Doris Kretzschmar

Age-Dependent Neurodegeneration and Alzheimer-Amyloid Plaque Formation in Transgenic Drosophila

β-Amyloid peptides that are cleaved from the amyloid precursor protein (APP) play a critical role in Alzheimers disease (AD) pathophysiology. Here, we show that in Drosophila, the targeted expression of the key genes of AD, APP, theβ-site APP-cleaving enzyme BACE, and the presenilins led to the generation of β-amyloid plaques and age-dependent neurodegeneration as well as to semilethality, a shortened life span, and defects in wing vein development. Genetic manipulations or pharmacological treatments with secretase inhibitors influenced the activity of the APP-processing proteases and modulated the severity of the phenotypes. This invertebrate model of amyloid plaque pathology demonstrates Aβ-induced neurodegeneration as a basic biological principle and may allow additional genetic analyses of the underlying molecular pathways.

Neurotoxic effects induced by the Drosophila amyloid-β peptide suggest a conserved toxic function

The accumulation of amyloid-beta (Abeta) into plaques is a hallmark feature of Alzheimers disease (AD). While amyloid precursor protein (APP)-related proteins are found in most organisms, only Abeta fragments from human APP have been shown to induce amyloid deposits and progressive neurodegeneration. Therefore, it was suggested that neurotoxic effects are a specific property of human Abeta. Here we show that Abeta fragments derived from the Drosophila orthologue APPL aggregate into intracellular fibrils, amyloid deposits, and cause age-dependent behavioral deficits and neurodegeneration. We also show that APPL can be cleaved by a novel fly beta-secretase-like enzyme. This suggests that Abeta-induced neurotoxicity is a conserved function of APP proteins whereby the lack of conservation in the primary sequence indicates that secondary structural aspects determine their pathogenesis. In addition, we found that the behavioral phenotypes precede extracellular amyloid deposit formation, supporting results that intracellular Abeta plays a key role in AD.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila.

Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila

Patients with polyglutamine expansion diseases, like Huntingtons disease or several spinocerebellar ataxias, first present with neurological symptoms that can occur in the absence of neurodegeneration. Behavioral symptoms thus appear to be caused by neuronal dysfunction, rather than cell death. Pathogenesis in polyglutamine expansion diseases is largely viewed as a cell‐autonomous process in neurons. It is likely, however, that this process is influenced by changes in glial physiology and, at least in the case of DRPLA glial inclusions and glial cell death, seems to be an important part in the pathogenesis. To investigate these aspects in a Drosophila model system, we expressed polyglutamine proteins in the adult nervous system. Glial‐specific expression of a polyglutamine (Q)‐expanded (n = 78) and also a nonexpanded (n = 27) truncated version of human ataxin‐3 led to the formation of protein aggregates and glial cell death. Behavioral changes were observed prior to cell death. This reveals that glia is susceptible to the toxic action of polyglutamine proteins. Neuronal expression of the same constructs resulted in behavioral changes similar to those resulting from glial expression but did not cause neurodegeneration. Behavioral deficits were selective and affected two analyzed fly behaviors differently. Both glial and neuronal aggregates of Q78 and Q27 appeared early in pathogenesis and, at the electron microscopic resolution, had a fibrillary substructure. This shows that a nonexpanded stretch can cause similar histological and behavioral symptoms as the expanded stretch, however, with a significant delay.

Loss-of-function mutations in PNPLA6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in Gordon Holmes syndrome.

CONTEXT Gordon Holmes syndrome (GHS) is characterized by cerebellar ataxia/atrophy and normosmic hypogonadotropic hypogonadism (nHH). The underlying pathophysiology of this combined neurodegeneration and nHH remains unknown. OBJECTIVE We aimed to provide insight into the disease mechanism in GHS. METHODS We studied a cohort of 6 multiplex families with GHS through autozygosity mapping and whole-exome sequencing. RESULTS We identified 6 patients from 3 independent families carrying loss-of-function mutations in PNPLA6, which encodes neuropathy target esterase (NTE), a lysophospholipase that maintains intracellular phospholipid homeostasis by converting lysophosphatidylcholine to glycerophosphocholine. Wild-type PNPLA6, but not PNPLA6 bearing these mutations, rescued a well-established Drosophila neurodegenerative phenotype caused by the absence of sws, the fly ortholog of mammalian PNPLA6. Inhibition of NTE activity in the LβT2 gonadotrope cell line diminished LH response to GnRH by reducing GnRH-stimulated LH exocytosis, without affecting GnRH receptor signaling or LHβ synthesis. CONCLUSION These results suggest that NTE-dependent alteration of phospholipid homeostasis in GHS causes both neurodegeneration and impaired LH release from pituitary gonadotropes, leading to nHH.

Swiss Cheese, a protein involved in progressive neurodegeneration, acts as a noncanonical regulatory subunit for PKA-C3.

The Drosophila Swiss Cheese (SWS) protein and its vertebrate ortholog Neuropathy Target Esterase (NTE) are required for neuronal survival and glial integrity. In humans, NTE is the target of organophosphorous compounds which cause a paralyzing axonal degeneration and recently mutations in NTE have been shown to cause a Hereditary Spastic Paraplegia called NTE-related Motor-Neuron Disorder. SWS and NTE are concentrated in the endoplasmic reticulum and both have been shown to have an esterase function against an artificial substrate. However, the functional mechanisms and the pathways in which SWS/NTE are involved in are still widely unknown. Here, we show that SWS interacts specifically with the C3 catalytic subunit of cAMP activated protein kinase (PKA-C3), which together with orthologs in mouse (Pkare) and human (PrKX) forms a novel class of catalytic subunits of unknown function. This interaction requires a domain of SWS which shows homology to regulatory subunits of PKA and, like conventional regulatory subunits, the binding of SWS to the PKA-C3 inhibits its function. Consistent with this result, expression of additional PKA-C3 induces degeneration and enhances the neurodegenerative phenotype in sws mutants. We also show that the complex formation with the membrane-bound SWS tethers PKA-C3 to membranes. We therefore propose a model in which SWS acts as a noncanonical subunit for PKA-C3, whereby the complex formation regulates the localization and kinase activity of PKA-C3, and that disruption of this regulation can induce neurodegeneration.

