Long-term safety of MRI-guided administration of AAV2-GDNF and gadoteridol in the putamen of individuals with Parkinson's disease.

Direct putaminal infusion of adeno-associated virus vector (serotype 2) (AAV2) containing the human glial cell line-derived neurotrophic factor (GDNF) transgene was studied in a phase I clinical trial of participants with advanced Parkinson's disease (PD). Convection-enhanced delivery of AAV2-GDNF with a surrogate imaging tracer (gadoteridol) was used to track infusate distribution during real-time intraoperative magnetic resonance imaging (iMRI). Pre-, intra-, and serial postoperative (up to 5 years after infusion) MRI were analyzed in 13 participants with PD treated with bilateral putaminal co-infusions (52 infusions in total) of AAV2-GDNF and gadoteridol (infusion volume, 450 mL per putamen). Real-time iMRI confirmed infusion cannula placement, anatomic quantification of volumetric perfusion within the putamen, and direct visualization of off-target leakage or cannula reflux (which permitted corresponding infusion rate/cannula adjustments). Serial post-treatment MRI assessment (n = 13) demonstrated no evidence of cerebral parenchyma toxicity in the corresponding regions of AAV2-GDNF and gadoteridol co-infusion or surrounding regions over long-term follow-up. Direct confirmation of key intraoperative safety and efficacy parameters underscores the safety and tissue targeting value of real-time imaging with co-infused gadoteridol and putative therapeutic agents (i.e., AAV2-GDNF). This delivery-imaging platform enhances safety, permits delivery personalization, improves therapeutic distribution, and facilitates assessment of efficacy and dosing effect.

Advancing gene therapies, methods, and technologies for Parkinson's Disease and other neurological disorders.

INTRODUCTION: Vector-based intracerebral gene therapies are being used to treat specific neurodegenerative conditions such as Parkinson's Disease (PD). This review presents a basis for central nervous system (CNS) gene therapy treatments of neurodegenerative diseases such as PD, as well as the need for novel skill sets and health delivery strategies within the clinical neurosciences (neurology and neurosurgery) to meet future demand for such therapies. STATE OF THE ART: Preclinical vector-based gene therapy approaches have been translated into clinical trials for PD and other neurodegenerative conditions. Unfortunately, such trials, and parallel efforts using other therapeutics, have yet to provide a breakthrough. Image-guided convection enhanced delivery (CED) optimises the parenchymal distribution of gene therapies applied within the CNS, and may ultimately provide such a breakthrough. CLINICAL IMPLICATIONS: Currently, image-guided CED and gene therapy are not part of training programmes for most neurosurgeons and neurologists. As a result, few medical centres and hospitals have sufficiently experienced teams to participate in gene transfer clinical trials for PD or other neurological conditions. If CNS gene therapies prove to be efficacious for PD and/or other conditions, the demand for such treatments will overwhelm the available number of experienced clinical neuroscience teams and treatment centres. FUTURE DIRECTIONS: Expanded indications and demand for CNS gene therapies will require a worldwide educational effort to supplement the training of clinical neuroscience practitioners. Initially, a limited number of Centres of Excellence will need to establish relevant educational training requirements and best practice for such therapeutic approaches. Advanced technologies, including robotics and artificial intelligence, are especially germane in this regard, and will expand the treatment team's capabilities while assisting in the safe and timely care of those afflicted.

Precision pharmacology for Alzheimer's disease.

