Expanding the Spectrum of AP5Z1‐Related Hereditary Spastic Paraplegia (HSP‐SPG48): A Multicenter Study on a Rare Disease

Amy C. Ogilvie, MS, Pedro Gonzalez-Alegre, MD, PhD,† and Jordan L. Schultz, PharmD†* Department of Psychiatry, Carver College of Medicine at the University of Iowa, Iowa City, Iowa, USA Department of Epidemiology, College of Public Health at the University of Iowa, Iowa City, Iowa, USA Department of Neurology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA Department of Neurology, Carver College of Medicine at the University of Iowa, Iowa City, Iowa, USA Division of Pharmacy Practice and Sciences, University of Iowa College of Pharmacy, Iowa City, Iowa, USA

presenting with neuropathy, ataxia, dystonia, and parkinsonism in addition to spastic paraplegia (SP). AP5Z1 codes for the ζsubunit of the AP-5 complex, implicated in vesicular-mediated intracellular sorting and trafficking of cargo proteins, 1 -Functional studies demonstrate the accumulation of multilamellar structures (endolysosomes) in SPG48 skin fibroblasts. 2 Here, we screened 2035 HSP patients from 3 tertiary centers (Athens, University of Athens [UOA]; London, University College London; and Paris, Paris, Sciences & Lettres [PSL], Assistance Publique-Hôpitaux de Paris [APHP]) for mutations in AP5Z1 and performed functional studies in 2 cases with pathogenic variants in AP5Z1. We also present a literature review for AP5Z1 cases, a pathway analysis, and follow-up data on previously reported patients, where available (Supplementary Material 1, 4, and 5).
In total, 9 patients from 8 unrelated families carrying biallelic pathogenic variants in AP5Z1 were identified ( Fig. 1; Supplementary Material 1). We show that AP5Z1-related disease usually presents with a combination of late-onset SP (mean: 54.3 ± 5.3 years) and axonal neuropathy. Other frequent clinical features in our cohort were urinary incontinence, hearing loss, and visual impairment. Interestingly, 1 patient had epileptic seizures. Brain magnetic resonance imaging (MRI) was available for 6 patients. Leukoencephalopathy and thinning of the corpus callosum (TCC) were present in 1 patient and "ears of the lynx" sign, a "moth-eaten" appearance of the basal ganglia, and TCC in another (Fig. 2). The remaining 4 patients had normal MRI.
Our In this study we expanded the phenotypic and genotypic spectrum of SPG48 showing that SPG48 is a slowly progressing, late-onset, complicated HSP manifesting with SP, axonal neuropathy, cognitive impairment in line with the SPG48 patients reported so far [and in Refs 1 and 3] and, interestingly, epileptic seizures (patient G, Supplementary Material 3). Epileptic seizures have not been previously reported in SPG48; however, they are well described in other HSP subtypes, such as SPG11 and SPG15, which are functionally related to SPG48 3 and in many lysosomal storage diseases. Indeed, our functional studies on SPG48 cell lines confirm defects in endosome and lysosome homeostasis. We also confirm here previously described neuroimaging findings ("ears of the lynx" sign, TCC, and white matter lesions) in a subgroup of patients.
To date, no specific therapies are approved for HSP. Of note, treatment strategies are proposed in complex forms of HSP such as cholesterol-lowering agents for HSP-CYP7B1 (SPG5A), as CYP7B1 gene is involved in the degradation of cholesterol into primary bile acids. 4 A randomizedcontrolled trial showed that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in the serum of SPG5 patients, 5 and evolocumab (PCSK9 inhibitor) is currently evaluated in a phase 2 clinical trial (NCT04101643). In addition, "tideglusib" (GSK3β inhibitor) was tested on iPSC neuronal lines of an SPG11 patient and decreased cell death. 6

FIG. 3.
Functional studies on SPG48 patient fibroblasts. (A) Whole-cell Western blots of patient-derived fibroblast lines (denoted by their mutation) including controls, loaded at equal protein levels and probed with antibodies against SPG11, SPG15, AP5Z1, AP1G1 (AP1G1 is used as a loading control). Note the loss of AP5Z1; (B) Immunofluorescence microscopy of SPG48 patient-derived fibroblasts doubled labeled with antibodies against EEA1 to mark early endosomes and LAMP1 to mark late endosomes/lysosomes. In AP5Z1-deficient patient lines, there is an increase in the brightness and size of the LAMP1-positive puncta. Scale bar = 20 μm; (C) Quantification of the increase in LAMP1 spot intensity was performed for 2 independent SPG48 patient-derived fibroblast lines using Volocity. At least 20 cells were quantified per condition. Data show mean of 3 independent experiments and results of a 2-tailed t test: (*P < 0.05; **P < 0.01). [Color figure can be viewed at wileyonlinelibrary.com] Our study strengthens the evidence supporting autophagic dysfunction as one of the underlying molecular pathways in HSP 7 and further expands the phenotypic spectrum of AP5Z1-related SPG48.