Rachel M. Bailey

The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins

A primary pathologic component of Alzheimers disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70-interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies.

Progranulin Mediates Caspase-Dependent Cleavage of TAR DNA Binding Protein-43

TAR DNA binding protein-43 (TDP-43) is the pathologic substrate of neuronal and glial inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTDL-U) and in amyotrophic lateral sclerosis (ALS). Mutations in the progranulin gene (PGRN) have been shown to cause familial FTLD-U. The relationship between progranulin and TDP-43 and their respective roles in neurodegeneration is unknown. We report that progranulin mediates proteolytic cleavage of TDP-43 to generate ∼35 and ∼25 kDa species. Suppression of PGRN expression with small interfering RNA leads to caspase-dependent accumulation of TDP-43 fragments that can be inhibited with caspase inhibitor treatment. Cells treated with staurosporine also induced caspase-dependent cleavage and redistribution of TDP-43 from its nuclear localization to cytoplasm. Altered cleavage and redistribution of TDP-43 in cell culture models are similar to findings in FTLD-U and ALS. The results suggest that abnormal metabolism of TDP-43 mediated by progranulin may play a pivotal role in neurodegeneration.

CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinsons disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.

Akt and CHIP coregulate tau degradation through coordinated interactions.

A hallmark of the pathology of Alzheimers disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PAR1/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimers disease.

LRRK2 phosphorylates novel tau epitopes and promotes tauopathy

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson’s disease (PD). The neuropathology of LRRK2-related PD is heterogeneous and can include aberrant tau phosphorylation or neurofibrillary tau pathology. Recently, LRRK2 has been shown to phosphorylate tau in vitro; however, the major epitopes phosphorylated by LRRK2 and the physiological or pathogenic consequences of these modifications in vivo are unknown. Using mass spectrometry, we identified multiple sites on recombinant tau that are phosphorylated by LRRK2 in vitro, including pT149 and pT153, which are phospho-epitopes that to date have been largely unexplored. Importantly, we demonstrate that expression of transgenic LRRK2 in a mouse model of tauopathy increased the aggregation of insoluble tau and its phosphorylation at T149, T153, T205, and S199/S202/T205 epitopes. These findings indicate that tau can be a LRRK2 substrate and that this interaction can enhance salient features of human disease.

Methods for gene transfer to the central nervous system.

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed.

In vivo functional brain mapping in a conditional mouse model of human tauopathy (tauP301L) reveals reduced neural activity in memory formation structures.

BackgroundTauopathies are characterized by intracellular deposition of the microtubule-associated protein tau as filamentous aggregates. The rTg4510 mouse conditionally expresses mutant human tau protein in various forebrain areas under the Tet-off expression system. Mice develop neurofibrillary tangles, with significant neuronal loss and cognitive deficits by 6 months of age. Previous behavioral and biochemical work has linked the expression and aggregates of mutant tau to functional impairments. The present work used manganese-enhanced magnetic resonance imaging (MEMRI) to investigate basal levels of brain activity in the rTg4510 and control mice.ResultsOur results show an unmistakable curtailment of neural activity in the amygdala and hippocampus, two regions known for their role in memory formation, but not the cortex, cerebellum, striatum and hypothalamus in tau expressing mice.ConclusionBehavioral impairments associated with changes in activity in these areas may correspond to age progressive mutant tauP301L-induced neurodegeneration.

Physiologically relevant factors influence tau phosphorylation by leucine-rich repeat kinase 2

Hyperphosphorylation and aggregation of tau are observed in multiple neurodegenerative diseases termed tauopathies. Tau has also been implicated in the pathogenesis of Parkinsons disease (PD) and parkinsonisms. Some PD patients with mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene exhibit tau pathology. Mutations in LRRK2 are a major risk factor for PD, but LRRK2 protein function remains unclear. The most common mutation, G2019S, is located in the kinase domain of LRRK2 and enhances kinase activity in vitro. This suggests that the kinase activity of LRRK2 may underlie its cellular toxicity. Recently, in vitro studies have suggested a direct interaction between tubulin‐bound tau and LRRK2 that results in tau phosphorylation at one identified site. Here we present data suggesting that microtubules (MTs) enhance LRRK2‐mediated tau phosphorylation at three different epitopes. We also explore the effect of divalent cations as catalytic cofactors for G2019S LRRK2‐mediated tau phosphorylation and show that manganese does not support kinase activity but inhibits the efficient ability of magnesium to catalyze LRRK2‐mediated phosphorylation of tau. These results suggest that cofactors such as MTs and cations in the cellular milieu have an important impact on LRRK2‐tau interactions and resultant tau phosphorylation.