The complex multifactorial nature of polygenic Alzheimer's disease (AD) presents significant challenges for drug development. AD pathophysiology is progressing in a non-linear dynamic fashion across multiple systems levels - from molecules to organ systems - and through adaptation, to compensation, and decompensation to systems failure. Adaptation and compensation maintain homeostasis: a dynamic equilibrium resulting from the dynamic non-linear interaction between genome, epigenome, and environment. An individual vulnerability to stressors exists on the basis of individual triggers, drivers, and thresholds accounting for the initiation and failure of adaptive and compensatory responses. Consequently, the distinct pattern of AD pathophysiology in space and time must be investigated on the basis of the individual biological makeup. This requires the implementation of systems biology and neurophysiology to facilitate Precision Medicine (PM) and Precision Pharmacology (PP). The regulation of several processes at multiple levels of complexity from gene expression to cellular cycle to tissue repair and system-wide network activation has different time delays (temporal scale) according to the affected systems (spatial scale). The initial failure might originate and occur at every level potentially affecting the whole dynamic interrelated systems within an organism. Unraveling the spatial and temporal dynamics of non-linear pathophysiological mechanisms across the continuum of hierarchical self-organized systems levels and from systems homeostasis to systems failure is key to understand AD. Measuring and, possibly, controlling space- and time-scaled adaptive and compensatory responses occurring during AD will represent a crucial step to achieve the capacity to substantially modify the disease course and progression at the best suitable timepoints, thus counteracting disrupting critical pathophysiological inputs. This approach will provide the conceptual basis for effective disease-modifying pathway-based targeted therapies. PP is based on an exploratory and integrative strategy to complex diseases such as brain proteinopathies including AD, aimed at identifying simultaneous aberrant molecular pathways and predicting their temporal impact on the systems levels. The depiction of pathway-based molecular signatures of complex diseases contributes to the accurate and mechanistic stratification of distinct subcohorts of individuals at the earliest compensatory stage when treatment intervention may reverse, stop, or delay the disease. In addition, individualized drug selection may optimize treatment safety by decreasing risk and amplitude of side effects and adverse reactions. From a methodological point of view, comprehensive "omics"-based biomarkers will guide the exploration of spatio-temporal systems-wide morpho-functional shifts along the continuum of AD pathophysiology, from adaptation to irreversible failure. The Alzheimer Precision Medicine Initiative (APMI) and the APMI cohort program (APMI-CP) have commenced to facilitate a paradigm shift towards effective drug discovery and development in AD.

Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media.

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 108 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

Identification of preclinical Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood exosomes: A case-control study.

BACKGROUND: Proteins pathogenic in Alzheimer's disease (AD) were extracted from neurally derived blood exosomes and quantified to develop biomarkers for the staging of sporadic AD. METHODS: Blood exosomes obtained at one time-point from patients with AD (n = 57) or frontotemporal dementia (FTD) (n = 16), and at two time-points from others (n = 24) when cognitively normal and 1 to 10 years later when diagnosed with AD were enriched for neural sources by immunoabsorption. AD-pathogenic exosomal proteins were extracted and quantified by enzyme-linked immunosorbent assays. RESULTS: Mean exosomal levels of total tau, P-T181-tau, P-S396-tau, and amyloid ß 1-42 (Aß1-42) for AD and levels of P-T181-tau and Aß1-42 for FTD were significantly higher than for case-controls. Step-wise discriminant modeling incorporated P-T181-tau, P-S396-tau, and Aß1-42 in AD, but only P-T181-tau in FTD. Classification of 96.4% of AD patients and 87.5% of FTD patients was correct. In 24 AD patients, exosomal levels of P-S396-tau, P-T181-tau, and Aß1-42 were significantly higher than for controls both 1 to 10 years before and when diagnosed with AD. CONCLUSIONS: Levels of P-S396-tau, P-T181-tau, and Aß1-42 in extracts of neurally derived blood exosomes predict the development of AD up to 10 years before clinical onset.

The critical need for defining preclinical biomarkers in Alzheimer's disease.

The increasing number of afflicted individuals with late-onset Alzheimer's disease (AD) poses significant emotional and financial burden on the world's population. Therapeutics designed to treat symptoms or alter the disease course have failed to make an impact, despite substantial investments by governments, pharmaceutical industry, and private donors. These failures in treatment efficacy have led many to believe that symptomatic disease, including both mild cognitive impairment (MCI) and AD, may be refractory to therapeutic intervention. The recent focus on biomarkers for defining the preclinical state of MCI/AD is in the hope of defining a therapeutic window in which the neural substrate remains responsive to treatment. The ability of biomarkers to adequately define the at-risk state may ultimately allow novel or repurposed therapeutic agents to finally achieve the disease-modifying status for AD. In this review, we examine current preclinical AD biomarkers and suggest how to generalize their use going forward.

Interventional MRI-guided putaminal delivery of AAV2-GDNF for a planned clinical trial in Parkinson's disease.

