Alpha-synuclein null mutation exacerbates the phenotype of a model of Menkes disease in female mice.

Genetic modifier screens provide a useful tool, in diverse organisms from Drosophila to C. elegans and mice, for recovering new genes of interest that may reduce or enhance a phenotype of interest. This study reports a modifier screen, based on N-ethyl-N-nitrosourea (ENU) mutagenesis and outcrossing, designed to increase understanding of the normal function of murine α-synuclein (Snca). Human SNCA was the first gene linked to familial Parkinson's disease. Since the discovery of the genetic link of SNCA to Parkinson's nearly three decades ago, numerous studies have investigated the normal function of SNCA protein with divergent roles associated with different cellular compartments. Understanding of the normal function of murine Snca is complicated by the fact that mice with homozygous null mutations live a normal lifespan and have only subtle synaptic deficits. Here, we report that the first genetic modifier (a sensitized mutation) that was identified in our screen was the X-linked gene, ATPase copper transporting alpha (Atp7a). In humans, mutations in Atp7a are linked to to Menkes disease, a disease with pleiotropic phenotypes that include a severe neurological component. Atp7a encodes a trans-Golgi copper transporter that supplies the copper co-factor to enzymes that pass through the ER-Golgi network. Male mice that carry a mutation in Atp7a die within 3 weeks of age regardless of Snca genotype. In contrast, here we show that Snca disruption modifies the phenotype of Atp7a in female mice. Female mice that carry the Atp7a mutation, on an Snca null background, die earlier (prior to 35 days) at a significantly higher rate than those that carry the Atp7a mutation on a wildtype Snca background ATPase copper transporting alpha. Thus, Snca null mutations sensitize female mice to mutations in Atp7a, suggesting that Snca protein may have a protective effect in females, perhaps in neurons, given the co-expression patterns. Although data has suggested diverse functions for human and mouse α-synuclein proteins in multiple cell compartments, this is the first demonstration via use of genetic screening to demonstrate that Snca protein may function in the ER-Golgi system in the mammalian brain in a sex-dependent manner.

Therapeutic Trial of anle138b in Mouse Models of Genetic Prion Disease.

Phenotypic screening has yielded small-molecule inhibitors of prion replication that are effective in vivo against certain prion strains but not others. Here, we sought to test the small molecule anle138b in multiple mouse models of prion disease. In mice inoculated with the RML strain of prions, anle138b doubled survival and durably suppressed astrogliosis measured by live-animal bioluminescence imaging. In knock-in mouse models of the D178N and E200K mutations that cause genetic prion disease, however, we were unable to identify a clear, quantifiable disease endpoint against which to measure therapeutic efficacy. Among untreated animals, the mutations did not impact overall survival, and bioluminescence remained low out to >20 months of age. Vacuolization and PrP deposition were observed in some brain regions in a subset of mutant animals but appeared to be unable to carry the weight of a primary endpoint in a therapeutic study. We conclude that not all animal models of prion disease are suited to well-powered therapeutic efficacy studies, and care should be taken in choosing the models that will support drug development programs. IMPORTANCE There is an urgent need to develop drugs for prion disease, a currently untreatable neurodegenerative disease. In this effort, there is a debate over which animal models can best support a drug development program. While the study of prion disease benefits from excellent animal models because prions naturally afflict many different mammals, different models have different capabilities and limitations. Here, we conducted a therapeutic efficacy study of the drug candidate anle138b in mouse models with two of the most common mutations that cause genetic prion disease. In a more typical model where prions are injected directly into the brain, we found anle138b to be effective. In the genetic models, however, the animals never reached a clear, measurable point of disease onset. We conclude that not all prion disease animal models are ideally suited to drug efficacy studies, and well-defined, quantitative disease metrics should be a priority.

Parkinson's Disease: Overview of Transcription Factor Regulation, Genetics, and Cellular and Animal Models.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, affecting nearly 7-10 million people worldwide. Over the last decade, there has been considerable progress in our understanding of the genetic basis of PD, in the development of stem cell-based and animal models of PD, and in management of some clinical features. However, there remains little ability to change the trajectory of PD and limited knowledge of the underlying etiology of PD. The role of genetics versus environment and the underlying physiology that determines the trajectory of the disease are still debated. Moreover, even though protein aggregates such as Lewy bodies and Lewy neurites may provide diagnostic value, their physiological role remains to be fully elucidated. Finally, limitations to the model systems for probing the genetics, etiology and biology of Parkinson's disease have historically been a challenge. Here, we review highlights of the genetics of PD, advances in understanding molecular pathways and physiology, especially transcriptional factor (TF) regulators, and the development of model systems to probe etiology and potential therapeutic applications.

Regional variability and genotypic and pharmacodynamic effects on PrP concentration in the CNS.

Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints.

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

Antisense oligonucleotides extend survival of prion-infected mice.

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.

Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration.

Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid-based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water (2H2O), distinct, newly synthesized 2H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2H2O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson's disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.

Exacerbated synucleinopathy in mice expressing A53T SNCA on a Snca null background.

Alpha-Synuclein is a major component of Lewy bodies, neuronal inclusions diagnostic for Parkinson's disease (PD). While an Ala53Thr mutation in alpha-synuclein can cause PD in humans, in mice the wildtype residue at position 53 is threonine, indicating that mice are either too short-lived to develop PD, or are protected by the six other amino acid differences between the proteins in these two species. Mice carrying an Ala53Thr human SNCA transgene driven by the mouse prion promoter show a mild movement disorder and only rarely develop severe pathology by 2 years of age. To determine whether the presence of mouse alpha-synuclein affects the pathogenicity of the human protein, the transgene was crossed into mice lacking endogenous alpha-synuclein. Mice that express only human alpha-synuclein developed a neuronopathy characterized by limb weakness and paralysis with onset beginning at 16 months of age. The neuronopathy is probably due to high levels of expression of the transgene in the ventral spinal cord leading to motor neuron damage and Wallerian degeneration of the ventral roots. These data suggest mouse alpha-synuclein is protective against the deleterious effects of the human mutant protein.

Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein.

Although the mutation of alpha-synuclein, a protein associated with presynaptic vesicles, is implicated in the etiology and pathogenesis of Parkinson's disease, the biological function of the normal protein is unknown. Mice that lack alpha-synuclein have been generated by homologous recombination in embryonic stem cells. Electron microscopic examination of hippocampal synapses revealed a striking selective deficiency of undocked vesicles without affecting docked vesicles. Field recording of CA1 synapses in hippocampal slices from the mutant mice demonstrated normal basal synaptic transmission, paired-pulse facilitation, and response to a brief train of high-frequency stimulation (100 Hz, 40 pulses) that exhausts only docked vesicles. In contrast, the alpha-synuclein knock-out mice exhibited significant impairments in synaptic response to a prolonged train of repetitive stimulation (12.5 Hz, 300 pulses) capable of depleting docked as well as reserve pool vesicles. Moreover, the replenishment of the docked vesicles by reserve pool vesicles after depletion was slower in the mutant synapses. Thus, alpha-synuclein may be required for the genesis and/or maintenance of a subset of presynaptic vesicles, those in the "reserve" or "resting" pools. These results reveal, for the first time, the normal function of endogenous alpha-synuclein in regulating synaptic vesicle mobilization at nerve terminals.