Postdoctoral Fellow, Macquarie University
Zebrafish could hold the key to better-understanding of Motor Neuron Disease (MND/ALS).
Accumulation of proteins into insoluble aggregates in neurons and glia is now recognized as a common pathological hallmark of many neurodegenerative diseases (e.g. in Alzheimer’s, and Parkinson’s disease). In Amyotrophic Lateral Sclerosis (ALS), the intracellular accumulation of proteins in neurons is also well established. The clinical data describes a focal onset and the subsequent spread of muscle paralysis to other regions over time (Ravits et al., 2007;Ravits and La Spada, 2009;Kanouchi et al., 2012). In line with these clinical observations, cell-culture studies show the secretion of ALS-proteins from neurons (Grad et al., 2014, Feiler 2016), and that aggregates isolated from patient tissue are internalized by neurons and can propagate further protein aggregation (Nonaka et al., 2013). Taken together, the evidence for a spread of these aggregates is beginning to emerge and is entirely limited to studies using cultured nerve cells (in-vitro studies).
For many years, non-neuronal cells (glia) were considered as static, structural components of the brain and spinal cord, whose main purpose was to provide metabolic and substrate support for neurons. Now it is recognised that glia represent a dynamic component of the nervous system, actively provide a range of essential functions (Hanisch and Kettenmann, 2007; Kettenmann et al., 2011; Morsch et al., 2015). Through this process, it is believed that microglia actively maintain homeostatic balance within the brain and spinal cord and abnormalities in microglial clearance are linked to worsened outcomes in neurodegenerative diseases (Block et al., 2007).
Our project investigates the nature of interactions between neurons and microglia that lead to motor neuron degeneration and potentially ALS. We have established a unique in-vivo platform to evaluate aggregate distribution and neurodegeneration throughout the nervous system in the zebrafish spinal cord. We can visualise the subcellular localisation of these ALS aggregates following the onset of degeneration as well as its redistribution during that process. Importantly, we now have uncovered that microglial clearance plays an important role early on during neurodegeneration.
The longer-term clinical significance of this would be the possible identification of therapeutic strategies that could delay progression of the disease. While not a “cure”, it could potentially preserve lifestyle of patients in the early, less debilitating stages of the disease.
Abstract: Microglia are the resident phagocytes of the brain that are responsible for the clearance of injured neurons, an essential step in subsequent tissue regeneration. How death signals are controlled both in space and time to attract these cells toward the site of injury is a topic of great interest. To this aim, we have used the optically transparent zebrafish larval brain and identified rapidly propagating Ca2+ waves that determine the range of microglial responses to neuronal cell death. We show that while Ca2+-mediated microglial responses require ATP, the spreading of intercellular Ca2+ waves is ATP independent. Finally, we identify glutamate as a potent inducer of Ca2+-transmitted microglial attraction. Thus, this real-time analysis reveals the existence of a mechanism controlling microglial targeted migration to neuronal injuries that is initiated by glutamate and proceeds across the brain in the form of a Ca2+ wave.
Pub.: 29 May '12, Pinned: 18 Nov '17
Abstract: The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons.
Pub.: 06 Jun '14, Pinned: 18 Nov '17
Abstract: Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.
Pub.: 29 Jun '14, Pinned: 18 Nov '17
Abstract: During early development of the central nervous system (CNS), a subset of yolk‐sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self‐renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue‐specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR‐124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR‐124 in the mpeg1 expressing yolk‐sac‐derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR‐124 was used to assess microglial development in a miR‐124 loss‐of‐function environment. Following the induction of miR‐124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR‐124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR‐124 overexpression in differentiated neurons. Conversely, expression of the miR‐124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR‐124 overexpression. This study provides in vivo evidence that miR‐124 activity has a key role in the development of functionally mature microglia. © 2015 Wiley Periodicals, Inc. Develop Neurobiol, 2015
Pub.: 25 Jul '15, Pinned: 18 Nov '17
Abstract: Microglial research has entered a fertile, dynamic phase characterized by novel technologies including two-photon imaging, whole-genome transcriptomic and epigenomic analysis with complementary bioinformatics, unbiased proteomics, cytometry by time of flight (CyTOF; Fluidigm) cytometry, and complex high-content experimental models including slice culture and zebrafish. Against this vivid background of newly emerging data, investigators will encounter in the microglial research literature a body of published work using the terminology of macrophage polarization, most commonly into the M1 and M2 phenotypes. It is the assertion of this opinion piece that microglial polarization has not been established by research findings. Rather, the adoption of this schema was undertaken in an attempt to simplify data interpretation at a time when the ontogeny and functional significance of microglia had not yet been characterized. Now, terminology suggesting established meaningful pathways of microglial polarization hinders rather than aids research progress and should be discarded.