Clinical trials involving direct infusion of neurotrophic therapies for Parkinson's disease (PD) have suffered from poor coverage of the putamen. The planned use of a novel interventional-magnetic resonance imaging (iMRI) targeting system for achieving precise, real-time convection-enhanced delivery in a planned clinical trial of adeno-associated virus serotype 2 (AAV2)-glial-derived neurotrophic factor (GDNF) in PD patients was modeled in nonhuman primates (NHP). NHP received bilateral coinfusions of gadoteridol (Gd)/AAV2-GDNF into two sites in each putamen, and three NHP received larger infusion volumes in the thalamus. The average targeting error for cannula tip placement in the putamen was <1 mm, and adjacent putamenal infusions were distributed in a uniform manner. GDNF expression patterns in the putamen were highly correlated with areas of Gd distribution seen on MRI. The distribution volume to infusion volume ratio in the putamen was similar to that in the thalamus, where larger infusions were achieved. Modeling the placement of adjacent 150 and 300 µl thalamic infusions into the three-dimensional space of the human putamen demonstrated coverage of the postcommissural putamen, containment within the striatum and expected anterograde transport to globus pallidus and substantia nigra pars reticulata. The results elucidate the necessary parameters for achieving widespread GDNF expression in the putamenal motor area and afferent substantia nigra of PD patients.

A retrotransposon storm marks clinical phenoconversion to late-onset Alzheimer's disease.

Recent reports have suggested that the reactivation of otherwise transcriptionally silent transposable elements (TEs) might induce brain degeneration, either by dysregulating the expression of genes and pathways implicated in cognitive decline and dementia or through the induction of immune-mediated neuroinflammation resulting in the elimination of neural and glial cells. In the work we present here, we test the hypothesis that differentially expressed TEs in blood could be used as biomarkers of cognitive decline and development of AD. To this aim, we used a sample of aging subjects (age > 70) that developed late-onset Alzheimer's disease (LOAD) over a relatively short period of time (12-48 months), for which blood was available before and after their phenoconversion, and a group of cognitive stable subjects as controls. We applied our developed and validated customized pipeline that allows the identification, characterization, and quantification of the differentially expressed (DE) TEs before and after the onset of manifest LOAD, through analyses of RNA-Seq data. We compared the level of DE TEs within more than 600,000 TE-mapping RNA transcripts from 25 individuals, whose specimens we obtained before and after their phenotypic conversion (phenoconversion) to LOAD, and discovered that 1790 TE transcripts showed significant expression differences between these two timepoints (logFC ± 1.5, logCMP > 5.3, nominal p value < 0.01). These DE transcripts mapped both over- and under-expressed TE elements. Occurring before the clinical phenoconversion, this TE storm features significant increases in DE transcripts of LINEs, LTRs, and SVAs, while those for SINEs are significantly depleted. These dysregulations end with signs of manifest LOAD. This set of highly DE transcripts generates a TE transcriptional profile that accurately discriminates the before and after phenoconversion states of these subjects. Our findings suggest that a storm of DE TEs occurs before phenoconversion from normal cognition to manifest LOAD in risk individuals compared to controls, and may provide useful blood-based biomarkers for heralding such a clinical transition, also suggesting that TEs can indeed participate in the complex process of neurodegeneration.

Optimal region of the putamen for image-guided convection-enhanced delivery of therapeutics in human and non-human primates.

Optimal results in the direct brain delivery of brain therapeutics such as growth factors or viral vector into primate brain depend on reproducible distribution throughout the target region. In the present study, we retrospectively analyzed MRI of 25 convection-enhanced delivery (CED) infusions with MRI contrast into the putamen of non-human primates (NHP). Infused volume (V(i)) was compared to total volume of distribution (V(d)) versus V(d) within the target putamen. Excellent distribution of contrast agent within the putamen was obtained in eight cases that were used to define an optimal target volume or "green" zone. Partial or poor distribution with leakage into adjacent anatomical structures was noted in 17 cases, defining "blue" and "red" zones, respectively. Quantitative containment (99±1%) of infused gadoteridol within the putamen was obtained when the cannula was placed in the green zone, 87±3% in the blue zone and 49±0.05% in the red zone. These results were used to determine a set of 3D stereotactic coordinates that define an optimal site for putaminal infusions in NHP and human putamen. We conclude that cannula placement and definition of optimal (green zone) stereotactic coordinates have important implications in ensuring effective delivery of therapeutics into the putamen utilizing routine stereotactic MRI localization procedures and should be considered when local therapies such as gene transfer or protein administration are being translated into clinical therapy.

Plasma Sphingomyelins in Late-Onset Alzheimer's Disease.

