Andrew J. Petersen

Dnr1 mutations cause neurodegeneration in Drosophila by activating the innate immune response in the brain

Significance Infection triggers the innate immune response in all metazoans, activating regulatory pathways that result in expression of effector proteins, including potent antimicrobial peptides. These pathways can also be activated in the brain by aging, stress, and injury. Although nominally protective, excessive neuroinflammatory responses may themselves contribute to neurodegenerative disease by mechanisms that remain unclear. We found that hyperactivation of innate immunity in the Drosophila brain as a result of mutation or bacterial injection causes neurodegeneration because of neurotoxic effects of antimicrobial peptides. These findings have important implications for the role of neuroinflammation in human neurodegenerative disease. A growing body of evidence in humans implicates chronic activation of the innate immune response in the brain as a major cause of neuropathology in various neurodegenerative conditions, although the mechanisms remain unclear. In an unbiased genetic screen for mutants exhibiting neurodegeneration in Drosophila, we have recovered a mutation of dnr1 (defense repressor 1), a negative regulator of the Imd (immune deficiency) innate immune-response pathway. dnr1 mutants exhibit shortened lifespan and progressive, age-dependent neuropathology associated with activation of the Imd pathway and elevated expression of AMP (antimicrobial peptide) genes. To test the hypothesis that overactivation of innate immune-response pathways in the brain is responsible for neurodegeneration, we demonstrated that direct bacterial infection in the brain of wild-type flies also triggers neurodegeneration. In both cases, neurodegeneration is dependent on the NF-κB transcription factor, Relish. Moreover, we found that neural overexpression of individual AMP genes is sufficient to cause neurodegeneration. These results provide a mechanistic link between innate immune responses and neurodegeneration and may have important implications for the role of neuroinflammation in human neurodegenerative diseases as well.

ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila

To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia–telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non–cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimers in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.

The Innate Immune Response Transcription Factor Relish Is Necessary for Neurodegeneration in a Drosophila Model of Ataxia-Telangiectasia

Neurodegeneration is a hallmark of the human disease ataxia-telangiectasia (A-T) that is caused by mutation of the A-T mutated (ATM) gene. We have analyzed Drosophila melanogaster ATM mutants to determine the molecular mechanisms underlying neurodegeneration in A-T. Previously, we found that ATM mutants upregulate the expression of innate immune response (IIR) genes and undergo neurodegeneration in the central nervous system. Here, we present evidence that activation of the IIR is a cause of neurodegeneration in ATM mutants. Three lines of evidence indicate that ATM mutations cause neurodegeneration by activating the Nuclear Factor-κB (NF-κB) transcription factor Relish, a key regulator of the Immune deficiency (Imd) IIR signaling pathway. First, the level of upregulation of IIR genes, including Relish target genes, was directly correlated with the level of neurodegeneration in ATM mutants. Second, Relish mutations inhibited upregulation of IIR genes and neurodegeneration in ATM mutants. Third, overexpression of constitutively active Relish in glial cells activated the IIR and caused neurodegeneration. In contrast, we found that Imd and Dif mutations did not affect neurodegeneration in ATM mutants. Imd encodes an activator of Relish in the response to gram-negative bacteria, and Dif encodes an immune responsive NF-κB transcription factor in the Toll signaling pathway. These data indicate that the signal that causes neurodegeneration in ATM mutants activates a specific NF-κB protein and does so through an unknown activator. In summary, these findings suggest that neurodegeneration in human A-T is caused by activation of a specific NF-κB protein in glial cells.

A Drosophila model of closed head traumatic brain injury

Significance Traumatic brain injury (TBI) occurs when a strong jolt to the head causes damage to brain cells, resulting in immediate and long-term consequences including physical, behavioral, and cognitive problems. Despite the importance of TBI as a major health issue, our understanding of the underlying cellular and molecular mechanisms is limited. To unravel these mechanisms, we have developed a model of TBI in the fruit fly, Drosophila melanogaster, where we can apply many powerful experimental tools. The main features of human TBI also occur in flies, suggesting that the underlying mechanisms are conserved. Our studies demonstrate the value of a fly model for understanding the consequences of TBI and may ultimately enable development of therapies for their prevention and treatment. Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of experimental tools available in flies. Closed head TBI was inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibited temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. Our data indicate that TBI results in death shortly after a primary injury only if the injury exceeds a certain threshold and that age and genetic background, but not sex, substantially affect this threshold. Furthermore, this threshold also appears to be dependent on the same cellular and molecular mechanisms that control normal longevity. This study demonstrates the potential of flies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.

PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte Pathology in Alzheimer's Disease

Summary Alzheimers disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased β-amyloid production, altered cytokine release, and dysregulated Ca2+ homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca2+ transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.

