three months after daily oral dosing, the animals were necropsied. RESULTS: We observed significant improvements in clinical rating score (P = 0.02) in the animals treated with 5 mg/kg compared to placebo. Behavioral recovery was associated with preservation of nigrostriatal dopaminergic markers, observed as higher tyrosine hydroxylase (TH) putaminal optical density (P = 0.011), higher stereological cell counts of TH-ir (P = 0.02) and vesicular monoamine transporter-2 (VMAT-2)-ir nigral neurons (P = 0.006). Stereological cell counts of Nissl stained nigral neurons confirmed neuroprotection (P = 0.017). Pioglitazone-treated monkeys also showed a dose-dependent modulation of CD68-ir inflammatory cells, that was significantly decreased for 5 mg/kg treated animals compared to placebo (P = 0.018). A separate experiment to assess CSF penetration of pioglitazone revealed that 5 mg/kg p.o. induced consistently higher levels than 2.5 mg/kg and 7.5 mg/kg. p.o. CONCLUSIONS: Our results indicate that oral administration of pioglitazone is neuroprotective when administered early after inducing a parkinsonian syndrome in rhesus monkeys and supports the concept that PPAR-γ is a viable target against neurodegeneration.