Pub.: 26 Jul '16, Pinned: 18 Nov '17
Abstract: In neurodegenerative diseases activation of immune cells is thought to play a major role. Microglia are the main immune cells of the central nervous system. When encountering disease related stimuli microglia adopt an activated phenotype that typically includes a rounded morphology. The exact role of microglia or other potentially infiltrating myeloid cells in different brain diseases is not fully understood. In this chapter we present techniques in zebrafish to induce degeneration of neurons, to activate the microglia, and to study activation phenotypes by immunohistochemistry and in vivo by fluorescence microscopic imaging.
Pub.: 08 Jan '17, Pinned: 18 Nov '17
Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative disorder that is characterized by loss of motor neurons and shows clinical, pathological, and genetic overlap with frontotemporal dementia (FTD). Activated microglia are a universal feature of ALS/FTD pathology; however, their role in disease pathogenesis remains incompletely understood. The recent discovery that ORF 72 on chromosome 9 (C9orf72), the gene most commonly mutated in ALS/FTD, has an important role in myeloid cells opened the possibility that altered microglial function plays an active role in disease. This Review highlights the contribution of microglia to ALS/FTD pathogenesis, discusses the connection between autoimmunity and ALS/FTD, and explores the possibility that C9orf72 and other ALS/FTD genes may have a "dual effect" on both neuronal and myeloid cell function that could explain a shared propensity for altered systemic immunity and neurodegeneration.
Pub.: 25 Jul '17, Pinned: 18 Nov '17
Abstract: Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic β-amyloid (Aβ)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.
Pub.: 21 Sep '17, Pinned: 18 Nov '17
Abstract: Transactive response DNA-binding protein-43 (TDP-43) is a multifunctional nucleic acid binding protein present in ubiquitinated inclusions in tissues of patients with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The ALS-associated mutations in the glycine-rich C-terminal domain of TDP-43 established a causal link between TDP-43 and disease, and conferred both loss- and gain-of-function properties in neurons. Since it has not been established whether these intra-neuronal changes are sufficient to cause ALS or whether non-cell autonomous neuronal-glial signaling could be involved, we investigated the extracellular effects of TDP-43 proteins on microglial activation and motoneuron toxicity. Wild-type, truncated 25kD C-terminal fragments, or mutant forms of TDP-43 all activated microglia and upregulated NOX2, TNF-α, and IL-1β, with WT forms being significantly less effective in activating microglia. This response to TDP-43 was mediated by its interaction with the microglial surface CD14 receptor and subsequent stimulation of the NF-κB and AP-1 pathways, as well as the intracellular inflammasome. At the cell surface, CD14 blocking antibodies suppressed microglial NF-κB activation and proinflammatory cytokine production mediated by TDP-43. Intracellularly, the NLRP3 inflammasome was induced and functional caspase-1 was produced augmenting the release of mature IL-1β. Further, TDP-43-mediated activation of microglia caused a proinflammatory cascade that was toxic to motoneurons. In the absence of microglia, TDP-43 was not toxic to motoneurons. The ability of TDP-43 to promote CD14-mediated activation of microglial NF-κB and AP-1 pathways, as well as the NLRP3 inflammasome, suggests the involvement of a non-cell autonomous proinflammatory signaling that enhances motoneuron injury, and may offer novel therapeutic targets in ALS.