Development of Intrathecal AAV9 Gene Therapy for Giant Axonal Neuropathy

An NIH-sponsored phase I clinical trial is underway to test a potential treatment for giant axonal neuropathy (GAN) using viral-mediated GAN gene replacement (https://clinicaltrials.gov/ct2/show/NCT02362438). This trial marks the first instance of intrathecal (IT) adeno-associated viral (AAV) gene transfer in humans. GAN is a rare pediatric neurodegenerative disorder caused by autosomal recessive loss-of-function mutations in the GAN gene, which encodes the gigaxonin protein. Gigaxonin is involved in the regulation, turnover, and degradation of intermediate filaments (IFs). The pathologic signature of GAN is giant axonal swellings filled with disorganized accumulations of IFs. Herein, we describe the development and characterization of the AAV vector carrying a normal copy of the human GAN transgene (AAV9/JeT-GAN) currently employed in the clinical trial. Treatment with AAV/JeT-GAN restored the normal configuration of IFs in patient fibroblasts within days in cell culture and by 4 weeks in GAN KO mice. IT delivery of AAV9/JeT-GAN in aged GAN KO mice preserved sciatic nerve ultrastructure, reduced neuronal IF accumulations and attenuated rotarod dysfunction. This strategy conferred sustained wild-type gigaxonin expression across the PNS and CNS for at least 1 year in mice. These results support the clinical evaluation of AAV9/JeT-GAN for potential therapeutic outcomes and treatment for GAN patients.

604. Comparison of Intra-Cisterna Magna and Lumbar Puncture Intrathecal Delivery of scAAV9 GeneTherapy for Giant Axonal Neuropathy

Giant axonal neuropathy (GAN) is a rare pediatric neurodegenerative disorder characterized by progressive neuropathy that presents as early as 3 years of age and with ultimate mortality during the second or third decade of life. GAN is caused by autosomal recessive loss-of-function mutations in the GAN gene that encodes for the gigaxonin protein. Gigaxonin plays a role in the organization/degradation of intermediate filaments (IFs) and GAN patients are pathologically characterized by large axonal swellings filled with disorganized aggregates of IFs. While GAN is primarily described as a progressive peripheral neuropathy, diffuse pathology from disorganized IFs is apparent throughout the central nervous system, enteric nervous system and other organ systems. An NIH-sponsored Phase I study is underway to test the safety of intrathecal lumbar puncture (LP) administration of scAAV9/JeT-GAN to treat the most severe aspects of GAN, namely the motor and sensory neuropathy. Gigaxonin gene transfer through a single LP injection is the first proposed therapy for GAN. Intra-cisterna magna (ICM) delivery of AAV9 vectors shows high transduction of the brain and spinal cord of animals; however, this method of vector delivery has not yet been tested for the treatment of GAN. This study compared the efficacy of using ICM or LP delivery of the scAAV9/JeT-GAN vector to treat GAN KO mice. GAN KO mice were injected with scAAV9/JeT-GAN at 15 months of age using ICM or LP delivery and motor performance was tested monthly. In agreement with our previous studies, we found that GAN KO mice have impaired motor performance around 20 months of age and that this deficit is attenuated with LP delivery of scAAV9/JeT-GAN. In contrast, GAN KO mice receiving ICM-delivered scAAV9/JeT-GAN did not have significantly improved motor function as compared to vehicle treated GAN KO mice. Analyses of disease-relevant pathologies in the brain, spinal cord, and peripheral nerves of these mice are ongoing. To date, antibodies are not available to reliably detect gigaxonin protein expression via immunohistochemical (IHC) analysis, so to directly compare the transduction of the two intrathecal delivery methods, we injected wild-type mice with ~4 × 1011 vg scAAV9/GFP via an ICM or LP injection, which is a higher dose than has been reported in any publication. Mice were harvested 4-weeks post-injection and the biodistribution of scAAV9 was analyzed via semi-quantitative PCR and IHC analysis of GFP. Compared to LP-injected mice, GFP expression was notably higher and more wide-spread in the brains of ICM-injected mice, while higher amounts of GFP were detected in the sciatic nerves of LP-injected mice as compared to ICM-injected mice. IHC analysis is ongoing and we will present a detailed map of the transduction of ICM- and LP-delivered scAAV9/GFP across the central and peripheral nervous systems and different organ systems. In conclusion, we report here that ICM-delivery of scAAV9/JeT-GAN does not attenuate motor deficits in GAN KO mice. Furthermore, the vector spread varies between ICM and LP delivery, suggesting that the site of intrathecal injection may have a critical impact on the therapeutic benefit of gene vectors for a given disease.