Department of Ophthalmology and Visual Sciences
University of Utah Health Science Center
Salt Lake City, UT

EMMETT A. HUMBLE RESEARCH PROJECT
INPP5E, phosphoinositides and retinal degeneration

Current Research Interests
Dr. Baehr is interested in understanding mechanisms leading to retina disease and in developing gene-based therapies to address photoreceptor degeneration. His research will study the role of INPP5E, a phosphatidylinositide phosphatase present in photoreceptors, in the pathogenesis of photoreceptors. INPP5E mutations are associated with Joubert syndrome, featuring ataxia (lack of muscle control), hyperpnea (abnormal breathing patterns), abnormal eye and tongue movements, polydactyly (more than ten digits) and retinitis pigmentosa RP). The project is a continuation of a RRF project started in 2018 on which his laboratory has made excellent progress and submitted a paper to J. Biol. Chem. entitled “Deletion of INPP5E in retina impairs axoneme formation and disc morphogenesis.”  Below is an abstract of the paper:

INPP5E (pharbin) is a ubiquitously-expressed, farnesylated phosphatidylinositol polyphosphate 5’-phosphatase which modulates the phosphoinositide composition of membranes. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome, a syndromic ciliopathy affecting multiple tissues including brain, liver, kidney and retina (RP and LCA). INPP5E is located in primary cilia and mutations or loss of INPP5E have been associated with ciliary dysfunction. We show that in contrast to primary cilia, INPP5E is absent in the photoreceptor outer segment, a modified primary cilium dedicated to phototransduction. We generated Inpp5eF/F;Six3-Cre (in short retInpp5e-/-) mice which exhibit a rapidly progressing rod-cone degeneration completed at P21 in the central retina. Mutant rod outer segments (ROS) are truncated at P10 and form neither axoneme nor disc structures. Mutant cone outer segments (COS) form spherical structures as early as P8. At P10, mutant OS contain all phototransduction components and structural proteins. retInpp5e-/- rod connecting cilia appear unaffected by loss of INPP5E, but IFT-B/A particles accumulate in the proximal OS blocking IFT. The results show that ablation of INPP5E does not impair the secretory pathway, but blocks axonemal extension, and prevents disc morphogenesis.

In March of this year, the manuscript Dr. Baehr references above that was submitted in November 2020, was published in the Journal of Biological Chemistry.  2021 Publications

Plans for 2021

Specific Aims:
Aim 1. Determination of INPP5E target membranes in the inner segment.
The precise localization of INPP5E in the inner segment/connecting cilium will be determined based on confocal microscopy employing monospecific antibodies. Changes of the Golgi apparatus and other endomembranes will be assessed by electron microscopy.
Aim 2. Assessment of PI4P and PI(4,5)P2 levels
Levels of PIPs are tightly controlled by phosphatases (e.g., INPP5E) (16). We will determine and statistically evaluate levels of PI4P and PIP2 in control and mutant photoreceptors with PIPspecific monoclonal antibodies. We will further determine whether changes in F-actin at the proximal outer segment will prevent disc morphogenesis and extension of the axoneme.

Progress in 2020

The role of Afr-like protein 2 (ARL2) in photoreceptors

Research Interests
The purpose of Dr. Baehr’s project was to generate photoreceptor-specific knockouts of ARL2 in the mouse model and study the consequences on connecting cilium and axoneme structure.  The connecting cilium connect the outer segment of rods and cones to the inner segment where proteins are synthesized.  The axoneme consists of micro-tubule doublets providing stability to the outer segment.  Recent evidence shows that  ARL2 forms a stable complex with chaperones that enable tublin folding.  Missense mutations in ARL2 lead to con-rod dystrophy.

Dr. Baehr’s hypothesis was that absence of ARL2 in rods and cones will interfere with axoneme formation causing retina disease.  He proposed to generate rod- and cone- specific deletions of ARL2 and assess their phenotypes by confocal microscopy, electron microscopy and electroretinography (ERG).  The long term aim was to generate an AAV virus expressing ARL2 to rescue photoreceptor degeneration by intravitreal or subretinal injection, similarly described as for cone rescue in the NPHP5 knockout.

Specific Aims:
Aim 1: The Arl2 gene contains five exons, Dr. Baehr will delete exon 2 which inactivetes the gene.  He will monitor changes in rod photoreceptor development using confocal microscopy, transmission electron microscopy and electroretinography (ERG), anticipating that a connecting cilium may6 develop but probably not extend, and outer segments may form, but will degenerate.  He expects that photoreceptor degeneration will proceed slowly allowing a window of 1-2 weeks for analysis.

Aim 2: Dr. Baehr proposes to delete ARL2 in the adult mouse using tamoxifen induction of Cre expression.  He will monitor the effect of ARL2 deletion every five days, it takes 10 days to renew an outer segment,  by confocal and electron microscopy.  this research will elucidate the length of the proximal axoneme and possible changes in the mutant, and it is expected that absence of ARL2 affects stability of tubules, resulting in shortening of the axoneme.

Dr. Baehr’s work resulted in two publications and one manuscript submission.

Progress in 2019

The Function of the Centrosomal Protein, CEP164, associated with Joubert syndrome and LCA

Recessive mutations of CEP164 are associated with nephronophthisis (NPHP) and retinal degeneration (LCA). Mechanisms leading to photoreceptor cell death are unknown. We will generate conditional knockouts to study the consequences of CEP164 deletion in retina, and in rods and cones. The long-term goal is devise gene replacement vectors for retina-specific gene therapy.

INPP5E is a inositol phosphatase that causes Joubert Syndrome with Leber Congenital Amaurosis when mutated.  Dr. Baehr generated a mouse model in which INPP5E was deleted during embryonic development in the retina. His research showed that Rhodopsin accumulates in the retInpp5e-/- ONL and outer segment (OS) that never reach normal length.  The results show that deletion of INPP5E impairs export of OS proteins from the endoplasmic reticulum, disables the Golgi apparatus, affects retrograde IFT and prevents disc morphogenesis, presumably through changing the distribution of the phosphoinositides.  A manuscript draft was submitted later in the year.

Progress in 2018
The road to understanding INPP5E-Joubert Syndrome

Dr. Wolfgang Baehr (right) with a fellow University of Utah researcher

The road to understanding INPP5E-Joubert Syndrome. A poster was presented at ARVO 2018, and a paper is in preparation. Additional papers were published in 2018.

Purpose of INPP5E project: Mutations in INPP5E are associated with Joubert syndrome and MORM disease (mental retardation, truncal obesity, retinal dystrophy and micropenis). Joubert syndrome is a syndromic ciliopathy that includes ataxia, hyperpnea, abnormal eye and tongue movements, polydactyly and retinitis pigmentosa. To date, the function of INPP5E is unclear. We sought to study INPP5E function by generating retina- and photoreceptor-specific knockouts. Our goal is to identify the onset and cause of degeneration and devise gene-based therapies to ameliorate or cure the retina disease.

Results: Using qRT/PCR, we found that INPP5E is abundantly expressed in the retina, ten- to twenty-fold higher than in trachea or testis. In WT mouse rods, INPP5E locates to the inner segments and is absent in the outer segment, connecting cilium, and basal body. As absence of INPP5E in the OS is controversial, we expressed EGFP-INPP5E in rods by neonatal electroporation. The results show that INPP5E is excluded from WT outer segments and distributes to the ER surrounding the nucleus and Golgi apparatus, colocalizing with the Golgi marker, anti-giantin. We hypothesize that INPP5E may be required for membrane vesicle trafficking and ciliogenesis.