Pub.: 30 Jul '15, Pinned: 17 Nov '17
Abstract: In the brain, neurons that fail to assemble into functional circuits are eliminated. Their clearance depends on microglia, immune cells that colonize the CNS during embryogenesis. Despite the importance of these cells in development and disease, the mechanisms that target and position microglia within the brain are unclear. Here we show that, in zebrafish, attraction of microglia into the brain exploits differences in developmental neuronal apoptosis and that these provide a mechanism for microglial distribution. Reducing neuronal cell death results in fewer microglia, whereas increased apoptosis enhances brain colonization, resulting in more microglia at later stages. Interestingly, attraction into the brain depends on nucleotide signaling, the same signaling system used to guide microglia toward brain injuries. Finally, this work uncovers a cell-non-autonomous role for developmental apoptosis. Classically considered a wasteful process, programmed cell death is exploited here to configure the immune-neuronal interface of the brain.
Pub.: 19 Jul '16, Pinned: 17 Nov '17
Abstract: Microglia, the resident macrophage precursors of the brain, are necessary for the maintenance of tissue homeostasis and activated by a wide range of pathological stimuli. They have a key role in immune and inflammatory responses. Early microglia stem from primitive macrophages, however the transition from early motile forms to the ramified mature resident microglia has not been assayed in real time. In order to provide such an assay, we used zebrafish transgenic lines in which fluorescent reporter expression is driven by the promoter of macrophage expressed gene 1 (mpeg1; Ellet et al. : Blood 117(4): e49-e56,). This enabled the investigation of the development of these cells in live, intact larvae. We show that microglia develop from highly motile amoeboid cells that are engaged in phagocytosis of apoptotic cell bodies into a microglial cell type that rapidly morphs back and forth between amoeboid and ramified morphologies. These morphing microglia eventually settle into a typical mature ramified morphology. Developing microglia frequently come into contact with blood capillaries in the brain, and also frequently contact each other. Up to 10 days postfertilization, microglia were observed to undergo symmetric division. In the adult optic tectum, the microglia are highly branched, resembling mammalian microglia. In addition, the mpeg1 transgene also labeled highly branched cells in the skin overlying the optic tectum from 8-9 days postfertilization, which likely represent Langerhans cells. Thus, the development of zebrafish microglia and their cellular interactions was studied in the intact developing brain in real time and at cellular resolution.
Pub.: 01 Jun '12, Pinned: 26 Sep '17
Abstract: Glioblastoma multiforme is the most common and deadliest form of brain cancer. Glioblastomas are infiltrated by a high number of microglia, which promote tumor growth and surrounding tissue invasion. However, it is unclear how microglia and glioma cells physically interact and if there are differences, depending on glioma cell type. Hence, we have developed a novel live imaging assay to study microglia-glioma interactions in vivo in the zebrafish brain. We transplanted well-established human glioblastoma cell lines, U87 and U251, into transgenic zebrafish lines with labelled macrophages/microglia. Our confocal live imaging results show distinct interactions between microglia and U87, as well as U251 glioblastoma cells that differ in number and nature. Importantly these interactions do not appear to be antitumoral as zebrafish microglia do not engulf and phagocytose the human glioblastoma cells. Finally, xenotransplants into the irf8(-/-) zebrafish mutant that lacks microglia, as well as pharmacological inhibition of the CSF-1 receptor (CSF-1R) on microglia, confirm a prominent role for zebrafish microglia in promoting human glioblastoma cell growth. This new model will be an important tool for drug screening and the development of future immunotherapeutics targeting microglia within glioma.
Pub.: 26 Oct '16, Pinned: 26 Sep '17
Abstract: Publication date: Available online 31 October 2016 Source:Methods in Cell Biology Author(s): K.R. Astell, D. Sieger Glioblastoma is the most frequent and aggressive primary malignant brain tumor. Gliomas exhibit high genetic diversity in addition to complex and variable clinical features. Glioblastoma tumors are highly resistant to multimodal therapies and there is significant patient mortality within the first two years after prognosis. At present clinical treatments are palliative, not curative. Glioblastomas contain a high number of microglia and infiltrating macrophages, which are positively correlated with glioma grade and invasiveness. Microglia are the resident macrophages of the central nervous system. These cells constantly scan the brain and react promptly to any abnormality, removing detrimental factors and safeguarding the central nervous system against further damage. Microglia and macrophages that have colonized the glioblastoma display protumoral functions and promote tumor growth. The optically transparent zebrafish larva facilitates imaging of fluorescently labeled cells at high spatial and temporal resolution in vivo. It is therefore an excellent model to investigate microglia-glioma cell interactions at the early stages of tumor development. Here we provide several methods that can be used to study the early stages of microglia-glioma cell interactions in the zebrafish. We present a technique for the xenotransplantation of mammalian oncogenic cells into the zebrafish brain and provide advice for image capture and analysis.
