A Failed Future

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156468/2/mds28130.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156468/1/mds28130_am.pd

The virtuous cycle of axon growth: Axonal transport of growth-promoting machinery as an intrinsic determinant of axon regeneration

Injury to the brain and spinal cord has devastating consequences because adult central nervous system (CNS) axons fail to regenerate. Injury to the peripheral nervous system (PNS) has a better prognosis, because adult PNS neurons support robust axon regeneration over long distances. CNS axons have some regenerative capacity during development, but this is lost with maturity. Two reasons for the failure of CNS regeneration are extrinsic inhibitory molecules, and a weak intrinsic capacity for growth. Extrinsic inhibitory molecules have been well characterised, but less is known about the neuron-intrinsic mechanisms which prevent axon re-growth. Key signalling pathways and genetic / epigenetic factors have been identified which can enhance regenerative capacity, but the precise cellular mechanisms mediating their actions have not been characterised. Recent studies suggest that an important prerequisite for regeneration is an efficient supply of growth-promoting machinery to the axon, however this appears to be lacking from non-regenerative axons in the adult CNS. In the first part of this review, we summarise the evidence linking axon transport to axon regeneration. We discuss the developmental decline in axon regeneration capacity in the CNS, and comment on how this is paralleled by a similar decline in the selective axonal transport of regeneration-associated receptors such as integrins and growth factor receptors. In the second part, we discuss the mechanisms regulating selective polarised transport within neurons, how these relate to the intrinsic control of axon regeneration, and whether they can be targeted to enhance regenerative capacity.ERA‐NET Neuron International Foundation for Research in Paraplegia Christopher and Dana Reeve Foundation. Grant Numbers: JFC‐2013(3), JFC‐2013(4) Gates Cambridge Trust Medical Research Council. Grant Numbers: G1000864 018556, MR/R004463/

Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

Delivering glial cell line-derived neurotrophic factor (GDNF) to the brain is a potential treatment for Parkinson'sDisease (PD). Here we use an implantable encapsulated cell technology that uses modified human clonal ARPE-19cells to deliver of GDNF to the brain. In vivostudies demonstrated sustained delivery of GDNF to the rat striatumover 6 months. Anatomical benefits and behavioral efficacy were shown in 6-OHDA lesioned rats where nigraldopaminergic neurons were preserved in neuroprotection studies and dopaminergicfibers were restored inneurorecovery studies. When larger, clinical-sized devices were implanted for 3 months into the putamen ofG\u20acottingen minipigs, GDNF was widely distributed throughout the putamen and caudate producing a significantupregulation of tyrosine hydroxylase immunohistochemistry. These results are thefirst to provide clear evidencethat implantation of encapsulated GDNF-secreting cells deliver efficacious and biologically relevant amounts ofGDNF in a sustained and targeted manner that is scalable to treat the large putamen in patients with Parkinson'sdiseas

Proteasome-targeted nanobodies alleviate pathology and functional decline in an α-synuclein-based Parkinson's disease model.

Therapeutics designed to target α-synuclein (α-syn) aggregation may be critical in halting the progression of pathology in Parkinson's disease (PD) patients. Nanobodies are single-domain antibody fragments that bind with antibody specificity, but allow readier genetic engineering and delivery. When expressed intracellularly as intrabodies, anti-α-syn nanobodies fused to a proteasome-targeting proline, aspartate or glutamate, serine, and threonine (PEST) motif can modulate monomeric concentrations of target proteins. Here we aimed to validate and compare the in vivo therapeutic potential of gene therapy delivery of two proteasome-directed nanobodies selectively targeting α-syn in a synuclein overexpression-based PD model: VH14*PEST (non-amyloid component region) and NbSyn87*PEST (C-terminal region). Stereotaxic injections of adeno-associated viral 5-α-syn (AAV5-α-syn) into the substantia nigra (SN) were performed in Sprague-Dawley rats that were sorted into three cohorts based on pre-operative behavioral testing. Rats were treated with unilateral SN injections of vectors for VH14*PEST, NbSyn87*PEST, or injected with saline 3 weeks post lesion. Post-mortem assessments of the SN showed that both nanobodies markedly reduced the level of phosphorylated Serine-129 α-syn labeling relative to saline-treated animals. VH14*PEST showed considerable maintenance of striatal dopaminergic tone in comparison to saline-treated and NbSyn87*PEST-treated animals as measured by tyrosine hydroxylase immunoreactivity (optical density), DAT immunoreactivity (optical density), and dopamine concentration (high-performance liquid chromatography). Microglial accumulation and inflammatory response, assessed by stereological counts of Iba-1-labeled cells, was modestly increased in NbSyn87*PEST-injected rats but not in VH14*PEST-treated or saline-treated animals. Modest behavioral rescue was also observed, although there was pronounced variability among individual animals. These data validate in vivo therapeutic efficacy of vector-delivered intracellular nanobodies targeting α-syn misfolding and aggregation in synucleinopathies such as PD

Evaluation of alpha-synuclein immunohistochemical methods for the detection of Lewy-type synucleinopathy in gastrointestinal biopsies

