Models Of Neurodegenerative Diseases Research Articles

Protective effect of Capsosiphon fulvescens on oxidative stress-stimulated neurodegenerative dysfunction of PC12 cells and zebrafish larva models

Reactive oxygen species (ROS) at high concentrations induce oxidative stress, an imbalanced redox state that is a prevalent cause of neurodegenerative disorders. This study aimed to investigate the protective effect of Capsosiphon fulvescens (CF) extract on oxidative stress-induced impairment of cognitive function in models of neurodegenerative diseases. CF was extracted with subcritical water and several solvents and H2O2 (0.25 mM) or aluminum chloride (AlCl3; 25 μM) as an inducer of ROS was treated in PC12 neuronal cells and zebrafish larvae. All statistical analyses were performed using one-way analysis of variance and Dunnett’s test using GraphPad Prism. H2O2 and AlCl3 were found to significantly induce ROS production in PC12 neuronal cells and zebrafish larvae. In addition, they strongly affected intracellular Ca2+ levels, antioxidant enzyme activity, brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) signaling, acetylcholinesterase (AChE) activity, and hallmarks of Alzheimer’s disease. However, treatment of H2O2-induced PC12 cells or AlCl3-induced zebrafish larvae with CF subcritical water extract at 90°C and CF water extract effectively regulated excessive ROS production, intracellular Ca2+ levels, and mRNA expression of superoxide dismutase, glutathione peroxide, glycogen synthase kinase-3 beta, β-amyloid, tau, AChE, BDNF, and TrkB. Our study suggested that CF extracts can be a potential source of nutraceuticals that can improve the impairment of cognitive function and synaptic plasticity by regulating ROS generation in neurodegenerative diseases.

Cell-based therapeutic strategies for treatment of spinocerebellar ataxias: an update.

Spinocerebellar ataxias are heritable neurodegenerative diseases caused by a cytosine-adenine-guanine expansion, which encodes a long glutamine tract (polyglutamine) in the respective wild-type protein causing misfolding and protein aggregation. Clinical features of polyglutamine spinocerebellar ataxias include neuronal aggregation, mitochondrial dysfunction, decreased proteasomal activity, and autophagy impairment. Mutant polyglutamine protein aggregates accumulate within neurons and cause neural dysfunction and death in specific regions of the central nervous system. Spinocerebellar ataxias are mostly characterized by progressive ataxia, speech and swallowing problems, loss of coordination and gait deficits. Over the past decade, efforts have been made to ameliorate disease symptoms in patients, yet no cure is available. Previous studies have been proposing the use of stem cells as promising tools for central nervous system tissue regeneration. So far, pre-clinical trials have shown improvement in various models of neurodegenerative diseases following stem cell transplantation, including animal models of spinocerebellar ataxia types 1, 2, and 3. However, contrasting results can be found in the literature, depending on the animal model, cell type, and route of administration used. Nonetheless, clinical trials using cellular implants into degenerated brain regions have already been applied, with the expectation that these cells would be able to differentiate into the specific neuronal subtypes and re-populate these regions, reconstructing the affected neural network. Meanwhile, the question of how feasible it is to continue such treatments remains unanswered, with long-lasting effects being still unknown. To establish the value of these advanced therapeutic tools, it is important to predict the actions of the transplanted cells as well as to understand which cell type can induce the best outcomes for each disease. Further studies are needed to determine the best route of administration, without neglecting the possible risks of repetitive transplantation that these approaches so far appear to demand. Despite the challenges ahead of us, cell-transplantation therapies are reported to have transient but beneficial outcomes in spinocerebellar ataxias, which encourages efforts towards their improvement in the future.

Profiling neuroprotective potential of trehalose in animal models of neurodegenerative diseases: a systematic review.

