In the La Spada laboratory, we apply the tools of molecular genetics, neuroscience, biochemistry, functional genomics, cell biology, pharmacology, and physiology to understand the cellular and molecular basis of neurodegenerative disease. Within the last decade, it has become clear that a key question in the neurodegenerative disease field is the selective vulnerability of different neuronal populations in the various diseases. Inherited disorders such as Huntington's disease are characterized by widespread expression of a mutant gene product throughout the central nervous system but display well circumscribed patterns of neuronal dysfunction and demise. This theme is also apparent in genetic examples of common neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and prion disease. Our basic research efforts are dedicated to understanding the basis for this selective vulnerability in different neurological disorders.
We work on three different inherited CAG-polyglutamine repeat diseases: Huntington's disease, X-linked spinal and bulbar muscular atrophy, and spinocerebellar ataxia type 7. We also are pursuing the mechanistic basis of common neurodegenerative disorders, focusing on ALS and Parkinson’s disease. We have generated numerous mouse models for these diseases and have studied transcriptional dysregulation and cellular pathology in these disorders, using a variety of genomics and proteomics approaches. We have generated cell culture and primary neuron models for neurodegenerative disorders, and we are adept at developing and characterizing stem cell models of these diseases by reprogramming primary cells from human patients into induced pluripotent stem cells (iPSCs), from which we derive different types of neurons, astrocytes, and skeletal muscle cells. Our latest stem cell modeling work involves co-culture of different cell types and generation of organoids.
By applying emerging a variety of techniques to the study of neurobiology and neuropathology, we have developed a significant translational and therapy development portfolio, based upon mechanistic insights and target pathways identified from our work. We currently are pursuing a variety of strategies to identify novel chemical matter and biological agents as treatments for neurodegenerative diseases, as we seek to delineate the molecular and cellular pathways by which neurons become dysfunctional and use this knowledge to devise rationale therapies for this class of diseases.
Spinal and bulbar muscular atrophy (SBMA)
X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration. While a graduate student, I identified the cause of SBMA as an expansion of a trinucleotide repeat in the androgen receptor gene. As the first disorder shown to be caused by an expanded polyglutamine repeat tract, this discovery of a novel type of genetic mutation led to the emergence of a new field of study.
To understand the molecular basis of SBMA disease pathogenesis, we introduced the entire AR gene with 100 CAG repeats on a yeast artificial chromosome (YAC) into mice, and successfully recapitulated the SBMA phenotype of neurogenic atrophy and motor neuron loss in a mouse model. Studies of the AR YAC CAG100 (AR100) mouse model indicated that transcription interference with CREB-binding protein (CBP) activation of vascular endothelial growth factor (VEGF) gene expression in the spinal cord may contribute to motor neuronopathy in our SBMA mouse model. Further investigation established that SBMA disease pathogenesis, both in the nervous system and the periphery, involves two simultaneous independent pathways: gain-of-function misfolded protein toxicity and loss of normal protein function.
To determine the cellular basis of SBMA disease pathogenesis, we created novel mouse models of SBMA, including BAC transgenic mice containing a floxed first exon (i.e. the BAC fxAR121 line) to permit cell-type specific excision of the AR transgene. After characterizing these mice and validating their utility for conditional termination of polyQ-AR transgene expression, we crossed BAC fxAR121 mice with Human Skeletal Actin (HSA)-Cre mice, and documented that excision of the AR121Q transgene from skeletal muscle prevented development of both systemic and neuromuscular SBMA disease phenotypes, revealing a crucial role for muscle expression of mutant polyQ-AR in SBMA motor neuron degeneration.
We have also uncovered autophagy dysregulation as a defining feature of SBMA by analyzing in vivo and in vitro models, including a human SBMA stem cell model. These studies revealed abnormalities of autophagosome maturation and fusion with lysosomes in SBMA cell culture models, transgenic mice, and neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs), thereby linking autophagy dysfunction to the onset of SBMA neurodegenerative disease phenotypes. To delineate the basis of this effect, we considered the transcriptional regulation of the autophagy pathway. This work revealed a physical and functional interaction between normal AR protein and transcription factor EB (TFEB), and indicated that TFEB dysregulation accounts for the autophagy defects in SBMA. Our findings suggest that skeletal muscle and autophagy pathway dysregulation are key cellular and molecular targets for motor neuron disease therapy development.
