Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C > T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with fronto-temporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement.

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor-Rakeb syndrome and Parkinson's disease (PD), providing protection against α-synuclein, Mn 2+ , and Zn 2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders.mitochondria | lysosome | flippase | α-synuclein | P5-type ATPase N euronal fitness depends on optimal lysosomal function and efficient lysosomal delivery of proteins and organelles by autophagy for subsequent breakdown (1, 2). Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of Parkinson's disease (PD) associated with dementia, which is caused by mutations in ATP13A2/PARK9 (3). Mutations in or knockdown (KD) of ATP13A2 lead to lysosomal dysfunctions, including reduced lysosomal acidification, decreased degradation of lysosomal substrates (4), impaired autophagosomal flux (4, 5), and accumulation of fragmented mitochondria (5, 6). By contrast, overexpression (OE) of Ypk9p (i.e., the yeast ATP13A2 ortholog) protects yeast against toxicity of α-synuclein (7), which is the major protein in Lewy bodies, the abnormal protein aggregates that develop inside nerve cells in PD. This protective effect of ATP13A2 on α-synuclein toxicity is conserved in yeast, Caenorhabditis elegans, and rat neuronal cells (7). Because ATP13A2 imparts resistance to Mn 2+ (7-9) and Zn 2+ (10-12), it was proposed that ATP13A2 may function as a Mn 2+ (7-9) and/or Zn 2+ transporter (10-12).ATP13A2 belongs to the P5 subfamily of the P-type ATPase superfamily, which comprises five subfamilies (P1-5) of membrane transporters. P-type ATPases hydrolyze ATP to actively transport inorganic ions across membranes or lipids between membrane leaflets (reviewed in ref. 13). During the transport cycle, a phosphointermediate is formed on a conserved aspartate residue (14). The human P5-type ATPases are divided into two groups, P5A (ATP13A1) and P5B (ATP13A2-5), but their transport specificity has not been established (14-16).P-type ATPases comprise a membrane-embedded core of six transmembrane (TM) helices (M1-6) that form the substrate binding site(s) and entrance/exit pathways for the transp...

Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. ATP13A1 complemented a deletion of the yeast P5A ATPase SPF1, while none of ATP13A2-5 could complement either the loss of SPF1 or that of the single P5B ATPase YPK9 in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.

Identification of the substrate of P5A-ATPases would throw light on an important general process in the ER that is still not fully understood. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.