BACKGROUND: Altered plasma levels of sphingolipids, including sphingomyelins (SM), have been found in mouse models of Alzheimer's disease (AD) and in AD patient plasma samples. OBJECTIVE: This study assesses fourteen plasma SM species in a late-onset AD (LOAD) patient cohort (n = 138). METHODS: Specimens from control, preclinical, and symptomatic subjects were analyzed using targeted mass-spectrometry-based metabolomic methods. RESULTS: Total plasma SM levels were not significantly affected by age or cognitive status. However, one metabolite that has been elevated in manifest AD in several recent studies, SM OHC14:1, was reduced significantly in pre-clinical AD and MCI relative to normal controls. CONCLUSION: We recommend additional comprehensive plasma lipidomics in experimental and clinical biospecimens related to LOAD that might advance the utility of plasma sphingomyelin levels in molecular phenotyping and interpretations of pathobiological mechanisms.

TGFß Drives Metabolic Perturbations during Epithelial Mesenchymal Transition in Pancreatic Cancer: TGFß Induced EMT in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy wherein a majority of patients present metastatic disease at diagnosis. Although the role of epithelial to mesenchymal transition (EMT), mediated by transforming growth factor beta (TGFß), in imparting an aggressive phenotype to PDAC is well documented, the underlying biochemical pathway perturbations driving this behaviour have not been elucidated. We used high-resolution mass spectrometry (HRMS) based molecular phenotyping approach in order to delineate metabolic changes concomitant to TGFß-induced EMT in pancreatic cancer cells. Strikingly, we observed robust changes in amino acid and energy metabolism that may contribute to tumor invasion and metastasis. Somewhat unexpectedly, TGFß treatment resulted in an increase in intracellular levels of retinoic acid (RA) that in turn resulted in increased levels of extracellular matrix (ECM) proteins including fibronectin (FN) and collagen (COL1). These findings were further validated in plasma samples obtained from patients with resectable pancreatic cancer. Taken together, these observations provide novel insights into small molecule dysregulation that triggers a molecular cascade resulting in increased EMT-like changes in pancreatic cancer cells, a paradigm that can be potentially targeted for better clinical outcomes.

Author Correction: Plasma microRNA markers of upper limb recovery following human stroke.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Community-Based Study Identifying Metabolic Biomarkers of Mild Cognitive Impairment and Alzheimer's Disease Using Artificial Intelligence and Machine Learning.

BACKGROUND: Currently, there is no objective, clinically available tool for the accurate diagnosis of Alzheimer's disease (AD). There is a pressing need for a novel, minimally invasive, cost friendly, and easily accessible tool to diagnose AD, assess disease severity, and prognosticate course. Metabolomics is a promising tool for discovery of new, biologically, and clinically relevant biomarkers for AD detection and classification. OBJECTIVE: Utilizing artificial intelligence and machine learning, we aim to assess whether a panel of metabolites as detected in plasma can be used as an objective and clinically feasible tool for the diagnosis of mild cognitive impairment (MCI) and AD. METHODS: Using a community-based sample cohort acquired from different sites across the US, we adopted an approach combining Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR), Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and various machine learning statistical approaches to identify a biomarker panel capable of identifying those patients with AD and MCI from healthy controls. RESULTS: Of the 212 measured metabolites, 5 were identified as optimal to discriminate between controls, and individuals with MCI or AD. Our models performed with AUC values in the range of 0.72-0.76, with the sensitivity and specificity values ranging from 0.75-0.85 and 0.69-0.81, respectively. Univariate and pathway analysis identified lipid metabolism as the most perturbed biochemical pathway in MCI and AD. CONCLUSION: A comprehensive method of acquiring metabolomics data, coupled with machine learning techniques, has identified a strong panel of diagnostic biomarkers capable of identifying individuals with MCI and AD. Further, our data confirm what other groups have reported, that lipid metabolism is significantly perturbed in those individuals suffering with dementia. This work may provide additional insight into AD pathogenesis and encourage more in-depth analysis of the AD lipidome.

GDNF and Parkinson's Disease: Where Next? A Summary from a Recent Workshop.

The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.

Real-time MR imaging of adeno-associated viral vector delivery to the primate brain.

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes.

Striatal volume differences between non-human and human primates.