Trehalose, a unique nonreducing crystalline disaccharide, is a potential disease-modifying treatment for neurodegenerative diseases associated with protein misfolding and aggregation due to aging, intrinsic mutations, or autophagy dysregulation. This systematic review summarizes the effects of trehalose on its underlying mechanisms in animal models of selected neurodegenerative disorders (tau pathology, synucleinopathy, polyglutamine tract, and motor neuron diseases). All animal studies on neurodegenerative diseases treated with trehalose published in Medline (accessed via EBSCOhost) and Scopus were considered. Of the 2259 studies screened, 29 met the eligibility criteria. According to the SYstematic Review Center for Laboratory Animal Experiment (SYRCLE) risk of bias tool, we reported 22 out of 29 studies with a high risk of bias. The present findings support the purported role of trehalose in autophagic flux and protein refolding. This review identified several other lesser-known pathways, including modifying amyloid precursor protein processing, inhibition of reactive gliosis, the integrity of the blood-brain barrier, activation of growth factors, upregulation of the downstream antioxidant signaling pathway, and protection against mitochondrial defects. The absence of adverse events and improvements in the outcome parameters were observed in some studies, which supports the transition to human clinical trials. It is possible to conclude that trehalose exerts its neuroprotective effects through both direct and indirect pathways. However, heterogeneous methodologies and outcome measures across the studies rendered it impossible to derive a definitive conclusion. Translational studies on trehalose would need to clarify three important questions: 1) bioavailability with oral administration, 2) optimal time window to confer neuroprotective benefits, and 3) optimal dosage to confer neuroprotection.

Genetic Interactions of Progranulin Across the ALS-FTD Spectrum and Beyond.

Progranulin (PGRN) is a growth factor in which mutations are one of the leading causes of frontotemporal dementia (FTD), and has been implicated in an assortment of neurodegenerative diseases. Conversely, higher levels of the protein have shown potential as a general neuronal protective factor. While examining its neuroprotective applications on a broader scale would be unfeasible in mammalian models, we turned to the nematode C. elegans to map the interactions of PGRN across multiple genetic models of neurodegenerative diseases. Our results indicate that while the overexpression of PGRN appears to be protective across all models tested, the loss of PGRN exacerbated the disease phenotypes of all but three of the models tested. Given the ease of genetic analysis in nematodes, we propose this model organism as an efficient tool to build a comprehensive map of PGRN's genetic interactions.

XperCT-guided Intra-cisterna Magna Injection of Streptozotocin for Establishing an Alzheimer’s Disease Model Using the Cynomolgus Monkey (Macaca fascicularis)

Till date, researchers have been developing animal models of Alzheimer's disease (AD) in various species to understand the pathological characterization and molecular mechanistic pathways associated with this condition in humans to identify potential therapeutic treatments. A widely recognized AD model that mimics the pathology of human AD involves the intracerebroventricular (ICV) injection with streptozotocin (STZ). However, ICV injection as an invasive approach has several limitations related to complicated surgical procedures. Therefore, in the present study, we created a customized stereotaxic frame using the XperCT-guided system for injecting STZ in cynomolgus monkeys, aiming to establish an AD model. The anatomical structures surrounding the cisterna magna (CM) were confirmed using CT/MRI fusion images of monkey brain with XperCT, the c-arm cone beam computed tomography. XperCT was used to determine the appropriate direction in which the needle tip should be inserted within the CM region. Cerebrospinal fluid (CSF) was collected to confirm the accurate target site when STZ was injected into the CM. Cynomolgus monkeys were administered STZ dissolved in artificial CSF once every week for 4 weeks via intracisterna magna (ICM) injection using XperCT-guided stereotactic system. The molecular mechanisms underlying the progression of STZ-induced AD pathology were analyzed two weeks after the final injection. The monkeys subjected to XperCT-based STZ injection via the ICM route showed features of AD pathology, including markedly enhanced neuronal loss, synaptic impairment, and tau phosphorylation in the hippocampus. These findings suggest a new approach for the construction of neurodegenerative disease models and development of therapeutic strategies.

Modeling of Neurodegenerative Diseases: ‘Step by Step’ and ‘Network’ Organization of the Complexes of Model Systems

Neurodegenerative diseases have acquired the status of one of the leading causes of death in developed countries, which requires creating new model systems capable of accurately reproducing the mechanisms underlying these pathologies. Here we analyzed modern model systems and their contribution to the solution of unexplored manifestations of neuropathological processes. Each model has unique properties that make it the optimal tool for modeling certain aspects of neurodegenerative disorders. We concluded that to optimize research, it is necessary to combine models into complexes that include organisms and artificial systems of different organizational levels. Such complexes can be organized in two ways. The first method can be described as "step by step", where each model for studying a certain characteristic is a separate step that allows using the information obtained in the modeling process for the gradual study of increasingly complex processes in subsequent models. The second way is a 'network' approach. Studies are carried out with several types of models simultaneously, and experiments with each specific type are adjusted in conformity with the data obtained from other models. In our opinion, the 'network' approach to combining individual model systems seems more promising for fundamental biology as well as diagnostics and therapy.