Amyloid precursor proteins are protective in Drosophila models of progressive neurodegeneration.

The processing of Amyloid Precursor Proteins (APPs) results in several fragments, including soluble N-terminal ectodomains (sAPPs) and C-terminal intracellular domains (AICD). sAPPs have been ascribed neurotrophic or neuroprotective functions in cell culture, although β-cleaved sAPPs can have deleterious effects and trigger neuronal cell death. Here we describe a neuroproprotective function of APP and fly APPL (Amyloid Precursor Protein-like) in vivo in several Drosophila mutants with progressive neurodegeneration. We show that expression of the N-terminal ectodomain is sufficient to suppress the progressive degeneration in these mutants and that the secretion of the ectodomain is required for this function. In addition, a protective effect is achieved by expressing kuzbanian (which has α-secretase activity) whereas expression of fly and human BACE aggravates the phenotypes, suggesting that the protective function is specifically mediated by the α-cleaved ectodomain. Furthermore, genetic and molecular studies suggest that the N-terminal fragments interact with full-length APPL activating a downstream signaling pathway via the AICD. Because we show protective effects in mutants that affect different genes (AMP-activated protein kinase, MAP1b, rasGAP), we propose that the protective effect is not due to a genetic interaction between APPL and these genes but a more general aspect of APP proteins. The result that APP proteins and specifically their soluble α-cleaved ectodomains can protect against progressive neurodegeneration in vivo provides support for the hypothesis that a disruption of the physiological function of APP could play a role in the pathogenesis of Alzheimers Disease.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photoreceptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

Identification of novel 1,4-benzoxazine compounds that are protective in tissue culture and in vivo models of neurodegeneration.

Neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, and Huntingtons disease and conditions such as ischemic stroke affect millions of individuals annually and exert an enormous financial burden on society. A hallmark of these conditions is the abnormal loss of neurons. Currently, there are no effective strategies to prevent neuronal death in these pathologies. We report that several 2‐arylidine and 2‐hetarylidin derivatives of the 1,4‐benzoxazines class of compounds are highly protective in tissue culture models of neurodegeneration. Results obtained using pharmcalogical inhibitors indicate that neuroprotection by these compounds does not involve the Raf–MEK–ERK or PI‐3 kinase–Akt signaling pathways nor other survival‐promoting molecules such as protein kinase A (PKA), calcium calmodulin kinase A (CaMK), and histone deacetylases (HDACs). We tested one of these compounds, (Z)‐6‐amino‐2‐(3′,5′‐dibromo‐4′‐hydroxybenzylidene)‐2H‐benzo[b][1,4]oxazin‐3(4H)‐one, designated as HSB‐13, in the 3‐nitropropionic acid (3‐NP)‐induced mouse model of Huntingtons disease. HSB‐13 reduced striatal degeneration and improved behavioral performance in mice administered with 3‐NP. Furthermore, HSB‐13 was protective in a Drosophila model of amyloid precursor protein (APP) toxicity. To understand how HSB‐13 and other 1,4‐benzoxazines protect neurons, we performed kinase profiling analyses. These analyses showed that HSB‐13 inhibits GSK3, p38 MAPK, and cyclin‐dependent kinases (CDKs). In comparison, another compound, called ASK‐2a, that protects cerebellar granule neurons against low‐potassium‐induced death inhibits GSK3 and p38 MAPK but not CDKs. Despite its structural similarity to HSB‐13, however, ASK‐2a is incapable of protecting cortical neurons and HT22 cells against homocysteic acid (HCA)‐induced or Aβ toxicity, suggesting that protection against HCA and Aβ depends on CDK inhibition. Compounds described in this study represent a novel therapeutic tool in the treatment of neurodegenerative diseases.

Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases?

There are 50 ways to leave your lover (Simon 1987) but many more to kill your brain cells. Several neurodegenerative diseases in humans, like Alzheimer’s disease, have been intensely studied but the underlying cellular and molecular mechanisms are still unknown for most of them. For those syndromes where associated gene products have been identified their biochemistry and physiological as well as pathogenic function is often still under debate. This is in part due to the inherent limitations of genetic analyses in humans and other mammals and therefore experimentally accessible invertebrate in vivo models, such as Caenorhabditis elegans and Drosophila melanogaster, have recently been introduced to investigate neurodegenerative syndromes. Several laboratories have used transgenic approaches in Drosophila to study the human genes associated with neurodegenerative diseases. This has added substantially to our understanding of the mechanisms leading to neurodegenerative diseases in humans. The isolation and characterization of Drosophila mutants, which display a variety of neurodegenerative phenotypes, also provide valuable insights into genes, pathways, and mechanisms causing neurodegeneration. So far only about two dozen such mutants have been described but already their characterization reveals an involvement of various cellular functions in neurodegeneration, ranging from preventing oxidative stress to RNA editing. Some of the isolated genes can already be associated with human neurodegenerative diseases and hopefully the isolation and characterization of more of these mutants, together with an analysis of homologous genes in vertebrate models, will provide insights into the genetic and molecular basis of human neurodegenerative diseases.