Pub.: 07 Nov '16, Pinned: 26 Sep '17
Abstract: Microglia coordinate various functions in the central nervous system ranging from removing synaptic connections, to maintaining brain homeostasis by monitoring neuronal function, and clearing protein aggregates across the lifespan. Here we investigated whether increased microglial phagocytic activity that clears amyloid can also cause pathological synapse loss. We identified TDP-43, a DNA-RNA binding protein encoded by the Tardbp gene, as a strong regulator of microglial phagocytosis. Mice lacking TDP-43 in microglia exhibit reduced amyloid load in a model of Alzheimer's disease (AD) but at the same time display drastic synapse loss, even in the absence of amyloid. Clinical examination from TDP-43 pathology cases reveal a considerably reduced prevalence of AD and decreased amyloid pathology compared to age-matched healthy controls, confirming our experimental results. Overall, our data suggest that dysfunctional microglia might play a causative role in the pathogenesis of neurodegenerative disorders, critically modulating the early stages of cognitive decline.
Pub.: 04 Jul '17, Pinned: 26 Sep '17
Abstract: Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells. Here, we used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Our findings argue that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of propagated misfolding of SOD1.
Pub.: 02 Mar '16, Pinned: 28 Aug '17
Abstract: Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43(+) inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS- and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Double-homozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.
Pub.: 05 Mar '13, Pinned: 24 Aug '17
Abstract: Mutation of Tar DNA-binding protein 43 (TDP-43) is linked to amyotrophic lateral sclerosis. Although astrocytes have important roles in neuron function and survival, their potential contribution to TDP-43 pathogenesis is unclear. Here, we created novel lines of transgenic rats that express a mutant form of human TDP-43 (M337V substitution) restricted to astrocytes. Selective expression of mutant TDP-43 in astrocytes caused a progressive loss of motor neurons and the denervation atrophy of skeletal muscles, resulting in progressive paralysis. The spinal cord of transgenic rats also exhibited a progressive depletion of the astroglial glutamate transporters GLT-1 and GLAST. Astrocytic expression of mutant TDP-43 led to activation of astrocytes and microglia, with an induction of the neurotoxic factor Lcn2 in reactive astrocytes that was independent of TDP-43 expression. These results indicate that mutant TDP-43 in astrocytes is sufficient to cause non-cell-autonomous death of motor neurons. This motor neuron death likely involves deficiency in neuroprotective genes and induction of neurotoxic genes in astrocytes.
Pub.: 30 May '13, Pinned: 24 Aug '17
Abstract: TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43(A315T) transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.
Pub.: 09 Oct '15, Pinned: 24 Aug '17
Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two progressive, fatal neurodegenerative syndromes with considerable clinical, genetic and pathological overlap. Clinical symptoms of FTD can be seen in ALS patients and vice versa. Recent genetic discoveries conclusively link the two diseases, and several common molecular players have been identified (TDP-43, FUS, C9ORF72). The definitive etiologies of ALS and FTD are currently unknown and both disorders lack a cure. Glia, specifically astrocytes and microglia are heavily implicated in the onset and progression of neurodegeneration witnessed in ALS and FTD. In this review, we summarize the current understanding of the role of microglia and astrocytes involved in ALS and FTD, highlighting their recent implications in neuroinflammation, alterations in waste clearance involving phagocytosis and the newly described glymphatic system, and vascular abnormalities. Elucidating the precise mechanisms of how astrocytes and microglia are involved in ALS and FTD will be crucial in characterizing these two disorders and may represent more effective interventions for disease progression and treatment options in the future.
