Real-time trajectory guide tracking for intraoperative MRI-guided neurosurgery.

PURPOSE: In current intraoperative MRI (IMRI) methods, an iterative approach is used to aim trajectory guides at intracerebral targets: image MR-visible features, determine current aim by fitting model to image, manipulate device, repeat. Infrequent updates are produced by such methods, compared to rapid optically tracked stereotaxy used in the operating room. Our goal was to develop a real-time interactive IMRI method for aiming. METHODS: The current trajectory was computed from two points along the guide's central axis, rather than by imaging the entire device. These points were determined by correlating one-dimensional spokes from a radial sequence with the known cross-sectional projection of the guide. The real-time platform RTHawk was utilized to control MR sequences and data acquisition. On-screen updates were viewed by the operator while simultaneously manipulating the guide to align it with the planned trajectory. Accuracy was quantitated in a phantom, and in vivo validation was demonstrated in nonhuman primates undergoing preclinical gene ( n = 5 $$ n=5 $$ ) and cell ( n = 4 $$ n=4 $$ ) delivery surgeries. RESULTS: Updates were produced at 5 Hz In 10 phantom experiments at a depth of 48 mm, the cannula tip was placed with radial error of (min, mean, max) = (0.16, 0.29, 0.68) mm. Successful in vivo delivery of payloads to all 14 targets was demonstrated across nine surgeries with depths of (min, mean, max) = (33.3, 37.9, 42.5) mm. CONCLUSION: A real-time interactive update rate was achieved, reducing operator fatigue without compromising accuracy. Qualitative interpretation of images during aiming was rendered unnecessary by objectively computing device alignment.

The role of nonhuman primate models in the development of cell-based therapies for Parkinson's disease.

Through the course of over three decades, nonhuman primate (NHP) studies on cell-based therapies (CBTs) for Parkinson's disease (PD) have provided insight into the feasibility, safety and efficacy of the approach, methods of cell collection and preparation, cell viability, as well as potential brain targets. Today, NHP research continues to be a vital source of information for improving cell grafts and analyzing how the host affects graft survival, integration and function. Overall, this article aims to discuss the role that NHP models of PD have played in CBT development and highlights specific issues that need to be considered to maximize the value of NHP studies for the successful clinical translation of CBTs.

Internalized α-synuclein fibrils become truncated and resist degradation in neurons while glial cells rapidly degrade α-synuclein fibrils.

Parkinson's disease (PD) and other α-synucleinopathies are characterized by the accumulation of α-synuclein (αS) pathology that can spread via the cell-to-cell transmission of αS aggregates. To better understand how various brain cells contribute to the spreading of αS pathology, we examined the metabolism of αS aggreges or pre-formed fibrils (PFFs) in neuronal and glial cells (microglia, astrocytes, and oligodendrocytes). In neurons, while the full-length αS rapidly disappeared following αS PFF uptake, truncated αS accumulated with a half-life of days rather than hours. Epitope mapping and fractionation studies indicate that αS PFF was truncated at the C-terminal region following uptake and remained insoluble/aggregated. In contrast, microglia and astrocytes rapidly metabolized αS PFF as the half-lives of αS PFF in these glial cells were <6 hours. Differential processing of αS by neurons was recapitulated in cell lines as differentiated CLU neuronal cell lines stably accumulate truncated αS while undifferentiated cells rapidly metabolize αS. Immunolocalization and subcellular fractionation studies show that internalized αS PFF is initially localized to endosomes followed by lysosomes. The lysosome is largely responsible for the degradation of internalized αS PFF as the inhibition of lysosomal function leads to the stabilization of αS in all cell types. Significantly, αS PFF causes lysosomal dysfunction in neurons. In summary, we show that neurons are inefficient in metabolizing internalized αS aggregates, partially because αS aggregates cause lysosomal dysfunction, potentially generating aggregation-prone truncated αS. In contrast, glial cells may protect neurons from αS aggregates by rapidly clearing αS aggregates.

Loss of tau expression attenuates neurodegeneration associated with α-synucleinopathy.