RETRACTED: Combined metabolic activators improve metabolic functions in the animal models of neurodegenerative diseases

BackgroundNeurodegenerative diseases (NDDs), including Alzheimer's disease (AD) and Parkinson's disease (PD), are associated with metabolic abnormalities. Integrative analysis of human clinical data and animal studies have contributed to a better understanding of the molecular and cellular pathways involved in the progression of NDDs. Previously, we have reported that the combined metabolic activators (CMA), which include the precursors of nicotinamide adenine dinucleotide and glutathione can be utilized to alleviate metabolic disorders by activating mitochondrial metabolism. MethodsWe first analysed the brain transcriptomics data from AD patients and controls using a brain-specific genome-scale metabolic model (GEM). Then, we investigated the effect of CMA administration in animal models of AD and PD. We evaluated pathological and immunohistochemical findings of brain and liver tissues. Moreover, PD rats were tested for locomotor activity and apomorphine-induced rotation. FindingsAnalysis of transcriptomics data with GEM revealed that mitochondrial dysfunction is involved in the underlying molecular pathways of AD. In animal models of AD and PD, we showed significant damage in the high-fat diet groups' brain and liver tissues compared to the chow diet. The histological analyses revealed that hyperemia, degeneration and necrosis in neurons were improved by CMA administration in both AD and PD animal models. These findings were supported by immunohistochemical evidence of decreased immunoreactivity in neurons. In parallel to the improvement in the brain, we also observed dramatic metabolic improvement in the liver tissue. CMA administration also showed a beneficial effect on behavioural functions in PD rats. InterpretationOverall, we showed that CMA administration significantly improved behavioural scores in parallel with the neurohistological outcomes in the AD and PD animal models and is a promising treatment for improving the metabolic parameters and brain functions in NDDs.

Identification of HSPB8 modulators counteracting misfolded protein accumulation in neurodegenerative diseases

AimsThe small Heat Shock Protein B8 (HSPB8) is the core component of the Chaperone-Assisted Selective Autophagy (CASA) complex. This complex selectively targets, transports, and tags misfolded proteins for their recognition by autophagy receptors and insertion into the autophagosome for clearance. CASA is essential to maintain intracellular proteostasis, especially in heart, muscle, and brain often exposed to various types of cell stresses. In neurons, HSPB8 protects against neurotoxicity caused by misfolded proteins in several models of neurodegenerative diseases; by facilitating autophagy, HSPB8 assists misfolded proteins degradation also counteracting proteasome overwhelming and inhibition. Materials and methodsTo enhance HSPB8 protective activity, we screened a library of approximately 120,000 small molecules to identify compounds capable of increasing HSPB8 gene transcription, translation, or protein stability. Key findingsWe found 83 active compounds active in preliminary dose-response assays and further classified them in 19 chemical classes by medicinal chemists' visual inspection. Of these 19 prototypes, 14 induced HSPB8 mRNA and protein levels in SH-SY5Y cells. Out of these 14 compounds, 3 successfully reduced the aggregation propensity of a disease-associated mutant misfolded superoxide dismutase 1 (SOD1) protein in a flow cytometry-based aggregation assay (Flow cytometric analysis of Inclusions and Trafficking (FloIT)) and induced the expression (mRNA and protein) of some autophagy receptors. Notably, the 3 hits were inactive in HSPB8-depleted cells, confirming that their protective activity is mediated by and requires HSPB8. SignificanceThese compounds may be highly relevant for a therapeutic approach in several human disorders, including neurodegenerative diseases, in which enhancement of CASA exerts beneficial activities.