Spinocerebellar ataxia type 7 (SCA7)
Spinocerebellar ataxia type 7 (SCA7) is a dominantly inherited neurodegenerative disorder characterized by cerebellar and retinal degeneration. A productive strategy for determining the molecular basis of a disease process and for providing an avenue for therapeutic advance has been the development of animal models of human diseases. We have applied this strategy in our studies of SCA7, and have used the mouse as a model system for the study of this disease. Using this approach, we successfully recapitulated the retinal and cerebellar degeneration in human SCA7 patients in the mouse, and used this model to obtain a mechanistic explanation for the rather selective cone-rod dystrophy retinal degeneration seen in human patients. Our model indicates that the polyglutamine-expanded version of ataxin-7 causes disease pathogenesis by interfering with the function of a transcription factor whose expression pattern is principally restricted to the photoreceptor nuclei and other retinal neuronal nuclei. This finding supported the concept that polyglutamine diseases involve transcription dysregulation as a key feature and suggests obvious modes of therapeutic intervention. We have also learned that ataxin-7 is a transcription factor, and have found evidence for a dominant negative effect of the polyglutamine expansion upon the transcription co-activator complexes of which ataxin-7 is a part. One major focus of our SCA7 work is to identify the genes whose expression is altered in this disease, due to impaired chromatin remodeling.
Our studies of SCA7 have also indicated that the cerebellar degeneration in this disorder is non- cell autonomous, as we have found that expression of mutant ataxin-7 protein in Purkinje cell neurons is not required for Purkinje cell degeneration. Of the possible mechanisms of non-cell autonomous Purkinje cell degeneration, we considered the hypothesis of glial dysfunction, and postulated that one target cell population was the Bergmann glia, whose cell bodies surround Purkinje neurons and ensheath Purkinje cell dendrites with their radial processes. When we analyzed Bergmann glia both immunohistochemically and ultrastructurally in SCA7 transgenic mice and in a SCA7 patient, we observed biochemical and histological abnormalities. We then showed that expression of mutant ataxin-7 protein in Bergmann glia surrounding the Purkinje cells is sufficient to cause ataxia and Purkinje cell degeneration in mice. In our most recent published work, we developed a powerful new mouse model wherein polyQ-ataxin-7 expression could be spatially and temporally regulated. In this model, an ataxin-7 92Q cDNA is flanked by loxP sites at the start site of translation in the murine PrP gene in a bacterial artificial chromosome (BAC). The PrP-floxed-SCA7-92Q BAC mice ubiquitously express mutant ataxin-7 protein in all neurons, including PCs, and develop cerebellar ataxia and histopathology. To determine which cell types are required for SCA7 disease pathogenesis, PrP-floxed-SCA7-92Q BAC mice were crossed with driver lines expressing Cre-recombinase in BG (Gfa2-Cre), or in PCs and IO (Pcp2-Cre). Gfa2-Cre;PrP-floxed-SCA7-92Q BAC and Pcp2-Cre;PrP-floxed-SCA7-92Q BAC bigenic mice each displayed a less severe phenotype than PrP-floxed-SCA7-92Q BAC littermates. Excision of ataxin-7-92Q from PCs and IO neurons not only protected against cerebellar atrophy, but also prevented BG pathology. Excision of ataxin-7-92Q from BG, however, did not prevent these pathological features, suggesting that BG pathology is non-cell autonomous in SCA7. When we created triple transgenic (i.e. Gfa2-Cre; Pcp2-Cre; PrP-floxed-SCA7-92Q BAC) mice, onset of cerebellar ataxia was delayed up to 20 weeks. Taken together, these findings indicate that BG, PCs, and IO neurons interact in an intimate and tightly interconnected network, and thus together contribute to SCA7.
We have also pursued therapy development for SCA7, and thus evaluated antisense oligonucleotide (ASO) knock-down as a treatment for SCA7 in representative mouse models. Through this work, we demonstrated that ataxin-7 ASO knock-down is a viable treatment for SCA7 retinal degeneration.