BACKGROUND: Neuronal dysfunction and degeneration linked to α-synuclein (αS) pathology is thought to be responsible for the progressive nature of Parkinson's disease and related dementia with Lewy bodies. Studies have indicated bidirectional pathological relationships between αS pathology and tau abnormalities. We recently showed that A53T mutant human αS (HuαS) can cause post-synaptic and cognitive deficits that require microtubule-associated protein tau expression. However, the role of tau in the development of αS pathology and subsequent neuronal dysfunction has been controversial. Herein, we set out to determine the role of tau in the onset and progression of αS pathology (α-synucleinopathy) using a transgenic mouse model of α-synucleinopathy lacking mouse tau expression. METHODS: Transgenic mice expressing A53T mutant HuαS (TgA53T) were crossed with mTau-/- mice to generate TgA53T/mTau-/-. To achieve more uniform induction of α-synucleinopathy in mice, we used intramuscular injections of αS preformed fibrils (PFF) in non-transgenic (nTg), TgA53T, TgA53T/mTau-/-, and mTau-/- mice. Motor behavior was analyzed at 70 days post inoculation (dpi) of PFF and tissues for biochemical and neuropathological analysis were collected at 40 dpi, 70 dpi, and end stage. RESULTS: Loss of tau expression significantly delayed the onset of motor deficits in the TgA53T model and the progression of α-synucleinopathy disease, as evidenced by a significant reduction in histopathological and behavioral markers of neurodegeneration and disease, and a significant improvement in survival. In vitro application of PFF to primary mouse hippocampal neurons demonstrated no changes in PFF uptake and processing or pS129 αS aggregation as a function of tau expression. However, PFF-induced neurotoxicity, including morphological deficits in nTg neurons, was prevented with tau removal. CONCLUSIONS: Collectively, our data suggest that tau is likely acting downstream of αS pathology to affect neuronal homeostasis and survival. This work further supports the investigation of tau in α-synucleinopathies to identify novel disease-modifying therapeutic strategies.

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.

Ablating Tau Reduces Hyperexcitability and Moderates Electroencephalographic Slowing in Transgenic Mice Expressing A53T Human α-Synuclein.

Abnormal intraneuronal accumulation of the presynaptic protein α-synuclein (α-syn) is implicated in the etiology of dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). Recent work revealed that mice expressing human α-syn with the alanine-53-threonine (A53T) mutation have a similar phenotype to the human condition, exhibiting long-term potentiation deficits, learning and memory deficits, and inhibitory hippocampal remodeling, all of which were reversed by genetic ablation of microtubule-associated protein tau. Significantly, memory deficits were associated with histological signs of network hyperactivity/seizures. Electrophysiological abnormalities are often seen in parkinsonian dementias. Baseline electroencephalogram (EEG) slowing is used as a supportive diagnostic feature in DLB and PDD, and patients with these diseases may exhibit indicators of broad network dysfunction such as sleep dysregulation, myoclonus, and seizures. Given the translational significance, we examined whether human A53T α-syn expressing mice exhibit endogenous-tau-dependent EEG abnormalities, as measured with epidural electrodes over the frontal and parietal cortices. Using template-based waveform sorting, we determined that A53T mice have significantly high numbers of epileptiform events as early as 3-4 months of age and throughout life, and this effect is markedly attenuated in the absence of tau. Epileptic myoclonus occurred in half of A53T mice and was markedly reduced by tau ablation. In spectral analysis, tau ablation partially reduced EEG slowing in 6-7 month transgenic mice. We found abnormal sleeping patterns in transgenic mice that were more pronounced in older groups, but did not find evidence that this was influenced by tau genotype. Together, these data support the notion that tau facilitates A53T α-syn-induced hyperexcitability that both precedes and coincides with associated synaptic, cognitive, and behavioral effects. Tau also contributes to some aspects of EEG slowing in A53T mice. Importantly, our work supports tau-based approaches as an effective early intervention in α-synucleinopathies to treat aberrant network activity.

[18F]FEPPA PET imaging for monitoring CD68-positive microglia/macrophage neuroinflammation in nonhuman primates.

