The La Spada Lab

at UC Irvine

Spinal and bulbar muscular atrophy

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

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 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.  Further studies revealed that SCA7 cerebellar degeneration involves glial dysfunction, as expression of mutant ataxin-7 in Bergmann glia can produce ataxia and Purkinje cell degeneration in mice.  We have also pursued dosage reduction as a therapy for SCA7 by identifying antisense oligonucleotides (ASOs) directed against the ataxin-7 gene, evaluated ataxin-7 ASO knock-down as a treatment for SCA7 retinal disease in mice, and found that intravitreal injection of ataxin-7 ASO is a viable treatment.  Using patient iPSCs and model mice, we have shown that SCA7 patient neural derivatives display altered mitochondrial function.  Through unbiased transcriptome analysis of SCA7 mice, we discovered coordinate down-regulation of genes controlling Ca++ flux, which we linked to impaired function of Sirtuin-1 (Sirt1), due to NAD+ depletion and PARP1 activation.  This work demonstrated that Sirt1 achieves neuroprotection by promoting calcium regulation, and that NAD+ dysregulation underlies Sirt1 dysfunction in SCA7, indicating cerebellar ataxias exhibit altered calcium homeostasis due to metabolic dysregulation, suggesting shared therapy targets.

In our most recent work on SCA7, we profiled the gene expression signatures in the cerebellum of SCA7 knock-in mice with a Purkinje cell-enriched single-nucleus RNA-seq method, highlighting early- and late-stage biological pathways uniquely impacting glial and neuronal populations.  The results of our snRNA-seq analysis of SCA7 266Q knock-in mouse cerebellum revealed significant expression alterations involving synapse organization genes, which we validated by directly examining synapse distribution and circuit function in SCA7 mice.  We detected a striking alteration of SCA7 PC zebrin-II subtype specification at the level of gene expression, and upon anatomical analysis, we discovered a dramatic obliteration of zebrin-II parasagittal striping in SCA7 knock-in mice.  When we evaluated zebrin-II parasagittal striping patterns in related polyglutamine SCAs, we discovered that this surprising dysregulation of Purkinje cell zebrin-II subtypes extends to other polyglutamine disease mouse models, suggesting that dysfunctional zebrin-II subtype maintenance is a defining feature of polyglutamine ataxias.  We expect that this work will be of broad interest, because it implicates altered development as a key factor in the pathogenic cascade of cerebellar neurodegenerative disorders, with implications for the neurodegenerative disease field.

Huntington’s disease and related disorders

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.  We then 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, improved protein and mitochondrial quality control, and decreased neuroinflammation due to microglia activation.  We have also linked PPARd dysregulation to pathogenesis of Parkinson’s disease and Alzheimer’s disease, and we have ongoing studies of pathogenesis using induced pluripotent stem cell-derived neurons, microglia, and astrocytes from patient and isogenic control lines.

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the rapid and progressive loss of motor neurons in the brain and spinal cord, with sparing of sensory neurons.   Overall, ALS shows familial inheritance in about 10% of cases, while the remaining 90% of patients are likely explained by a combination of environmental and genetic factors, with heritability estimated at about 50%.  ALS is thus a heterogeneous group of progressive motor neuron disorders that typically lead to death from respiratory collapse two to five years after disease onset, although dominantly inherited ALS4 is a juvenile-onset familial form of ALS characterized by very slowly progressive motor neuronopathy.  ALS4 is caused by mutations in the gene for Senataxin, a 2,677 amino acid protein with a highly conserved DNA/RNA helicase domain.  To determine the mechanistic basis for SETX motor neuron toxicity, we developed ALS4 mouse models and documented how SETX mutant mice recapitulate nuclear clearing of TDP-43, accompanied by TDP-43 cytosolic mislocalization, which is the hallmark pathology observed in human ALS patients.

We have also considered the role of SETX as a modifier of the most common form of familial ALS, which is due to a hexanucleotide repeat expansion in the C9orf72 gene.  We found that decreased SETX expression enhances C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, while transgenic human SETX is a potent suppressor of toxicity resulting from mutant C9orf72 expression and associated arginine dipeptide repeat production.  Finally, we discovered that SETX model mice display an immunological signature consisting of clonally activated CD8 T cells, and replacement of SETX mutant bone marrow with bone marrow transplanted from a normal donor is sufficient to rescue neuromuscular disease phenotypes.  All of these findings reveal how SETX performs myriad roles in cellular and molecular processes relevant to motor neuron health and molecular homeostasis.

In a parallel line of investigation, we are seeking genetic modifiers of ALS disease risk and have identified variants that regulate alternative polyadenylation as a highly significant determinant of likelihood to develop sporadic ALS.  One high priority variant reduces the expression of a gene whose protein product appears to modulate TDP-43 homeostasis and thereby places carriers at greatly increased risk of developing ALS.  These parallel lines of investigation are thus yielding novel therapy targets which may pave the way toward future treatments for this devastating disorder.

MAP4K3 as a central node in mTORC1 nutrient sensing regulation

Neurons are particularly dependent on robust quality control mechanisms to maintain cellular homeostasis and functionality throughout their long lifetime.  Autophagy is an evolutionarily conserved lysosomal degradation pathway that maintains cellular homeostasis by recycling damaged organelles, aggregated proteins, and aged cellular components. In recent years, the molecular mechanisms that regulate autophagy have garnered tremendous scientific interest due to the importance of this cellular process for neural development, function, and survival. Indeed, dysregulation of autophagy is implicated in many neurodegenerative diseases.  Activation of autophagy can be induced in response to cellular nutrient deprivation or stress.  Amino acid depletion is a particularly powerful activator of autophagy, as autophagy-mediated protein degradation yields free amino acids for protein synthesis and energy production.  The mechanistic target of rapamycin complex 1 (mTORC1) negatively regulates autophagy in response to nutrient sensing, and excessive activation of mTORC1 is thought to play a role in aberrant autophagy associated with various pathological conditions.

Our lab recently identified a member of the mitogen activated protein kinase (MAPK) family, MAP4K3, as an amino acid-dependent negative regulator of mTORC1, and ongoing studies are seeking the regulatory pathway by which MAP4K3 activates mTORC1 in the CNS.  We have also discovered that MAP4K3 is a major regulator of autophagy, functioning upstream of mTORC1.  In response to amino acid starvation, MAP4K3 phosphorylates TFEB at serine 3 in the cytosol. We found that loss of MAP4K3 is sufficient for TFEB nuclear localization, transcription of TFEB-regulated lysosomal genes, and productive autophagy.  To determine the physiological relevance of MAP4K3 phosphorylation of TFEB at serine 3, we recently employed genome engineering to derive knock-in mice expressing a phospho-null (TFEB S3A) or phospho-mimetic (TFEB S3E) version of TFEB.  Studies of TFEB S3E and TFEB S3A mice are currently underway.

One main goal of this work is to develop drug inhibitors of MAP4K3.  To achieve this goal, we performed in silico screening for small molecules that interfere with MAP4K3 open pocket dimerization, and evaluated 13 compounds in a series of secondary and tertiary assays, identifying three promising hits.  We will build on our MAP4K3 inhibitor translational drug discovery work by honing in on the most promising lead compounds through structure-activity relationship generation of a compound series, coupled with an independent in silico screen and kinase inhibitor potency testing followed by a critical path of validation assays, and we will test if our lead MAP4K3 inhibitor(s) can rescue relevant disease phenotypes in appropriate mouse models.