Autophagy regulation, nutrient-sensing, and mitochondrial homeostasis
The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving the retina and cerebellum, with a phenotype of ataxia and blindness. We discovered that loss-of-function of a zinc carboxypetidase protein (Nna1) leads to the rapid degeneration and death of Purkinje cell neurons in pcd mice. To determine the basis for pcd neurodegeneration, we obtained loss-of-function alleles of the Drosophila Nna1 orthologue (NnaD) and discovered that reduced NnaD function yields a semi-lethal phenotype, with survivors displaying phenotypes that mirror those observed in pcd mice. We then linked Nna1-NnaD proteins with basic mitochondrial metabolic functions by combining quantitative proteomics studies of pcd mice with biochemical studies and ultrastructural analysis of NnaD loss-of-function flies and pcd mice.
Our discovery of a role for Nna1 loss-of-function in mitochondrial dysfunction and our documentation of increased mitophagy in degenerating Purkinje cell neurons suggest mechanistic relationships between altered mitochondrial quality control and the regulation of mitochondrial autophagy. In light of its essential role in cellular homeostasis, we hypothesized that autophagy would be subject to sophisticated transcriptional regulatory control. To delineate regulation of autophagy in CNS, we developed a novel culture system for autophagy induction in primary cortical neurons, and we used this system to identify microRNA let-7 as a potent repressor of mTORC1 activity through suppression of the so-called amino acid sensing pathway. To further examine the link between the amino acid sensing pathway and autophagy, we studied MAP4K3, a kinase whose activation is dictated by amino acid status, and found that MAP4K3 phosphorylation of TFEB to inhibit TFEB nuclear localization and autophagy activation. Our results indicate that MAP4K3 lies upstream of mTORC1 in regulation of TFEB, constituting a powerful brake on autophagy.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor and cognitive impairment, accompanied by personality change and psychiatric illness. The motor abnormality stems from dysfunction of medium spiny neurons in the striatum. HD is caused by a CAG repeat expansion in the huntingtin (htt) gene, and is thus one of nine neurodegenerative disorders due to polyglutamine (polyQ) tract expansions in unrelated proteins. An intriguing feature of HD pathogenesis is the enhanced vulnerability of the striatum and certain regions of the cerebral cortex to neurodegeneration. An understanding of why subsets of neurons in the striatum and cortex preferentially degenerate in HD - despite widespread expression of mutant htt throughout the CNS - remains elusive. Determining the basis of cell-type specific neurodegeneration and neuron loss in HD, however, may be crucial to development of effective therapies for this currently untreatable disorder.
An important clue to selective vulnerability in HD has been the detection of specific mitochondrial oxidative phosporylation defects in the striatum of HD patients and modeling of HD in rodents by administration of mitochondrial toxins such as 3-nitropropionic acid. Another key feature of HD disease pathogenesis has been the production of truncated polyQ-expanded huntingtin peptide fragments that localize to the nucleus and there disrupt transcription. We discovered that Huntington’s disease mice display deranged thermoregulation, and traced the molecular basis of this phenotype to altered function of PPAR-g co-activator 1-a (PGC-1a), a transcription co-activator. To test the hypothesis that PGC-1a is a major factor in HD neurological dysfunction and neurodegeneration, we set out to determine if genetic over-expression of PGC-1a could compensate for the documented interference with PGC-1a function. We established an inducible transgenic system for PGC-1a, and used this approach to create HD transgenic mice that express increased levels of PGC-1a. We found that not only does PGC-1a ameliorate HD neurological phenotypes, but PGC-1a also virtually eradicates htt protein aggregates in the brains of HD mice. Further investigation of PGC-1a’s ability to enhance proteostasis led us to identify TFEB, a master regulator of the autophagy-lysosomal pathway, as a key target of PGC-1a.
These studies led my group to focus on the role of the PPAR’s. We identified PPARd transcription dysregulation as the basis for HD mitochondrial dysfunction stemming from PGC-1a interference, and established that PPARd is an essential regulator in CNS. Based upon this discovery, we repurposed a potent, selective PPARd agonist (KD3010), and documented its utility as a treatment for HD in a preclinical trial and in medium spiny neurons derived from HD patient iPSCs. We found that activation of RXR, a transcription factor that heterodimerizes with PPARd, is similarly capable of neuroprotection in HD and determined that PPARd neuroprotection stems from enhanced energy production and improved protein and mitochondrial quality control in the CNS.