Amyloid-beta and tau pathologies act synergistically to induce novel disease stage-specific microglia subtypes

BackgroundAmongst risk alleles associated with late-onset Alzheimer’s disease (AD), those that converged on the regulation of microglia activity have emerged as central to disease progression. Yet, how canonical amyloid-β (Aβ) and tau pathologies regulate microglia subtypes during the progression of AD remains poorly understood.MethodsWe use single-cell RNA-sequencing to profile microglia subtypes from mice exhibiting both Aβ and tau pathologies across disease progression. We identify novel microglia subtypes that are induced in response to both Aβ and tau pathologies in a disease-stage-specific manner. To validate the observation in AD mouse models, we also generated a snRNA-Seq dataset from the human superior frontal gyrus (SFG) and entorhinal cortex (ERC) at different Braak stages.ResultsWe show that during early-stage disease, interferon signaling induces a subtype of microglia termed Early-stage AD-Associated Microglia (EADAM) in response to both Aβ and tau pathologies. During late-stage disease, a second microglia subtype termed Late-stage AD-Associated Microglia (LADAM) is detected. While similar microglia subtypes are observed in other models of neurodegenerative disease, the magnitude and composition of gene signatures found in EADAM and LADAM are distinct, suggesting the necessity of both Aβ and tau pathologies to elicit their emergence. Importantly, the pattern of EADAM- and LADAM-associated gene expression is observed in microglia from AD brains, during the early (Braak II)- or late (Braak VI/V)- stage of the disease, respectively. Furthermore, we show that several Siglec genes are selectively expressed in either EADAM or LADAM. Siglecg is expressed in white-matter-associated LADAM, and expression of Siglec-10, the human orthologue of Siglecg, is progressively elevated in an AD-stage-dependent manner but not shown in non-AD tauopathy.ConclusionsUsing scRNA-Seq in mouse models bearing amyloid-β and/or tau pathologies, we identify novel microglia subtypes induced by the combination of Aβ and tau pathologies in a disease stage-specific manner. Our findings suggest that both Aβ and tau pathologies are required for the disease stage-specific induction of EADAM and LADAM. In addition, we revealed Siglecs as biomarkers of AD progression and potential therapeutic targets.

HSP70-HSP90 Chaperone Networking in Protein-Misfolding Disease.

Molecular chaperones and their associated co-chaperones are essential in health and disease as they are key facilitators of protein-folding, quality control and function. In particular, the heat-shock protein (HSP) 70 and HSP90 molecular chaperone networks have been associated with neurodegenerative diseases caused by aberrant protein-folding. The pathogenesis of these disorders usually includes the formation of deposits of misfolded, aggregated protein. HSP70 and HSP90, plus their co-chaperones, have been recognised as potent modulators of misfolded protein toxicity, inclusion formation and cell survival in cellular and animal models of neurodegenerative disease. Moreover, these chaperone machines function not only in folding but also in proteasome-mediated degradation of neurodegenerative disease proteins. This chapter gives an overview of the HSP70 and HSP90 chaperones, and their respective regulatory co-chaperones, and explores how the HSP70 and HSP90 chaperone systems form a larger functional network and its relevance to counteracting neurodegenerative disease associated with misfolded proteins and disruption of proteostasis.

Mushroom Natural Products in Neurodegenerative Disease Drug Discovery

The variety of drugs available to treat neurodegenerative diseases is limited. Most of these drug's efficacy is restricted by individual genetics and disease stages and usually do not prevent neurodegeneration acting long after irreversible damage has already occurred. Thus, drugs targeting the molecular mechanisms underlying subsequent neurodegeneration have the potential to negate symptom manifestation and subsequent neurodegeneration. Neuroinflammation is a common feature of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis, and is associated with the activation of the NLRP3 inflammasome, which in turn leads to neurodegeneration. Inflammasome activation and oligomerisation is suggested to be a major driver of disease progression occurring in microglia. With several natural products and natural product derivatives currently in clinical trials, mushrooms have been highlighted as a rich and largely untapped source of biologically active compounds in both in vitro and in vivo neurodegenerative disease models, partially supported by successful clinical trial evaluations. Additionally, novel high-throughput methods for the screening of natural product compound libraries are being developed to help accelerate the neurodegenerative disease drug discovery process, targeting neuroinflammation. However, the breadth of research relating to mushroom natural product high-throughput screening is limited, providing an exciting opportunity for further detailed investigations.