The observation showing that Lewy type synucleinopathy (LTS), the pathological hallmark of Parkinson’s disease (PD), is found in the gut of almost all PD subjects led to a substantial amount of research to develop a diagnostic procedure in living patients based on endoscopically obtained gastrointestinal biopsies. However, the existing studies have provided conflicting results regarding the sensitivity and specificity of gastrointestinal biopsies for the detection of LTS. We therefore undertook a multi-center staining and blinded judging of a common set of slides from colonic biopsies to determine the optimal protocol for the detection of LTS. Four different immunohistochemical methods, developed in four separate expert laboratories, were evaluated for their sensitivity and specificity to detect enteric LTS. Test sets of formalin-fixed, paraffin-embedded sections from biopsies of 9 PD subjects and 3 controls were stained with the 4 methods and graded by 4 different observers. Four types of staining morphology (granular staining in the lamina propria, perivascular/vascular wall staining in the submucosa, lacy-granular pattern in the submucosa and epithelial cell nuclear staining) were variably observed in the slides stained by the 4 methods. Positive alpha-synuclein staining was observed by all 5 judges in most of the slides from control cases, regardless of the staining methods that were used. Moreover, none of the tested method or staining pattern had a specificity and sensitivity more than 80 % regarding to PD. Overall, our study suggest that the tested methods are not adequate for the prediction of PD using gastrointestinal biopsies. Future studies are warranted to test new immunostaining methods

Mapping meningeal vasculature in non-human primates

Background. The blood-brain barrier has been the focus of most prior work examining intracranial vasculature in the context of various brain diseases.1 Recently, meningeal vasculature has become more widely recognized as a key contributor to brain clearance and its immune function.2 Meninges are highly vascularized and complex tissue. Vessels of the outer dural layer comprise an extensive, parallel intracranial vascular bed, which lies outside the brain and subarachnoid space. In addition to the blood vasculature, meninges harbor lymphatic channels that potentially provide extra capacity for clearance of proteinaceous fluid and immune cell trafficking. Most of our knowledge of the meningeal vasculature, including lymphatics, comes from rodent models. Rodent meninges are readily available, small, thin and optically transparent. These characteristics permit imaging in whole-mount flat preparations.3 Technical barriers, however, remain high for imaging studies of the meninges of larger mammals. This is especially true for primates, and ultimately humans. Non-human primate (NHP) and human dura is large, thick and opaque, with a high content of connective tissue. These characteristics limit options for routine high-resolution imaging and leave unanswered questions about the architecture of blood and lymphatic vessels in primate dura. So far, the presence of lymphatic vessels in primates has been demonstrated by non-invasive techniques like magnetic resonance imaging (MRI) or on sections of paraffin-embedded specimens. Neither of the techniques fully addresses spatial and phenotypical features of the vascular networks. In our work, we provide solutions for these technical barriers using new clearing and imaging protocols to successfully visualize blood and lymphatic vessels in NPH dura in their entirety. Methods. Here we used novel approaches to tissue clearing and resonance scanning confocal imaging of large areas with sickness over 1000 M. Results. Our approach revealed extensive and dense vascular networks in NHP dura probed with vascular marker CD31 (Figure 1). Image clarity and resolution is sufficient for visualization of the smallest vessels. In the dura, blood vessels are mostly represented by veins. Vascular networks can be further analyzed with semi-automated tracing and quantitative metrics in 3D space. We showed that lymphatic vessels in NPH dura are located similarly to that in rodents: in the area of the superior sagittal sinus (SSS) and along the middle meningeal artery (MMA). They are also present in the major dural fold, tentorium cerebelli, which is underdeveloped in rodents. Unlike previously described in mice, these vessels are negative for LYVE-1 lymphatic marker but strongly positive for podoplanin. In the area of SSS, there is a large plexus of branching irregular blind-ended sacs with a wide range of diameters. Vessels in the MMA region have a different appearance. Two vessels always run along the veins flanking MMA that follow the artery branching. Our protocol also permits imaging of the extracellular matrix and the cells that reside in the dural environment. Conclusions. We developed clearing, mounting and imaging protocols that permitted panoramic fluorescence-based microscopy of NPH dura. These new techniques are directly applicable to primate models of neurodegenerative diseases with a focus on the complex interplay between meningeal arteries, veins, and lymphatics

Is axonal degeneration a key early event in Parkinson’s disease?

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here by permission of IOS Press for personal use, not for redistribution. The definitive version was published in Journal of Parkinson's Disease 6 (2016): 703-707, doi:10.3233/JPD-160881.Recent research suggests that in Parkinson’s disease the long, thin and unmyelinated axons of dopaminergic neurons degenerate early in the disease process. We organized a workshop entitled ‘Axonal Pathology in Parkinson’s disease’, on March 23rd, 2016, in Cleveland, Ohio with the goals of summarizing the state-of-the-art and defining key gaps in knowledge. A group of eight research leaders discussed new developments in clinical pathology, functional imaging, animal models, and mechanisms of degeneration including neuroinflammation, autophagy and axonal transport deficits. While the workshop focused on PD, comparisons were made to other neurological conditions where axonal degeneration is well recognized

Enhanced Efficacy of the CDNF/MANF Family by Combined Intranigral Overexpression in the 6-OHDA Rat Model of Parkinson's Disease

Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.Molecular Therapy (2014); doi:10.1038/mt.2014.206