PURPOSE: The aim of this study was to examine whether the translocator protein 18-kDa (TSPO) PET ligand [18F]FEPPA has the sensitivity for detecting changes in CD68-positive microglial/macrophage activation in hemiparkinsonian rhesus macaques treated with allogeneic grafts of induced pluripotent stem cell-derived midbrain dopaminergic neurons (iPSC-mDA). METHODS: In vivo positron emission tomography (PET) imaging with [18F]FEPPA was used in conjunction with postmortem CD68 immunostaining to evaluate neuroinflammation in the brains of hemiparkinsonian rhesus macaques (n = 6) that received allogeneic iPSC-mDA grafts in the putamen ipsilateral to MPTP administration. RESULTS: Based on assessment of radiotracer uptake and confirmed by visual inspection of the imaging data, nonhuman primates with allogeneic grafts showed increased [18F]FEPPA binding at the graft sites relative to the contralateral putamen. From PET asymmetry analysis of the images, the mean asymmetry index of the monkeys was AI = - 0.085 ± 0.018. Evaluation and scoring of CD68 immunoreactivity by an investigator blind to the treatment identified significantly more neuroinflammation in the grafted areas of the putamen compared to the contralateral putamen (p = 0.0004). [18F]FEPPA PET AI showed a positive correlation with CD68 immunoreactivity AI ratings in the monkeys (Spearman's ρ = 0.94; p = 0.005). CONCLUSION: These findings reveal that [18F]FEPPA PET is an effective marker for detecting increased CD68-positive microglial/macrophage activation and demonstrates sufficient sensitivity to detect changes in neuroinflammation in vivo following allogeneic cell engraftment.

In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson's Disease in Common Marmosets.

Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.

α-Synuclein Expression Is Preserved in Substantia Nigra GABAergic Fibers of Young and Aged Neurotoxin-Treated Rhesus Monkeys.

α-Synuclein (α-syn) is a small presynaptic protein distributed ubiquitously in the central and peripheral nervous system. In normal conditions, α-syn is found in soluble form, while in Parkinson's disease (PD) it may phosphorylate, aggregate, and combine with other proteins to form Lewy bodies. The purpose of this study was to evaluate, in nonhuman primates, whether α-syn expression is affected by age and neurotoxin challenge. Young adult (n = 5, 5-10 years old) and aged (n = 4, 23-25 years old) rhesus monkeys received a single unilateral carotid artery injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three months post-MPTP the animals were necropsied by transcardiac perfusion, and their brains extracted and processed with immunohistochemical methods. Quantification of tyrosine hydroxylase (TH)-positive substantia nigra (SN) neurons showed a significant 80-89% decrease in the side ipsilateral to MPTP administration in young and old animals. Optical density of TH- immunoreactivity (-ir) in the caudate and putamen presented a 60-70% loss compared with the contralateral side. α-Syn-ir was present in both ipsi- and contra- lateral MPTP-treated nigra, caudate, and putamen, mostly in fibers; its intracellular distribution was not affected by age. Comparison of α-syn-ir between MPTP-treated young and aged monkeys revealed significantly higher optical density for both the ipsi- and contralateral caudate and SN in the aged animals. TH and α-syn immunofluorescence confirmed the loss of nigral TH-ir dopaminergic neurons in the MPTP-treated side of intoxicated animals, but bilateral α-syn expression. Colabeling of GAD67 and α-syn immunofluorescence showed that α-syn expression was present mainly in GABAergic fibers. Our results demonstrate that, 3 months post unilateral intracarotid artery infusion of MPTP, α-syn expression in the SN is largely present in GABAergic fibers, regardless of age. Bilateral increase of α-syn expression in SN fibers of aged, compared with young rhesus monkeys, suggests that α-syn-ir may increase with age, but not after neurotoxin-induced dopaminergic nigral cell loss.

In Vitro Modeling of Leucine-Rich Repeat Kinase 2 G2019S-Mediated Parkinson's Disease Pathology.