Cell sorting after tracer injection to elucidate allocation of cellular FDG uptake in different cell types and different microglial activation states

AbstractBackground2‐deoxy‐2‐[18F]fluoro‐d‐glucose positron‐emission‐tomography (FDG‐PET) is widely used to study cerebral glucose metabolism. Radiosorting revealed that the FDG‐PET signal is directly influenced by microglial glucose uptake in mouse models and we found similar associations in patients with neurodegenerative diseases. Now, we questioned the allocation of cellular FDG uptake in the rodent brain.MethodSingle cell [18F]FDG uptake as assessed by magnetic activated cell sorting (MACS) was multiplied with published total cell numbers in the mouse brain, i.e. 71e+6 neurons or 7% (≈ 7.6e+6) microglia cells to estimate the overall uptake per cell fraction. Estimated cellular microglial [18F]FDG uptake as assessed by MACS was cross‐validated with observed %‐FDG‐PET reductions after microglia depletion. Interstitial and vessel [18F]FDG was estimated by interstitial compartment volume and assumed equilibrium of non‐phosphorylated extra‐ and intracellular [14C]Deoxyglucose. Synaptic [18F]FDG uptake was estimated via an enriched synaptosome fraction relative to [18F]FDG uptake of whole‐brain homogenate. A microglia proliferation index of 1.3 was assumed for the whole brain in the Aβ mouse model.ResultMicroglia cells accounted for 8.6% of the overall brain [18F]FDG uptake in wild‐type mice and for 29.9% of the overall brain [18F]FDG uptake in PS2APP mice, confirmed by similar alterations after microglia depletion in PET. Synapses contributed to 13.2‐17.1% of the overall brain [18F]FDG uptake whereas interstitial (5.9‐7.7%) and vessel (2.6‐3.4%) [18F]FDG uptake was of minor magnitude. Neuronal and astrocyte cell bodies accounted for <10% of the overall brain [18F]FDG uptake, but a proportion of 42‐55% was not recovered and likely allocated in lost cell processes, oligodendrocytes and other cell types.ConclusionMicroglial FDG uptake alterations are the source of FDG‐PET changes in models of neurodegenerative diseases. Our data should increase the awareness to consider glia cells as key contributors of FDG uptake and the detailed proportions of these cellular contributions in healthy brains and under disease conditions deserve deeper investigation.

Phonemic restoration in Alzheimer’s disease and semantic dementia

AbstractBackgroundIn daily life, spoken messages are often interrupted by extraneous sounds. However, we generally perceive such messages as continuous: our brains automatically and efficiently ‘repair’ interrupted speech signals by phonemic restoration, a fundamental physiological process whereby speech sounds that are obscured by noise are ‘filled‐in’ perceptually to reconstitute the underlying signal. As a dynamic, integrative process, phonemic restoration is potentially affected by neurodegenerative pathologies but this has not been clarified.MethodHere we studied this phenomenon in patients with typical Alzheimer’s disease and semantic dementia, relative to healthy age‐matched controls. Participants heard isolated sounds, spoken real words and pseudowords in which noise bursts either overlaid a consonant or replaced it; a tendency to hear replaced (missing) speech sounds as present signified phonemic restoration.ResultAll groups perceived isolated noises normally and showed phonemic restoration of real words, most marked in the Alzheimer’s disease group. For pseudowords, healthy controls showed no phonemic restoration, patients with Alzheimer’s disease showed a marked suppression of phonemic restoration and patients with semantic dementia showed phonemic restoration comparable to real words.ConclusionOur findings provide the first evidence that phonemic restoration is preserved or even enhanced in neurodegenerative diseases, with distinct syndromic profiles that may reflect the relative integrity of bottom‐up phonological representation and top‐down lexical disambiguation mechanisms in different diseases. This work has theoretical implications for predictive coding models of language and neurodegenerative disease, and for understanding cognitive ‘repair’ processes in dementia.