Pub.: 19 Nov '15, Pinned: 24 Aug '17
Abstract: The progression of amyotrophic lateral sclerosis (ALS) through the brain has recently been staged using independent neuropathological and neuroimaging modalities. The two schemes tie into the concept of pathological spread through corticofugal axonal transmission that stems from observation of oligodendrocyte pTDP-43 aggregates along with neuronal inclusions. Here, we aimed to assess evidence of transmission along axonal pathways by looking for pTDP-43 oligodendrocyte pathology in involved white matter tracts, and to present a first validation of the neuropathological staging scheme. pTDP-43 immunohistochemistry was performed in select white matter tracts and grey matter regions from the staging scheme in postmortem-confirmed ALS cases (N = 34). Double-labelling immunofluorescence was performed to confirm co-localisation of pTDP-43 immunoreactivity to oligodendrocytes.While pTDP-43 immunoreactive oligodendrocytes were frequent in the white matter under the motor and sensory cortices, similar assessment of the white matter along the corticospinal tract and in the corpus callosum and cingulum bundle of the same cases revealed no pTDP-43 pathology, questioning the involvement of oligodendrocytes in pathological propagation. The assessment of Betz cell loss revealed that the lack of deep white matter pTDP-43 oligodendrocyte pathology was not due to an absence of motor axons. Assessment of the propagation of pathology to different grey matter regions validated that all cases could be allocated to one of four neuropathological stages, although Stage 4 cases were found to differ significantly in age of onset (~10 years older) and disease duration (shorter duration than Stage 3 and similar to Stage 2).Four stages of ALS neuropathology can be consistently identified, although evidence of sequential clinical progression requires further assessment. As limited pTDP-43 oligodendrocyte pathology in deep corticospinal and other white matter tracts from the motor cortex was observed, the propagation of pathology between neurons may not involve oligodendrocytes and the interpretation of the changes observed on neuroimaging should be modified accordingly.
Pub.: 29 Jul '15, Pinned: 24 Aug '17
Abstract: Amyotrophic Lateral Sclerosis is characterized by a focal onset of symptoms followed by a progressive spread of pathology that has been likened to transmission of infectious prions. Cell-to-cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated.We demonstrate in this paper that SOD1 aggregates interact with the cell surface triggering activation of Rac1 and subsequent membrane ruffling permitting aggregate uptake via stimulated macropinocytosis. In addition, other protein aggregates, including those associated with neurodegenerative diseases (TDP-43, Httex146Q, α-synuclein) also trigger membrane ruffling to gain entry into the cell. Aggregates are able to rupture unstructured macropinosomes to enter the cytosol allowing propagation of aggregation to proceed.Thus, we conclude that in addition to basic proteostasis mechanisms, pathways involved in the activation of macropinocytosis are key determinants in the spread of pathology in these misfolding diseases.
Pub.: 02 Nov '15, Pinned: 24 Aug '17
Abstract: Frontotemporal dementia is a devastating neurodegenerative disease causing stark alterations in personality and language. Characterized by severe atrophy of the frontal and temporal brain lobes, frontotemporal dementia (FTD) shows extreme heterogeneity in clinical presentation, genetic causes, and pathological findings. Like most neurodegenerative diseases, the initial symptoms of FTD are subtle, but increase in severity over time, as the disease progresses. Clinical progression is paralleled by exacerbation of pathological findings and the involvement of broader brain regions, which currently lack mechanistic explanation. Yet, a flurry of studies indicate that protein aggregates accumulating in neurodegenerative diseases can act as propagating entities, amplifying their pathogenic conformation, in a way similar to infectious prions. In this prion-centric view, FTD can be divided into three subtypes, TDP-43 or FUS proteinopathy and tauopathy. Here, we review the current evidence that FTD-linked pathology propagates in a prion-like manner and discuss the implications of these findings for disease progression and heterogeneity. Frontotemporal dementia (FTD) is a progressive neurodegenerative disease causing severe personality dysfunctions, characterized by profound heterogeneity. Accumulation of tau, TDP-43 or FUS cytoplasmic aggregates characterize molecularly distinct and non-overlapping FTD subtypes. Here, we discuss the current evidence suggesting that prion-like propagation and cell-to-cell spread of each of these cytoplasmic aggregates may underlie disease progression and heterogeneity. This article is part of the Frontotemporal Dementia special issue.