Convection-enhanced delivery (CED) has recently entered the clinic and represents a promising new delivery option for targeted gene therapy in Parkinson's disease (PD). The prime stereotactic target for the majority of recent gene therapy clinical trials has been the human putamen. The stereotactic delivery of therapeutic agents into putamen (or other subcortical structures) via CED remains problematic due to the difficulty in knowing what volume of therapeutic agent to deliver. Preclinical studies in non-human primates (NHP) offer a way to model treatment strategies prior to clinical trials. Understanding more accurately the volumetric differences in striatum, especially putamen, between NHP and humans is essential in predicting convective volume parameters in human clinical trials. In this study, magnetic resonance images (MRI) were obtained for volumetric measurements of striatum (putamen and caudate nucleus) and whole brain from 11 PD patients, 13 aged healthy human subjects, as well as 8 parkinsonian and 30 normal NHP. The human brain is 13-18 times larger than the monkey brain. However, this ratio is significantly smaller for striatum (5.7-6.5), caudate nucleus (4.6-6.6) and putamen (4.4-6.6). Size and species of the monkeys used for this comparative study are responsible for differences in ratios for each structure between monkeys and humans. This volumetric ratio may have important implications in the design of clinical therapies for PD and Huntington's disease and should be considered when local therapies such as gene transfer, local protein administration or cellular replacement are translated based on NHP research.

Infuse-as-you-go convective delivery to enhance coverage of elongated brain targets: technical note.

OBJECTIVE: To develop and assess a convective delivery technique that enhances the effectiveness of drug delivery to nonspherical brain nuclei, the authors developed an occipital "infuse-as-you-go" approach to the putamen and compared it to the currently used transfrontal approach. METHODS: Eleven nonhuman primates received a bilateral putamen injection of adeno-associated virus with 2 mM gadolinium-DTPA by real-time MR-guided convective perfusion via either a transfrontal (n = 5) or occipital infuse-as-you-go (n = 6) approach. RESULTS: MRI provided contemporaneous assessment and monitoring of putaminal infusions for transfrontal (2 to 3 infusion deposits) and occipital infuse-as-you-go (stepwise infusions) putaminal approaches. The infuse-as-you-go technique was more efficient than the transfrontal approach (mean 35 ± 1.1 vs 88 ± 8.3 minutes [SEM; p < 0.001]). More effective perfusion of the postcommissural and total putamen was achieved with the infuse-as-you-go versus transfronatal approaches (100-µl infusion volumes; mean posterior commissural coverage 76.2% ± 5.0% vs 32.8% ± 2.9% [p < 0.001]; and mean total coverage 53.5% ± 3.0% vs 38.9% ± 2.3% [p < 0.01]). CONCLUSIONS: The infuse-as-you-go approach, paralleling the longitudinal axis of the target structure, provides a more effective and efficient method for convective infusate coverage of elongated, irregularly shaped subcortical brain nuclei.

Development of a novel frameless skull-mounted ball-joint guide array for use in image-guided neurosurgery.

OBJECTIVE: Successful convection-enhanced delivery of therapeutic agents to subcortical brain structures requires accurate cannula placement. Stereotactic guiding devices have been developed to accurately target brain nuclei. However, technologies remain limited by a lack of MRI compatibility, or by devices' size, making them suboptimal for direct gene delivery to brain parenchyma. The goal of this study was to validate the accuracy of a novel frameless skull-mounted ball-joint guide array (BJGA) in targeting the nonhuman primate (NHP) brain. METHODS: Fifteen MRI-guided cannula insertions were performed on 9 NHPs, each targeting the putamen. Optimal trajectories were planned on a standard MRI console using 3D multiplanar baseline images. After cannula insertion, the intended trajectory was compared to the final trajectory to assess deviation (euclidean error) of the cannula tip. RESULTS: The average cannula tip deviation was 1.18 ± 0.60 mm (mean ± SD) as measured by 2 independent reviewers. Topological analysis showed a superior, posterior, and rightward directional bias, and the intra- and interclass correlation coefficients were > 0.85, indicating valid and reliable intra- and interobserver evaluation. CONCLUSIONS: The data demonstrate that the BJGA can be used to reliably target subcortical brain structures by using MRI guidance, with accuracy comparable to current frameless stereotactic systems. The size and versatility of the BJGA, combined with a streamlined workflow, allows for its potential applicability to a variety of intracranial neurosurgical procedures, and for greater flexibility in executing MRI-guided experiments within the NHP brain.