Leucine-rich repeat kinase 2 (LRRK2) G2019S (glycine to serine) is the most common mutation associated with sporadic and familial Parkinson's disease (PD) with 80% penetrance by age 70. This mutation is found worldwide, with up to 40% of individuals in the North African Arab population carrying the mutation. Induced pluripotent stem cells derived from fibroblasts of patients carrying the LRRK2 G2019S mutation have been a critical source of cells for generating dopaminergic neurons and studying G2019S-related pathology. These studies have elucidated LRRK2-related mechanisms of mitochondrial dysregulation, increased reactive oxygen species, truncated and simplified neurites, and cell death. These phenotypes are thought to result from the G2019S mutation increasing substrate access and therefore increasing the catalytic rate of the serine/threonine kinase. In this article, we critically review the contributions of in vitro modeling to the current knowledge on LRRK2 G2019S. We also analyze the role of patient-derived cell lines for the identification and validation of therapeutic targets, emphasizing their importance as part of a 3R approach to translational research and personalized medicine.

Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Adult Common Marmoset Fibroblasts.

The common marmoset monkey (Callithrix jacchus; Cj) is an advantageous nonhuman primate species for modeling age-related disorders, including Parkinson's disease, due to their shorter life span compared to macaques. Cj-derived induced pluripotent stem cells (Cj-iPSCs) from somatic cells are needed for in vitro disease modeling and testing regenerative medicine approaches. Here we report the development of a novel Cj-iPSC line derived from adult marmoset fibroblasts. The Cj-iPSCs showed potent pluripotency properties, including the development of mesodermal lineages in tumors after injection to immunocompromised mice, as well as ectoderm and endoderm lineages after in vitro differentiation regimens, demonstrating differentiated derivatives of all three embryonic layers. In addition, expression of key pluripotency genes (ZFP42, PODXL, DNMT3B, C-MYC, LIN28, KLF4, NANOG, SOX2, and OCT4) was observed. We then tested the neural differentiation capacity and gene expression profiles of Cj-iPSCs and a marmoset embryonic stem cell line (Cj-ESC) after dual-SMAD inhibition. Exposure to CHIR99021 and sonic hedgehog (SHH) for 12 and 16 days, respectively, patterned the cells toward a ventralized midbrain dopaminergic phenotype, confirmed by expression of FOXA2, OTX2, EN-1, and tyrosine hydroxylase. These results demonstrate that common marmoset stem cells will be able to serve as a platform for investigating regenerative medicine approaches targeting the dopaminergic system.

Real-Time Intraoperative MRI Intracerebral Delivery of Induced Pluripotent Stem Cell-Derived Neurons.

Induced pluripotent stem cell (iPSC)-derived neurons represent an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson's disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. We have previously developed a trajectory guide system for real-time intraoperative magnetic resonance imaging (RT-IMRI) delivery of infusates, such as viral vector suspensions for gene therapy strategies. Intracerebral delivery of iPSC-derived neurons presents different challenges than viral vectors, including limited cell survival if cells are kept at room temperature for prolonged periods of time, precipitation and aggregation of cells in the cannula, and obstruction during injection, which must be solved for successful application of this delivery approach. To develop procedures suitable for RT-IMRI cell delivery, we first performed in vitro studies to tailor the delivery hardware (e.g., cannula) and defined a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Then we performed an in vivo study to evaluate the feasibility of applying the system to nonhuman primates. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral cell delivery that are compatible with cell survival.

α-Synuclein and nonhuman primate models of Parkinson's disease.

Accumulation of α-synuclein (α-syn) leading to the formation of insoluble intracellular aggregates named Lewy bodies is proposed to have a significant role in Parkinson's disease (PD) pathology. Nonhuman primate (NHP) models of PD have proven essential for understanding the neurobiological basis of the disease and for the preclinical evaluation of first-in-class and invasive therapies. In addition to neurotoxin, aging and intracerebral gene transfer models, a new generation of models using inoculations of α-syn formulations, as well as transgenic methods is emerging. Understanding of their advantages and limitations will be essential when choosing a platform to evaluate α-syn-related pathology and interpreting the test results of new treatments targeting α-syn aggregation. In this review we aim to provide insight on this issue by critically analyzing the differences in endogenous α-syn, as well as α-syn pathology in PD and PD NHP models.