Pub.: 10 Aug '16, Pinned: 24 Aug '17
Abstract: Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and >95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded 'prion-like' protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.
Pub.: 04 Sep '16, Pinned: 24 Aug '17
Abstract: The most common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, are all protein-misfolding diseases and are characterized by the presence of disease-specific protein aggregates in affected neuronal cells. Recent studies have shown that, like tau and α-synuclein, TAR-DNA binding protein of 43 kDa (TDP-43) can form aggregates in vitro in a seed-dependent, self-templating, prion-like manner. Insoluble TDP-43 prepared from the brains of patients has been classified into several strains, which can be transferred from cell to cell in vitro, suggesting the involvement of mechanisms reminiscent of those by which prions spread through the nervous system. The idea that aberrant TDP-43 aggregates propagate in a prion-like manner between cells presents the possibility of novel therapeutic strategies to block spreading of these aggregates throughout the brain.
Pub.: 22 Jan '17, Pinned: 24 Aug '17
Abstract: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2-5 years after disease onset. Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords, and rare mutations in the gene encoding TDP-43 can cause ALS. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases. Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1) with ASOs against Sod1 has been shown to substantially slow disease progression. However, as SOD1 mutations account for only around 2-5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions. Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies, and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS. We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy. First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS, targeting ataxin-2 could represent a broadly effective therapeutic strategy.
Pub.: 14 Apr '17, Pinned: 24 Aug '17
Abstract: ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent propagation, accumulation and deposition in motor neurons may explain such a pattern of pathology. Previous work has suggested that the internalization of aggregates triggers stress granule formation. Given the close association of stress granules and TDP-43, we wondered whether internalisation of SOD1 aggregates stimulated TDP-43 cytosolic aggregate structures. Addition of recombinant mutant G93A SOD1 aggregates to NSC-34 cells was found to trigger a rapid shift of TDP-43 to the cytoplasm where it was still accumulated after 48 h. In addition, SOD1 aggregates also triggered cleavage of TDP-43 into fragments including a 25 kDa fragment. Collectively, this study suggests a role for protein aggregate uptake in TDP-43 pathology.
Pub.: 01 Jun '17, Pinned: 24 Aug '17
Abstract: Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.
Pub.: 18 Sep '15, Pinned: 24 Aug '17
Abstract: Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and, therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and discover effective therapeutics. We have generated a Tol2 Gateway-compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores, and compatibility for the introduction of genes of interest or disease-linked genes. This toolbox will advance the rapid and flexible generation of zebrafish models to discover the biology of the nervous system and the disease processes that lead to neurodegeneration.
Pub.: 16 Sep '16, Pinned: 24 Aug '17
Abstract: Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.
Pub.: 13 Feb '17, Pinned: 24 Aug '17
Abstract: TDP-43 is the major component protein of ubiquitin-positive inclusions in brains of patients with frontotemporal lobar degeneration (FTLD-TDP) or amyotrophic lateral sclerosis (ALS). Here, we report the characterization of prion-like properties of aggregated TDP-43 prepared from diseased brains. When insoluble TDP-43 from ALS or FTLD-TDP brains was introduced as seeds into SH-SY5Y cells expressing TDP-43, phosphorylated and ubiquitinated TDP-43 was aggregated in a self-templating manner. Immunoblot analyses revealed that the C-terminal fragments of insoluble TDP-43 characteristic of each disease type acted as seeds, inducing seed-dependent aggregation of TDP-43 in these cells. The seeding ability of insoluble TDP-43 was unaffected by proteinase treatment but was abrogated by formic acid. One subtype of TDP-43 aggregate was resistant to boiling treatment. The insoluble fraction from cells harboring TDP-43 aggregates could also trigger intracellular TDP-43 aggregation. These results indicate that insoluble TDP-43 has prion-like properties that may play a role in the progression of TDP-43 proteinopathy.
Pub.: 09 Jul '13, Pinned: 24 Aug '17