Drosophila innate immune response pathways moonlight in neurodegeneration

In this Extra View, we highlight recent Drosophila research that has uncovered a new role for the innate immune response. The research indicates that, in addition to combating infection, the innate immune response promotes neurodegeneration. Our publication (Petersen et al., 2012) reveals a correlative relationship between the innate immune response and neurodegeneration in a model of the human disease Ataxia-telangiectasia (A-T). We also found that glial cells are responsible for the innate immune response in the A-T model, and work by others implicates glial cells in neurodegeneration. Additionally, publications by Chinchore et al. (2012) and Tan et al. (2008) reveal a causative role for the innate immune response in models of human retinal degenerative disorders and Alzheimer disease, respectively. Collectively, these findings suggest that activation of the innate immune response is a shared cause of neurodegeneration in different human diseases.

Generation of a rod-specific NRL reporter line in human pluripotent stem cells

Reporter lines generated in human pluripotent stem cells can be highly useful for the analysis of specific cell types and lineages in live cultures. We created the first human rod reporter line using CRISPR/Cas9 genome editing to replace one allele of the Neural Retina Leucine zipper (NRL) gene with an eGFP transgene in the WA09 human embryonic stem cell (hESC) line. After confirming successful targeting, three-dimensional optic vesicle structures were produced to examine reporter specificity and to track rod differentiation in culture. The NRL+/eGFP hESC line robustly and exclusively labeled the entirety of rods throughout differentiation, eventually revealing highly mature structural features. This line provides a valuable tool for studying human rod development and disease and testing therapeutic strategies for retinitis pigmentosa.

A CRISPR/Cas9 based strategy to manipulate the Alzheimer\'s amyloid pathway

The gradual accumulation of amyloid-β (Aβ) is a neuropathologic hallmark of Alzheimer’s disease (AD); playing a key role in disease progression. Aβ is generated by the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases, with BACE-1 (β-site APP cleaving enzyme-1) cleavage as the rate limiting step 1–3. CRISPR/Cas9 guided gene-editing is emerging as a promising tool to edit pathogenic mutations and hinder disease progression 4,5,6 However, few studies have applied this technology to neurologic diseases 7–9. Besides technical caveats such as low editing efficiency in brains and limited in vivo validation 7, the canonical approach of ‘mutation-correction’ would only be applicable to the small fraction of neurodegenerative cases that are inherited (i.e. < 10% of AD, Parkinson’s, ALS); with a new strategy needed for every gene. Moreover, feasibility of CRISPR/Cas9 as a therapeutic possibility in sporadic AD has not been explored. Here we introduce a strategy to edit endogenous APP at the extreme C-terminus and reciprocally manipulate the amyloid pathway – attenuating β-cleavage and Aβ, while up-regulating neuroprotective a-cleavage. APP N-terminus, as well as compensatory APP homologues remain intact, and key physiologic parameters remain unaffected. Robust APP-editing is seen in cell lines, cultured neurons, human embryonic stem cells/iPSC-neurons, and mouse brains. Our strategy works by limiting the physical association of APP and BACE-1, and we also delineate the mechanism that abrogates APP/BACE-1 interaction in this setting. Our work offers an innovative ‘cut and silence’ gene-editing strategy that could be a new therapeutic paradigm for AD.

A CRISPR/CAS9 BASED STRATEGY TO ATTENUATE THE β-AMYLOID PATHWAY

C-terminus

HUMAN IPSC-DERIVED ALZHEIMER’S DISEASE ASTROCYTES RECAPITULATE DISEASE-RELATED PHENOTYPES

brains. However, other genes on human chr21 likely modulate the age of onset, severity and modality of the clinical picture, as DS individuals have later or absent onset of clinical AD, and less intracerebral haemorrhage pathology, than euploid individuals with the familial early onset AD caused by a duplication of APP gene (dupAPP). Our aim is to identify such modulator genes on chr21 using cellular models. Methods:Neurons derived from isogenic hiPSCs we generated from a mosaic DS individual, unpublished iPSCs from segmental trisomy 21 DS and non-DS individuals, as well as from DS individuals with extremely early or late dementia onset are used in 2D, 3D and cerebral organoid paradigms. Optogenetic stimulation of Channelrhodopsin-2 engineered hiPSCs is used to determine the neuronal activity-dependent cellular phenotype modulation. CRISPR/Cas9 mediated reduction from trisomy to disomy for individual selected candidate genes on chr21 are performed on isogenic T21 iPSCs. Results:We detect T21-caused neurodevelopmental delay, increased b-amyloid and phospho-Tau presence, complex mitochondrial dysfunction, accelerated DNA damage and abnormal endosomes, and some of these phenotypes exacerbate in 3D and cerebral organoid paradigms, and/or can be reproduced in primary human neuroectodermal cells expanded and differentiated in vitro. Conclusions: Cellular phenotypes relevant for AD pathology caused by trisomy21 can be reproduced and quantitatively assessed in iPSC-derived and primary NSC-derived neurons and differences sharpened by the use of cerebral organoid technology. Isogenic iPSCs allow detection of subtle changes in phenotypes and evaluation of single gene’s trisomy contribution using CRISPR/Cas9 editing. Segmental trisomy 21 iPSCs allow for assignment of phenotype effects to a region of chromosome 21, and can faster eliminate some candidate genes.