Contribution of intraocular pressure‐dependent and pressure‐independent glaucoma models to neuroprotection

AbstractGlaucoma is considered a heterogeneous group of degenerative optic neuropathies characterized mainly by an ongoing loss of retinal ganglion cells (RGCs) which causes progressive deterioration of the visual field and irreversible blindness. The aetiology and pathophysiology of glaucoma are not known in detail and are a matter of debate. Although the most significant risk factor in the development and progression of glaucoma correlates with elevated intraocular pressure (IOP), at present the only treatment available to prevent or delay its development is to reduce IOP by medication or surgery to slow its detrimental effects. But reducing IOP alone does not stop the progression of the disease. Therefore, IOP may not be the only motive for glaucomatous retinopathy and optic neuropathy, and there could be other hidden mechanisms contributing to the loss of RGCs.A suitable animal model of neurodegenerative disease, such as glaucoma, should be easy to induce and reproduce, feasible and predictable, and allow detection and quantification at the biochemical, morphological and functional levels of the induced pathological changes. However, no model emulates all aspects of a pathology such as glaucoma, which is likely to have more than one aetiology and manifest itself through different pathological mechanisms. Hence, the interest and need to establish and use different experimental models in rodents to improve our understanding of the pathophysiology of glaucoma and to investigate possible neuroprotective agents. In general, experimental models of glaucoma established in rodents involve a transient or chronic increase in IOP above normal values, which allows us to study the pathological changes associated with ocular hypertension. However, elevated IOP may not be the only cause of glaucomatous retinopathy and optic neuropathy, hence the need for models that do not involve an increase in intraocular pressure to deal with specific issues of the evolution of this pathology, such as optic nerve injury, retinal ischaemia/reperfusion, intravitreal injection of excitatory amino acids or endothelin‐induced optic neuropathy.Results obtained in different experimental models of glaucoma mimicking ocular hypertension or optic nerve damage have confirmed the existence of morphofunctional differences in the time course of RGC loss, both between different types of RGCs and between different lesions. As well as the effects on other retinal cell populations. In this regard, in the outer retina, there is a significant contrast between damage caused by ocular hypertension and optic nerve injury. Both axotomy and ocular hypertension induce a selective loss of RGCs, but ocular hypertension also promotes a loss of photoreceptors.Thanks to the different glaucoma models studied, the findings obtained have contributed to improving our knowledge of glaucomatous pathology and to the development of future neuroprotective therapies.

Translational Inhibition of Multiple Neurotoxic Proteins Leads to Improved Cognition in Alzheimer’s Disease

AbstractBackgroundOverexpression of neurotoxic proteins drives downstream events that dysregulate axonal transport, lead to inflammation, nerve cell death, and loss of function. By inhibiting the translation of amyloid precursor protein, tau and alpha‐synuclein, our drug restored axonal transport, lowered inflammation, and protected nerve cells from dying in animal models. Buntanetap’s reversal of the toxic cascade leading to nerve cell death did preserve and/or restore full function in 7 different animal models of neurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s disease (PD).MethodIn a double blind, placebo controlled Phase2a study, early AD patients were randomized into 80mg Buntanetap (n = 10) or placebo (n = 5) QD for 25±2 days. We evaluated the safety, pharmacokinetics of Buntanetap, biomarkers, and its efficacy.ResultIn clinical trials, Buntanetap was safe in over 200 subjects, both healthy volunteers and AD/PD patients. In our recent double‐blind, placebo controlled Phase2a study, patients randomized to either 80mg QD Buntanetap or placebo we measured biomarkers in AD patients and showed that Buntanetap can lower neurotoxic proteins, improve axonal integrity and synaptic function, and lower inflammation. Buntanetap has also improved AD patients’ cognitive functions as measured by ADASCog11 and WAIS coding test after 25±2 days treatment.ConclusionThese encouraging data support our further development of Buntanetap as a potential treatment for Alzheimer’s disease. We are planning three double‐blind, placebo‐controlled studies, a short 3‐month dose‐finding trial and two long‐term 12‐ and 18‐month trials in early and moderate AD patients.

Translational Inhibition of Multiple Neurotoxic Proteins Leads to Improved Motor Function in Parkinson’s Disease

AbstractBackgroundParkinson’s disease (PD) is a neurodegenerative disorder that affects predominately dopaminergic neurons in substantia nigra. Although alpha‐synuclein (αSYN) is the main component of Lewy bodies, TDP‐43, Aβ and tau are also detected in PD. Buntanetap has been shown to inhibit the translation of multiple neurotoxic proteins such as amyloid precursor protein, tau and alpha‐synuclein. It been shown in animal models to restore axonal transport, lower inflammation and protect nerve cells from dying. By reversing the toxic cascade leading to nerve cell death, Buntanetap preserves and restores full function in 7 different animal models of neurodegenerative diseases including PD.MethodIn our double‐blind, placebo‐controlled, dose‐finding Phase 2a trial, 5 different doses of Buntanetap (5mg, 10mg, 20mg, 40mg and 80mg QD) were tested compared to control after25±2 days treatment. Buntanetap’s safety, pharmacokinetics, biomarkers and efficacy were evaluated.ResultIn clinical trials, Buntanetap was safe in over 200 subjects, both in healthy volunteers and in AD/PD patient. In PD patients, Buntanetap lowers neurotoxic proteins, improves axonal integrity and synaptic function, and lowers inflammation. Buntanetap improves PD patients’ mobility shown by MDS‐UPDRS and WAIS coding test after 25±2 days treatment. Of the 5 different doses, 10 & 20mg Buntanetap QD showed the best clinical outcomes.ConclusionThese data supported us getting regulatory approval to develop next stage pivotal studies in larger population and for longer durations. The FDA consented us to move the development into two phase 3 clinical studies – one in early and one in advanced PD. Our first pivotal trial intends to treat 450 early PD patients at 1:1:1 ration for 10mg, 20mg Buntanetap QD or placebo for 6 months duration.

Relevance of the pre‐clinical models in TBI

AbstractIn the absence of any effective translation from preclinical models to clinical application, what has been achieved by laboratory models of TBI? Analysis and assimilation of results from preclinical TBI studies is confounded by the heterogeneity of the models and the TBI paradigms employed. Variance in animal, injury location, severity, number, frequency, age of animal at injury and age of animal at analyses, not to mention the research focus of the investigators themselves, all contribute to the difficulties in data integration and interpretation. Nonetheless, preclinical TBI research has identified areas of focus and enabled creation of timelines of TBI pathobiology. Ultimately, validation of any potential therapeutic targets will require exploitation of the heterogeneity of preclinical models in order to facilitate clinical translation, given the heterogeneity of the patient population.In the course of modeling TBI paradigms in (primarily) mouse models, the failure of recapitulation of TBI‐dependent neurodegenerative pathology representing AD, PD, ALS etc. has prompted the use of mouse models of neurodegenerative disease, expressing genetic risk factors or humanized for proteins implicated in human neurodegenerative diseases, in efforts to understand how TBI can exacerbate or even precipitate the neurodegenerative pathobiology. These investigations have generated many interesting observations, not always easily consolidated into any clear pathogenic mechanism, but some overarching themes have emerged. Some of the published TBI approaches, and findings, will be reviewed, and knowledge gaps and future directions will be discussed.

New models of Parkinson's like neuroinflammation in human microglia clone 3: Activation profiles induced by INF-γ plus high glucose and mitochondrial inhibitors.

Microglia activation and neuroinflammation have been extensively studied in murine models of neurodegenerative diseases; however, to overcome the genetic differences between species, a human cell model of microglia able to recapitulate the activation profiles described in patients is needed. Here we developed human models of Parkinson's like neuroinflammation by using the human microglia clone 3 (HMC3) cells, whose activation profile in response to classic inflammatory stimuli has been controversial and reported only at mRNA levels so far. In fact, we showed the increased expression of the pro-inflammatory markers iNOS, Caspase 1, IL-1β, in response to IFN-γ plus high glucose, a non-specific disease stimulus that emphasized the dynamic polarization and heterogenicity of the microglial population. More specifically, we demonstrated the polarization of HMC3 cells through the upregulation of iNOS expression and nitrite production in response to the Parkinson's like stimuli, 6-hydroxidopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the latter depending on the NF-κB pathway. Furthermore, we identified inflammatory mediators that promote the pro-inflammatory activation of human microglia as function of different pathways that can simulate the phenotypic transition according to the stage of the pathology. In conclusion, we established and characterized different systems of HMC3 cells activation as in vitro models of Parkinson's like neuroinflammation.

3-Dimensional Immunostaining and Automated Deep-Learning Based Analysis of Nerve Degeneration

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.

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.